XXIV Fungal Genetics Conference at Asilomar

Cell Biology (1-75)
Population and Evolutionary Biology (76-130)
Genomics and Proteomics (131-234)
Industrial Biology and Biotechnology (235-254)
Biochemistry and Secondary Metabolism (255-290)
Gene Regulation (291-356)
Developmental Biology (358-411)
Animal and Plant Pathogens; Fungal-Host Interactions (412-565)
Model systems (566-568)
Other (571-601)

Plenary session abstracts

CELL BIOLOGY

1. Metarhizium anisopliae perilipin homolog MPL1 regulates lipid metabolism, appressorial turgor pressure and virulence. Chengshu Wang and Raymond J. St. Leger. Department of Entomology, University of Maryland, College Park, MD, 20742, USA. stleger@umd.edu

Cells store lipids in droplets. Studies addressing the control of lipid-based energy homeostasis of mammals identified proteins of the PAT-domain family, such as perilipin that surround the lipid droplets. Factors that mediate lipid storage in fungi are still unknown. Here we describe a gene (Mpl1) in the insect pathogen M. anisopliae that has structural similarities to mammalian perilipins. Consistent with a role in lipid storage, Mpl1 is predominantly expressed when M. anisopliae is engaged in accumulating lipids and ectopically expressed GFP-tagged MPL1 localized to lipid droplets. Mutant M. anisopliae lacking MPL1 have thinner hyphae, fewer lipid droplets, particularly in appressoria and a decrease in total lipids. Mpl1 therefore acts in a perilipin-like manner with an evolutionary conserved function in lipid metabolism. However, animal and fungal proteins have also been selected to cope with different tasks. Thus, turgor generation by Mpl1 appressoria is dramatically reduced indicating that lipid droplets are required for solute accumulation. This was linked with reduced ability to breach insect cuticle so that Mpl1 is a pathogenicity determinant. Blast searches of fungal genomes revealed that perilipin homologs are found only in pezizomycotinal ascomycetes and occur as single copy genes. Expression of Mpl1 in yeast cells, a fungus that lacks a perilipin-like gene, blocked their ability to mobilize lipids during starvation conditions.

2. Characterization of AtmA and PrpA reveals a correlation between ROS and apical dominance in Aspergillus nidulans. Camile P. Semighini¹, Gustavo H. Goldman² and Steven D. Harris¹. ¹Plant Pathology, University of Nebraska Lincoln; ² Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Brazil Presenter’s e-mail: csemighini2@unl.edu

The DNA damage response (DDR) is a protective mechanism that ensures the maintenance of genomic integrity. DDR is composed of many conserved proteins such as the Ataxia telangiectasia mutated (ATM) kinase and the Poly (ADP)- ribose polymerases (PARP). We are using the filamentous fungus Aspergillus nidulans as a model system and we believe that certain aspects of the DDR in this organism may reflect the unique organization of fungal hyphae. We have characterized the function of the A. nidulans ATM (AtmA) and PARP (PrpA) homologues. Besides their expected roles in DDR, those homologues also make an unexpected contribution to hyphal morphogenesis. In the absence of DNA damage, the deltaatmA strain is defective in polarized hyphal growth while the deltaprpA^+/- mutant produces an increased number of germ tubes per spore and displays an increase in the formation of lateral branches. Staining of a wild type strain with Nitro Blue Tetrazolium (NBT) revealed that a gradient of Reactive Oxygen Species (ROS) localize to the hyphal tip. NBT staining of deltaatmA and deltaprpA^+/- strains correlated their hyphal morphology defect with disruption of ROS’ specific localization. Our results suggest that ROS gradient may enforce apical dominance.

3. Roles of Aspergillus nidulans cdc42 and rac1 homologues in polarized growth. Aleksandra Virag and Steven Harris. Plant Science Initiative, N235 Beadle Center, University of Nebraska, Lincoln, NE 68588. E-mail: avirag2@unlnotes.unl.edu

In Saccharomyces cerevisiae the Rho GTPase Cdc42 is required for polarity establishment and maintenance. In contrast, Cdc42 homologues in filamentous Euascomycetes and Basidiomycetes do not appear to be important for polarity establishment, but rather for polarity maintenance and the establishment of secondary polarity axes-branches. The latter fungi also possess another Rho GTPase, Rac1, which is important for polarization but is absent from S. cerevisiae. Here we utilized the model Euascomycete Aspergillus nidulans to characterize the connection between Cdc42 and Rac1, and their presumed downstream components and effectors. Potential effectors include putative polarisome components (SpaA and SepA), as well as proteins required for microfilament and microtubule organization. Results suggest that Cdc42 and Rac1 have overlapping functions in polarity establishment. Preliminary genetic observations have identified, in addition to SpaA, other downstream effectors such as two distinct paxillin homologues. Based on our results we propose that Cdc42 and Rac1 cooperate in polarity-related processes.

4. Pheromone receptor Bar2 of the homobasidiomycete Schizophyllum commune. Susann Jezewski* & Erika Kothe. Friedrich-Schiller-University, Dept. of Microbiology, Neugasse 25, 07743 Jena, Germany; E-mail: Susann.Jezewski@uni-jena.de

The tetrapolar mating system of Schizophyllum commune allows easy discrimination of mating types and is associated with two genetic systems, which contain the A and B genes. Each B locus codes for one pheromone receptor, belonging to the family of G protein coupled seven transmembrane domain receptors, and several pheromones, small lipopeptide ligands. The interaction of a pheromone receptor and non-self ligands results in nuclear migration and clamp cell fusion, which are part of sexual development. There is no induction of B gene dependent sexual development by pheromones of self-specificity, of which several are encoded within each locus. The recognition of pheromones by multi-specific pheromone receptors and the distinction of self and non-self are necessary for a compatible mating process. The function of the pheromone/receptor system implies the localization of the receptor protein in the tip of growing hyphae or in clamp cells of S. commune. The visualization of the Balpha2 receptor protein (Bar2) should be possible by a fusion of the intracellular C-terminus of Bar2 with fluorescent proteins, like GFP or DsRed. However, the transient process of clamp cell formation and the low expression of mating type genes make the determination of receptor molecules complicate. By means of real time PCR, a quantification of pheromone receptor mRNA will be possible in different mutant backgrounds under special conditions.

5. Does the single Dothideomycete MAT1-1-1 protein harbor domains for activities performed by multiple MAT1-1 proteins from Sordariomycetes? Banu Metin¹, Shun-Wen Lu², B. Gillian Turgeon³. ¹Department of Food Engineering, Middle East Technical University, 06531 Ankara, Turkey, e102950@metu.edu.tr. ²Department of Plant Pathology, Cornell University, Ithaca, NY 14853, USA, swl2@cornell.edu ³Department of Plant Pathology, Cornell University, Ithaca, NY 14853, USA, bgt1@cornell.edu

In filamentous ascomycetes, mating type loci exist as two alternate forms, MAT1-1 and MAT1-2, defining two mating types. While MAT1-2 contains a single gene with an HMG box domain in all ascomycetes examined so far, MAT1-1 shows differences. In Sordariomycetes, the MAT1-1 idiomorph contains three genes: MAT1-1-1 encoding a protein with an alpha 1 domain, MAT1-1-2 encoding a protein with an HPG domain, and MAT1-1-3 encoding a protein with an HMG box domain. In contrast, in Dothideomycetes, the MAT1-1 idiomorph harbors a single gene, which has been believed to contain only the alpha 1 domain. Careful comparison of the MAT1-1-1 and MAT1-2-1 protein sequences, however, reveals conserved motifs, including one resembling the HMG box sequence, in both proteins. To determine if this second HMG motif is functionally important in both MAT1-1-1 and MAT1-2-1, point mutations were made in MAT1-1-1 (W152A, G160F, V186A, and Y198*) and MAT1-2-1 (W138A, I158A, H170E) sequences of Cochliobolus heterostrophus. These mutated genes are being used to transform the MAT-deleted C. heterostrophus strain C4-41.7 and the strains will be tested for mating competence. Effects of the mutations on mating ability will address the question of whether the single MAT1-1-1 proteins of Dothideomycetes have evolved to carry out all activities provided by multiple MAT1-1 proteins of Sordariomycetes.

6. Analysis of a protein complex required for septation in the filamentous ascomycete Ashbya gossypii. Andrea Walther and Juergen Wendland. Carlsberg Laboratory, Yeast Biology, DK-2500 Valby, Copenhagen, Denmark

Hyphae of filamentous fungi are compartmentalized in regular intervals by septation. Key steps for septation are (i) the selection of a septal site, (ii) the formation of protein complexes at this site and (iii) actin-myosin ring constriction, which is accompanied by chitin deposition. Previously we have analyzed the role of the A. gossypii BUD3 gene in septum formation. Here we present evidence that Bud3 belongs to a protein complex at septal sites that involves septins and Bud4. We have analyzed the function of the septin encoding gene CDC3 and the A. gossypii homolog of the Saccharomyces cerevisiae BUD4 gene. Deletion of CDC3 yields viable albeit slow growing mutants that exhibit a penetrant septation defect. Aberrant chitin deposition at random cortical positions is found in the cdc3 mutant bearing similarity to the septation defect found in bud3 strains. In contrast to cdc3 strains, bud3 hyphae also show correct septation. Mutant bud3 strains are able to sporulate while cdc3 strains do not form spores. Both, Cdc3 and Bud4 were localized at septal sites using GFP-tagged proteins. Using the S. cerevisiae two-hybrid system we established protein-protein interactions among these proteins. This establishes a network of proteins required for correct septum formation and further on may be used as a landmark to direct lateral branching.

7. Characterization of Protein O-mannosyltransferases in Aspergillus nidulans. Thanyanuch Kriangkripipat and Michelle Momany*. Department of Plant Biology, University of Georgia, Athens, Georgia 30602 USA tkriang@plantbio.uga.edu

Protein O-mannosyltransferases (PMTs) are found in bacteria, fungi, and animals but are not present in plants. In fungi, PMTs are divided into three subfamilies, PMT1, PMT2 and PMT4 and each species has 3-7 PMTs. Aspergillus nidulans possesses three PMTs, Pmt1, Pmt2 and Pmt4. Single pmt deletion mutants are viable. Each Δpmt mutant exhibits different phenotypes when characterized by growth at different temperatures, morphology and sensitivity to chemicals disturbing cell wall synthesis. Double mutants show additive phenotypes. The Δpmt1 mutant has hyphal tip lysis and produces aberrant conidiophores at 42^OC. The Δpmt2 mutant cannot send out germ tubes at 42^OC. The

Δpmt4 mutant has swollen hyphae and produces aberrant conidiophores at 42^OC. The Δpmt1Δpmt2 double mutant is viable and has additive phenotypes of Δpmt1 mutant and Δpmt2 mutant. The Δpmt2Δpmt4 double mutant is viable but very sick and forms a microcolony only when an osmotic stabilizer is added to the medium. Lower temperatures and osmoticum can partially restore wildtype hyphal growth and conidiation of these Δpmt mutants except for the Δpmt2Δpmt4 double mutant. Our results suggest that protein O-mannosylation is important for cell wall integrity of A. nidulans.

8. The autophagy gene, atgA, is required for conidiophore development and starvation-induced hyphal growth in Aspergillus fumigatus. Daryl Richie, Michael Miley, David Askew . University of Cincinnati, Pathology and Laboratory Medicine

Autophagy is a highly conserved eukaryotic process in which cytoplasmic constituents are sequestered within a double membrane vesicle and delivered to the vacuole for the recycling of macromolecules. The purpose of this study was to determine the role of autophagy in the opportunistic human pathogen Aspergillus fumigatus. Autophagy was disrupted by deletion of the A. fumigatus atgA gene, which encodes the homolog of Saccharomyces cerevisiae Atg1p, a serine threonine kinase needed for induction of autophagy. The autophagy mutant showed abnormal conidiophore development and reduced conidiation, which could be corrected by supplementing the medium with additional nitrogen. When hyphal plugs of the autophagy mutant were transferred from rich medium to starvation medium they displayed reduced radial growth as compared to wild type. However, the addition zinc or manganese to starvation medium allowed the autophagy mutant to grow to the same degree as wild type. In rich medium, the autophagy mutant was hypersensitive to EDTA. However, the growth of the autophagy mutant in EDTA could also be restored to wild-type levels by the addition of excess zinc and manganese to the medium. Our results suggest that A. fumigatus uses autophagy to recycle intracellular stores of nitrogen to support optimal conidiation in a nitrogen limiting environment. Furthermore, our results suggest that autophagy in A. fumigatus allows for the recycling of limiting trace elements needed for optimal radial growth.

Coprinus cinereus fruit body exhibits remarkable stipe elongation in the final phase of development: the stipe elongates 8 times in one night. A dominant elongationless1-1 (eln1-1) mutant defective in stipe elongation was first induced from the dikaryon. A recessive elongationless1-2 (eln1-2) mutant was then induced from the monokaryotic fruiting strain, CopD5-12. During stipe elongation in the wild type, the cylindrical component cells of the stipe elongate with their diameter unchanged, resulting in slender stipe cells. In the mutants, however, the cylindrical cells increase in diameter and the rate of elongation is much reduced, resulting in globular stipe cells. Genetic analysis mapped the eln1 locus 0.3 cM apart from his5 on chromosome I. We transformed an eln1-2 mutant with BACs assigned near his5, and found that two BAC clones, s2F7 and s14B9, have the complementing activity. We then defined the genomic region of the eln1 gene within about 30 kb by testing subclones from s14B9 for their complementing activity. We are now trying to identify the eln1 gene by testing PCR-amplified fragments from the 30-kb subclone.

10. A Rho1 Homologue From Ustilago maydis, Uro1, is Required for Normal Cell Growth. Zhanyang Yu¹, Cau D. Pham¹, Scott E. Gold², and Michael H. Perlin¹. ¹University of Louisville, Department of Biology, Program on Disease Evolution, Louisville, Kentucky, USA and ²University of Georgia, Department of Plant Pathology, Athens, Georgia, USA

In the pathogen of maize, U. maydis, a carefully orchestrated network of signaling is used to ensure that development proceeds in an orderly fashion. Uro1 is a homologue of the Schizosaccharomyces pombe and Saccharomyces cerevisiae Rho1 proteins, members of the family of Rho GTP-binding proteins that includes Cdc42 and Rac1. We have cloned and characterized the uro1 gene encoding this U. maydis homologue. Over- expression of either wild type or constitutively-active mutant Uro1 caused a slight reduction in mating efficiency. A diploid strain in which a single copy of uro1 was disrupted, similarly showed reduced “Fuz” reaction on charcoal agar relative to the wild type progenitor strain. This singly-disrupted diploid was unable to produce galls when injected into maize. To circumvent the likely lethality of disruption in haploid strains, the uro1 gene was placed under control of the crg promoter, so as to allow its expression in arabinose media or its shut down in glucose. When uro1 expression was shut off, cells failed to reproduce; however, they could recover if transferred to arabinose medium up to 72 h after the initial shut off by glucose. Several possible interactors of Uro1 were identified by yeast 2- hybrid, including Ump2 (an ammonium transporter), Smu1 (the Pak-like kinase involved in mating), a CDC24 homologue (a RhoGEF), and, quite interestingly, PTEN (phosphatase and tensin homologue deleted on chromosome 10), which acts as a tumor suppressor gene in mammalian cells. We propose that, among its several possible roles, Uro1 acts as an inhibitor of PTEN in U. maydis, and normally prevents the latter’s action to promote arrest of cell division and/or apoptosis sequelae. We will provide additional experimental data which address this hypothesis.

11. The Nuclear Migration Protein NUDF Associates with BNFA and NUDC at Spindle Pole Bodies in Aspergillus nidulans. Kerstin Helmstaedt, Karen Meng, Silke Busch, Özgür Bayram, Oliver Valerius and Gerhard H. Braus. Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen, Grisebachstr. 8, D-37077 Göttingen, Germany. Tel. +49-551-3919693, Fax +49-551-393820, khelmst@gwdg.de

In Aspergillus nidulans, nuclear division depends on microtubuli, the motor dynein and nuclear distribution proteins like NUDF. Applying tandem affinity purification, we isolated a unique NUDF-associated protein, which we named BNFA (Binding of NUDF). An A. nidulans bnfA deletion strain did not show a nud phenotype indicating that a protein with redundant function might exist. A GFP-BNFA fusion localized to spindle pole bodies (SPBs) throughout the cell cycle. This position was depended on NUDF, since in a nudF6 strain BNFA localized mainly to dots in the cytoplasm. In a yeast two-hybrid screen using BNFA as bait, we found that BNFA is a dimer and that a link might exist to the septation signalling pathway. In a candidate approach, we analysed the putative NUDC-NUDF interaction in A. nidulans. Although NUDC-GFP alone was localized to immobile dots at the cortex, we found a direct interaction between NUDF and NUDC in yeast two-hybrid experiments, which depended on NUDF's WD40 domain. Applying bimolecular fluorescence complementation microscopy, we showed that in vivo NUDF and NUDC interact also at spindle pole bodies throughout the cell cycle and at immobile dots at the cortex.

12. Regulation of hyphal morphogenesis and the DNA damage response by the Aspergillus nidulans ATM homolog, ATMA. Iran Malavazi¹, Camile P. Semighini², Marcia Regina von Zeska Kress¹, Steven D. Harris², Gustavo H. Goldman¹. ¹Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, São Paulo, Brazil; ²Plant Science Initiative and Department of Plant Pathology, University of Nebraska, Lincoln, USA.

Ataxia telangiectasia (A-T) is an inherited disorder characterized by progressive loss of motor function and susceptibility to cancer. The most prominent clinical feature observed in A-T patients is the degeneration of Purkinje motor neurons. Numerous studies have emphasized the role of the affected gene product, ATM, in the regulation of the DNA damage response. However, in Purkinje cells, the bulk of ATM localizes to the cytoplasm and may play a role in vesicle trafficking. The nature of this function, and its involvement in the pathology underlying A-T, still remain unknown. Here we characterize the homolog of ATM (AtmA) in the filamentous fungus Aspergillus nidulans. In addition to its expected role in the DNA damage response, we find that AtmA is also required for polarized hyphal growth. We demonstrate that an atmA mutant has a severe defect in the control of nuclear proliferation and fails to generate a stable axis of hyphal polarity. Notably, cytoplasmic microtubules display aberrant cortical interactions at the hyphal tip. Our results suggest that AtmA regulates the function and/or localization of landmark proteins required for the formation of a polarity axis in A. nidulans. We propose that a similar function may contribute to the establishment of neuronal polarity. Financial Support: FAPESP and CNPq Brazil, Nebraska Research Foundation, USA

Endocytosis is a process through which substrates enter a cell without passing through the plasma membrane but via invagination of membrane and formation of intracellular vesicle. Rab7 regulates the trafficking between early and late endosomes as well as late endosomes to lysosomes in the endocytic pathway. We have isolated Le.Rab7, from Lentinula edodes, that is highly homologous to Rab7 (78% similarity). Two candidates, receptor for activated C kinase-1 (Le.RACK1) and Rab5 GTPase (Le.Rab5) were isolated as interacting partners of Le.Rab7 through in vivo and in vitro protein interaction assays. Le.Rab7 and Le.RACK1 express strongest in primordium, while Le.Rab5 expressed constitutively in all stages. In situ hybridization showed that the three transcripts were mainly localized at active growth regions of fruiting bodies. They strongly expressed at the outer region of trama cells and subhymenium in the hymenophore of gill tissue and at the prehymenophore in the ¡§eye organ¡¨ of young fruiting body. The protein localization of Le.Rab7 was also found in fruiting bodies. The existence of endocytosis in mycelium and gill tissue was shown by the internalization of endocytic dye, which co-localized with Le.Rab7 protein. It is the first report on the presence of endocytosis in basidiomycete. Le.Rab7, Le.Rab5 and Le.RACK1 may contribute to the production of basidiospores and the differentiation of mycelial cells to different cell types at the gill tissue.

14. Analysis of two cell end marker proteins, TeaA and TeaR, in Aspergillus nidulans. Norio Takeshita, Sven Konzack, Yuhei Higashitsuji and Reinhard Fischer. Applied Microbiology, University of Karlsruhe, Germany.

The interplay of the actin and the microtubule (MT) cytoskeleton in polarized growth of fungi has recently been revealed. In Schizosaccharomyces pombe, Tea1 is a key protein in this process. Tea1 is transported to the plus ends of MTs by the kinesin Tea2, and is delivered to cell ends by hitchhiking with the growing MTs. Mod5, which is posttranslationally modified by prenylation, anchors Tea1 at the cell ends. These three proteins were indentified by screening for polarity mutants. At the cell ends, Tea1 recruits formin which initiates actin assembly and the establishment of cell polarity. Tea1 and Tea2 homologues were identified in the Aspergillus nidulans genome (named TeaA and KipA), whereas Mod5 could not be identified due to sequence similarity. kipA mutants showed mislocalization of the Spitzenkorper and hence curved hyphae. GFP-KipA accumulated at the MT plus ends. teaA mutants showed a similar but not identical phenotype to kipA mutants. GFP-TeaA localized to one point in the apex of hyphal tips. To test whether the function of S. pombe Mod5 was conserved, we searched the A. nidulans genome for proteins with a C-terminal prenylation motif (CAAX). From 22 identified proteins one (536 amino acids) was likely to serve a TeaA-anchorage function. We named it TeaR. teaR mutants indeed displayed a phenotype similar to the kipA mutant. GFP-tagged TeaR localized to the membrane at hyphal tips. Their putative interactions will be analyzed.

15. The Aspergillus nidulans putative kinase KfsA plays a role in septation and is required for efficient asexual spore formation. Norio Takeshita, Anne Blumenstein, KayVienken and Reinhard Fischer Applied Microbiology, University of Karlsruhe, Germany

In eukaryotes cytokinesis has to be a well-controlled process to guaranty that each cell receives a nucleus. The process is well characterized in Saccharomyces cerevisiae, where a safeguard-machinery (MEN = mitotic exit network) has evolved to prevent cytokinesis before proper nuclear segregation. One important component is the kinase Kin4, which inhibits the MEN and prevents precautious cell division when the spindle position checkpoint is activated. In Aspergillus nidulans, nuclear division and cytokinesis are not coupled in hyphae, resulting in multinucleate compartments. In contrast, metulae, phialides and conidia are uninucleate. Here, we describe the role of a putative Kin4- related kinase, KfsA (kinase for septation) in the control of septum formation in A. nidulans. KfsA showed similarity to Kin4 only in the kinase domain but is conserved among filamentous fungi. kfsA mutants did not show any obvious phenotype in hyphal growth but produced less conidia. Conidiophores of kfsA mutants often contained several septa and a certain number of metulae harboured two nuclei and the corresponding phialides remained anucleate. The latter result suggests a septum-control mechanism of KfsA during nuclear distribution in the yeast-like growth of metulae, which guarantees that cytokinesis only occurs if both cells contain a nucleus. A GFP-KfsA fusion protein localized to septa in hyphae and the conidiophore and to the cortex of hyphae.

16. Nuclear anarchy: asynchronous nuclear division in Ashbya gossypii. Amy S. Gladfelter. Dartmouth College, Department of Biological Sciences amy.gladfelter@dartmouth.edu

Multinucleated cells are essential for disease processes such as tumorigenesis and fungal infection, but little is known about how the unique organization of multinucleated cells influences the control of mitosis. We have found that nuclei divide asynchronously in the filamentous ascomycete Ashbya gossypii. In these cells, neighboring nuclei are in different stages of the nuclear division cycle despite being only a few micrometers apart and bathed in the same cytoplasm. Similar asynchronous nuclear division also has been observed in multinucleated tumors and other filamentous fungi, but how independent nuclear behavior is established in the context of a common cytoplasm remains to be discovered. Using in vivo time-lapse microscopy, automated image analysis, and molecular genetic approaches we are currently testing two main hypotheses to determine when and how nuclear asynchrony develops: 1.) Nuclear asynchrony emerges in the G1/S transition due to the unequal distribution of cell cycle regulators among nuclei and 2.) The unequal localization of cell cycle regulators among nuclei arises due to differences in Spindle Pole Body age and/or Nuclear Pore Complexes between neighboring nuclei. With these studies we are identifying mechanisms regulating the G1/S transition and delineating how nuclear asymmetries are established in multinucleated cells.

17. CRE-1, a regulator of catabolite repression is involved in determining PKA-dependent polarity of N. crassa Carmit Ziv, Rena Gorovits and Oded Yarden. Dept. of Plant Pathology and Microbiology, The Otto Warburg Minerva Center for Agricultural Biotechnology Faculty of Agricultural, Food and Environmental Quality Sciences The Hebrew University of Jerusalem Rehovot 76100, Israel

Polar hyphal elongation is a complex event in fungal growth and is affected by multiple factors. The Neurospora crassa mcb gene encodes a regulatory subunit of the cAMP-dependent protein kinase A (PKA). We have identified a 19bp deletion at the 5' UTR of mcb temperature-sensitive allele which we propose impairs transcript integrity of the regulatory subunit and thus may result in increase PKA activity and a complete loss of growth polarity at restrictive temperatures. As a link between the PKA pathway and carbon source sensing has been established in Aspergillus and yeasts, we tested the effect of different carbon sources on mcb morphology. Surprisingly, when grown in the presence of fructose, but not in either glucose or sucrose, the typical bulbous mcb phenotype was completely suppressed. As glucose and fructose equally support N. crassa wild-type growth we concluded that the differential effect on mcb involves a regulatory, rather than a metabolic, mechanism. To determine the involvement of carbon catabolite repression (CCR) in determining PKA-dependent polarity, we inactivated a key CCR regulator, cre- 1. The delta-cre-1 strain showed reduced growth on glucose vs. fructose, in addition to altered hyphal morphology and impaired CCR, as evident by changes in inv and adh-1 expression and amylase and beta- gal activity. The deletion of cre-1 in an mcb background partially restored polarity when grown on glucose, indicating that downstream CRE-1-regulated elements can compensate for polarity defects caused by mis-regulated PKA activity. These results suggest a role for the CRE-1-mediated cellular response to carbon source in determining PKA- dependent polar growth in N. crassa.

18. Altering COT1 kinase phosphorylation sites affects its function in N. crassa Carmit Ziv, Rena Gorovits and Oded Yarden. Dept. of Plant Pathology and Microbiology, The Otto Warburg Minerva Center for Agricultural Biotechnology Faculty of Agricultural, Food and Environmental Quality Sciences The Hebrew University of Jerusalem Rehovot 76100, Israel

The Neurospora crassa COT1 kinase is the founding member of the conserved NDR protein kinase family. Defects in cot-1 confer a highly pleotropic phenotype typified by a restricted, colonial, growth. In vitro analysis of mammalian NDR demonstrated the involvement of conserved phosphorylation sites in the regulation of NDR kinase activity. Using antibodies against the phosphorylated human NDR protein we detected the presence of the two conserved phosphorylation sites in N. crassa COT1: Ser-419, a putative MOB2-stimulated autophosphorylated site and Thr- 591, a putative Ste20-like kinase-phosphorylated site required for NDR activity. To determine the significance of the phosphorylation states of COT1 on cell morphology in vivo, we analyzed strains harboring a 5xmyc::cot- 1 fusion allele at the cot-1 locus, in which these conserved regulatory residues have been substituted by either Glu or Ala to mimic the phosphorylated and unphosphorylated states, respectively. The resulting 4 strains (designated S419E, S419A, T591E and T519A) were viable, yet some presented altered morphology. The importance of Ser-419 was evident by a reduced growth rate of both S419E and S419A strains. In addition S419A exhibited a dramatic abnormal, hyper-branched, morphology and produced a ropy-like colony. Altering Thr-591 resulted in a milder effect; strain T591E showed reduced aerial hyphae and enhanced carotenoid production, while T591A displayed a reduced growth rate. Determining COT1 kinase activity levels, localization and interaction with additional proteins in these strains will help elucidate the in vivo role of phosphorylation in governing specific functions of COT1.

19. The BEM46-like protein is essential for hyphal growth from ascospores of Neurospora crassa and targeted to the ER. Mercker M, Alves S, Weiland N, and F. Kempken. Botanisches Institut, Christian-Albrechts-Universität zu Kiel, Olshausenstr. 40, 24098 Kiel, Germany; fkempken@bot.uni-kiel.de

BEM46 proteins are evolutionary conserved members of the α/β-hydrolase super family. However, to date, their exact role remains unknown. Therefore, to better understand the cellular role of BEM46 and its homologs, we used the model organism Neurospora crassa in conjunction with bem46 RNAi, overexpression vectors, and repeat induced point (RIP) mutation analyses. These studies clearly demonstrated that BEM46 is required for cell-type- specific hyphal growth and indicate a role of BEM46 in maintaining polarity. For example, vegetative hyphae, perithecia, and ascospores develop normally, but hyphae germinating from ascospores exhibit a strong loss-of-polarity phenotype. Moreover, we found that the BEM46 protein is targeted to the ER, and localizes at or close to the plasma membrane. BEM46 thus being a new ER marker for filamentous fungi, and the first for the model organism Neurospora crassa. Thus our data suggest BEM46 plays a role in a signal transduction pathway that is involved in determining or maintaining cell-type-specific polarity. This finding also implies a higher degree of differentiation of fungal hyphae than currently expected.

20. Functional analysis of histidine-containing phosphotransmitter gene ypdA in Aspergillus nidulans. Natsuko Sato, Kentaro Furukawa, Tomonori Fujioka, Osamu Mizutani, and Keietsu Abe. Molecular and Cell Biology, Tohoku University, Sendai, Japan.

The high-osmolarity glycerol (HOG) response pathway responding to osmotic stimuli has been well studied in Saccharomyces cerevisiae. Sln1p-Ypd1p-Ssk1p proteins organize a two-component signalling (TCS) unit in the upstream of the HOG pathway, and negatively regulate the downstream Hog1p mitogen-activated protein kinase (MAPK) cascade. We previously revealed that a filamentous fungus Aspergillus nidulans possesses all counterparts of the components of S. cerevisiae HOG pathway. Deletion of Ypd1p, the TCS histidine-containing phosphotransmitter of S. cerevisiae, is known to cause lethality because of constitutive activation of Hog1 MAPK. While, S. cerevisiae possesses only one set of TCS unit consisted of Sln1p and Ypd1p, A. nidulans has been predicted to have 15 histidine kinases and some of them are thought to interact with the unique YpdA. Thus, the TCS pathway of A. nidulans might be more complex and robust than that of yeast . In addition, YPD1 is essential in S. cervisiae but not in Shizosaccharomyces pombe. In the present study, in order to examine in vivo functionality of A. nidulans ypdA gene, we constructed an ypdA delta strain conditionally expressing the ypdA gene under the control of A. nidulans alcA promoter and investigated its phenotypes under the ypdA-repressed condition. Downregulation of ypdA transcription caused sever growth inhibition. We observed a constitutive phosphorylation of HogA MAPK in A. nidulans ypdA delta. These results suggest that YpdA is a essential component of the upstream of A. nidulans HOG (AnHOG) pathway, and the growth inhibition caused by ypdA delta would be attributed to disorder of signalling through the AnHOG pathway.

21. Calcium plays an essential role in the toxicity of the Penicillium chrysogenum antifungal protein PAF in Aspergillus nidulans. Ulrike Binder¹, Sairah Saeed², Andrea Eigentler¹, Diana Bartelt², Florentine Marx¹. ¹Biocenter, Division of Molecular Biology, Innsbruck Medical University, Fritz-Pregl Strasse 3, A-6020 Innsbruck, Austria ²Dept. of Biological Sciences, Institute for Biotechnology, St. John's University, 8000 Utopia Parkway, Queens, NY 11439

The small, basic and cysteine-rich protein PAF is secreted by Penicillium chrysogenum and exhibits growth inhibitory activity against various members of filamentous ascomycetes including human and plant pathogens. We are interested in the elucidation of the mechanism of action of this antifungal protein. Previous investigations showed that PAF toxicity is related to plasma membrane hyperpolarization and the activation of ion channels. We hypothesize that PAF toxicity results at least in part in the perturbation of the calcium homeostasis. In this study we show, that minimal concentrations of calcium neutralize PAF activity. Furthermore, we detected a PAF–induced elevation of the intracellular calcium concentration in an Aspergillus nidulans strain that expresses the calcium sensitive photoprotein aequorin. This increase in intracellular calcium concentration was abrogated by the addition of the extracellular calcium chelator EGTA, which indicates that an influx of extracellular calcium is responsible for this effect. Verapamil, an L-type calcium channel blocker, positively influenced the growth of PAF–exposed A. nidulans. Further studies are presently carried out to characterize the role of calcium homeostasis in the toxicity of PAF in detail. This work is supported by the Austrian National Bank and the Austrian Science Foundation to F.M. and in part by NIGMS R15GM077345 to DB.

22. Functional Comparison of CDC42 and RAC1 in the Dimorphic Fungus Ustilago maydis. Andrea Hlubek and Michael Bölker. Fachbereich Biologie, Universität Marburg, Karl-von-Frisch-Str. 8, 35032 Marburg, Germany; email: ahlubek@web.de

Cdc42 and Rac1 are small GTPases, which regulate various cellular processes like morphogenesis, vesicle trafficking, cytokinesis and cell polarity. Guanine nucleotide exchange factors (GEFs) convert the GTPases to their active form, GTPase activating proteins (GAPs) stimulate the GTPase activity, thereby leading to the formation of the inactive state. In the dimorphic fungus U. maydis the GTPases Cdc42 and Rac1 are highly similar but trigger distinct cellular responses. We could show that U. maydis Cdc42, but not Rac1, complements the lethal phenotype of the temperature sensitive cdc42-1 allele in S. cerevisiae. To define the regions that are responsible for the specificity of these proteins, we generated a set of chimeric proteins, which were checked for complementation of both U. maydis and S. cerevisiae mutants. These experiments demonstrate that the region between amino acids 41 and 56 is necessary and sufficient to determine the specificity of Cdc42 and Rac1. In vivo and in vitro experiments let us hypothesize that specificity is regulated by interaction with the GEFs, which we are currently characterizing. Another important spatial regulator of GTPases is the Guanine dissociation inhibitor RhoGDI (Rdi1), which is important for proper localization of GTPases and therefore necessary for distinct cellular responses. Interestingly, the deletion of rdi1 in U. maydis interferes with filament formation induced by Rac1 overexpression.

23. The Role of UncA, an Unc104-related kinesin motor protein, in Aspergillus nidulans. Nadine Zekert, Daniel Veith and Reinhard Fischer. Applied Microbiology, University of Karlsruhe, Germany.

We have previously shown that in Aspergillus nidulans conventional kinesin, KinA, is required for fast hyphal growth and the Kip2 family motor, KipA, for the establishment and maintenance of hyphal polarity. Here, we characterize UncA, an Unc-104 related motor protein. UncA (1631 amino acids) contains a motor domain, a forkhead association domain and a PH domain. A homologue of this motor was shown to be required for mitochondrial motility along microtubules in Neurospora crassa. However, in A. nidulans mitochondrial movement rather depends on actin. Deletion of uncA in A. nidulans caused slower hyphal extension with more branching, suggesting a role in polar growth. The fact that uncA deletion was not lethal suggested redundant functions of other kinesins. KinA was a good candidate for this, but surprisingly, a kinA-uncA double mutant was viable and displayed the same branching phenotype as the uncA mutant while its growth phenotype resemble the kinA mutant. UncA- GFP fusion displayed a spot-like distribution in the cytoplasm. The spots showed rapid bidirectional movement. The introduction of a rigor-mutation into uncA inhibited this movement and led to a rod-like staining pattern. Experiments to show whether these rods are microtubules or e.g. mitochondria are under way. A second Unc104-related motor was additionally found in the genome and named UncB (671 amino acids). The function will be characterized by deletion and subcellular localization studies.

24. Aspergillus fumigatus rasA: a non-essential Ras homolog that regulates germination, mitosis and hyphal morphology. Jarrod Fortwendel and Judith Rhodes. University of Cincinnati, Cincinnati, Ohio, USA.

Ras subfamily genes are known to control growth and differentiation in a wide variety of microorganisms. The majority of Ras homologs in pathogenic organisms act as molecular switches that regulate morphogenesis and, in some cases, virulence. The A. fumigatus genome contains two members of the prototypical Ras family of small GTPases, rasA and rasB. Of these Ras sub-family genes, rasA shares 98% homology to the essential rasA gene of Aspergillus nidulans. In this study, we report that deletion of A. fumigatus rasA caused delayed germination, nearly absent radial outgrowth, and decreased hyphal mass. Growth of the rasA deletion mutant on solid agar revealed an extremely compact colony morphology with markedly decreased conidiation. Although conidiation was reduced and delayed, the conidial viability is at wild-type levels. Growth in liquid media caused development of meandering hyphae with increased diameter, when compared to wild type. The rasA deletion mutant was resistant to protoplast preparation, even when using twice the levels of cell wall digestion enzymes that are sufficient for the wild type organism. Normal mitotic events in the mutant were also disturbed, as staining with propidium iodide revealed many small nuclei that were irregularly positioned throughout the hyphae. These data show that rasA is important to a wide range of cellular processes during the complex growth patterns of the important human pathogen A. fumigatus.

25. A PTS2 peroxisomal targeting sequence in ApsB is required for functioning of non-nuclear MTOCs in Aspergillus nidulans. Daniel Veith, Nadine Zekert, Rüdiger Suelmann and Reinhard Fischer. Applied Microbiology, University of Karlsruhe, Germany.

Peroxisomes are a diverse class of organelles involved in different physiological processes in eukaryotic cells. A special class of peroxisomes is represented by the fungal Woronin body, in which one protein, Hex1 in Neurospora crassa, forms a hexagonal crystal and serves a structural function in plugging septal pores after damage of hyphae. Here, we suggest a new class of peroxisomes, which is involved in microtubule formation. In Aspergillus nidulans, septum- associated microtubule-organizing centres (MTOCs) are polymerizing cytoplasmic microtubules in addition to MTOCs at the spindle pole bodies (SPBs). Previously, we identified a novel MTOC-associated protein in A. nidulans, ApsB, whose absence affected microtubule formation from septal MTOCs more than from SPBs, suggesting different organization of the two protein complexes. In a yeast-two-hybrid screening, we identified the peroxisomal protein HexA as an interacting protein of ApsB and discovered that ApsB is targeted to peroxisomes via a PTS2 peroxisomal targeting sequence or by a piggy-bag mechanism together with HexA. Peroxisomal localization of ApsB was necessary for normal function of septal MTOCs. ApsB translocated into Woronin bodies was not active, suggesting two distinct populations of septal peroxisomes, the Woronin bodies and the ones required for MTOC activity.

26. Evolution of the mechanisms that regulate cellular morphogenesis in fungi: a comparative genomics approach. Haoyu Si, Kaimei Xu, and Steven D. Harris*. Plant Science Initiative and Department of Plant Pathology, University of Nebraska, Lincoln, NE, 68588-0660. *Contact: Sharri1@unlnotes.unl.edu

The defining feature of fungal cells is polarized growth, whereby cell wall deposition is confined to a discrete location on the cell surface. The annotation of multiple fungal genome sequences has revealed that the signaling modules and morphogenetic machinery involved in polarized growth are largely conserved across the fungal kingdom. Nevertheless, fungal cells exhibit a diverse variety of shapes that are largely based on two growth patterns: hyphae and yeast. We suggest that these different patterns reflect variation in the mechanisms that spatially and temporally regulate cellular morphogenesis. To test our hypothesis, we are using a comparative genomics approach to examine the functional evolution of the regulatory modules that specify budding patterns in S. cerevisiae. The landmarks that specify the axial budding pattern (Bud3, Bud4, and Axl2) are weakly conserved in the Pezizamycotina. We are in the process of deleting and charactering these homologues in A. nidulans, Neurospora crassa, and Fusarium graminearum. Our preliminary results show that Bud3 and Bud4 are required for septum formation and, suggest that Bud3 may serve as a GEF (guanine nucleotide exchange factor) for the GTPase Rho4. We propose that the ancestral function of the axial markers may be the regulation of septin organization, which, upon the loss of hyphal growth in S. cerevisiae, allowed them to be co-opted into the module that regulates axial budding. By contrast, only two of the landmarks that specify the bipolar budding pattern (Rax1 and Rax2) are conserved in the Pezizamycotina. Moreover, our deletion analyses have failed to uncover an obvious role for these homologues in hyphal morphogenesis.

27. Survival following exposure to hydrogen peroxide (H₂O ₂) is developmental stage- dependent in Aspergillus nidulans. Sairah Saeed and Diana Bartelt. Dept. of Biological Sci., St. John’s University, Queens, NY 11439

Among the earliest antimicrobial defense mechanisms is the release of H₂O₂ formed during the respiratory burst into the phagosome during the degranulation process. Aspergillus sp. are opportunistic pathogens, causing a high rate of mortality in immunosuppressed patients. The ability to survive exposure to hydrogen peroxide is important in the onset of invasive Aspergillosis . We have found that survival of A. nidulans after exposure to hydrogen peroxide is dependent upon the stage of development, the density of the culture and the concentration of H₂O ₂. Exposure of 10 hr. mycelial cultures to 10 mM peroxide causes greater than 90 percent killing while greater than 90 percent of cells in an 18 culture at the same density survive. Stability of H₂O ₂ in the medium is also dependent upon the density of the culture, the stage of development and H₂O ₂ concentration. There appears to be a positive correlation between viability and the ability of the culture to destroy H₂O ₂. The induction of catalase and/or peroxidase activity following exposure to H₂O ₂ is examined. [Supported by NIGMS R15GM077345-01A1].

28. Genetic and molecular analysis of a temperature-sensitive mutant un-17 carrying a mutation in the poly (A) polymerase gene in Neurospora crassa. S. Tanaka, N. Takayanagi, K. Murasawa, C. Ishii, and H. Inoue. Lab. of Genet., Fac. of Sci., Saitama Univ., Saitama, Japan.

un-17 mutant was isolated as a temperature-sensitive (ts) mutant in Neurospora crassa. This mutant shows several interesting phenotypes. In restriction temperature (37 C^o), the growth immediately stops and the cells are going to die. This death is suppressed by adding of cycloheximide or conditions repressing growth. Even in permissive temperature, it shows female sterility and is deficient in production of extracellular superoxide dismutase SOD4. To know the function of the un-17 gene, we tried to clone the gene. We mapped a position of the mutation to right of linkage group III, and searched a DNA fragment which complements the ts phenotype of un- 17 mutant from a N. crassa genome library. The cloned gene had homology to yeast poly (A) polymerase (PAP), and the PAP-homolog gene of un-17 had a mutation of one base substitution. New strains which express a recombinant PAP derived from either wild-type or un-17 strain were constructed and analyzed. The recombinant PAP of wild-type indicated the activity of PAP, and un-17 strains which were induced a constitutive expression of wild-type PAP recovered mutant phenotypes of original un-17 strains. These data suggest that un-17 encodes the essential PAP gene.

29. Two-component response regulators, ChSsk1p and ChSkn7p, additively regulate high-osmolarity adaptation and fungicide sensitivity in Cochliobolus heterostrophus. Kosuke Izumitsu. Kyoto University, Graduate School of Agriculture, Kyoto, Japan

Filamentous ascomycetous fungi possess many histidine kinases and two conserved response regulators, Ssk1p and Skn7p, in their two-component signaling systems. We previously reported that the fungi-unique group III histidine kinase regulates high-osmolarity adaptation and iprodione/fludioxonil fungicide sensitivity by controlling the phosphorylation of Hog1-type mitogen-activated protein kinase (MAPK) in filamentous ascomycetes. Here, we have characterized the response regulator genes ChSsk1 and ChSkn7 in the Southern corn leaf bright fungus Cochliobolus heterostrophus. Both Chssk1- and Chskn7-disrupted mutants showed little sensitivity to high-osmolarity stress and moderate resistance to the iprodione/fludioxonil fungicides. The phosphorylation of Hog1-type MAPK BmHog1p induced by high-osmolarity stress and fungicide treatments was only regulated by ChSsk1p, indicating that ChSkn7p has roles in high-osmolarity adaptation and fungicide sensitivity that are independent from the activation of BmHog1p. The ChSsk1/ChSkn7 double mutants clearly showed higher sensitivity to osmolar stress and higher resistance to fungicides than the single mutants. The dose responses of the double mutants fit well with those of the group III histidine kinase-deficient strain. These results suggest that in filamentous ascomycetes, the Ssk1-type and Skn7-type response regulators control high-osmolarity adaptation and fungicide sensitivity additively with differential mechanisms under the regulation of the group III histidine kinase. This study provides evidence that filamentous fungi have a unique two-component signaling system that is different from yeast and is responsible for high-osmolarity adaptation and fungicide sensitivity.

30. Functional topology of the growing hyphal tip. Michael Köhli¹, Philipp Knechtle¹, Virginie Galati¹, Kamila Boudier¹, Robert Roberson² and Peter Philippsen¹. ¹ Biozentrum, University of Basel, Klingelbergstrasse 50/70, 4056 Basel, Switzerland. ² School of Life Sciences, Arizona State University, Tempe AZ 85287, USA.

Growth of fungal hyphae depends on initial definition of a polarity axis, tip-directed intracellular transport and efficient polarized secretion. A complex protein network whose components are conserved in eukaryotes coordinates these processes. While molecular data have become available only recently, a hallmark of polarized growth in filamentous fungi was described long time ago: the Spitzenkörper, a vesicle-based, dynamic structure that is found in the tip of growing hyphae. In order to identify proteins colocalizing with the Spitzenkörper, we monitored GFP-fusions to 14 polarity factors of Ashbya gossypii. Interestingly, in several cases, a given GFP-fusion showed localization patterns that differed between hyphae of the same mycelium, even if these hyphae were spaced less than 50 µm apart from each other. We show that these differences in localization correlate with hyphal growth speed. In slow growing hyphae, the investigated polarity factors mostly localized to the apical cortex independently of the factor’s identity. Contrary, in fast growing hyphae, at least four functional zones defined by distinct sets of polarity factors were observed: the Spitzenkörper, the central apical cortex, the entire apical cortex and the extended apical cortex. The putative polarisome components AgSpa2, AgPea2 and AgBni1, the Rab-GTPase AgSec4 and the exocyst components AgSec3 and AgExo70 were found to localize to the Spitzenkörper. These findings suggest that the Spitzenkörper is directly involved in regulation of the actin cytoskeleton and secretion but not in polarity maintenance or establishment per se.

31. Variation in nuclear DNA content and chromosomes among parent isolates and progeny in Phytophthora infestans. Mursel Catal, Pavani Tumbalam, Willie Kirk, Gerard Adams. Department of Plant Pathology, Michigan State University, East Lansing, MI 48824.

The DNA content of nuclei of various isolates of P. infestans and progeny from controlled matings was assessed by laser flow cytometry. Variation in size of chromosomes among isolates was evaluated with Southern hybridizations, and some virulence genes were located on chromosome fragments. Progeny varied in virulence, race, mating type, cold tolerance and genetic markers. In each controlled mating a small proportion of progeny were unusual, showing secondary homothallism, loss of virulence, and the combined markers of both parents. Variation in DNA content of nuclei among parents and, more interestingly, among the unusual progeny are presented and discussed in relation to chromosome patterns and ploidy.

32. The Aspergillus nidulans nuclear pore complex protein An-Nup-2 plays a novel role in mitosis but is not essential for nuclear transport. Sarine Markossian and Stephen A. Osmani. The Ohio State University, Columbus, Ohio, USA. markossian.1@osu.edu

The nuclear pore complex (NPC) regulates nuclear trafficking and is composed of ~30 subunits called nucleoporins (Nups). In yeast, Nup2p has been shown to facilitate nuclear transport. Unlike yeast Nup2p, Aspergillus nidulans Nup2 (An-Nup2) localizes to chromatin during mitosis but to the NPC during interphase. This indicates An-Nup2 may play a role during mitosis. An-nup2 is essential and its deletion causes mitotic defects. We therefore speculate that the localization of An-Nup2 to mitotic chromatin is important for mitosis. To test this hypothesis, a domain study was performed to define the An-Nup2 domain responsible for its mitotic translocation to chromatin and an antibody was generated against An-Nup2. A domain spanning from aa 400 to aa 1200, which encompasses a basic stretch of amino acids, a coiled coil region, and two potential nuclear localization sequences (NLS), is sufficient to locate An-Nup2-GFP to the NPC during interphase and to DNA during mitosis. The An-Nup2 antibody was used for immunofluorescence and successfully stained the nuclear periphery during interphase and chromatin during mitosis confirming the An-Nup2-GFP localization. Most importantly, the heterokaryon rescue technique was used to define if nuclear transport and/or mitosis is defective without An-Nup2. An-Nup2 deleted cells were not deficient in nuclear transport of NLS-dsRed suggesting that the lethality caused by An-Nup2 deletion is due to mitotic defects and not nuclear transport defects. In conclusion, there is emerging evidence that the localization of An-Nup2 to mitotic chromatin is essential for mitosis although An-Nup2 may not be essential for nuclear transport.

33. Cell biology of the mode of action on Neurospora crassa of an antifungal agent from Hypocrea atroviridis. Verena Seidl^{1, 2}, Christian P. Kubicek² and Nick D. Read ¹. ¹Institute of Cell Biology, University of Edinburgh, UK, ²Institute of Chemical Engineering, Vienna University of Technology, Austria.

The well characterized model system, Neurospora crassa, was used as a host for the necrotrophic mycoparasite Hypocrea atroviridis (Trichoderma atroviride) to get new insights into the mechanisms underlying mycoparasitism by Hypocrea/Trichoderma spp. Although mycoparasitic Hypocrea/Trichoderma spp. have a wide host range, their interactions are mostly studied with plant pathogens, and many aspects of the mycoparasitic process are not well understood. Hypocrea/Trichoderma spp. produce highly effective antifungal compounds and antibiotics, but very little is known about their specific modes of action. H. atroviridis displayed antagonism against N. crassa in plate confrontation assays. Before making contact, N. crassa hyphae showed a severely stressed phenotype characterized by slow, bud-like growth and hyperbranching. Spore germination was also strongly inhibited. To characterize the stress response in more detail we are studying the effect of the inhibitory compound on various subcellular processes, including Spitzenkörper formation, endocytosis, membrane potential and mitochondrial activity. The inhibitory compound is constitutively secreted by H. atroviridis independent of the presence of a host. It was isolated and purified and could be shown to be a small, non-volatile organic molecule and it is currently being characterized by mass spectrometry.

34. Identification of novel genes involved in hyphal fusion in filamentous fungi. Alex Lichius and Nick D. Read. Fungal Cell Biology Group, Institute of Cell Biology, University of Edinburgh, UK. (A.Lichius@sms.ed.ac.uk)

Vegetative hyphal fusion is essential for the establishment of an interconnected colony network, and is a key feature of the filamentous fungi. A small number of proteins involved in regulating hyphal fusion have been identifed in Neurospora crassa. Hyphal fusion and appressorium formation in fungal pathogens share a number of features in common, including adhesion, cell swelling, secretion of cell wall degrading enzymes, penetration and MAP kinase signalling. We are asking the question: how much does the machinery involved in appressorium formation share in common with that involved in hyphal fusion? To answer this question and identify new proteins involved in these processes, we are screening 42 knockout mutants of genes in N. crassa which are orthologs of genes encoding proteins involved in appressorium formation and/or are pathogenicity determinants in the rice blast fungus, Magnaporthe oryzae. Processes in which these Magnaporthe proteins play a role include surface recognition, cell adhesion, appressorium induction, appressorium development, and host cell penetration. Conidial anastomosis tube (CAT) fusion is being used as simple and convenient experimental system for this genetic screen. We are also assessing hyphal fusion in mature colonies and use a wide range of live-cell analytical techniques to characterize the mutants. Acknowledgements: AL is funded by the School of Biological Sciences studentship of the University of Edinburgh.

35. A Cytoplasmic Nuclear Remnant is involved in the Segregation of Nucleolar proteins in Aspergillus nidulans. Leena Ukil, Colin De Souza, Hui-Lin Liu and Stephen A. Osmani, Department of Molecular Genetics, The Ohio State University, 484 W 12th Ave, Columbus, OH 43210, Email: leenaukil@yahoo.com

The nucleolus is a prominent nuclear structure whose mitotic segregation is poorly understood. During yeast mitosis the nucleolus segregates intact with rDNA. In contrast, during open mitosis the nucleolus is disassembled then reassembled. Aspergillus nidulans nuclei undergoes partially open mitosis, which is an evolutionary intermediate between open and closed mitosis. We therefore determined how A. nidulans nucleoli are segregated during mitosis. Unlike Saccharomyces cerevisiae, few A. nidulans nucleolar proteins segregate with DNA. Instead we have defined two patterns by which different nucleolar proteins segregate during mitosis: (1) Dispersal into the cytoplasm at the onset of mitosis but with some protein remaining bound to DNA, (2) A novel pattern in which nucleolar proteins remain in a nuclear remnant, distinct from daughter nuclei, before re-accumulating into daughter nucleoli during G1. Dual labeling of nucleolar proteins and nuclear envelope markers reveal that the nucleolar remnant is generated as a result of a double nuclear envelope fission event. This double fission occurs around a nucleolar protein mass during telophase. This mechanism generates two transport competent daughter nuclei and a very transient nucleolar remnant containing class 2 nucleolar proteins. This study indicates A. nidulans undergoes mitotic disassembly then reassembly of its nucleolus, as do higher eukaryotes, and that generation of daughter nuclei occurs via a double fission mechanism, not a single fission as occurs in yeasts. We suggest the novel mitotic nuclear remnant we have defined serves as a storage pool from which equal distribution of nucleolar proteins occur. It may also serve as a sink for unwanted cytoplasmic proteins or RNAs that gain access to nuclei during mitosis and may be a positional cue for the double fission.

36. Genetic dissection of phosphate acquisition in Neurospora crassa. Wayne K. Versaw, Yuan Jin, Lauren Baber and Kelly Guido. Dept of Biology, Texas A&M University, College Station, TX 77843

Depending on environmental conditions, N. crassa obtains phosphate via a constitutive, low-affinity uptake system or a phosphate-repressible, high-affinity uptake system. The high affinity system consists of two structurally unrelated transporters, PHO-4 and PHO-5. Homologous transporters with similar functional attributes have been well described in the yeast Saccharomyces cerevisiae. Low affinity phosphate transporters also have been described in yeast but based on mutant analyses, the single corresponding N. crassa homolog, PHO-6, does not appear to have a significant role in phosphate uptake. We describe a putative transporter, PHO-7, and evidence suggesting that PHO-7 constitutes the low-affinity phosphate transport system in N. crassa. PHO-7 represents a novel class of transport proteins that is conserved in filamentous fungi but is not represented in the yeasts.

37. The Aspergillus nidulans snxA1 and nimA5 mutations interact to affect mitotic spindle structure. Yulon Stewart, Ryan Day, Kirk Jackson, Michael Jackson, and Sarah Lea McGuire. Millsaps College, 1701 N. State St., Jackson, MS, 39210.

Both the nimA and snxA genes interact with nimX^cdc2 to affect mitosis in Aspergillus nidulans. nimA affects the nuclear import of nimX^cdc2, while the snxA1 mutation is a cold-sensitive suppressor of the nimX2^cdc2 mutation. snxA1 leads to abnormal nuclear morphology at 17^oC. To better understand effects of the snxA1 mutation on cells and the relationship between snxA and nimA, we generated strains expressing GFP-tubA (alpha-tubulin) and various combinations of snxA and nimA mutations. At 17^oC snxA1/GFP-tubA cells had severe nuclear defects, thickened hyphae, abnormal spindle structures, and abnormal interphase microtubule arrays. Mitotic spindles were highly variable in length. Some spindles had no nuclei attached to them, while others were bifurcated or trifurcated and had fragmented, variably condensed nuclei along their lengths. Similar abnormal nuclei and spindle structures were observed when snxA1/nimA1/GFP-tubA cells and snxA1/nimA5/GFP-tubA cells were germinated at 32^oC and upshifted to 44^oC for 3 hours, suggesting that the effects of snxA1 on nimA are not allele-specific. After 3 hours at 44^oC, 69% of snxA1/nimA1/GFP-tubA cells had abnormal nuclei, and 56% had abnormal spindles; similar results were obtained with snxA1/nimA5/GFP-tubA cells. Confocal microscopy of the abnormal spindles shows highly unusual spindle structures, which are more severe in cells carrying the snxA1/nimA5 double mutant; snxA1/nimA1 double mutant cells often have significantly shortened spindles. Efforts to clone the snxA gene are ongoing and should aid in the understanding of the interactions of the snxA and nimA genes in mitotic control. Supported by NIH R15GM55885 and NIH RR016476 from the MFGN INBRE program of the NCRR.

38. Withdrawn

39. Cell Biology of Biotrophic Blast Invasion by the Rice Blast Fungus Magnaporthe grisea Prasanna Knakanala¹, Kirk Czymmek², Barbara Valent¹. ¹Kansas State University, Manhattan, KS ²University of Delaware, Newark, DE

The filamentous ascomycete fungus, Magnaporthe grisea is a hemibiotroph that causes rice blast disease accounting for 60 million tons of grain loss annually. The blast fungus produces intracellular Invasive Hyphae (IH) to colonize the plant, but detailed cellular and molecular mechanisms by which this occurs were not studied. We applied live- cell imaging to characterize spatial and temporal development of IH and plant responses inside successively-invaded rice cells. Early loading experiments with the endocytotic tracker, FM4-64, showed dynamic plant membranes around IH. These hyphae showed remarkable plasticity and recruited plant cell components. IH exhibited pseudohyphal growth and were sealed in plant membrane, termed the Extra-Invasive Hyphal Membrane (EIHM). The fungus spent up to 12 hours in the first cell, often tightly packing it with IH. IH that moved into neighboring cells were biotrophic, although they were initially thinner and grew more rapidly. IH in neighboring cells were wrapped in EIHM with distinct membrane caps at the hyphal tips. Time-lapse imaging showed IH scanning plant cell walls before crossing them, and transmission electron microscopy showed crossing occurring at pit fields. This and additional evidence strongly suggest that IH co-opt plasmodesmata for cell-to-cell movement. Our studies have revealed insights into a novel hemibiotrophic strategy employed by the blast fungus. Analysis of biotrophic blast invasion will significantly contribute to characterization of secreted fungal effectors that impact normal plant processes. To further understand the molecular basis of this infection strategy we have employed laser microdissection to identify the effectors involved in these processes.

40. Identification of transcription factor regulated by OS-2 MAP kinase in Neurospora crassa. Azusa Shiozawa¹, Shinpei Banno², Kazuhiro Yamashita¹, Setsuko Watanabe¹, Fumiyasu Fukumori¹, and Makoto Fujimura¹. ¹ Dept of Life Sciences, Toyo Univ, Gunma, Japan. ²PRRC., Toyo Univ, Gunma, Japan.

Expression of the genes for glycerol synthesis (gcy-1, gcy-3, and dak-1), catalase (ctt-1), and gluconeogenic key enzymes (fbp-1 and pck-1) are stimulated by osmotic stress and fludioxonil in OS-2 MAP kinase dependent manner in Neurospora crassa. To identify the transcriptional factor regulated by OS-2 MAP kinase, we isolated three disruptants for atf-1 (CREB-type), msn-1 (STRE-type), and nap-1 (AP-1-type) genes, which are orthologs of SKO1, MSN2/4 and YAP1 of budding yeast, respectively. The msn-1 disruptant stimulated aerial hyphae on agar medium and showed increased resistance to oxidative stress such as menadione and t-butyl hydroperoxide. In contrast, the nap-1 disruptant was sensitive to oxidative stress. No gene disruption including atf-1 affected sensitivity to osmotic stress and fludioxonil. However, fludioxonil-induced activation of the OS-2-dependent genes was almost thoroughly cancelled in the atf-1 mutant but not in the msn-1 and nap-1 mutants. The atf-1 mutant grew well on the agar medium, but no progeny with atf-1 was obtained when the atf-1 mutant was crossed with the wild-type strain. These data suggested that the transcription factor ATF-1 may acts downstream of OS-2 MAP kinase, and probably plays an important role in the viability of ascospore.

41. Expression of the glucose-repressible grg-1 gene is stimulated by osmotic stress and fludioxonil in OS-2 MAP kinase dependent manner. Setsuko Watanabe¹ , Shinpei Banno² , Azusa Shiozawa¹ , Noriyuki Ochiai³ , Makoto Kimura³ , Makoto Fujimura¹. ¹Dept of Life Sciences , Toyo University , Gunma , Japapan. ²PRRC., Toyo University , Gunma , Japapan. ³Env.Mol.Biol., RIKEN , Yokohama , Japan.

Two-component histidine pathway consisting of OS-1 (histidine kinase) and OS-2 (MAP kinase) plays an important role in osmotic regulation in Neurospora crassa. The cAMP-PKA pathway, comprised of MCB/HAH (regulatory subunit) and PKAC-1 (catalytic subunit), participates in the switching of conidiation and filamentation probably in response to carbon sources. The hah;os-2 double mutant was more sensitive to osmotic stress than the os-2 mutant. In contrast, the pkac-1 mutation partially suppressed osmotic sensitivity of the os-2 mutant, suggesting crosstalk between these pathways. Furthermore, we found that expression of the grg-1 (glucose- repressible gene) gene was regulated under the OS pathway. Real-time PCR analysis demonstrated that transcription of the grg-1 gene was stimulated by osmotic stress and fludioxonil in the wild-type strain but not in the os-2 mutant. The GFP fluorescence intensity of the strain with the Pgrg-1-sgfp fused gene was significantly increased by osmotic stress and fludioxonil. There are several elements in the promoter region of the grg-1 gene such as CRE (cAMP-Responsive Element) and STRE (Stress Responsive Element). Promoter analysis is currently being undertaken.

42. Withdrawn

Checkpoint is an important mechanism for both DNA repair and chromosome maintenance. This mechanism has been studied in many organisms including yeast and human cells. In Neurospora crassa, various DNA repair genes have been studied in, but any studies concerning the DNA damage checkpoint had not been done. Recently, the clock gene prd-4 in Neurospora was identified as a homologue of Human Chk2. It is meaning that a relationship between DNA damage checkpoint and circadian clock exists. But data about the role of prd-4 toward both DNA damage checkpoint and DNA repair was not so much. So, we investigated three genes, that is one human Chk1 homologue and two Chk2 homologues. In general, checkpoint-deficient mutants show higher sensitivity against many mutagens than the wild type. In the S.cerevisiae RAD53 (Chk2 homologue ) mutant is sensitive to some mutagens containing camptothecin (CPT), which causes replication fork collapse, and hydroxy urea (HU), that induces stall of replication fork. The Neurospora Chk1 (ncchk1 ) disruptant indicated sensitivity to both CPT and HU. And two Neurospora Chk2 (nccds1 and prd-4 ) disruptants indicated sensitivity to CPT but not to HU. Moreover, we tried to make the relationship between these genes and other checkpoint genes clear. Surprisingly, CPT sensitivity of mus- 9 (Neurospora ATR ) was suppressed by nccds1 mutation. In yeast and human, CHK2 activation is dependent on ATR or ATM kinase activity, but such a genetic relationship observed in Neurospora had not be reported. These results indicate that Neurospora crassa has a unique mechanism in regulation of the DNA damage checkpoint.

44. A single amino acid determines GAP-specificity of duplicated Rho-GTPases in Ashbya gossypii. Michael Köhli¹, Sabrina Buck² and Hans-Peter Schmitz^1,2. ¹Applied Microbiology, Biozentrum Universität Basel, Klingelbergstr. 50-70, 4056 Basel, Switzerland ²Department of Genetics, University of Osnabrück, Barbarastr. 11, 49076 Osnabrück, Germany, hans- peter.schmitz@biologie.uni-osnabrueck.de, Tel.: +49 541 969 2289, Fax: +49 541 969 2293

While it is a known fact that gene duplication is a driving force of evolution, the resulting functional diversification is often unknown. We investigated the changes in function of the tandem duplicated homologs AgRho1a and AgRho1b of the Saccharomyces cerevisiae RHO1 gene in the filamentous fungus Ashbya gossypii. We could show that the main differences between the two duplicated copies are due to a single amino acid mutation in the switch I region of one of the two AgRHO1 genes. This exchange alters the specificity for the two Rho1-GAPs AgSac7 and AgLrg1 as shown by mutant analysis, interaction studies and GAP-assays with purified proteins. Our results explicitly show an example how gene duplication and single amino acid changes can rewire signal transduction networks during evolution.

45. Role of Microtubule Organizing Centers for the Distribution, Oscillation and Division of Nuclei in the Filamentous Fungus Ashbya gossypii. Claudia Birrer, Sandrine Grava, Tineke van den Hoorn, Dominic Hoepfner and Peter Philippsen, Molecular Microbiology, Biozentrum, University of Basel, Klingelbergstrasse 50/70, 4056 Basel, Switzerland, Claudia.lang@stud.unibas.ch

Nuclear positioning is important for normal growth and development of all eukaryotes including filamentous fungi. Among those, Ashbya gossypii is a particularly attractive organism to study nuclear distribution in multi-nucleated hyphae. Its 9 MB genome is completely sequenced and the annotation revealed a high degree of synteny to Saccharomyces cerevisiae. Using GFP-labled histone H4 nuclear dynamics including oscillation, bypassing and division have already been studied earlier (Alberti-Segui et al. 2001, Gladfelter et al. 2006). These processes are most likely co-regulated by the activities of microtubule organizing centers (MTOCs) embedded in the nuclear membrane and the dynamics of an elaborated microtubule (MT) network. We monitored MTOCs in A. gossypii hyphae carrying YFP-labled Tub4, the A. gossypii homolog of gamma-tubulin. Furthermore we studied the locations of re-emerging MTs after nocodazole treatment. Our data exclude hyphal tips or septa as locations for MTOCs and confirm A. gossypii nuclear-localized MTOCs as places of nucleating MTs. By in vivo labeling of alpha-tubulin with GFP and immuno-staining we could show that MTOCs nucleate two kinds of MTs: long ones interconnecting nuclei and short ones contacting the cell cortex. We also analyzed the function of AgSpc72, another component of A. gossypii MTOCs. Its homolog in S. cerevisiae is part of the outer, cytoplasmic layer of MTOCs. Yeast cells lacking this component only generate very short MTs. We show, that AgSPC72-deleted hyphae still form long cytoplasmic MTs, short MTs were not observed. Nuclear migration is substantially disturbed: nuclei are clustered distally from the tip, and nuclear oscillations are abolished. Moreover, the fraction of mitotic nuclei is twofold increased compared to wild type. To gain further insight into the controlled distribution and movement of nuclei we have started to investigate the role of other components on nuclear dynamics in A. gossypii.

46. Mitochondrial transport in Neurospora crassa by the motor proteins NKIN2 and NKIN3. Daniela Justa¹, Jolante Reth², Guenther Woehlke² and Stephan Seiler¹. ¹ Institute of Microbiology and Genetics, Goettingen, Germany ² Institute for Cell Biology, Munich, Germany

The most widely used mechanism for intracellular transport involves molecular motor proteins that carry cargo directionally along cytoskeletal tracks (myosins along actin filaments and kinesins and dyneins along microtubules). Several proteins that interact with motor proteins have been identified, but our knowledge about how the motor-cargo interaction takes place and how it is regulated is still limited. Recently, it has been shown that two kinesins of the kin3 family (named KIN2 and KIN3) are associated with mitochondria in vitro. To further characterize this interaction, we have generated mutants in both kinesins. Phenotypic analysis suggests that KIN2 is the main motor responsible for mitochondrial motility in vegetative hyphae, but also that its loss can be compensated by KIN3. The inability of kin-3 deletion strains to produce spores implicates an important function of this motor during sexual development. Currently, we are generating a kin-2;kin-3 double mutant to analyze the partially overlapping functions of the two motors in more detail. Life imaging of GFP-tagged versions of KIN2 and KIN3 is being developed to study the dynamic localization of the motor proteins. To further dissect the interacting domains of the motors and the responsible mitochondrial receptors, we are generating myc-tagged constructs of both kinesins to purify associated components.

47. Polar tip extension is coordinated by POD6 and COT1 in a motor protein-dependent manner in Neurospora crassa. Sabine Maerz¹, Nico Vogt¹, Carmit Ziv², Oded Yarden² and Stephan Seiler¹. ¹University of Goettingen, Germany ²The Hebrew University of Jerusalem, Israel

Members of the Ste20 and NDR protein kinase families are important for cell differentiation and morphogenesis in various organisms. We characterized POD6 (NCU02537.2), a member of the GCK family of Ste20 kinases that is essential for hyphal tip extension and coordinated branch formation in N. crassa. pod-6 and the NDR kinase mutant cot-1 exhibit indistinguishable growth defects, characterized by cessation of cell elongation, hyperbranching, and altered cell-wall composition. We suggest that POD6 and COT1 act in the same genetic pathway, based on the fact that both pod-6 and cot-1 can be suppressed by 1) environmental stresses, 2) altering PKA activity, and 3) common extragenic suppressors. Unlinked noncomplementation of cot-1/pod-6 alleles indicates a potential physical interaction between the two kinases, which is supported by coimmunoprecipitation, yeast two hybrid studies, partial colocalization of both proteins in wild-type cells, and their common mislocalization in dynein/kinesin mutants. We conclude that POD6 acts together with COT1 and is essential for polar cell extension in a motor protein-dependent manner in N. crassa. We are currently developing enzyme assays for both kinases and are in the process of establishing an interaction network on the basis of yeast two hybrid studies and co-affinity purifications.

48. Spitzenkörper localization and intracellular traffic of GFP-labeled class I and class VI chitin synthases in living hyphae of Neurospora crassa. M. Riquelme¹, S. Bartnicki-Garcia¹, J.M. González-Prieto², E. Sánchez- León¹, J. A. Verdín-Ramos¹, A. Beltrán-Aguilar¹, and M. Freitag³. ¹Department of Microbiology. Center for Scientific Research and Higher Education of Ensenada (CICESE). Km 107 Ctra. Tijuana-Ensenada. 22860 Ensenada, Baja California, México; ²Center for Genomic Biotechnology. Blvd. del Maestro s/n. 88710 Cd. Reynosa, Tamaulipas, México; ³Department of Biochemistry and Biophysics ALS 201. Oregon State University, Corvallis, OR 97331-7305, USA.

Chitin synthases (CHS) are essential enzymes involved in cell wall synthesis in fungi and arthropods. Little is known about their intracellular movement in growing fungal hyphae. Here, we present data on the localization of two selected CHS, CHS-3 and CHS-6, from Neurospora crassa. Both CHS-3 and CHS-6 were labeled at their carboxyl terminus with green fluorescent protein (GFP). We used high-resolution laser scanning confocal microscopy (LSCM) to analyze the localization and trafficking of CHS-3 and CHS-6 in growing hyphae. CHS-3-GFP and CHS-6-GFP showed similar distribution patterns along hyphae. In distal regions (beyond 45 µm from the tip), CHS-GFP was found mainly in a highly stained network of large endomembranous compartments and in nearby developing septa. At the subapex, the fluorescence was observed in numerous vesicles or groups of vesicles that moved predominantly forward until reaching the most proximal subapical region (15-20 µm from the tip). At the tip, the fluorescence congregated into a conspicuous single body corresponding to the location of the Spitzenkörper (Spk). Co-labeling of the Spk with the fluorescent marker FM4-64, showed CHS-GFP localized in the inner core of the Spk, the same region in which microvesicles were previously detected by transmission electron microscopy. Analysis of fluorescence recovery after photobleaching (FRAP) suggested that fluorescence at the Spk stems from the immediately surroundings vesicles. There was no co-localization of CHS-GFP fluorescence with stained secretory compartments fluorescence suggesting that CHS-3 and CHS-6 are not transported via the classical ER to Golgi to cell surface secretory pathway.

49. The Phycomyces blakesleeanus madB gene is a member of the White Collar 2 family. Catalina Sanz¹ and Arturo P. Eslava¹. ¹Ãrea de Genetica, Centro Hispano-Luso de Investigaciones Agrarias (CIALE), Universidad de Salamanca, Spain catsof@usal.es, eslava@usal.es

Phycomyces blakesleeanus is a filamentous fungus that has been used as a model in several studies of environmental signals as light, chemicals, wind, gravity and adjacent objects. The P. blakesleeanus genome sequence has led to the identification of three putative homologous genes belonging to the White Collar 1 family of blue- light photoreceptors and four putative homologous genes (wctA, wctB, wctC and wctD) from the White Collar 2 family. In a recent work, we have found that mutations in one of those White Collar 1 homologous genes are responsible for the blind phenotype observed in the madA strains, and as such, this gene has been named madA (Idnurm et al., 2006). In the present work we have analyzed the White Collar 2 family (wct genes) in several P. blakesleeanus strains that exhibit an abnormal sensitivity to light (from madA to madJ) in order to detect possible mutations. All the analyzed madB strains were affected in the wctA gene and present the same G+906A mutation, so it seems likely that all the strains are from the same origin. No other mutations in the White Collar 2 gene family were found among the rest of the mad strains. The G to A change presumably prevents the splicing of the first intron of the gene. We have amplified the cDNA copy of the wctA gene in the madB mutants by PCR and have observed that these cDNAs are partially unspliced. The cDNAs from the madB strains C111, C112 and C109 have been cloned and sequenced to ensure that the mutation does indeed prevent the splicing of the first intron, and to show that there are no secondary splicing events. We have also analyzed some genetic crosses of the wild type strain with some madB strains and have looked for recombinants to confirm whether the wild type recombinants do have the wild type copy of the gene, while the madB recombinants have the G to A change. As all the madB strains show impaired light-sensing this phenotype seems to be link to the mutation found, therefore we have decided to name the wctA gene madB.

50. The role of the exocyst in growth and morphogenesis of Neurospora crassa hyphae. A., Beltrán-Aguilar¹, S., Seiler², and M., Riquelme¹. ¹Center for Scientific Research and higher Education of Ensenada (CICESE), Department of Microbiology. Km. 107 Ctra. Tijuana-Ensenada, 22860, Baja California, México. ² Institut für Mikrobiologie und Genetik Abt. Molekulare Mikrobiologie und GenetikGeorg-August- Universität Göttingen Grisebachstr. 8D-37077 Göttingen, Germany.

Exocytosis is the last step of secretion and it consists on vesicle incorporation to the plasma membrane. A high fidelity protein-protein interaction (mediated by Sec proteins, SNAREs, GTPases and the exocyst) helps to provide specific recognition at the exocytosis sites. The secretory machinery involved in this process has not been fully described in filamentous fungi, but most of its components are evolutionary conserved among eukaryotes from yeast to mammals’ neurons. The exocyst complex plays an important role in the vesicle-plasma membrane interactions previous to SNAREs assembly and it is compossed of eight proteins (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84). Using a Neurospora crassa sec5 mutant strain we have analyzed the importance of this component for Spitzenkörper formation, polarized growth and hyphal morphology. At low magnification N. crassa sec5 showed a button like colony morphology, with hyperbranched and distorted hyphae. The mutant growth rate was 2.03% that of the wild type strain. At higher magnification the intracellular organization was severely affected. One of the key differences between the wild type and the mutant was the lack of a Spitzenkörper in the latter under high-resolution phase-contrast video- microscopy. A vector containing a sec5::sgfp the fusion was introduced into a N. crassa wild type strain and the localization and traffic of SEC5-GFP was analyzed by laser scanning confocal microscopy. Fluorescence did not accumulate at the tip, but was observed in a punctuate pattern all along the hyphae. This study establishes the basis for future analyses directed to elucidate the role of the exocyst in the apical growth of filamentous fungi.

51. Putative mannose transporters complement a branching/septation defect in Aspergillus nidulans. Loretta Jackson-Hayes, Lauren Fay, Terry W. Hill and Darlene M. Loprete. Departments of Biology and Chemistry, Rhodes College, Memphis, TN 38112. jacksonhayesl@rhodes.edu

In order to identify novel genes affecting cell wall integrity, we have generated mutant strains of the filamentous fungus Aspergillus nidulans, which show hypersensitivity to the chitin synthase inhibitor Calcofluor White (CFW). The phenotype of one of these strains (R205) also shows morphological abnormalities related to branching and septation. We have cloned two DNA fragments from an A. nidulans genomic DNA library which improve resistance to CFW and restore a more normal phenotype. One fragment is gene AN8848.3, "MT1", which shows homology to GDP-mannose transporters. The second fragment is gene AN9298.3, "MT2", which is a similar but distinct gene also homologous to GDP-mannose transporters. When separately cloned, the putative GDP-mannose transporters restore normal phenotype including full restoration of subapical hyphal compartment length and branch density in the mutant. Sequencing reveals a genetic lesion in Exon 5 of MT1 which causes an alanine to proline substitution and no mutation in MT2 in mutant strain R205. Cloned R205 MT1 containing the Exon 5 mutation does not complement the R205 phenotype. Attempts to produce null mutants of MT1 did not produce viable transformants, suggesting that AN8848.3 is an essential gene. MT2 null mutants grow normally under normal growth conditions and show wild type CFW resistance.

52. Constitutive PKA activity prevents growth arrest of Aspergillus fumigatus conidia. Kevin Fuller, Wei Zhao, Lauren Fox, Judith Rhodes. University of Cincinnati, Cincinnati, OH

We have previously generated a mutant of A. fumigatus deficient in the regulatory subunit of PKA and described that conidia are hypersensitive to various forms of oxidative stress. We also noted that the mutant conidia are larger than those of wt in distilled water. Both swelling and increased sensitivity to oxidative stress accompany metabolic activation after exposure of resting conidia to a germinant. Accordingly, we wanted to determine if the increase in conidial size of the pkaR deletion mutant was due to an unexpected initiation of germination in water. To determine the metabolic state of the conidia, we evaluated mitochondrial activity with Mitotracker Orange. Whereas it required 4 hours in a rich medium for the wt and complemented strains to stain positively, the delta pkaR conidia were positive at earlier time points, even in the absence of a germinant. Furthermore, the mutant conidia were killed more readily by hydrogen peroxide when compared to the resting conidia of the other isogenic strains. However, after the 4 hour time point, all members of the isogenic set showed equivalent killing by the treatment. We, therefore, conclude that the hypersensitivity to hydrogen peroxide seen with the conidia of the pkaR deletion mutant is due to failure of resting conidia to maintain growth arrest.

53. Ras signaling in the human pathogen Cryptococcus neoformans. Connie B. Nichols and J. Andrew Alspaugh. Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA. connie.nichols@duke.edu

Cryptococcus neoformans is an opportunistic human fungal pathogen that is the causative agent of cryptococcosis, a life-threatening infection. The ability to grow at high temperature is an important requirement of C. neoformans to establish disease. Previously we found that Ras1 was required for growth at high temperature and for sexual differentiation. We have now identified the downstream components of Ras1 that mediate the response to high temperature, including Cdc24, Cdc42, and Ste20. In the fission yeast Schizosaccharomyces pombe, Ras mediates morphology and sexual differentiation using different upstream activators and downstream effectors. To understand how Ras1 mediates both high temperature growth and sexual differentiation in C. neoformans we have generated a series of ras1 mutant strains including dominant negative, dominant active, farnesylation-defective, and palmitoylation-defective ras1 mutant strains. In addition we have used genetic epistasis experiments to characterize the role of the Cdc24-Cdc42-Ste20 branch of Ras1 signaling in sexual differentiation in C. neoformans.

54. Two-component signaling system for osmotic regulation and fungicide sensitivity in Cochliobolus heterostrophus. Chihiro Tanaka, Kosuke Izumitsu, and Akira Yoshimi* Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, JAPAN. *Present address: New Industry Creation Hatchery Center (NICHe), Tohoku University, Aoba, Sendai 980-8579, JAPAN. E-mail: chihiro@remach.kais.kyoto-u.ac.jp

We have studied the mode of action of dicarboximide/phenylpyrrole fungicides, using resistant mutants of a maize pathogen Cochliobolus heterostrophus. In this fungus, the resistant mutants always showed hyperosmotic sensitivity, as reported in N. crassa. And dic1 encoding a histidine kinase (HK) was characterized as a responsible gene for fungicide resistance and osmotic adaptation. We also elucidated that exposure to the fungicides led to Dic1p mediated improper activation of Hog1-MAPK (BmHog1p). HK and MAPK are core components in two- component signaling and MAPK signaling systems, respectively. These results suggested that both signaling systems were involved in the mode of the action of the fungicides and high-osmorality adaptation in this fungus. To investigate further, we isolated mutants of ChSln1 (HK, whose orthologue is a sole HK in yeast and involved in its osmo-adaptation), ChSsk1 (response regulator: RR) and ChSkn7 (another RR) and compared their phenotypes with a wild- type, dic1 and Bmhog1. ChSln1 disruptants showed no apparent phenotypic changes with respect to osmo-sensitivity. However, both of Chssk1 and Chskn7 mutants showed moderate resistance to the fungicides, and some sensitivity to high osmorality. The phenotype comparisons and phosphorylation analyses of Hog1- MAPK in these mutants revealed that C. heterostrophus has two streams of two-component signaling pathways for the osmotic adaptation and fungicide sensitivity: one is Dic1p-ChSsk1p driving Hog1-MAPK cascade, another is Dic1p-ChSkn7p, and both streams were controlled by Dic1p.

Total internal reflection fluorescence (TIRF) microscopy is an optical technique that uses evanescent waves to excite fluorophores within 200 nm of the specimen and coverslip interface. This effectively eliminates background fluorescence from other regions of the cell and improves spatial resolution by increasing the signal-to-noise ratio. Furthermore, living cells can be viewed over extended time periods due to low phototoxic effects. Here, we used TIRF microscopy to study cortical microtubule (MT) dynamics in living primary hyphae of Neurospora crassa expressing β-tubulin- GFP. Detection of plus-end MT dynamics was much improved with this approach compared to confocal and widefield fluorescence microscopy methods. Surprisingly, MTs in N. crassa polymerize twice as fast as those reported in Aspergillus nidulans, while MT depolymerization rates in both species were similar. During this study, we observed events of MT fragmentation and fragment motility. These behaviors were compared to those observed in MT-motor mutant strains in order to address the role(s) played by dynein/dynactin and kinesin in MT behavior.

56. Micro and nano morphology of Aspergillus species in response to carbon starvation. Mark R. Marten, Judith Kadarusman, Liming Zhao, Youghyun Kim, Bill Moss Zebulon Jones and David Schaefer². UMBC, Chemical & Biochemical Engineering, Baltimore, MD 21784, ²Towson Univ., Physics Dept, Towson, MD 21252

We are using Aspergillus species as models to characterize fungal morphological response to carbon starvation. To study micro-morphology we use a parallel-plate flow chamber, with a narrow (50 micron) gap, forcing two dimensional fungal growth. This chamber is perfused with oxygenated growth medium, and mounted on a microscope stage, allowing us to follow morphological development of individual mycelial as a function of time and growth environment. In response to carbon deprivation (i.e., step change, glucose⁺ to glucose^-), A. oryaze enters a lag phase where extention ceases, vacuolation increases, and duration depends on the initial size of the mycelium. When complete, regrowth begins at a relatively constant rate independent of mycelial size. When this behavior is compared to that during rapamycin-induced autophagy we find quantitative differences implying autophagy may comprise only a portion of a more complicated starvation stress response. To study nano-morphology we use an atomic force microscope (AFM). We have developed the ability to use the AFM for testing material properties of fungal cell walls as a function of axial position. We will discuss development of this technique and how we are using it to study changes in A. nidulans morphology during carbon deprivation.

57. Withdrawn

58. A functional analysis of the DopA protein of A. nidulans and A. fumigatus. Holger Hannemann and Bruce Miller. University of Idaho, MMBB. Moscow, Idaho

Aspergillus nidulans (An) is a widely used model organism whereas A. fumigatus (Af) is an airborne pathogen, causing mostly fatal invasive mycoses in immunocompromised patients. Polarized growth is one of the key factors of fungal pathogenesis. DopA, whose homologs are found in fungi, worms and chordata, is a protein involved in polarization of cells. An An strain expressing a temperature sensitive DopA protein (DopAts) displays aberrant cellular morphology of all cell types, asynchronous cell pattern formation and loss of both asexual and sexual reproductive cycles at the restrictive temperature. Successive shortening of the An and Af dopA open reading frames demonstrated that the leucine zippers, located at the C-terminal end of the proteins, serve different functions in these two organisms. DopA-GFP fusions in both An and Af revealed that the proteins are localized to the Golgi and the endosomal membrane networks in both species. Genetic interactions between An dopA1ts and An rasA suggest that DopA modulates signaling through a RasA-mediated signal transduction pathway that controls cell polarity and morphogenesis. Additionally, the N-terminal dopey domain of An DopA interacts physically with BemA, the S. cerevisiae (Sc) Bem3 homolog. Therefore, the interactions between An RasA and An DopA, An BemA and An DopA and signaling interactions between Sc Ras and the Sc Cdc42 regulatory module suggests that the DopA/Dop1 protein plays a central role in integrating signal transduction and mechanisms regulating cell polarity in the fungi.

59. The Aspergillus nidulans snoA inhibitor of cell division associates with the BRDF checkpoint domain of nimO^Dbf4. Steve James, James Barra, Megan Campbell, and Matthew Denholtz. Biology Department, Gettysburg College, Gettysburg, PA. sjames@gettysburg.edu

In Aspergillus nidulans, nimO^Dbf4 and cdc7 encode regulatory and catalytic subunits of the conserved DBF4-dependent kinase (DDK). DDK initiates DNA synthesis by phosphorylating the replicative DNA helicase to trigger DNA unwinding at origins of replication. In addition, DBF4 plays an important role in the DNA damage response. This role is mediated by an N-terminal BRDF motif (BRCT and DBF4 similarity domain), as revealed by mutations in yeast homologs that confer enhanced sensitivity to DNA damage agents and failure to restrain DNA synthesis during genotoxic stress. In Saccharomyces cerevisiae, RAD53/CHK2 kinase is the only checkpoint mediator known to associate directly with the DBF4 BRDF motif. We identified a novel inhibitor of nimO^Dbf4 called snoA (suppressor-of-nimO). Loss of snoA rescues nimO18 ts-lethality and hypomorphic nimO+ expression. Conversely, snoA overexpression confers a dose-dependent, lethal interphase cell cycle arrest in nimO18 cells. Here we report a novel interaction between the nimO BRDF motif and snoA. Using yeast two-hybrid analysis, we demonstrate that a short (~100 amino acid) serine- and proline-rich region in the snoA C-terminus can associate with the nimO BRDF. This novel discovery suggests that snoA may act to regulate normal DNA synthesis or to exert S phase checkpoint control by direct association with the A. nidulans DBF4-dependent kinase. (Supported by NSF-RUI #01-14446 to SJ)

60. Where is calcium in the cell and how does it get there? Barry Bowman, Stephen Abreu, Marija Draskovic and Emma Jean Bowman. Department of MCD Biology, University of California, Santa Cruz, CA 95060 email:bowman@biology.ucsc.edu

Calcium gradients are hypothesized to have a central role in polar growth. However, little is known about the distribution of calcium within fungal cells or the proteins that transport it. By fractionating cells we have found that mitochondria, vacuoles, and unidentified light organelles contain roughly equal amounts of calcium. The Neurospora crassa genome contains at least four P-type ATPases that appear to pump calcium - nca-1, nca-2, nca-3, and pmr-1. In addition seven genes encode transport proteins that may exchange calcium for H⁺ or Na⁺, cax being the best characterized. We have characterized nine mutant strains that lack a calcium transporter and have fused several transporters to GFP. The types of transporters and the distribution of calcium are more complex in N. crassa than in S. cerevisiae, the best studied system. Disruption of the cax gene causes the complete loss of calcium from the vacuoles, but does not affect other compartments in N. crassa. Yeasts lack a homolog of the endoplasmic reticulum Ca-ATPase of animal cells (the SERCA ATPase), but N. crassa has a homolog, nca-1. Interestingly, nca-1::GFP localizes largely to the nuclear envelope, suggesting the nuclear envelope forms a major part of the endoplasmic reticulum in fungi. Two of the mutant strains, pmr-1 and one of the homologs of cax, are significantly altered in hyphal morphology. Do they help to generate calcium gradients at the hyphal tip?

61. Withdrawn

62. Withdrawn

63. Two-component response regulators, ChSsk1p and ChSkn7p, additively regulate high-osmolarity adaptation and fungicide sensitivity in Cochliobolus heterostrophus. Kosuke Izumitsu, Akira Yoshimi* and Chihiro Tanaka. Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, JAPAN. *Present adress: New Industry Creation Hatchery Center (NICHe), Tohoku University, Aoba, Sendai 980-8579, JAPAN.

Filamentous fungi possess many histidine kinases and two conserved response regulators, Ssk1p and Skn7p, in their two- component signaling systems. We have characterized the response regulator genes ChSsk1 and ChSkn7 in the Southern corn leaf bright fungus Cochliobolus heterostrophus. Both Chssk1- and Chskn7- disrupted mutants showed little sensitivity to high-osmolarity stress and moderate resistance to the iprodione/fludioxonil fungicides. The phosphorylation of Hog1-type MAPK BmHog1p induced by high-osmolarity stress and fungicide treatments was only regulated by ChSsk1p, indicating that ChSkn7p has roles in high-osmolarity adaptation and fungicide sensitivity that are independent from the activation of BmHog1p. The ChSsk1/ChSkn7 double mutants clearly showed higher sensitivity to osmolar stress and higher resistance to fungicides than the single mutants. The dose responses of the double mutants fit well with those of the group III histidine kinase-deficient strain. These results suggest that in filamentous ascomycetes, the Ssk1-type and Skn7-type response regulators control high-osmolarity adaptation and fungicide sensitivity additively with differential mechanisms under the regulation of the group III histidine kinase.

64. The role of cAMP-receptor-like genes in Neurospora crassa development. Svetlana Krystofova and Katherine Borkovich, Plant Pathology, University of California Riverside, Riverside, CA 92521, svetlana@ucr.edu

G-protein coupled receptors (GPCRs) are 7-transmembrane proteins that sense a variety of extracellular stimuli, such as light, odor, chemoattractants, peptides, neurotrasmitters, hormones and lipids. Putative GPCRs have been identified in the Neurospora crassa genome sequence. Based on phylogenetic analysis, three of these genes, gpr-1, gpr-2 and gpr-3, belong to a cAMP-receptor-like gene family that includes Arabidopsis GCR1, Dictyostelium crlA-crlC, Cryptococcus neoformans gpr4 and Aspergillus nidulans GprH. Deletion of gpr-1 leads to very specific defects during sexual development (lack of ostioles), while asexual development is not affected. The absence of ostioles in mature perithecia negatively contributes to the ability to release ascospores by an ejection mechanism. Moreover, perithecia are frequently ruptured leading to the release of inner material. The functions of gpr-2 and gpr-3 remain unknown. The expression profiles indicate that these genes are expressed in early developmental stages. Deletion mutants have been isolated and phenotypically analyzed. The possibility of redundancy between gpr-1, gpr-2 and gpr-3 has been investigated. Both genetic and biochemical approaches have been used to identify possible downstream effectors of N. crassa cAMP-receptor-like proteins.

65. Role of cytoplasmic bulk flow and microtubule-related motor proteins in nuclear displacement in Neurospora crassa mature hyphae. Silvia L. Ramos-Garcia¹, Rosa R. Mouriño-Pérez¹, Michael Freitag², Salomon Bartnicki-Garcia¹. ¹Departamento de Microbiologia. Centro de Investigación Científica y Educación Superior de Ensenada. Ensenada B. C. Mexico. rmourino@cicese.mx. ² Department of Biochemistry and Biophysics, Center for Genome Research and Biocomputing. Oregon State University. Corvallis, USA.

Nuclear movement and localization are multifactorial events that have been intensely studied over the last two decades. Here we report the results of a study to evaluate nuclear dynamics in the subapical region of Neurospora hyphae. Nucleus movement may be controlled by many factors, for example, cytoplasmic bulk flow, the cytoskeleton (microtubules and actin microfilaments) and motor proteins (dynein/dynactin complex and conventional kinesin). By confocal microscopy, the nuclear displacement of five strains of Neurospora crassa that express histone H 1 tagged with GFP was compared. We included a control strain, ropy-1, ropy-3, nkin and a ropy-1; nkin double mutant. Additionally, we treated the H1-GFP control strain with benomyl to inhibit microtubule formation and with cytochalasin A to inhibit actin microfilaments. Anterograde and retrograde movement of nuclei were observed in all strains, except the double mutant. The displacement rate of nuclei was highly correlated to hyphal elongation rate. The exception was the nkin mutant and cells treated with cytochalasin A. The shape of nuclei in growing hyphae was different: In the control, almost all nuclei were elongated, while in all other strains varying numbers of spherical nuclei were observed. The distance between the hyphal apex and the first nucleus varied between all strains and treatments: The ropy-1 mutant showed the largest exclusion region and the cytochalasin A-treated cells completely lacked this nucleus exclusion zone. In conclusion, the movement and distribution of nuclei in mature hyphae appear determined by a combination of forces, with cytoplasmic bulk flow being a major determinant. Nevertheless, motor proteins bind nuclei to microtubules and actin microfilaments appear to act as a scaffolding to move all components in unison with the cytoplasm.

Schizosaccharomyces pombe has the SCD1 signaling pathway regulated by RAS1. This pathway is necessary for proper morphogenesis such as polarized growth or actin cytoskeleton, and pheromone response. In this pathway, SCD1 that is GDP/GTP exchange factor (GEF) for CDC42 (small GTPase) interacts with SCD2 (scaffolding protein). Then, GTP-bound CDC42 and SHK1 (p-21 activated kinase) also interact with SCD2. This complex transfers phosphate signals from up stream to down stream. Therefore, SCD2 and SHK1 function in the same pathway, both proteins are important factors mediate this pathway. Neurospora crassa also has the homologous of fission yeast SCD2 and SHK1, so we each named these genes ncSCD2 and ncSHK1, respectively. Then, we constructed strains defective in ncSCD2 and ncSHK1 to characterize and analyze these genes function. Both null mutants grew more slowly than wild type strain, and displayed morphological defects that appear to have abnormal branching pattern and frequently septations. Moreover, both null mutants couldn't fertilize. But there were some differences in phenotype between ncSCD2 mutant and ncSHK1 mutant. SHK1 mutant resisted osmotic stress more strongly than ncSCD2 mutant. This data suggests that N.crassa also has the SCD1 signaling pathway, and that ncSCD2 and ncSHK1 function resemble to fission yeast scd2 or shk1. In addition, the fact that SHK1 mutant had resisted high osmotic stress implied possibility that the SCD1 signaling pathway link to the HOG (High-osmolarity glycerol) pathway in N.crassa.

67. Spatially segregated SNARE protein interactions in filamentous fungus. Mari Valkonen¹, Markku Saloheimo¹, Merja Penttilä¹, Nick D. Read², and Rory R. Duncan³, ¹VTT Biotechnology, P.O. Box 1000, FIN-02044 VTT, Finland. ²Fungal Cell Biology Group, Institute of Cell Biology, University of Edinburgh, Edinburgh EH9 3JH, UK. ³Membrane Biology Group, University of Edinburgh, George Square, EH8 9XD, UK

The machinery for trafficking proteins through the secretory pathway is well conserved in eukaryotes, but remains poorly characterized in filamentous fungi. We describe the isolation of the snc1 and sso1 genes encoding exocytic SNARE proteins from Trichoderma reesei. The encoded SNCI protein can complement Snc protein depletion in S. cerevisiae whilst the T. reesei SSOI protein was unable to complement depletion of its yeast homologues. The localization and interactions of the T. reesei SNARE proteins were studied with advanced fluorescence imaging methods using fluorescent fusions of the SNARE proteins. The SSOI and SNCI proteins co-localized in sterol-independent clusters on the plasma membrane in sub-apical but not apical hyphal regions. The v-SNARE SNCI localized to the apical vesicle cluster within the Spitzenkörper of the growing hyphal tips when expressed under the T. reesei cbh1 promoter. Using fluorescence lifetime imaging microscopy (FLIM) and fluorescence energy transfer (FRET) analysis, we quantified the interactions between these proteins with high spatial resolution in living cells. Our data showed that the site of SNARE complex formation between these proteins is on the plasma membrane of non- growing hyphae in old sub-peripheral regions of the colony, but that there is no interaction between the proteins in growing hyphal tips at the colony margin.

68. A RasGAP protein involved in polarity establishment and maintenance in Aspergillus nidulans. Laura Harispe^1,2,3, Lisette Gorfinkiel², Cecilia Portela², Miguel A. Peñalva³, and Claudio Scazzocchio¹. ¹IGM, Univ. Paris-Sud XI. Bat 409 Centre d´Orsay. 91405 Orsay(France). ²F. Ciencias, Univ. de la República. Iguá 4525. 11400 Montevideo(Uruguay) ³CIB, CSIC, Ramiro de Maeztu 9. 28040 Madrid(Spain)

Filamentous fungi represent an extreme example of polarised growth^(a). We report on the identification and characterisation of GapA, an A. nidulans RasGAP involved in polarity establishment and maintenance. GapA was identified after serendipitously isolating a partial loss-of-function mutation, designated gapA1, in a genetic screen. Phenotypic characteristics resulting from gapA deletion include compact colony morphology, a marked delay in polarity establishment during conidial germination, impairment of polarised hyphal extension, a conspicuous developmental defect typically manifested by the absence of one layer of sterigmata in the conidiophore and a defect in the otherwise polarised distribution of the actin cystoskeleton. GapA-GFP protein fusion expressed from a gene replacement allel appears localises to hyphal tips and septa. This localisation suggests that a Ras protein(s), whose activity is antagonised by GapA plays a role in the regulation of the actin cytoskeleton at the hyphal tip and that this abnormal regulation underlies the polarity phenotypes associated with gapA loss-of-function. ^(a)Momany M. (2002),Curr. Opinion in Microbiology, 5:580–585.

69. Optical tweezer micromanipulation of living fungal cells. Graham Wright¹, Jochen Arlt², Wilson Poon² & Nick Read¹. ¹Institute of Cell Biology, University of Edinburgh, Edinburgh, UK. ²School of Physics, University of Edinburgh, Edinburgh, UK graham@fungalcell.org

Optical tweezers facilitate the non-invasive micromanipulation of both inert and biological microscopic particles solely by using light. We are using optical tweezers in a range of cell biological applications. They have been used in various experimental live-cell studies to manipulate intracellular organelles, hyphal growth and branching, and whole cells. Optically trapped microbeads have been used for the localized delivery of chemicals and to mechanically stimulate cells. Finally, the effects of optical trapping on fungal cell viability have been assessed.

70. Characterization of radC, the Aspergillus nidulans homolog of RAD52, a key gene for DNA repair by homologous recombination. Michael Lynge Nielsen, Gaëlle Lettier, Jakob Blæsbjerg Nielsen and Uffe Hasbro Mortensen. Technical University of Denmark, BioCentrum-DTU

Repair of DNA double-strand breaks (DSBs) is crucial for maintaining genome integrity and failure to repair even a single DNA DSB is lethal as it causes loss of part of a chromosome during cell division. In the yeast, Saccharomyces cerevisiae, Rad52 plays a fundamental role in the repair of DSBs by homologous recombination (HR) and among genes involved in DNA DSB repair and HR deletion of RAD52 produces the most dramatic phenotype. Hence, S. cerevisiae cells lacking Rad52 are impaired in DSB repair and all types of HR including targeted integration of transforming DNA. Orthologs of RAD52 have been identified in other organisms, including Schizosaccharomyces pombe, Aspergillus nidulans, chicken, mouse, and human. However, in contrast to S. cerevisiae, the absence of RAD52 in higher eukaryotes does not result in a severe phenotype, due to the evolution of additional repair pathways and the role of Rad52 in DNA DSB repair and HR remains unclear. To address the role of Rad52 in higher eukaryotes, we have recently constructed a radC (homolog of RAD52) deletion mutant in A. nidulans and performed an initial characterization of the mutant strain. Unlike in S. cerevisiae, where Rad52 is essential for the repair of all types of DNA lesions that require HR, we show that in A. nidulans it is only required for the repair of a subset of these lesions. Hence, in an evolutionary perspective, HR repair in A. nidulans may represent an intermediate state between S. cerevisiae and human and A. nidulans may therefore be a useful model to further the understanding of HR in higher eukaryotes.

71. A new cell type produced by conidia that is involved in sexual reproduction in Neurospora crassa. Hsiao- Che Kuo, Gabriela Roca, Chris Jeffree and Nick D. Read. Institute of Cell Biology, University of Edinburgh, UK

We describe a new cell type, the conidial sex tube (CST), which is produced by macroconidia and arthroconidia of Neurospora crassa. It is morphologically and physiologically distinct from germ tubes and conidial anastomosis tubes (CATs), and under separate genetic control. CSTs are characterized by being long, thin (thinner than germ tubes), straight and unbranched. They contain few nuclei and do not avoid or grow towards each other. The CST surface texture and chemistry is different to that of germ tubes and CATs. CSTs require the presence of protoperithecia of opposite mating type to be induced (germ tube and CAT formation are inhibited under these coniditions). Trichogynes show strong chemotropisms towards CSTs but CSTs do not grow towards trichogynes. On making contact, trichogynes coil around CSTs. Trichogynes are not attracted to or coil around CSTs in mutants not producing sex pheromones when male only and trichogynes are not attracted to CSTs in mutants lacking sex pheromone receptor when female only. Nuclear division is inhibited in the presence of CSTs. CST formation is weakly conidium density dependent in contrast to CAT formation which is strongly conidium density dependent and germ tube formation which is conidium density independent.

72. Organelle organization and behavior during CAT fusion and heterokaryon formation in Neurospora crassa. Gabriela Roca¹, Michael B. Freitag² & Nick D. Read¹. ¹Inst. of Cell Biology, University of Edinburgh, UK. ²Dept. of Biochemistry and Biophysics, Oregon State University, USA

Conidia of Neurospora crassa form conidial anastomosis tubes (CATs) which are specialized hyphae that are distinct from germ tubes. CAT induction, homing and fusion were analyzed using strains expressing different GFP- or mRFP-fusion proteins, and compatible heterokaryons resulting from the fusion of these strains. Various organelle- selective dyes were also used. Cytoplasm passed through fused CATs prior to microtubules, and were followed by mitochondria and then nuclei. When heterokaryons were generated in which one of the parent homokaryons expressed either H1::GFP or H1::mRFP, it took > 3 h for nuclei in the unlabelled homokaryon to become labeled. Mitosis occured more slowly in ungerminated macroconidia (~ 1.5 h) than in germ tubes (~ 15 min). Mitosis was arrested during CAT homing and nuclei did not enter CATs. We also analysed heterokaryons resulting from CAT fusion between vegetatively incompatible strains differing in mating type. Nuclear immobilization and cell vacuolarization started 6 h after fusion but was not immediately followed by cell death. Heterokaryon incompatibility following CAT fusion is therefore delayed in contrast to the rapid cell death that follows incompatible fusions in mature colonies. This suggests that N. crassa possesses a mechanism to escape from vegetative incompatibility during the early stage of colony establishment.

73. Intracelular Analysis of Neurospora crassa Germlings by Confocal Microscopy. Castro-Longoria E., Araujo-Palomares C. and Riquelme M. ecastro@cicese.mx. Department of Microbiology, CICESE, Km. 107 C. Tij-Ensenada, 22860 Ensenada, B. C., México.

By laser scanning confocal microscopy and using the amphiphilic styril dye FM4-64 and Neurospora crassa wild type strain whose microtubules are labeled with GFP we have analyzed the intracellular changes prior to the appearance of a mature Spk. Observations began at the early stages of spore germination and were carried out until a conspicuous Spk could be observed. Before the Spk appearance, germ tubes ( <150 µm) displayed a uniform distribution of organelles along the length of the cells. Once the germlings reached approximately 66.4 + 4.5 µm in length, a small accumulation of vesicles stained with FM4-64, but not detected by phase-contrast microscopy could be observed at the apex of the cell. When germlings reached more than 150 µm in length visible organelles such as mitochondria and nuclei experienced a displacement towards the subapical region of the cell and a small exclusion zone free of organelles formed at the apex. The position of this exclusion zone within the apex seemed to determine the germling growth direction, which was highly erratic. Few minutes after it first appeared, upon growth of the germling, the exclusion zone started to become occupied by an accumulation of material that gradually concentrated into a light gray body that we describe as an immature Spk, the accumulation of FM4-64 colocalized with the position the immature Spk. Our preliminary observations on microtubule organization during germ tube elongation showed that during the early stages of germination of N. crassa, cytoplasmic microtubules were less abundant, shorter and differently organized when compared to those previously reported in mature vegetative hyphae. At later stages, when a mature Spk was observed, cytoplasmic microtubules were more abundant, longer, and were distributed along the main longitudinal axis of growth of the germ tube. The formation of a mature Spk and a longitudinal and abundant distribution of microtubules coincided with the stabilization of the growth direction of the germling, therefore suggesting that it is at this stage when the transition from germling to vegetative hypha occurs.

74. Reversion Analysis of the Cytoplasmic Dynein Motor: a Trip Around the Ring. Michael Plamann, David Madole, Robert Schnittker, and Elizabeth Wulff. School of Biological Sciences, University of Missouri-Kansas City

Cytoplasmic dynein is a large, microtubule-associated motor complex that facilitates minus-end-directed transport of various cargoes. Dynein heavy chain (DHC) is >4000 residues in length, with the last two-thirds of the heavy chain forming the motor head. Six domains within the dynein motor exhibit varying degrees of homology to the AAA+ superfamily of ATPases. These domains are followed by a distinct C-terminal domain and together form a ring-like structure from which a microtubule-binding domain (MTBD) protrudes. Using a genetic assay, we have isolated over 50 DHC mutants of Neurospora that produce full-length proteins that are defective in function. We have identified DHC point mutations in nearly all domains within the dynein motor head. We have now isolated revertants for a subset of these DHC mutants and have identified the respective intragenic suppressor mutations in >100 revertants. Interestingly, we have found that two mutations within the MTBD are suppressed by mutations in various domains around the ring. These results suggest that the conformational states of the MTBD and motor head are tightly coupled. Most DHC mutations examined to date revert exclusively by intragenic suppression. However, 30 to 90% of the AAA#6 and C- terminal domain DHC revertants contain extragenic suppressors. These results suggest that AAA#6 and C-terminal domain mutations are readily bypassed by mutations in other genes.

75. Endocytosis and polarized growth in Aspergillus nidulans. Araújo-Bazán L., Peñalva M.A & Espeso E.A. CIB, C.S.I.C., Ramiro de Maeztu, 9 28040 Madrid (Spain).

In Aspergillus nidulans polarity maintenance during hyphal growth involves the polarised deposition of secretory vesicles at the extending tip. Cortical actin patches are present along the hyphal tube but strongly predominate in the apical region One third of the endocytic proteins directly regulate actin assembly and/or bind to actin. The unique Abp1 and SlaB orthologue in Aspergillus were found using bioinformatics and being designated as abpA and slaB, respectively. After cDNA sequencing, we determined that AbpA shows strong similarity with Abp1p having one conserved cofilin domain and two, instead of one, SH3 domains. AbpA localises to highly motile and transient peripheral punctuate structures overlapping with actin patches. SlaB also localises to peripheral patches, which are markedly more abundant and cortical than those of AbpA but their motility is notably restricted as compared to AbpA patches. Similar subcellular localisation has been determined for actin and the amphiphysin orthologue AnRvs167. AbpA patches show random distribution during the isotropic growth phase preceding polarity establishment, but polarise as soon as a germtube primordium emerges from the swelled conidiospore. Thus, while endocytosis can occur along hyphae, the apical predominance and spatial organization of these endocytic machinery proteins as a slightly subapical ring strongly suggest that tight spatial coupling of secretion and compensatory endocytosis underlies hyphal growth.

Population and Evolutionary Biology

76. Recombination hotspots flank the Cryptococcus mating-type locus: implications for the evolution of a fungal sex chromosome. Yen-Ping Hsueh, Alexander Idnurm, and Joseph Heitman Department of Molecular Genetics and Microbiology, Duke University, Durham, North Carolina

Recombination increases dramatically during meiosis to promote genetic exchange and generate recombinant progeny. Interestingly, meiotic recombination is unevenly distributed throughout genomes and as a consequence genetic and physical map distances do not have a simple linear relationship. Recombination hotspots and coldspots have been described in many organisms and often reflect global features of chromosome structure. In particular, recombination frequencies are often distorted within or outside sex determining regions of the genome. Here we report that recombination is elevated adjacent to the mating-type locus (MAT) in the pathogenic basidiomycete Cryptococcus neoformans. Among fungi, C. neoformans has an unusually large MAT locus, and recombination is suppressed between the two >100 kb mating-type specific alleles. When genetic markers were introduced at defined physical distances from MAT, we found the meiotic recombination frequency to be ~20% between MAT and a flanking marker at 5, 10, 50 or 100 kb from the right border. As a result, the physical/genetic map ratio in the regions adjacent to MAT is distorted ~10- to 50- fold compared to the genome-wide average. Moreover, recombination frequently occurred on both sides of MAT and negative interference between crossovers was observed. Sequence analysis revealed a correlation between high G + C content and these hotspot regions. We hypothesize that the presence of recombinational activators may have driven several key events during the assembly and reshaping of the MAT locus, and may have played similar roles in the origins of both metabolic and biosynthetic gene clusters. Our findings suggest that during meiosis the MAT locus may be exchanged onto different genetic backgrounds and therefore have broad evolutionary implications with respect to mating-type switching in both model and and pathogenic yeasts.

77. Survey of mating-type (MAT) gene distribution in the Penicillia. Carly Eagle¹, Rob A. Samson², Paul S. Dyer^{1 1}School of Biology, University Park, University of Nottingham, Nottingham, NG7 2RD ²Fungal Biodiversity Centre, CBS, P.O. Box 85167, 3508 Utrecht, The Netherlands sbxcee@nottingham.ac.uk

The genus Penicillium contains various species that are important in medicine, industry and food spoilage. This genus is polyphyletic, containing four subgenera; Biverticillium, Furcatum, Aspergilloides and Penicillium. Studies are underway to investigate the genetic basis of reproductive mode in the Penicillia, initially involving a screen of mating-type (MAT) genes in asexual and sexual taxa. Penicillium marneffei has previously been shown to contain either a MAT1-1 or MAT1-2 locus. This species is classified within the subgenus Biverticillium which is closely related to the sexual genus Talaromyces. The focus of current work is the subgenus Penicillium which contains the asexual species Penicillium chrysogenum (produces penicillin) and P. griseofulvum (produces griseofulvin), and is closely related to the sexual genus Eupencillium. Using a degenerate PCR approach, MAT1-1 and MAT1-2 loci have been amplified and sequenced from a number of species belonging to this subgenera.

78. Reproductive strategy and genetic polymorphism in Pleurotus pulmonarius populations in Russia. Alla Shnyreva. Department of Mycology and Algology, Moscow State University, Moscow, Russia, e-mail: shnyreva@herba.msu.ru

The genetic structure of populations of P. pulmonarius was inferred from variation among 157 individuals at 14 polymorphic allozyme loci and RAPD-profiles to determine the extent of geographic differentiation in this widespread mushroom species in Central Russia. Population genetic analysis was carried out by employing homokaryotic and dikaryotic strains (parents – offspring). To define interbreeding fungal populations, crosses between homokaryons were performed. Genetic exchange was shown to be not only limited to the sexual cycle and to occur in heterokaryon- homokaryon mating (he-ho-mating). The majority of the genetic variation was contained within populations; however, considerable genetic differentiation was observed among populations from three distant regions (F_ST = 0.750). The low level of inbreeding (F_IS = 0.018) suggested that the populations were panmictic. The most common type of mating events was confirmed to be a spore-mediated outcrossing reflected in spatial distribution of mating type factors. However, gene flow between geographic localities was restricted. Clustering analysis demonstrated that genetic distance was correlated with geographic distance and that a large component of the genetic variation was due to allele frequency differences among populations. Population structure of P.pulmonarius will be discussed with a special reference to sexual reproduction, clonality, and outcrossing events. The research was supported by RFBR grant 06- 04-08161.

Several clades of unicellular organisms diverged near the animal/fungal divide. Of these, the Mesomycetozoans or Ichthyosporids are perhaps the most diverse and are comprised of approximately 70 spp. of animal commensuals and parasites. Mesomycetozoans have been isolated from animals ranging from arthropods and molluscs to vertebrates but very few have been propagated in culture. We have successfully cultured five new species isolated from the gut contents of various marine invertebrates. We characterized the species using ribosomal DNA sequences and have studied their life- cycles and ultrastructure using microscopy. We documented and photographed four mechanisms of dispersal and the asexual life-cycles of three species. One species, which we plan to name ‘Creolimax fragrantissima,’ disperses via numerous slug-shaped amoebae and produces multicellular clusters. Further details of amoeboid behaviour, phylogenetic placement and ultrastructure of this new species will be presented. Understanding of the biology of these closely related fungus-like protists is integral to our understanding of how fungus-like forms evolved and why the true fungi became such a successful multicellular kingdom.

80. Evolutionary Aspects of the Alkaloid Biosynthesis in Claviceps sp. Nicole Lorenz¹, Caroline Machado², Ella Konnova², Chris Schardl², Paul Tudzynski¹. ¹Institute of Botany, WWU Muenster, Germany. ²Department of Plant Pathology, University of Kentucky, USA

The genus Claviceps consists of many phytopathogenic ascomycets producing ergot alkaloids during a specific stage of their lifecycle. Ergot alkaloids show high homology to some neurotransmitters and bind to the same receptors in the CNS leading to an application in a variety of clinical conditions. The genes encoding for the enzymes needed for alkaloid production are organized in a cluster. To study the evolution of the alkaloid biosynthesis the work focused on a comparison of the cluster sequences of three Claviceps species producing different kinds of alkaloids as endproducts of their pathways. While C. purpurea is able to synthesize ergopeptides the other species fulfill only a truncated pathway leading to an accumulation of species-specific intermediates. C. fusiformis shows a corresponding cluster including the genes for the early steps within the pathway. Two additional genes for further synthesis are present but complementation of the corresponding knock-out mutants of C. purpurea failed (the genes seem to be not functional due to a rearrangement event) whereas the implementation of a functional gene of C. purpurea results in a modified alkaloid spectrum of C. fusiformis. C. hirtella synthesizes ergometrine, a simple ergopeptide and should only lack the large NRPS genes. First sequence analysis confirmed these assumptions.

81. Rapid evolution by genetic decay of mating type (mat) genes in homothallic Neurospora. Lotta Wik, Magnus Karlsson, Hanna Johannesson Dept of Evolutionary Biology, Uppsala University Hanna.Johannesson@ebc.uu.se

In filamentous ascomycetes, three basic sexual reproductive modes are present: heterothallism, homothallism and pseudohomothallism. The mat locus is the sole determinant of the mating type in Neurospora, and is the master-regulator of sexual reproduction in this genus. We sequenced the four mat-genes from Neurospora taxa with different reproductive systems, and used likelihood-based methods to reveal that two mat-genes (mat a-1 and mat A-2) evolve under positive selection in heterothallic taxa, while all four mat-genes in homothallic taxa are subjected to an evolutionary decay. In all four genes a lower ratio of non-synonymous to synonymous substitutions (dN/dS) was found in heterothallic than in homothallic taxa. This is due to a higher proportion of conserved sites in the heterothallic dataset and a higher proportion of neutrally evolving sites among the homothallic species. While the heterothallic taxa show a codon or domain specific selective pressure, the codons with elevated dN/dS were more evenly distributed along the genes in the homothallic dataset. This indication of mat-gene degeneration is further supported by the observations of a disrupted ORF of mat a-1, mat A-2 and mat A-3 in homothallic taxa, and the finding of mat A-2 being silenced in N. africana and N. galapagosensis, thus becoming a pseudogene in these two taxa. Our result indicate that mat-genes evolve in an adaptive manner in heterothallic taxa, while in homothallic taxa they are not needed to be functionally conserved.

82. Many Globally Isolated AD Hybrid Strains of Cryptococcus neoformans Originated in Africa. Anastasia P. Litvintseva, Irka Templeton, Joseph Heitman, and Thomas G. Mitchell. Duke University Medical Center, Durham, USA

AD strains of C. neoformans are hybrids between C. neoformans var. grubii (serotype A) and C. neoformans var. neoformans (serotype D). While most isolates of serotype A and D are haploid, AD strains are diploid or aneuploid, contain two sets of chromosomes and two mating type alleles, MATa and MATalpha, one from each of the serotypes. The global population of serotype A is dominated by isolates with the MATalpha mating type; however, about half of the globally analyzed AD strains possess the extremely rare serotype A MATa allele. Previously we described an unusual population of serotype A in Botswana, in which 25% of the strains contain the rare MATa allele. Here we utilized two methods, phylogenetic analysis of three genes and genotyping by AFLP, and discovered that AD hybrid strains possessing the rare serotype A MATa allele are clonally related and cluster with isolates of serotype A from Botswana, whereas AD hybrids that possess the MATalpha serotype A allele cluster with cosmopolitan isolates of serotype A. We also determined that AD hybrids are more resistant to UV radiation than haploid serotype A strains from Botswana. These findings support two hypotheses: (i) AaDalpha strains originated in Botswana from a cross between strains of serotype A and D; (ii) This fusion produced strains with increased fitness, enabling the Botswanan serotype A MATa genome, which is otherwise geographically restricted, to propagate globally.

Although horizontal gene transfer (HGT) is a major aspect of prokaryotic evolution and is becoming increasingly documented between prokaryotes and eukaryotes, very few cases of HGT of nuclear genes from eukaryote to eukaryote have been documented so far. Several obstacles impede the unequivocal demonstration of HGT. The detection of foreign genes is facilitated if the recipient and donor genomes are from two different kingdoms, but if they are relatively closely related then observations suggestive of HGT may be equally consistent with alternative scenarios of gene duplication and gene loss. Aspergillus oryzae is a filamentous fungus that has a large gene complement compared to its annotated relatives. Here we test three alternative explanations for the existence of these extra genes in Aspergillus oryzae: recent gene duplication, whole genome duplication, and HGT. We show using gene location, evolutionary rate, and phylogeny based methods that HGT took place from the close sister subphylum Sordariomycetes into an ancestor of Aspergillus oryzae. Finally, we investigate the role of the transferred genes and show that transfer played a role in enriching Aspergillus oryzae's gene repertoire, specifically by expanding its hydrolytic enzyme gene complement.

84. The influence of CAT fusion on colony establishment in Neurospora crassa. M. Gabriela Roca ^1*, Natalia Angarita-Jaimes², Catherine Towers², David Towers² and Nick Read¹ 1 Institute of Cell Biology, University of Edinburgh, UK 2 School of Mechanical Engineering, University of Leeds, UK * Gabriela.Roca@ed.ac.uk

Hyphal network formation is a key feature of filamentous fungi. We are using Neurospora crassa as a model to study hyphal networks which are formed during colony establishment by means of conidial anastomosis tube (CAT) fusion. The role of CAT fusion in colony establishment is presently unclear. We have addressed this issue by recording germling growth and nuclear dynamics during the first 10 h following conidial germination. Nuclei were labeled with H1::GFP and imaged by confocal microscopy. Advanced image processing techniques were used to track nuclear motion and determine germ tube growth rates. Nuclei were identified by a particle mask correlation method and then tracked with a particle tracking velocimetry (PTV) algorithm. Germling growth was monitored by means of a level set algorithm for centerline extraction. Germ tubes within microcolonies derived from single conidia grew significantly more slowly ( < 0.03 ìm/sec) than colonies formed from conidia that had fused via CATs ( < 0.085 ìm/sec). Nuclei in microcolonies derived from fused conidia achieved higher velocities ( < 1.2 ìm/sec) than those formed from single unfused conidia (< 0.8 ìm/sec). Our initial results provide evidence for cooperative behavior between fused conidial germlings; CAT fusion seems to stimulate germ tube growth and actively promote nuclear mixing.

85. Molecular characterization of the Fusarium graminearum species complex in Japan. H. Suga¹, G.W. Karugia¹, T. Ward², L.R. Gale³, K. Tomimura⁴, T. Nakajima⁴, A. Miyasaka⁴, S. Koizumi⁴, K. Kageyama¹, and M. Hyakumachi¹. ¹Gifu University, Gifu, Japan. ²Agricultural Research Service, USDA, Peoria, USA. ³University of Minnesota, St. Paul, USA. ⁴National Agricultural Research Center, Tsukuba Japan.

We collected 298 strains of the the Fusarium graminearum species complex from wheat or barley from 2001 to 2004 in Japan and investigated species and trichothecene chemotype compositions. Species-diagnostic PCR-RFLP revealed the presence and differential distribution of Fusarium graminearum s. str. and Fusarium asiaticum in Japan. F. graminearum s. str. is predominant in the north, while F. asiaticum is predominant in southern regions. In the Tohoku area, distinct co-occurrence of these species was observed. Chemotyping by multiplex PCR revealed significantly different chemotype compositions of these species. All 50 strains of F. graminearum s. str. were of a 15- or 3-acetyl deoxynivalenol type, while 173 out of 246 strains of F. asiaticum were of a nivalenol type. The possibility of gene flow between the two species was investigated by PCR-RFLP markers, as interspecies hybridization of F. graminearum s. str. and F. asiaticum was previously achieved in vitro. However, no obvious hybrids were detected from 98 strains examined, including strains collected from regions where both species co-occur.

86. Withdrawn

87. Phytophthora database: An integrated resource for detecting, monitoring, and managing Phytophthora diseases. Seogchan Kang¹, Jaime Blair¹, Dave Geiser¹, Izabela Makalowska¹, Sook-Young Park¹, Bongsoo Park¹, Mike Coffey², Kelly Ivors³, Yong-Hwan Lee⁴, Jong-Seon Park⁴, and Frank Martin⁵. ¹Penn State, University Park, PA. ²UC, Riverside, CA. ³NCSU, Raleigh, NC. ⁴Seoul National Univ., Seoul, Korea. ⁵USDA-ARS, Salinas, CA.

High virulence of Phytophthora species and their ability to spread rapidly establishes Phytophthora as one of the most important groups of plant pathogens. The ability to accurately and rapidly identify the causal agent of a disease is crucial for developing effective regulatory and management strategies against Phytophthora. The Phytophthora database project ( Link here ) was initiated to enhance our capability of rapid detection and diagnosis of Phytophthora spp. by archiving known genotypic and phenotypic diversity in a highly integrative database. To compliment the species morphological descriptions and serve as a molecular reference for isolate identification, ~2000 accessions representing 71 morphological species maintained in the WPC Genetic Resource Collection have been genetically characterized. A seven-locus phylogeny of the whole genus supports the division of Phytophthora into approximately eight major groups. In addition to these nuclear genes, four mitochondrially encoded genes are being sequenced from the same isolates to construct a mitochondrially based phylogenetic framework. This project is on-going; sequence and species data are continually being deposited, and new data analysis and visualization tools are being developed to increase utility and breadth of this database. An overview of database functions will be presented.

The cooperative behaviors of filamentous fungi have long fascinated mycologists. In these experiments we use micromanipulations of individual conidia to demonstrate that time to germination is significantly reduced when two or more conidia are grouped. Single conidia take longer to germinate and both the length of the germ tube, as well as the perimeter of a colony, are significantly smaller as compared to germ tubes and colony perimeters of two or more conidia. A similar result is found with two wildtype strains as well as the soft mutant (which lacks the ability to fuse), proving that the pattern is a general one and is likely to be controlled by early signals working independently of the fusion process. Experiments with isolates from geographically distant locations suggest that this kind of cooperation functions even when isolates are genetically distinct, but whether one isolate is co-opting the mechanism for its own benefit remains to be tested.

89. Interspecific transfer of host-specific toxin genes in Stagonospora nodorum. Richard P Oliver¹, Peter S. Solomon¹, James Hane¹, Eva H. Stukenbrock², Bruce A McDonald², Zhaohiu Liu³, Justin D Faris³, Timothy L Friesen³. ¹ACNFP, Murdoch University, Australia ²ETH, Zurich, Switzerland ³USDA and NDSU, ND

The host-specific toxin ToxA, produced by P. tritici-repentis, confers virulence on wheat genotypes carrying Tsn1. The Stagonospora nodorum gene, SnToxA was identified from a reannotation of the genome sequence and is nearly identical to PtrToxA. Disruption of SnToxA reduced disease on wheat lines carrying Tsn1. ToxA genes from a large isolate collection from P. tritici-repentis were identical but those from S. nodorum were highly variable. P. tritici-repentis was first identified in 1902 but it was not until 1941 that typical tan spot symptoms were first described. Nowadays, P. tritici- repentis is a regular and abundant pathogen. Our evidence strongly suggests that ToxA was horizontally transferred to P. tritici-repentis some time prior to 1941. These new forms appear to have rapidly spread around the world, probably in grain shipments (Nature Genetics 38 953-956). Our work indicates that S. nodorum produces several other toxins. The presence of multiple host-specific toxins and their transfer between species raises many interesting evolutionary questions; 1. How does toxin possession and expression affect the fecundity of fungal strains carrying the toxins? 2. How can we explain the expression of toxin receptor genes? 3.What are the mechanisms of sequence diversification in toxin genes?

90. Explosively launched ascospores are shaped to minimize drag. Marcus Roper, Michael P. Brenner and Anne Pringle, Harvard University Cambridge MA 02138, pringle@oeb.harvard.edu

Many species of ascomycete fungi have a sexual phase in which ascospores are forcibly ejected clear of the fruiting body. Reproductive success is dependent upon the spores germinating away from the parent fungus, and thus the spores must be ejected at very large launch speeds in order to pass through the layer of still air surrounding the fruiting body, into the vigorous air currents beyond. To do this these fungi have evolved mechanisms for applying initial accelerations that are unmatched in the plant or animal kingdoms. Spores are tiny, fast moving objects, so the distance that they can travel in still air is severely limited by fluid drag. We present evidence that drag minimisation is a primary determinant of spore shape. This results leads to several predictions, including upper and lower bounds upon spore size (spores are predicted to be no smaller than 1.8 micrometers and no larger than 30 micrometers) and a constant launch speed of 2 m/s across the phylum.

91. Sexual mating of B. cinerea (fuckeliana) illustrates PRP8 intein HEG activity. Annika A.M. Bokor and Russell T.M. Poulter, University of Otago, Dunedin, New Zealand

Inteins are proteins that are removed post-translationally from a host protein. This is an autocatalytic splicing process necessary for the maturation of the host protein. Inteins typically contain not only the necessary splicing domains but also a homing endonuclease (HEG). The HEG enables the intein encoding sequence to spread through the gene pool of a species and potentially to also undergo horizontal transmission to other species. In a diploid, heterozygous for the presence/absence of the intein gene, the HEG recognises a 30-50bp target sequence at the ‘empty’ allelic site. It introduces a double-stranded DNA break at this target. This double-strand break triggers the host’s recombinational repair system, which in the repair process copies the full-length intein into the target site. Our group has discovered several nuclear full-length inteins (located in the critical PRP8 gene) in species including the human fungal pathogens Aspergillus fumigatus, Histoplasma capsulatum and the plant pathogen Botrytis cinerea. To demonstrate intein replication a species must have a sexual cycle and there must be polymorphism for presence/absence of the intein in the gene pool of the species. We have demonstrated that B. cinerea is polymorphic for presence/absence of the PRP8 intein. Characterisation of HEG activity in B. cinerea sexual cycle will be presented. This is the first mycelial system in which the intein replication cycle has been demonstrated.

The basidiomycetous yeast Cryptococcus gattii causes life-threatening disease mainly in immunocompetent patients. Four molecular types have been identified: VGI/AFLP4-VGIV/AFL7. Two subtypes of molecular type VGII/AFLP6 have emerged in 2000 as a primary pathogen on Vancouver Island, Canada, causing a large scale cryptococcosis outbreak in humans and animals: VGIIa/AFLP6A, the major and more virulent and VGIIb/AFLP6B, the minor and less virulent genotype. A global molecular epidemiological study of more than 160 VGII strains recovered since 1986 was conducted using PCR fingerprinting, AFLP and MLST analysis (8 polymorphic loci: ACT1, CAP59, IDE, IGS, LAC1, PLB1, SXI1alpha, URA5). VGIIa is also present in Colombia, Brazil, Venezuela, Argentina, USA, Thailand and Greece. Australian VGII isolates clustered with VGIIb. Brazilian VGIIa isolates are identical with the Vancouver Island outbreak strains. Colombian VGIIa isolates are closely related but not identical to the Vancouver Island outbreak strains and are avirulent. The vast majority of Colombian isolates are MATa compared with the Vancouver Island outbreak isolates (all MATalpha). In vitro mating experiments showed that the avirulent Colombian MATa strains mate with the virulent VGII MATalpha strains from Brazil and Vancouver. The fact that there are VGIIa and VGIIb isolates recovered as early as in 1986 in South America indicates that these genotypes may have been present for a long time in the Americas rather than being a result of a recent recombination event between a less virulent genotype introduced to North America from Australia and an unknown mating partner as suggested by Farser et al. (Nature 437,2005).

93. Evolution and phylogeny of Candida species inferred from multigene analyses. Clement Tsui ¹, Heide-Marie Daniel ³, Wieland Meyer^1,2. ¹Molecular Mycology Research Laboratory, Center for Infectious Diseases and Microbiology, Westmead Hospital, Westmead, Australia; email: clementsui@gmail.com ²Western Clinical School, Department of Medicine, The University of Sydney, Australia; ³BCCM/MUCL culture collection, Université catholique de Louvain, Unite de Microbiologié, Louvain-la- Neuve, Belgium.

Candida species are ubiquitous human pathogens, plant endophytes and insect symbionts. Phylogenies based on rDNA and actin sequences suggested that the genus is not monophyletic, and did not clearly resolved the relationships among related genera and species. Protein coding genes are useful to resolve taxonomic positions among a broad range of fungi. We selected over 70 taxa from Candida and allied genera, and investigated their phylogenetic relationships using nuclear sequences of the largest subunit (RPB1) and second largest subunit (RPB2) of RNA polymerase II gene, actin, mitochondrial cytochrome oxidase subunit 2 (COX2) gene, and LSU rDNA. The DNA sequences were subjected to maximum parsimony and Bayesian analyses. Four major phylogenetic groups were recognized according to Bayesian probabilities. Group A contained six pathogenic species of Candida, seemed to derive from non-pathogenic species, while Group B contained species of Clavispora, Metschinikowia, and Pichia guilliermondii. Species of Debaryomyces were related to groups A and B but formed an independent clade. Group C consisted species in the Saccharomycetaceae, in sibling relationship with a clade containing Pichia jadinii. Pichia fermentans and other environmental species concentrated in Group D. The phylogenetic relationships of species and the evolution of codon usage would be discussed.

94. Phylogeny of clusters of genes with predicted roles in biosynthesis of epipolythiodioxopiperazine toxins in ascomycetes. Nicola Patron¹, Anton Cozijnsen¹, Bill Nierman², Ross Waller¹ and Barbara Howlett¹. ¹School of Botany, the University of Melbourne, Australia; ² The Institute for Genomic Research, USA

Epipolythiodioxopiperazines (ETPs) are toxins made by phylogenetically diverse filamentous fungi. Ten clustered genes are responsible for biosynthesis of the core ETP moiety. Clusters with most or all the core genes are present in Magnaporthe grisea, Chaetomium globosum, Fusarium graminaerum, Trichoderma virens, T.reesei, Leptosphaeria maculans, Penicillium lilacinoechinulatum, Aspergillus terreus, A.clavatus, A.flavus, A.oryzae, A.fumigatus, Neosartorya fischeri. Each of six cluster genes examined is closely related phylogenetically to non-cluster paralogues within filamentous fungi. Similar relationships were inferred for all six genes, each one sharing a common ancestry to the exclusion of non-cluster paralogues, excepting cluster genes from C.globosum and F.graminarium. This suggests that most ETP gene clusters have a common origin and that the cluster was inherited relatively intact, rather than assembling independently in the different ascomycete lineages. Internal topologies of clusters do not reflect phylogeny of ascomycete lineages. Gene clusters fall into three phylogenetic classes, which do not necessarily reflect the structures of toxins produced. The disjunct heredity of these clusters may arise from multiple independent losses of cluster class or lateral gene transfer of clusters between lineages.

95. Horizontal Gene Transfer and Recombination in Histone 3 Gene Evolution of Alternaria. Soon Gyu Hong, and Barry M. Pryor. Korea Polar Research Institute, Incheon, Korea.

Evolution of H3 gene was analyzed by comparing H3 gene phylogeny with reference phylogeny constructed by alt a 1, gpd, and ITS sequences in Alternaria and related species. Several H3 gene types defined by intron presence and absence were recovered and distribution of each gene type on the phylogeny was very complex. We postulated four alternative hypotheses to explain the findings: 1) maintenance of different types of H3 gene in a strain with one of them as a major copy and the others as minor copies; 2) repeated insertion and deletion of introns during evolution of H3 genes; 3) maintenance of multiple copy of the gene in the ancestral species and independent loss of different type of genes in each descendent; 4) exchange of genetic materials among strains leading to horizontal transfer of the gene and recombination between different copies of the gene to achieve concerted evolution. Among them, the fourth hypothesis of horizontal gene transfer and recombination was chosen as the most plausible explanation for the evolution of H3 gene in Alternaria and related species. The first hypothesis of maintaining multiple types of H3 gene could explain the multiple PCR bands of radicina species-group and Stemphylium group.

There have been debates in defining species boundary in fungi with emphasis on phenotypic or genotypic characteristics. To test the extent of genetic diversity in lichen-forming fungus, Parmelia laevior, sequence variations of three genes (ITS, RPBII and mtSSU rDNA) were analyzed from 20 spore-germinated cultures from an identical fruiting body. All of the isolates contained identical mtSSU rDNA sequences, whereas two types of ITS and RPBII sequences were observed. Two types of ITS sequences showed 7 nucleotide difference among 639 bases (98.9% similarity). Two types of RPBII sequences showed 16 nucleotide difference among 1192 bases (98.83% similarity). To discriminate mixed cultures from homogeneous single spore cultures, two types of single-copy RPBII gene was specifically detected by oligonucleotide ligation assay (OLA) based upon the single nucleotide polymorphism. As a result, 13 isolates turned out as mixed cultures by showing both types of RPBII sequences. To detect incomplete concerted evolution of ITS sequences, OLA was applied for ITS sequences and it was found that all of the single-spore isolates had only one type of ITS sequences. RPBII and ITS alleles were segregated together in all of the single-spore isolates, implying that they are closely linked on the chromosome. From the identical sequences of mtSSU rDNA and high variation of ITS and RPBII sequences, it is concluded that boundary of species in Parmalia laevior is broader than have been expected from previous researches.

97. The evolution of mating type idiomorphs in the genus Mycosphaerella. Mahdi Arzanlou^1,2, Lute-Harm Zwiers¹, and Pedro W. Crous^1,2. ¹Evolutionary Phytopathology Group, CBS Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands. ²Laboratory of Phytopathology, Wageningen University, Binnenhaven 5, 6709 PD Wageningen, The Netherlands. arzanlou@cbs.knaw.nl

The genus Mycosphaerella is one of the largest genera of ascomycetes, comprising several thousand species. Despite the diverse ecological habitats inhabited by species of Mycosphaerella, many species are specialized plant pathogens, occurring on a wide range of hosts ranging from monocots, dicots to even gymnosperms. The fact that different Mycosphaerella species can co-occur on a single host and even within a single lesion, as well as their ability to jump between hosts, makes Mycosphaerella a good model to study the (co) evolution of closely related species. The mating strategy for the majority of Mycosphaerella species is yet unknown; for a few species a heterothallic or homothallic mating system has been observed. However, for the majority of the Mycosphaerella species the understanding of sex remains a puzzle. A large number of species in this genus are known only from their teleomorphs, while for many other species no Mycosphaerella state has been discovered, and only the anamorphic stage is known. In order to examine the importance of sexual reproduction in evolution and speciation of Mycosphaerella species occurring on banana, we analyzed the structure of the mating type idiomorphs of several additional Mycosphaerella species. Analysis of the structure of the mating type genes from homothallic and heterothallic members of this genus showed unique features in the mating type loci. In all mating type idiomorphs additional open reading frames (“ORFs”) can be distinguished which are seemingly unique to the Mycosphaerellaceae. Furthermore, the obtained data suggest that heterothallism is in most cases the ancestral state, and evidence exists that that some heterothallic species have evolved from a homothallic ancestor.

98. Molecular Analysis and Evolution of Gibberellin Biosynthetic Gene Clusters in Fungi. Christiane Bömke and Bettina Tudzynski. WWU Münster, Institut für Botanik, Schlossgarten 3, Germany

Although gibberellins (GAs) are ubiquitous diterpenoid plant hormones, they were first identified as secondary metabolites of Fusarium fujikuroi. Today it is known that they are also produced by several bacteria and some other fungi. Since a horizontal gene transfer of the GA genes from plants to fungi can be excluded due to fundamental differences at the chemical (pathway), biochemical (enzymes) and genetic level, we are interested in the origin of the fungal GA cluster genes. To elucidate this question, we focus on the distantly related GA producing fungus Sphaceloma manihoticola. The spectrum of the GAs present suggests that the GA biosynthesis in Sphaceloma follows the pathway known for Fusarium. However, in contrast to F. fujikuroi, the biosynthesis does not go to the end product GA₃ but stops two steps earlier with GA₄. The assumption that the responsible genes (P450-3 and des) are missing was confirmed after cloning of the S. manihoticola GA gene cluster: homologues of P450-3 and des which flank the gene cluster in Fusarium are missing in this cluster. The order of the other five genes is similar but not identical to F. fujikuroi due to gene inversions. The functional analysis of the genes was done by a combination of knock-outs and complementation of the corresponding F. fujikuroi-mutants. A heterologous gene expression of Ff-P450-3 and Ff-des will show if the complete pathway can be restored.

99. An Unprecedented Shift in the Composition of Fusarium Head Blight Pathogens in North America due to the Rapid Spread of a Highly Toxigenic Population of Fusarium graminearum. Todd J. Ward¹, Randall M. Clear², Alejandro P. Rooney¹, Kerry O’Donnell¹, Don Gaba², Jeannie Gilbert³, Susan Patrick², and David E. Starkey⁴. ¹Microbial Genomics and Bioprocessing Research Unit, Agricultural Research Service, United States Department of Agriculture, Peoria, Illinois, USA. ²Grain Research Laboratory, Canadian Grain Commission, Winnipeg, Manitoba, Canada. ³Agriculture and Agri-Food Canada, Winnipeg, Manitoba, Canada. ⁴University of Central Arkansas, Conway, Arkansas, USA.

To date, a fraction of the combined species and chemotype diversity of Fusarium head blight (FHB) pathogens is present in North America. However, our active surveillance with a unique multilocus genotyping assay revealed for the first time that 3ADON-producing isolates of F. graminearum are prevalent in North America. In addition, we identified a dramatic East-West (3ADON-15ADON) chemotype cline in Canada, and documented a recent and unprecedented shift in FHB pathogen composition in North America by demonstrating that the 3ADON chemotype frequency in Canadian prairie provinces increased 14-fold between 1998 and 2004. Analyses of population structure based on nine VNTR markers demonstrated that 3ADON and 15ADON isolates from western Canada represent distinct populations (F_ST = 0.4, P < 0.001) with low levels of gene flow evidenced by the identification of a small number of admixed isolates. On average, isolates from the 3ADON population produce significantly (P < 0.001) higher levels of trichothecene toxins, grow faster, and produce more spores in vitro than isolates from the 15ADON population. Combined with the rapid and significant shift in chemotype frequency that we have documented, these data suggest that a recently introduced or emergent population of F. graminearum with a 3ADON chemotype has a selective advantage and is rapidly changing the FHB landscape in North America. In addition, the demonstration that highly toxigenic FHB pathogens with a novel trichothecene profile have rapidly displaced less toxigenic isolates has significant implications for food safety and disease control efforts.

Exhibiting permanent geochemical stratification, Green Lake near Fayetteville New York, is an example of a meromictic lake. The lake’s physical environment is structured like a layer-cake: two chemically distinct ecozones— an upper oxic mixolimnion and lower anoxic monimolimnion— remain separated by a stable redox transition zone, or “chemocline”. Previous diversity studies have been conducted at other meromictic lakes. While related research has focused on bacterial and archaeal assemblages; few, if any, attempts have been made to characterize fungal diversity in meromictic environments. The goal of the present study is to determine how fungal diversity in Green Lake varies with depth and the associated changes in water chemistry. Our geochemical analysis revealed distinct stratification, with dramatic shifts in pH, temperature, oxidation-reduction potential, and specific conductance occurring within the chemocline. We expect that niche differentiation and community structure will exhibit vertical stratification in association these changes in water chemistry. In June of 2005, Microbes were collected from Green Lake via sterile syringes deployed at 23 depths spanning the chemocline. Sampling intervals varied from 5 m in chemically uniform regions of the mixolimnion and monimolimnion to 25 cm across the chemocline. A 500bp region of fungal 18s rDNA was selectively amplified using taxon-specific primers. We are currently in the process of using Denaturing Gradient Gel Electrophroesis (DGGE) to profile fungal diversity from water samples collected at various depths. Following DGGE, unique 18s rDNA amplicons will be sequenced to determine community composition. Preliminary DGGE of a chemocline community indicates a richness of 8 to 12 fungal species. Depth-related trends in fungal diversity will be presented.

101. Structural and Functional Evolution of Non-Ribosomal Peptide Synthetases. Kathryn E. Bushley¹ and B. Gillian Turgeon¹. ¹Department of Plant Pathology, Cornell University.

Non-ribosomal peptide synthetases (NRPSs) are multimodular enzymes that make non-ribosomal peptides (NRPs) through a thiotemplate mechanism independent of ribosomes. These structurally diverse molecules have important roles in basal and niche-specific developmental success of filamentous fungi. In addition to roles they play in their producers’ lives, NRPs have diverse biological effects on other organisms including antibiotic, immunosuppressant, antitumor and virulence-promoting activities. Previous studies suggested that NPS genes have a highly discontinuous distribution in filamentous ascomycetes (Lee et al., Eukaryotic Cell, 2005). Comparative analyses of a larger dataset suggest that NPSs show varying levels of functional and structural conservation. Genes with functions critical to the fungal cell (stress resistance, involvement in sexual and asexual reproduction, virulence, morphology, etc) are highly conserved. For example, Cochliobolus heterostrophus NPS6, responsible for extracellular siderophore biosynthesis (Oide at al., Plant Cell, 2006) is important for virulence and resistance to oxidative stress and is conserved both structurally and functionally across all fungi examined to date. Homologs of ChNPS2, responsible for biosynthesis of an intracellular siderophore, are also found in most fungi but show a history of duplication into two clades, NPS2 and NPS1/SidC/Sid2 (represented by Fusarium graminearum NPS1/Aspergillus nidulans SidC/Ustilago maydis Sid2), with corresponding differences in function. At the other end of the spectrum are taxon-specific genes such as ChNPS1 and ChNPS3 which are intact in only a few closely related Cochliobolus species. We are using a variety of phylogenetic tools to address the roles of recombination, natural selection, and duplication/loss, in generating diversity of NPSs and their corresponding peptide products.

102. Evolution of the Cryptococcus Mating-type Locus: a Comparative Genomics Approach. Keisha Findley¹, James Fraser², and Joseph Heitman¹.Department of Molecular Genetics and Microbiology¹, Duke University, Durham, North Carolina, School of Molecular and Microbial Sciences², University of Queensland, Brisbane, Australia. kmf13@duke.edu

Many basidiomycetous fungi have tetrapolar mating-type systems in which mating is regulated via two unlinked loci that control and establish cell identity and both must differ for sexual reproduction. One MAT locus encodes pheromones and pheromone receptors, while the other MAT locus encodes homeodomain transcription factors. In contrast, the fungus Cryptococcus exhibits a bipolar mating system with a single locus that encodes the pheromones, receptors, and transcription factors that establish mating-type. The ability of this fungus to reproduce sexually has been studied extensively and underlies development and is linked to pathogenicity. To characterize the evolution of the mating-type locus of this pathogenic fungal species complex, a comparative genomics approach is being applied with distantly and closely related species. This analysis involves cloning and sequencing the MAT locus from the closely related species Tsuchiyaea wingfieldii, Filobasidiella depauperata, Bullera dendrophila, Cryptococcus amylolentus, Cryptococccus heveanensis and the more distantly related species Cryptococcus laurentii and Cryptococcus albidus. To identify genes associated with MAT in these species, degenerate primers are applied for two genes flanking MAT (FAO1 and NOG2) and two highly conserved genes within MAT (RPO41 and LPD1). High-density filters of genomic fosmid libraries are produced and probed with these genes to identify MAT-specific fosmids. Positive clones are confirmed by Southern analysis and pooled to generate sequencing libraries. Thus far, preliminary assemblies have been generated for T. wingfieldii and C. heveanensis. Our results reveal that homologs of the homeodomain proteins Sxi1α and Sxi1a are both present and divergently transcribed similar, to bE and bW in Ustilago maydis. A second unlinked gene cluster encodes pheromone and pheromone receptor homologs and many other MAT associated genes. This analysis reveals that the translocation event that collapsed a tetrapolar to a bipolar system, and loss of one or the other homeodomain genes, occurred recently, perhaps concomitant with the emergence of this pathogenic species complex. Further MAT sequencing and studies on sexual reproduction potential are in progress.

103. Cryptic Sexuality in Aspergillus parasiticus and A. flavus. Jorge H. Ramirez-Prado¹, Geromy G. Moore¹, Bruce W. Horn², Ignazio Carbone¹. ¹Center for Integrated Fungal Research, Department of Plant Pathology, North Carolina State University, Raleigh, NC. ²National Peanut Research Laboratory, USDA, ARS, Dawson, GA.

Ascomycetous fungi of the genus Aspergillus comprise a wide variety of species of biotechnological importance (e.g. A. sojae, A. oryzae, A. niger) as well as pathogens and toxin producers (e.g. A. flavus, A. parasiticus, A. fumigatus, A. nidulans). With the exception of A. nidulans, which is a homothallic fungus, these species were previously thought to be strictly asexual. Recent studies report A. fumigatus to be heterothallic and possibly undergoing sexual reproduction. Our population genetic analysis of the aflatoxin gene cluster in a Georgia population of A. parasiticus showed evidence of four distinct recombination blocks each showing a pattern of significant linkage disequilibrium and shared evolutionary history, thus providing indirect evidence of the possibility of sexual recombination in nature. We therefore investigated whether compatible mating type “MAT” genes (MAT1-1 alpha box gene and MAT1-2 HMG gene) were present. We found that each distinct A. parasiticus chemotype lineage (OMST and G₁ dominant) was heterothallic and possessed a single MAT locus, containing either a MAT1-1 (G₁ dominant) or a MAT1-2 (OMST) idiomorph. We observed a similar heterothallic organization of the MAT locus in A. flavus sampled from the same Georgia field but there was no association of mating type idiomorph with a specific chemotype lineage. The existence of both mating type idiomorphs in equal proportions in A. parasiticus and A. flavus populations indicates the potential for a cryptic sexual state in these agriculturally important species.

104. What makes a pathogenic fungus? A comparative genomic analysis of Coccidiodies reveals the evolutionary rise of pathogenicity. Thomas J. Sharpton, Jason E. Stajich and John W. Taylor. Plant and Microbial Biology, University of California, Berkeley, CA 94720-3102, USA email: sharpton@berkeley.edu

Knowledge regarding the evolutionary origins of pathogenicity is currently limited, but ultimately necessary to tackle disease. We employed evolutionary comparative genomic techniques to evaluate the origins of the pathogenic phenotype of Coccidioides, a Eurotiomycete capable of causing disease in healthy human adults. Comparisons of the pathogenic genomes of C. immitis, C. posadasii and the close, non-pathogenic relative Uncinocarpus reesei revealed genes unique to the pathogens and genes rapidly evolving under selective pressure, perhaps to escape the host immune system. This analysis reveals putative antigens and pathogenicity genes that will be investigated further in vivo. Additionally, surveys of gene family size across the fungal kingdom reveal unique characteristic expansions and contractions of certain families in the Onygenales, which contains several dimorphic pathogens including Coccidoides. Strikingly, families that experience significant reduction in the dimorphic pathogens appear to play a role in plant pathogenicity. Additionally, Coccidioides appears to have undergone expansions in gene families known to contribute to pathogenicity in Coccidioides as well as non-Onygenalen pathogens. This analysis should serve to identify possible drug and vaccine targets for Coccidioides and ultimately clarify the origins of the genetic architecture that enables a pathogenic phenotype in the Onygenales.

105. Evidence of sexual reproduction in the Septoria speckled leaf blotch pathogen, Septoria passerinii, in the upper Great Plains. Seonghee Lee and Stephen M. Neate. Department of Plant Pathology, North Dakota State University, Fargo 58105- 5012, USA. Email: sul22@psu.edu

Fifty seven polymorphic AFLP markers and mating type idiomorphs of Septoria passerinii were used to examine the sexual reproduction of the barley pathogen under field conditions. A total of 496 Septoria passerinii isolates were collected from 19 widely distributed barley fields in North Dakota and western Minnesota. The level of genetic and genotypic diversity was low within a lesion, but the higher level was found within a leaf and field. Population differentiation was low among field populations, and a significant correlation between geographical distance and genetic distance was not found. In addition, two mating types of Septoria passerinii, MAT-1 and MAT-2, were frequently found in isolates taken from individual leaves. The chi-square test revealed that equal frequencies of both mating types were observed in a leaf and field population. Moreover, when the data for all 19 sites was pooled, the overall ratios for mating types did not statistically differ from 1:1 ratio. The equal frequencies of both mating types are consistent with a sexually reproducing fungal population. Thus, we conclude that sexual recombination is occurring in S. passerinii under field conditions in the upper Great Plains.

106. Comparative functional genomics: Reconstructing the evolution of central carbon metabolism in 13 fungal species. Dawn Thompson¹ and Aviv Regev^1,2. ¹Broad Institute of MIT and Harvard and ²Department of Biology, MIT, Cambridge, MA USA

Molecular networks are the information processing devices of cells and organisms, transforming extra- and intra-cellular signals into coherent cellular responses. Networks are also remarkably flexible and can re-configure in an adaptive response to perturbation. We seek to understand the mechanisms by which molecular networks accommodate changes by analyzing genomics data through the unifying abstraction of the functional module. Specifically, we focus on evolutionary changes that occur in the organization and regulation of central carbon metabolism in Ascomycota fungi. This is the cornerstone metabolic module of yeast cells. In free-living Ascomycete yeast species the constantly changing environment dictates that nutrient availability is the major factor controlling growth and development. To survive in a highly competitive ecosystem yeasts have evolved to take up and metabolize a variety of carbohydrates, the most important of which is glucose. In the model organism Saccharomyces cerevisiae the central carbon metabolic network has been clarified in great detail, but the regulatory network is less well characterized. Although central carbon metabolism follows the same general theme in all yeasts, important biochemical, genetic and regulatory variations exist. For example, physiologically yeasts can be classified into respiro-fermentative and respiratory during aerobic growth on glucose. The former is characterized by high glucose uptake and ethanol secretion rates, and low biomass yield (Saccharomyces cerevisiae and close relatives) and the latter by low glucose uptakes and high biomass yield (e.g. Kluyveromyces). In addition, for species other than Saccharomyces cerevisiae, many of which have important industrial uses and some are human pathogens, basic knowledge of this and other fundamental pathways is limited. We are setting up a genomics platform to study a set of 13 fully sequenced fungal species spanning over 300 million years of evolution. We grow each organism under a variety of steady state conditions (e.g. rich glucose, alternative carbon sources) and along transitions from one condition to another. We will then measure a variety of phenotypes, most notably growth characteristics and genome wide expression profiles (followed by metabolomics and proteomics). We will use our novel computational algorithms for orthology mapping (SYNERGY) and regulatory network reconstruction (CISPROF), along with novel algorithmic development, to reconstruct the regulatory modules in each extant species, and the modules that existed in ancestral (extinct) ones. We will then mine those reconstructions for general patterns of how module content and regulation evolves and how changes occur at different molecular levels

The blast fungus Magnaporthe oryzae is composed of host-specific subgroups. We examined the population structure of a subgroup, Eleusine isolates, using DNA makers (MGR586, rDNA-ITS, and grasshopper). The population of Eleusine isolates was divided into two groups, A and B. Interestingly, this grouping corresponded to the distribution of avirulence gene PWL1; all the B group isolates carried PWL1 whereas the A-group isolates did not. Furthermore, the presence/absence of PWL1 in Eleusine isolates was correlated with their pathogenicity toward weeping lovegrass (Eragrostis curvula); the A group, non-carrier of PWL1, was pathogenic on Eragrostis species. We assumed that the A group may be derived from an Eragrostis pathogen. To test this hypothesis, we constructed phylogenetic trees of M. oryzae isolates using rDNA-ITS, NUT1, Mdim2, beta-tubulin, Calmodulin, and EF-1alpha genes. In a combined tree the A group was clustered with Eragrostis isolates, not with the B group., as expected. Then, we cloned flanking regions of PWL1 in the A group, the B group, and Eragrostis isolates, and compared their structures. The A group and Eragorstis isolates carried a gene for a hypothetical protein (Acc. XM_369238) in a region corresponding to PWL1. In the B group isolates, however, most of the DNA fragment for the hypothetical protein was substituted with PWL1. These results suggest that the B group may have acquired PWL1 by horizontal transfer or recombination. The two-groups in the Eleusine isolates seem to have evolved through different courses.

108. Homing in on sexual recombination and spore production in the Cryptococcus neoformans species complex. Nathan Saul*^, Tien Bui†, Mark Krockenberger^, Richard Malik^ and Dee Carter†. *Molecular & Microbial Biosciences, and ^Faculty of Vet. Science, University of Sydney, NSW, Australia. d.carter@mmb.usyd.edu.au

Cryptococcosis, caused by Cryptococcus neoformans and C. gattii initiates with the inhalation of an infectious propagule. This is likely to be a basidiospore as yeast cells are encapsulated and unable reach lung alveoli. Spores can be produced sexually via the union of MATalpha and MATa cells, however most populations are overwhelmingly MATalpha. Recent studies have found recombinant spores can also be formed following MATalpha-MATalpha unions. Finally, some MATalpha strains can produce asexual spores, but there is little evidence of this in the genotypes responsible for most cryptococcal infections. A population genetics approach was used to investigate whether sexual recombination, and by implication the production of recombinant spores, had occurred in populations consisting of MATa and MATalpha cells, and in those that were MATalpha only. C. gattii isolates were obtained from a single Eucalyptus camaldulensis tree hollow that contained both mating types, and from a hollow in an area where only MATalpha cells occur. C. neoformans var. grubii isolates obtained from infected animals known to live exclusively in Sydney where MATa cells have never been reported, were also investigated. Sexual recombination was evident in all populations. Key to this finding were 1) selection of populations restricted in time and geographic space; and 2) preliminary phylogenetic analysis to identify recombining subpopulations within larger populations that initially appeared clonal.

109. Genome structure impacts molecular evolution at the AvrLm1 avirulence locus of Leptosphaeria maculans. L. Gout^1,3, M.L. Kuhn¹, L. Vincenot¹, L. Cattolico², S. Bernard-Samain², A. D’Armaillé¹, M.-H. Balesdent¹, and T. Rouxel¹. ¹INRA, PMDV, Route de St Cyr, 78016 Versailles, France. ²Genoscope-centre national de séquençage, 2 rue Crémieux, 91057 Evry, France. ³Protection des Plantes, INA-PG, 78850 Thiverval-Grignon, France

The Dothideomycete Leptosphaeria maculans is the most damaging disease of oilseed rape (Brassica napus) worldwide. In France, disease control relies mainly on the use of disease-resistant cultivars. The Rlm genes effectively control the disease as long as the corresponding avirulent allele (AvrLm) dominates in the pathogen population. However, L. maculans has the ability to rapidly adapt to the selection pressure exerted by a novel resistance gene as exemplified by the 3-year evolution towards virulence at the AvrLm1 locus in French field conditions. AvrLm1 was recently cloned and shown to be a solo gene within a 269-kb non-coding, heterochromatin-like region consisting of mosaics of degenerated repeats. We fully or partly sequenced the AvrLm1 genomic region in one avirulent and two virulent isolates. The gain of virulence was linked in both cases with a 260 kb deletion of a chromosomal segment spanning AvrLm1 and deletion breakpoints were identical or similar for both the virulent isolates. Among 343 field isolates analyzed, a similar large deletion leading to chromosome length polymorphism was evidenced by multilocus haplotype analysis in > 90% of the virulent isolates. Deletion breakpoints were strongly conserved in most of the virulent isolates leading to the hypothesis that a unique event of large deletion leading to the avrLm1 virulence has diffused in populations or is strongly constrained by the genome environment. Surprisingly, even though a large chromosomal fragment was lost to gain virulence, no effect on fungal and pathogenic fitness could be evidenced, either in vitro or under natural field infection conditions.

110. Intron evolution in basidiomycete laccase genes. Sreedhar Kilaru ¹, Pierre-Emmanuel Courty ², Patrik J. Hoegger ³, Timothy Y. James ⁴, Francis Martin ² and Ursula Kües ³. ¹ School of Biological Sciences, University of Bristol, Bristol, UK ² Centre INRA de Nancy, Champenoux, France ³ Molecular Wood Biotechnology, Georg-August-University, Göttingen, Germany* ⁴ Department of Biology, Duke University, Durham, NC, USA

Most but not all homobasidiomycetes possess laccase genes. Coprinopsis cinerea was shown to contain 17 different laccase genes that could be divided into two gene subfamilies by intron positions. Here, we analyze a total of 835 introns in 74 different homobasidiomycete laccase genes related to the C. cinerea subfamily 1 laccase genes. These introns distribute over 75 different positions over the length of the genes. An intron phase-analysis shows a 2:1:1 distribution for phase-zero, phase-one and phase-two insertion within codons and thus a bias towards phase-zero. Two ancient introns occur in phase-zero position of histidine codons, regardless of the taxonomical order. Other ancient introns were lost from individual orders whilst new ones were apparently acquired at different time points during laccase gene evolution. Some of these other intron positions distinguish genes from Agaricales, Aphyllophorales and Ceratobasidiales. Within an order, there are also species- or genus-specific intron positions. The data suggest that particularly in the Agaricales, laccase genes duplicated several-fold after division into families, genera or species. A phylogenetic tree constructed by intron position conservation grouped laccase genes primarily according to taxonomy. In contrast, in a phylogenetic tree based on protein similarity, laccases clustered partially by the fungal life-style (white rot versus litter-decomposing species). *Work in our laboratory is supported by the DBU (Deutsche Bundesstiftung Umwelt).

Mycosphaerella graminicola (Fückel) J. Schröt. in Cohn is a haploid ascomycete that causes septoria tritici blotch in durum ( Triticum turgidum L.) and in bread wheat (T. aesitivum L.). Two high-density genetic linkage maps with over 2000 Diversity Arrays Technology (DArT), AFLP and SSR markers were generated from two mapping populations derived from crosses between the sequenced isolate IPO323 and either isolate IPO94269 or IPO95052. Graphical genotyping revealed that some of the progenies lacked one or more linkage groups that were present in both parental isolates. PCR’s with primers derived from the SSR and DArT markers positioned on these linkage groups were used to verify the absence of these linkage groups in off spring isolates. We confirmed the absence of six linkage groups in the IPO323 x IPO94269 and IPO323 x IPO95052 progenies. We could not conclusively confirm the absence of two additional linkage groups, maybe due to the presence of translocations. We studied the origin and effect of this phenomenon. Our results demonstrate that the chromosomes were lost during meiosis possibly by non disjunction. The loss of chromosomes did not seem to affect viability, pathogenicity or fertility as back crosses between these progeny isolates and the parents produced significant numbers of off-spring isolates. The finished genome sequence of isolate IPO323 and the aligned DArT markers from the high-density map enabled us to identify the missing chromosomes containing apparently redundant genes. Interestingly, we also identified isolates in the IPO323 x IPO94269 progeny that were diploid for one linkage group. We hope our data will contribute to a better understanding of the genetics in Mycosphaerella and redundancy of its genomic content.

112. Simple sequence repeat (SSR) markers for Pythium aphanidermatum, P. irregulare, and P. cryptoirregulare. Seonghee Lee and Gary W. Moorman. Department of Plant Pathology, The Pennsylvania State University, University Park, 16802, PA. USA. Email: sul22@psu.edu

Six microsatellite-enriched genome libraries from three Pythium species, P. aphanidermatum, P. irregulare, and P. cryptoirregulare were constructed to develop simple sequence repeat (SSR) markers. Four synthetic di-, (AG)₁₂, (AC)₁₂, (GT)₁₂, and (CT)₁₂, and three trinucleotides repeats, (GGT)₆, (AAG)₈, and (AAC)₆, were used to screen microsatellite loci in the three Pythium species. Approximately 600 positive recombinant clones for each Pythium species were selected and sequenced. About 35 % in P. aphanidermatum, 17 % in P. irregulare, and 25 % clone sequences in P. cryptoirregulare contained the unique simple sequence repeats (> 4 repeats). Total 110 SSR primer pairs for P. aphanidermatum, 73 SSRs for P. cryptoirregulare, and 82 SSRs for P. irregulare were developed and tested for amplifications and polymorphisms on four isolates of each Pythium species. Twenty three in P. aphanidermatum, 39 in P. irregulare, and 41 polymorphic SSRs in P. cryptoirregulare were found. After screening the polymorphic SSR markers to 8 isolates of each species, the most polymorphic and reproductive SSR markers were developed for each Pythium species; 9 in P. aphanidermatum, 18 in P. irregulare, and 23 in P.cryptoirregulare. These newly developed SSR markers can be useful for monitoring the introduced isolates and population genetic studies.

113. Development of Phlebiopsis gigantea as a biocontrol agent for annosum root disease in the southeastern United States. Brian Higgins and Sarah F. Covert. Warnell School of Forestry and Natural Resources, University of Georgia, Athens, GA

The white-rot basidiomycete Heterobasidion annosum causes root and butt rot in conifers across temperate climate zones worldwide. In freshly thinned pine stands in the southeastern U.S., the ability of H. annosum to spread from cut stumps to live trees often results in significant volume loss. Phlebiopsis gigantea, another white- rot basidiomycete, has been used successfully across Europe to control H. annosum, but the U.S. Environmental Protection Agency (EPA) has not approved the use of P. gigantea in the U.S for this purpose. In order to develop a P. gigantea biocontrol product that will meet EPA regulatory approval, we have collected 64 isolates of P. gigantea from 14 sites in the southeastern U.S. Our early results suggest that P. gigantea isolates from the southeastern U.S. make sufficient spores in culture for commercial application of a spore suspension to cut stumps, and they are at least as effective in controlling H. annosum infection in southern pine wood as two commercially available P. gigantea isolates from Europe, known as RotStop (Finland) and PG1 (U.K.). We have developed 8 microsatellite markers to measure P. gigantea genetic diversity. These data will indicate how commercial application of selected P. gigantea strains is likely to impact local P. gigantea population structure. Current data on P. gigantea genetic diversity also be presented.

114. Relationships between Alleles at Lineage Diagnostic Loci in Fusarium graminearum. L.L. Anderson¹, Y-.W. Lee², R.L. Bowden³, and J.F. Leslie¹. ¹Department of Plant Pathology, Kansas State University, Manhattan, KS; ²School of Agricultural Biotechnology and Center for Agricultural Biomaterials, Seoul National University, Seoul, Korea; ³USDA-ARS Plant Science and Entomology Research Unit, Manhattan, KS.

DNA sequences of three nuclear genes (MAT-1-1-3, TRI101 and RED) were examined from over 500 isolates of Fusarium graminearum collected from South American and Korean wheat, maize and sorghum. Genetic networks were developed for each gene that illustrate the relationships between the DNA sequences of isolates in this study based on the minimum number of base pair changes that separate the isolates. Some lineage diagnostic single nucleotide polymorphisms (SNPs) are not conserved in this strain set. The lack of dichotomous branching results in polytomies, which suggests that the lineages did not evolve in a stepwise fashion. This is further supported by the inconsistent location of lineages within the networks. For example, in the MAT network, Lineage 3 is in close proximity to the center of the network, whereas in TRI101 it is one of the most distal. Additionally, several of the genetic networks cannot be resolved without cycles, which is consistent with recent gene flow between the lineages.

115. Genetic dissection of Neurospora crassa circadian clock using two independent QTL mapping analyses. Tae Sung Kim¹, Benjamin Logsdon², Jason Mezey², Kwangwon Lee¹. ¹Dept of Plant Pathology, ²Dept of Biological Statistics and Computational Biology, Cornell University, Ithaca NY 14853

Neurospora crassa has been a successful model organism in the circadian clock study for the past four decades. In an attempt to identify novel genetic components that are contributing the clock phenotypes, we relied on the quantitative genetics approach, Quantitative Trait loci (QTL) analysis. We constructed three independent mapping populations, whose parents are from geologically isolated locations. Two circadian clock phenotypes, period and phase, were evaluated in the 188 F1 progenies of each mapping population for the QTL analyses. Simple sequence-repeat- markers-based linkage maps that cover the whole genome with an average 13 cM interval were developed for the current study. To identify the clock QTLs, we performed two independent QTL mapping analyses, composite interval mapping (CIM) and Bayesian QTL mapping (BQM). For the two clock phenotypes, the CIM analysis identified 17 genetic loci from the three populations. BQM analysis confirmed all the 17 QTLs from CIM and furthermore detected additional 23 QTLs, suggesting that BQM has more power in identifying QTLs than CIM. 15 QTLs were co-localized with the previously identified clock genes, however, 25 of the identified QTLs were not co-localized with any previously identified clock genes. Our findings demonstrate that QTL analysis is a powerful tool identifying novel genetic elements on complex phenotypes.

116. Mites act in distribution of fungal spores in Coprinopsis cinerea. Monica Navarro-Gonzalez ¹, Wassana Chaisaena ¹, Olivia Sanchez-Hernandez¹, Stefan Schütz² and Ursula Kües ¹. ¹Institute of Forest Botany, and ²Institute of Forest Zoology and Forest Conservation, Georg- August-University, Göttingen, Germany

Mites are attracted to fungal cultures of Coprinopsis cinerea in order to graze on the mycelium. Both monokaryotic and dikaryotic mycelium are eaten by the mites including asexual spores (oidia). In vegetative cultures, only the melanized sclerotia are left, supporting a function in nature for duration: on fresh media, these sclerotia germinate. Upon fruiting body induction, primordia are also refused by the mites. When fruiting bodies matures, one can observe mites to climb up the stipe to feed on the autolysing cap tissues with the basidiospores. Stipes are refused as the sclerotia. Basidiospores are ingested but not digested. Faecal pellets are formed with around 400 basidiospores. These spores germinate and can easily form a dikaryon since the high number of eaten spores ensures that all four possible mating types arising from meiosis are represented. The fast moving mites thus contribute to the distribution of basidiospores in the environment and possibly to mixing populations in the nature. Mites lay eggs in close vicinity to spore pellets. This raises the idea that there might be a form of symbiotic interaction between mites and the fungus since germinated fungal colonies will serve as food for the hatching larvae. MNG, WC and OSH were funded by CONACYT (Mexico), the Rajamangala University of Technology and the DAAD.

117. Adaptive evolution of the blast pathogen Magnaporthe oryzae populations on cereal hosts in Africa. S. Sreenivasaprasad¹, J. P. Takan¹, S. Muthumeenakshi¹, J. Chipili¹, N. J. Talbot², E. O. Manyasa³ and Y. Sere⁴. ¹Warwick HRI, University of Warwick, Warwickshire, CV35 9EF, UK. ²University of Exeter, UK. ³ICRISAT, Kenya. ⁴ARC-WARDA, Benin.

Blast caused by Magnaporthe oryzae is a major production constraint to key cereals such as rice and finger millet in sub-Saharan Africa. We have used molecular, biological and pathological assays to characterise the pathogen populations on these hosts. In West Africa, where intensive rice cultivation is relatively recent (less than 500 years), blast pathogen populations showed typical lineage based structure established by MGR586 fingerprinting. Skewed distribution of the two mating types MAT1-1 and MAT1-2, high female sterility and low fertility were observed. Pathogen isolates revealed clear differences in compatibility on rice differentials exhibiting R gene-type interactions. In contrast, on finger millet, domesticated in East Africa a few thousand years ago, more than 190 blast pathogen haplotypes were identified. The pathogen populations revealed continuous variation (lack of clonality) reflecting sexual recombination. These indigenous blast populations were genetically distinct to those in Asia, and showed a near equal distribution of the mating types and high fertility. All 70 isolates tested showed basic compatibility to a range of finger millet varieties suggesting polygenic quantitative interactions. Thus the M. oryzae populations on rice and finger millet reveal distinctive adaptive evolutionary patterns related to crop domestication and agricultural intensification in sub-Saharan Africa.

118. Evolution of Mitogen-activated Protein (MAP) Kinase Cascade Components in Filamentous Fungi. C. Greenwald and H. H. Wilkinson. Texas A&M University, Plant Pathology and Microbiology, College Station, TX

All filamentous fungi contain three paralogous MAP kinase signaling cascades within their genomes. Each cascade consists of three phosphorylating enzymes (MAPKKK, MAPKK, and MAPK), which act sequentially to transduce and amplify an environmental signal from a receptor protein to downstream genes that are directly involved in phenotypic responses. Mutations in fungal cascade components are well known for dramatic phenotypic consequences including effects on pathogenicity, mating, asexual development, abiotic stress response, etc. We predicted that since MAP kinase cascades are central to fungal development and life history there would be evidence for strong conservation of these proteins. We compared the gene family trees for MAP kinase pathway components from eight filamentous fungal genomes representing a diversity of filamentous fungi (Neurospora crassa, Aspergillus nidulans, Fusarium graminearum, Magnaporthe grisea, Botrytis cinerea, Chaetomium globosum, Sclerotinia sclerotiorum, Aspergillus flavus). The analysis has revealed a higher rate of synonymous vs. non-synonymous mutations in the cascade component genes, from which we inferred they have undergone a history of purifying selection. As one might expect, evolution of genes upstream (e.g. environmental sensors) and downstream (e.g. transcription factors) from these cascades do not mirror this degree of conservation, in fact, orthologous genes are seldom maintained in all eight species.

Coccidioides immitis and C. posadasii are the causative agents of coccidioidomycosis, also known as Valley fever, a respiratory disease endemic to the desert southwestern US, along with parts of Mexico, and Central and South America. It is caused by inhalation of asexual spores that undergo differentiation to initiate the parasitic phase in host lungs and, if not controlled, disseminates to other parts of the body. Coccidioides is only known to propagate asexually by segmentation of hyphae to produce arthroconidia, although details of its growth in the environment are limited. However, each species of Coccidioides has a recombined population structure, suggesting sexual reproduction. The presence of mating-type genes is necessary, although not sufficient, for fungal sexual reproduction. In order to investigate the potential for sexual reproduction in Coccidioides we analyzed genome sequences for potential mating-type loci and have identified idiomorphs in Coccidioides indicative of a typical heterothallic ascomycete. Data will be presented that define the MAT1-1 and MAT1-2 idiomorphs of both C. immitis and C. posadasii, and show that they are highly conserved between the two species. The idiomorphs in Coccidioides are the largest reported for an ascomycete; MAT1-1 is 8 kb and MAT1-2 is 9 kb in length. The MAT1-1 idiomorph contains four putative ORFs, and the MAT1-2 idiomorph has five putative ORFs. Analyses of ~400 isolates of C. posadasii and C. immitis revealed that they contain either a MAT1-1 or a MAT1-2 locus, and that they are distributed in a 1:1 ratio. A detailed analysis of the putative ORFs, their expression and a comparison of the MAT loci to those of related fungi will be presented.

120. Characterizing the mating-type locus of the graminicolous Colletotrichum: Patterns of sex, selection and host specialization. Jo Anne Crouch¹, Michael R. Thon², Michele Groenner-Penna³, Bruce B. Clarke¹, Adlane Vilas-Boas³, Lisa J. Vaillancourt⁴ and Bradley I. Hillman¹. ¹Rutgers University, New Brunswick, NJ; ² Texas A&M University, College Station, TX; ³Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; ⁴ University of Kentucky, Lexington, KY.

While most filamentous ascomycetes have a one locus, two allele (idiomorph) mating system, extensive sampling of Colletotrichum species (teleomorph=Glomerella, order Phyllacorales) has identified only a single idiomorph (MAT1-2, with HMG box), regardless of reproductive lifestyle, i.e., heterothallic (self sterile) or homothallic (self fertile). Genetic analysis of C. graminicola has suggested the Colletotrichum mating system is governed by at least two unlinked loci. In order to explore its unique mating strategy, we have cloned and characterized the entire ~17-kb MAT locus from representatives of the falcate-spored, grass-inhabiting Colletotrichum group. First, using multi-locus phylogenetics (3,400-bp, 4 genes), we identified 11 unique species in a highly resolved tree topology that was almost entirely congruent with host plant association. Next, using a combination of transposon-mediated cosmid sequencing, cosmid chromosome walking and PCR amplification, we identified 6 genes at the MAT locus, including the MAT1-2, DNA lyase, and putative anaphase-promoting complex. Locus organization was identical in all 11 species, irrespective of homothallism (e.g. C. falcatum) or heterothallism (e.g. C. graminicola), with no evidence for a MAT1-1 idiomorph. Comparison of the MAT locus with the distantly related species C. gloeosporioides, a cosmopolitan pathogen of dicots, showed that this region has been broadly conserved across evolutionary time, with all genes arranged in the same chromosomal orientation, although several differences in gene length and the number of introns/exons were detected. An examination of polymorphism, divergence and selection pressures on the locus are currently in progress and will be discussed.

121. Evolution of an avirulence homolog (Avh) gene subfamily in Phytophthora ramorum, causal agent of sudden oak death. Erica M. Goss, Caroline M. Press, and Niklaus J. Grunwald Horticultural Crops Research Laboratory, USDA ARS, Corvallis, OR gosse@science.oregonstate.edu

Pathogen effectors can serve a virulence function on behalf of the pathogen or trigger a rapid defense response in resistant hosts. Sequencing of the Phytophthora ramorum genome and subsequent analysis identified a diverse superfamily of approximately 350 genes that share two protein motifs (RXLR and dEER) with the four known effectors in plant pathogenic oomycetes. These have been termed Avh (avirulence homolog) genes. While as a whole the genes in this superfamily share modest sequence similarity, small groups of closely related genes can be identified. We have investigated the molecular evolution of one such group of seven Avh genes. Microarray data suggests that four of these genes are expressed in isolate Pr-102. We sequenced the full coding region (approximately 400 bp) and flanking noncoding regions of each gene in the three P. ramorum lineages. The number of polymorphic sites within P. ramorum genes ranges from 1 to 12, suggesting different evolutionary pressures among genes. Analysis indicates that these genes contain both amino acids under purifying selection (e.g. in the signal peptide and RXLR and dEER motifs) and under positive selection. We have also been able to obtain the sequence of homologous Avh genes in the sister taxa P. hibernalis and P. lateralis, allowing for examination of the evolution of these genes across species.

122. Withdrawn

123. Evaluating genetic diversity of Fusarium proliferatum from orchids in Hawaii. Cassandra Swett¹, Laurel Anderson², John F. Leslie², and Janice Y. Uchida¹. ¹Department of Plant and Environmental Protection Sciences, University of Hawaii, Manoa, HI, USA. ²Department of Plant Pathology, Kansas State University, KS, USA.

Fusarium proliferatum is a recently introduced pathogen to the Hawaiian Islands, where it causes disease on orchids in commercial nurseries. The pathogen origin is unknown, although south-east Asia has been suggested as a source. In this study, we evaluated the genetic diversity of F. proliferatum haplotype populations from diseased orchids in three nurseries, one on the Island of Oahu, and two on Hawaii. Amplified fragment length polymorphisms (AFLP’s) were generated for between 29 and 49 single spore isolates per nursery, using three primer sets. Total genetic diversity of F. proliferatum is described for the state of Hawaii, and haplotype population diversity compared within and among nurseries. This work can be used to monitor the F. proliferatum strain populations, to evaluate if introduction is still occurring, and for future comparisons to strains populations from potential sources, to establish pathogen origins.

The evolution of eukaryotic genomes can be greatly influenced by the activities of transposable elements; conversely, transposons can serve as valuable tools that may increase our understanding of genome evolution and diversification. Here, we report the discovery of five transposon species from the two major lineages (clades A and B) of Colletotrichum cereale: the DNA transposons Collect1 and Collect2 (pogo and Tc1/mariner families, respectively), the long terminal repeat (LTR) retrotransposons Ccret1 and Ccret2 (Pseudoviridae and Metaviridae) and the non-LTR retrotransposon Ccret3. Using computational tools, our data show that 21 of 35 unique transposon loci display the signature pattern of recurring rounds of repeat-induced point mutation (RIP), a genome defense mechanism unique to filamentous fungi that mutates duplicated sequences by inducing GC-to-AT transitions. Analysis of over 100-kb of sequence data showed the RIPped transposons possess a skewed A+T base composition (genome=51%; RIPped=68%), increased incidence of TA, CT and AG dinucleotides, and decreased representation of the dinucleotide pairs TG, CA, CG, GA and TC. RIP index values were also consistent with those observed for RIPped transposons from Neurospora, Fusarium, Podospora and Aspergillus species, with CA, CG and TG the most common context for GC-to-AT transitions. Although our data supported the identification of RIP mutation acting only on transposons in the C. cereale clade B genome, none of the clade A transposon sequences displayed any evidence of RIP activity. Nevertheless, identification of a RIPped Ccret2 element in the closely related species C. falcatum suggests that RIP mutation predated cladogenesis between these taxa.

125. Patterns of recombination and population differentiation among Colletotrichum cereale isolates from 27 host plant genera. Jo Anne Crouch, Bruce B. Clarke and Bradley I. Hillman. Rutgers University, New Brunswick, NJ

In populations of the turfgrass anthracnose fungus Colletotrichum cereale, the prevailing mode of reproduction is thought to occur clonally, as inferred primarily through the lack of a teleomorph. Coupled with the visual absence of a sexual state, previous reports of clonal genotypes from RAPD and isozyme analyses contribute to the notion that C. cereale has endured throughout history as an asexual organism. Because a pathogen’s mode of reproduction strongly influences the course of its evolution and the alternative hypothesis has never been adequately tested, we are evaluating patterns of variation in this species. In the present study, we used an extensive nucleotide sequence dataset (4 genes, 3,400 nucleotides) to examine a large, worldwide sampling of pathogenic isolates of C. cereale from turfgrass and their counterparts isolated from cereal crops (wheat, oats, barley) and prairie grasses. This dataset illustrated that C. cereale is subdivided into several lineages, each composed of primarily (but not exclusively) either turfgrass or non- turfgrass derived isolates. Next, RFLP analysis of three transposon species provided evidence for recombination by C. cereale, even in relatively small populations. Furthermore, 21 of 35 unique transposon loci, when evaluated for dinucleotide compositional bias, displayed the signature pattern of repeat-induced point mutation (RIP), a genome defense mechanism that functions only during meiosis. Taken together, our data rejects the presumption of clonality for C. cereale and, given the severe losses sustained in turfgrass systems due to this pathogen, strongly emphasizes the need for additional inquiry into the biology, mating and dispersal mechanisms of C. cereale.

126. Examining diversity in populations of Anisogramma anomala. Sara N. Baxer, Jo Anne Crouch, C. Reed Funk, Thomas J. Molnar, Bradley I. Hillman. Department of Plant Biology, Rutgers University, New Brunswick, NJ

While Anisogramma anomala (Gnomoniaceae) is a relatively harmless inhabitant of the native American hazelnut (Corylus americana), this fungus is a destructive pathogen of the introduced, commercially important European hazelnut (C. avellana). Using systematic greenhouse and field inoculations, we have shown the breakdown of several sources that have provided long-term resistance to A. anomala in the Pacific Northwest. These findings raise questions about the possibility of geographically isolated populations of the fungus and whether variability in the fungus could be responsible for the breakdown of resistance. To address these questions, we have collected and are evaluating 185 isolates of A. anomala from 37 sites across North America. Our initial survey by sequence analysis at 4 gene regions (ITS, EF-1alpha, BT2, CAL; 1865 nucleotides) demonstrated an extremely low level of genetic variation between the isolates, despite the fact that A. anomala is only known to reproduce sexually. Using a hybrid-capture protocol, we are currently assembling a genomic DNA library enriched for microsatellite marker sequences to examine diversity in A. anomala populations at high levels of resolution.

127. Modern arbuscular mycorrhizal fungi are clonal: evidence based on population structure of Glomus etunicatum. Henk C. den Bakker¹, Joseph B. Morton², Teresa E. Pawlowska¹. ¹Department of Plant Pathology, 334 Plant Science Building, Cornell University, Ithaca, NY 14853-5904; ² Division of Plant and Soil Sciences, 1090 Agricultural Sciences Building, West Virginia University, Morgantown, WV 26506-6108

Arbuscular mycorrhizal fungi (phylum Glomeromycota) are generally believed to represent a group of ancient asexual organisms. To test the hypothesis of clonality, we explored the population structure of globally distributed isolates of Glomus etunicatum with a particular focus on two intensely sampled populations from California, USA. We used 9 anonymous markers, two protein coding loci (Beta tubuline and GAPDH) and ribosomal markers to (1) infer the phylogenetic relationship of G. etunicatum and its closest relatives, (2) to detect recombination and (3) to infer global and local haplotype distribution. (1) Phylogenetic analyses showed that G. etunicatum represents a monophyletic group, that is nested within an undescribed species Glomus ‘yellow’. (2) Gene trees inferred from the individual markers exhibited phylogenetic concordance. Based on this phylogenetic concordance and the outcome of various tests of recombination we have to conclude that G. etunicatum reproduces in a clonal fashion. Our population genetic study revealed 11 distinct haplotypes among the globally distributed isolates of G. etunicatum. Eight of these haplotypes were found in tropical and subtropical regions, one haplotype was only found in the Californian populations, while two haplotypes were found to occur worldwide, without any distinct geographic pattern. We hypothesize that these haplotypes may represent two clonal lineages that have dispersed rapidly all over the world due to human agricultural activities.

Coccidioides consists of two species: C. immitis, proposed to be restricted to California, and C. posadasii in Arizona, Texas, Mexico, and Latin America. Their geographical separation is putative, as some Californian patients were infected with C. posadasii and some non-Californian patients were infected with C. immitis. Geographical separation has also been observed at the population level within each species. Atypical infections may be due to patients traveling to and being exposed in other regions than where they were diagnosed, or due to long distance wind dispersal of infective arthroconidia. Alternatively, the ranges of the two species and their constituent populations may overlap. To test these alternative hypotheses, we determined the genotypes of 65 isolates obtained from soil samples in Tucson, Arizona, and genotypes of isolates from domesticated and wild animals from Tucson. These new genotypes were added to the database of all previously characterized isolates, and submitted to phylogenetic and population genetic analyses. Without exception, soil and animal isolates from Tucson are C. posadasii. Additionally, we have not yet observed any obvious sub-structuring among isolates that infect humans, domesticated and wild animals, and those found in the soil. These Tucson isolates encompass a significant amount of the genetic diversity of C. posadasii, suggesting that gene flow is sufficient to maintain high diversity within the Tucson subpopulation. There also appears to be greater gene flow than previously observed between Arizona and adjacent Mexico, suggesting that the strongest population structuring of C. posadasii may occur between the Sonoran and Chihuahuan Deserts.

129. Evolutionary genomics of the ectomycorrhizal fungus Paxillus involutus. Anders Tunlid, Tomas Johansson, Antoine LeQuere, Kasper Astrup Eriksen, Andres Schutzendubeln, Balaji Rajashekar, Björn Canbäck, Jenny Hedh, Peter Samson, Susanne Erland Department of Microbial Ecology, Lund University, SE 223 62 Lund, Sweden (anders.tunlid@mbioekol.lu.se)

It is well known that ectomycorrhizal (ECM) fungi can differ markedly in their ability to form mycorrhizae and to promote the growth of the host plant. Generally such phenotypic differences could be the result of variations in gene content, quantitative differences in gene expression, and structural differences in gene products. We have uced cDNA microarrays to compare the trancriptome and genomes of strains of Paxillus involutus . The analyses included Nau, that is not compatible with birch and poplar, and the two compatible strains Maj and ATCC200175. The array contained reporters for 1075 putative unique genes in P. involutus , derived from a collection of expressed sequence tags (ESTs). On genomic level, Nau and Maj were very similar. Only 16 out of 1,075 genes analyzed by microarray-based hybridizations had signals indicating differences in gene copy numbers. In contrast, 66 out of the 1,075 genes were differentially expressed in Maj compared to Nau after contact with birch roots. Thirty-seven of these symbiosis-regulated genes were also differentially expressed in the ATCC strain. Comparative analysis of DNA sequences of the symbiosis-regulated genes showed that two of them have evolved at an enhanced rate in Nau due to relaxed or positive selection.

130. Phylogenetic markers for the Genus Aspergillus developed from complete genome sequences. S.M. Witiak¹, R.A. Samson², J. Varga², A. Rokas³ and D.M. Geiser¹. ¹Department of Plant Pathology, Penn State University, University Park, PA 16802 USA, ²Centraalbureau voor Schimmelcutures, 3508 AD Utrecht, NETHERLANDS, ³ Broad Institute, 7 Cambridge Center, Cambridge MA 02142 USA. email: dgeiser@psu.edu

The genus Aspergillus comprises tremendous biological and ecological diversity, and comparative genomic research in the genus would be aided greatly by a detailed multilocus phylogeny. Complete genome sequences of eight Aspergillus species were used as a guide to identify gene regions suitable for multilocus phylogenetic analysis of the genus. Amino acid sequences identified as conserved among animal and fungal species were used to design PCR primers, based on DNA sequences aligned among the Aspergillus genomes. Primers were then screened among seventeen or more Aspergillus species representative of the known phylogenetic breadth of the genus, as well as Penicillium outgroups, to determine the breadth of their utility. To date, ten new loci have been amplified and/or sequenced successfully. These loci will be used to infer phylogenetic relationships at the genus level, as well as among closely related species within sections and subgenera. Their utility as markers for multilocus sequence typing (MLST) and for inference of species boundaries will also be assessed.

Genomics and Proteomics

131. Comparative genomics of Candida glabrata clinical strains and closely related species. Cécile Fairhead¹*, Christiane Bouchier², Christophe Hennequin³, Laurence Ma², Héloïse Muller¹ and Bernard Dujon¹ 1 and 2: Génétique Moléculaire des Levures and Plate-Forme Génomique, Institut Pasteur, 25 rue du Dr Roux 75015 Paris, France 3: Parasitologie-Mycologie, Faculté de Médecine St-Antoine, 27 rue Chaligny 75012 Paris, France *cfair@pasteur.fr

The hemiascomycete Candida glabrata is currently the second agent of candidiasis in humans after C. albicans. The genome of C. glabrata strain CBS138 was released in 2004. This species is in fact closely related to Saccharomyces cerevisiae and they share the same ancestor which has undergone a whole genome duplication. Although it has apparently retained the genes of the mating pathway; C. glabrata is only isolated as a haploid and no mating has been observed in this species. We have studied several isolates of C. glabrata by microsatellite analysis, and also by obtaining and examining Random Sequence Tags from three of them. The same strains were also examined for several features, such as electrophoretic karyotype and mating type. We will present evidence for the mainly clonal reproduction of this species, and discuss the mechanisms that lead to chromosome length polymorphism. We have also produced and examined Random Sequence Tags from three species that are described as the closest to C. glabrata: K. delphensis, C. castellii and K. bacillisporus. We will report on the divergence between these species and evolution of their gene functions and genomes.

Within the Génolevures project, we have initiated a large scale genomic survey of the entire phylum of Hemiascomycetes. Genetic maps were found to be poorly conserved. Rates and types of genome rarrangements appeared conserved between lineages but highly variable between lineages. This suggested that distinct evolutionary mechanisms operated in each phylogenetic branch within this otherwise homogeneous phylum. To confirm these findings, we choose to populate clades that were currently less scrutinized than the Saccharomyces or the Candida albicans clades. We focussed on the Kluyveromyces clade, which contains several species of applied interest. We compared the genomes of K. thermotolerans and Zygosaccharomyces rouxii to those of K. lactis, K. waltii and Eremothecium gossyppii. The proximity of these genomes, the absence of recent whole genome duplication and the overall conservation of large syntenic blocks facilitated whole genome alignments thus permitting robust annotation. Phylogenetic relationships between these species were revised using conserved othologues. Clusters of orthologous proteins were individually examined for possible cases of accelerated evolution, showing that species with high or low gene evolution rates coexist in the same clade.

133. Analysis of the fumitremorgin gene cluster in a human pathogen, Aspergillus fumigatus. Shubha Maiya¹, Alexander Grundmann², Kosalec I ³, Shu-Ming Li² and Geoffrey Turner¹. ¹Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 3QX, UK; ²Eberhard- Karls-Universität Tübingen, Pharmazeutische Biologie, Auf der Morgenstelle 8, 72076 Tübingen, Germany. ³ Department of Microbiology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb g.turner@shef.ac.uk.

Aspergillus fumigatus strains have been reported to produce a variety of secondary metabolites, including toxic prenylated alkaloids such as fumitremorgin C,and many putative secondary metabolic gene clusters have been identified in the genome reference strain Af293 following the completion of the genome sequence.A gene ftmA encoding a dimodular non-ribosomal peptide syntetase (NRPS) was found within one of the putative secondary metabolic gene clusters. ftmA was overexpressed in strain Af293 by insertion of multiple copies,and in the naïve host Aspergillus nidulans, which lacks the equivalent gene cluster,under the control of the alcA promoter.Though neither fumitremorgins nor the dipeptide intermediate brevianamide F,cyclo-L-Trp- L-Pro,could be detected in wild-type strains,brevianamide F accumulated in liquid cultures of both species following increased expression of the NRPS gene.The cyclic dipeptide brevianamide F is the precursor of a variety of prenylated alkaloids,including fumitremorgins A,B,C,tryprostatin B and verruculogen.RT-PCR indicated that some of the genes in this cluster are poorly transcribed in Af293.We have also observed that overexpressing both ftmA and ftmB,a prenyl transferase,under a regulatable promoter alcA in A. fumigatus and in A.nidulans led to the accumulation of tryprostatin B which is the next intermediate in the fumitremorgin pathway.In order to determine the function of other genes in the cluster and to understand why it is poorly expressed in Af293,we have begun analysis of a fumitremorgin producing strain Af6460.

134. Transposons in biotechnologically relevant strains of Aspergillus niger and Penicillium chrysogenum. Ilka Braumann¹, Marco van den Berg² & Frank Kempken¹. ¹Abteilung für Botanische Genetik und Molekularbiologie, Botanisches Institut und Botanischer Garten, Christian-Albrechts-Universität zu Kiel, Olshausenstraße 40, 24098 Kiel, Germany ²DSM Anti-Infectives, Alexander Fleminglaan 1, 2613 AX, Delft, The Netherlands

Despite of the relatively late discovery of fungal transposons and despite of the small fraction of repetitive DNA in fungal genomes (1.4%, 2.9%, and 3% in the genomes of Aspergillus oryzae, A. fumigatus and A. nidulans (Galagan et al., 2005)), today virtually all types of eukaryotic transposons have been identified in fungi (reviewed in Pöggeler and Kempken 2004). However, the impact of transposons on their fungal hosts’ genomic development is still a matter of debate. Transposition might play a role in enhancing recombination in asexual fungi (Rep et al., 2006). However, it often failed to identify actively transposing mobile elements in the genomes of fungal laboratory strains, possibly because of ongoing selection for phenotypic stability (Daboussi and Capy, 2003) . It is particularly interesting that relatively little is known about the role transposons have in industrial producing strains. We set out to investigate the transposon content in the genomes of biotechnologically important strains of the two fungi Aspergillus niger and Penicillium chrysogenum using the annotated genome sequences of strains CBS 513.88 and ATCC 28089. A compilation of transposon-like sequences identified in the two genomes is given. Single sequence sets have been analysed in more detail: For example the P. chrysogenum class II element PeTraII was identified, to our knowledge the first transposon known in this fungus. We further compared the distribution of selected elements in a set of different fungal strains, all emanating from each other, to investigate transposon mobility in classical strain improvement programs. As an additional approach to test for transposon activity we performed transposon trap analysis for both fungi. Moreover we are currently developing a system which allows us to test the activity of putative transposable elements identified during database analysis. References: Daboussi MJ, Capy P (2003) Annu Rev Microbiol 57, 275-99; Galagan JE, Calvo SE, Cuomo C et al. (2005), Nature 438, 1105-15; Pöggeler S, Kempken F (2004) In: The Mycota II, Genetics and Biotechnology, 2nd edition (ed. Kück U). Springer Verlag, Heidelberg, New York, Tokyo; Rep M, van der Does HC, Cornelissen BJC (2005) Fungal Genet Biol 42, 546-553

135. Horizontal gene transfer as the source of extra genes in Aspergillus oryzae. Nora Khaldi^*, Kenneth H. Wolfe. Smurfit Institute of Genetics. University of Dublin Trinity College Dublin 2. Ireland.

Although horizontal gene transfer (HGT) is a major aspect of prokaryotic evolution and is becoming increasingly documented between prokaryotes and eukaryotes, very few cases of the HGT of nuclear genes from eukaryote to eukaryote have been documented so far. Several obstacles impede the unequivocal demonstration of HGT. The detection of foreign genes is facilitated if the recipient and donor genomes are from two different kingdoms, but if they are relatively closely related then observations suggestive of HGT may be equally consistent with alternative scenarios of gene duplication and gene loss. Aspergillus oryzae is a filamentous fungus that has a large gene complement compared to its annotated relatives. Here we test three alternative explanations for the existence of these extra genes in Aspergillus oryzae: recent gene duplication, whole genome duplication, and HGT. We show using gene location, evolutionary rate, and phylogeny based methods that HGT took place from the close sister subphylum Sordariomycetes into an ancestor of Aspergillus oryzae. Finally, we investigate the role of the transferred genes and show that transfer played a role in enriching Aspergillus oryzae’s gene repertoire, specifically by expanding its hydrolytic enzyme gene complement.

136. Characterisation of the Trichoderma reesei proteasome. L. Kautto^1,2, J. Grinyer1, P. L. Bergquist^1,2,3 , V. S. J. Te’o^1,2, and K.M. H. Nevalainen^{1,2 1}Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney NSW 2109, Australia. ²Macquarie University Biotechnology Research Institute, Macquarie University, Sydney NSW 2109, Australia. ³Department of Molecular Medicine and Pathology, University of Auckland Medical School, Auckland, New Zealand

The filamentous fungus Trichoderma reesei is one of the most efficient eukaryotic cell factories available. Considering its extraordinary secretion capacity, between 40 – 100 grams L-1 of protein, this species can be characterised as “a professional” protein secretor. High-level secretion proposes a challenge for the cellular machinery responsible for protein quality control, which is mainly carried out by UPR and ERAD. Ubiquitin-proteasome pathway plays a key role in the post –translational regulation in eukaryotic cells. Proteins that are not folded correctly or not fully assembled are recognised in the early secretory pathway featuring a large (approximately 2.4 MDa) multicatalytic protease, the proteasome. We are especially interested in exploring the function of the proteasome in ERADication of misfolded proteins from the secretory pathway by using mutant forms of an endogenous efficiently secreted cellulase enzyme as experimental molecules. We have produced a 2D master map for the T. reesei 20S proteasome. From this reference map, 13 of the 14 20S proteasome subunits and many interactive proteins that were co-purified with the 20S proteasome have been identified. We have prepared a series of mutant forms of the main cellobiohydrolase enzyme CBHI to trace their secretion and presumed degradation in the proteasome. We are currently in a process of characterising T. reesei transformants expressing the mutant proteins.

137. Systems Biology of the Biological Clock. Dong, Wubei Xiaojia Tang, Yihai Yu, Roger Nilsen, James Griffith, Jonathan Arnold, and H.-Bernd Schuttler. University of Georgia, Athens, GA 30602. arnold@uga.edu.

A model-driven discovery process called computing life is used to guide a series of 3 microarray experiments. As much as 25% of the N. crassa transcriptome is found to be circadian. A clock mechanism involving the genes wc-1, wc-2, and frq appears to have many different kinds of outputs including genes whose products are in DNA metabolism, RNA metabolism, cell cycle, protein metabolism, transport, carbon metabolism, development, isoprenoid (i.e., including carotenoid) biosynthesis, development, and varied signaling processes. The known clock mechanism involving the FRQ oscillator appears to explain only about 43% of the genes, which are circadian and light-responsive. Only about 58% of the White-Collar Complex (WCC)- controlled genes appear to be circadian or light-responsive, implying other control functions for WCC. In each of three cycles of microarray experiments through the computing life paradigm microarray data support that wc-1 and wc-2 should be autoregulated by WCC. Among 11,000 genes in the N. crassa genome a total of 295 genes, including a large fraction of phosphatases/kinases, appear to be under the immediate control of the FRQ oscillator as validated by 3 independent microarray experiments.

138. Withdrawn

139. Comparative genome hybridization to examine genome variability in Cryptococcus species. GG Hu, I Liu, A Sham, JW Kronstad. Michael Smith Laboratories, The University of British Columbia, Vancouver, BC, Canada

The basidiomyceteous fungal pathogen Cryptococcus neoformans includes four serotypes, A, B, C, and D, based on antigenic differences in the polysaccharide capsule that is the major virulence factor. The A and D serotypes mainly infect immunocompromised individuals, while B and C are now classified as a separate species, C. gattii, which infects both immunocompromised and immunocompetent patients. Strains with hybrid serotypes (AD and AB) have also been identified from clinical and environmental sources. There are also two mating types (MAT alpha and MATa) in each serotype, although strains of the alpha mating type are more frequently isolated and only genomes for MAT alpha strains have been sequenced. Genome sequence information is now available for five Cryptococcus strains (serotype A strain H99, serotype D strains JEC21 and B3501, and serotype B strains R265 and WM276). In the present study, we used the complete genome sequences of H99, JEC21 and WM276 to obtain high-density oligonucleotide genomic microarrays for comparative genome hybridization (CGH) experiments. We first examined the genomes of the MATa strain NIH433 and the MATalpha strain NIH12 that were the original parents of a cross that generated the strain JEC21. The hybridization of these genomes to a JEC21 array allowed the mapping of putative recombination sites and a comparison of the MAT regions. We then examined the genomic differences between MATalpha and MATa strains in serotype A strains (H99, 125.91, BT63), and serotype B strains (WM276 and E566). The CGH data for the MATalpha and MATa regions allowed us to establish the relationship between the CGH signal log ratio and sequence identity, and to interpret additional differences in genome content between the strains. The differences for a set of these regions were confirmed by PCR and sequencing. We further examined genome content of the AD hybrid strains KW5, CDC228 and CDC304 using both H99 (serotype A) and JEC21 (serotype D) genomic arrays. Interestingly, the CGH data indicated that chromosome 1 of all three AD hybrid strains and chromosomes 6 and 7 of KW5 most likely originated only from a serotype A genome. In addition, chromosome 5 of CDC304 and chromosome 8 of KW5 appear to originate from a serotype D genome. These conclusions are supported by PCR-RFLP analysis of selected sites on these chromosomes. Taken together, the CGH data provide the first insight into the differences in whole genome content among MATalpha and MATa strains and AD hybrids.

140. Transcriptome analysis of Lentinula edodes mature fruiting bodies by novel pyrosequencing. W.W.Y. Chum, I.S.W. Kwok, T.C.H. Au, H.S. Kwan. The Chinese University of Hong Kong. Shatin, HKSAR

Developmental processes of basidiomycetes from vegetative mycelium to mature fruiting bodies for sporulation are dramatic morphological changes. We attempted to understand the biological mechanisms through transcriptome analysis of fruiting bodies of Lentinula edodes using high-throughput Genome Sequencer 20 System (454 Life Science). Combined cDNA from mature fruiting bodies before and during sporulation of L. edodes were sequenced to isolate a total of 7481 cDNA contigs. Each contig was generated from a number of reads of cDNA that represent the number of cDNA fragments aligned for the contigs. Numbers of reads/length may be proportional to gene expression levels of the contigs revealed by comparison with LongSAGE results of L. edodes. All contigs were homology searched by BLASTX. However, there is no genome data available for L. edodes studies, we developed a program to match the contigs from other databases followed by categorization with Gene Ontology (GO). Among a total 1202 annotated contigs, 909 contigs matched to sequences in database and their biological processes were classified by GO. Top 100 contigs in numbers of reads/length were analyzed and some of them may be related to mature fruiting body development or spore formation. About 50 contigs were novel. To our knowledge, this is the first study in sequencing fungi cDNA by using the GS20 for transcriptome analysis. The results greatly advance our knowledge in biological mechanisms of mature fruiting body and sporulation of mushroom.

141. Transcriptome analysis reveals differentially expressed genes involved in increased nuclear kinetics and polar growth in the Aspergillus nidulans ATM (atmA) null mutant. Iran Malavazi and Gustavo H. Goldman, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Brazil

Ataxia telangiectasia (AT) is an inherited disorder characterized by progressive loss of motor function and susceptibility to cancer. Numerous studies have emphasized the role of the affected gene product, ATM, in the regulation of the DNA damage response. Recently, in addition to its expected role in the DNA damage response, we found that the Aspergillus nidulans homologue, AtmA, is also required for nuclear proliferation and polarized hyphal growth. For a comprehensive evaluation of genes that have their expression modulated by ATM, here by using a microarray hybridization approach, we performed two sets of comparisons of the mutant with the wild type strain: (i) growth in complete medium for 60, 90, and 120 minutes, and (ii) HU block-release experiments for polar growth (60, 90, and 120 minutes release after 5 hours HU-growth). The first set of experiments revealed that several genes involved in DNA replication and in the pentose phosphate pathway (ppp) are more expressed in the delta atmA mutant. We constructed double mutants for delta atmA and two mutations impaired in the ppp, pppA (probably encoding a transaldolase) and pppB. The double mutants have a decreased nuclear proliferation rate when compared to the delta atmA mutant, suggesting that the increased expression of the ppp genes could be important for increased nuclear proliferation in the delta atmA mutant. The second set of experiments with global expression associated to polar growth revealed a decreased mRNA expression for the following group of genes in the delta atmA mutant: (i) genes involved in the formation of a polarized hyphae (e.g., cdc42) and control of polar growth; (ii) genes involved in the synthesis of phosphatidic acid (1,2-diacyl-sn-glycerol 3-phosphate); and (iii) genes involved in the ergosterol biosynthesis (plasma membrane microdomains, lipid rafts). We are currently investigating some of these genes by constructing double mutants with them and delta atmA. Financial support: FAPESP and CNPq, Brazil

142. Transcriptional profiling of cross pathway control in Neurospora crassa: Comparative analysis of the Gcn4p and CPC1 regulogs. Chaoguang Tian, Takao Kasuga and N. Louise Glass. Plant and Microbial Biology Department, University of California, Berkeley, CA 94720

Identifying and characterizing transcriptional regulatory networks is an important task in elucidating gene function and how these regulatory networks evolve. In Saccharomyces cerevisiae one of the most intensely studied transcription factors is the bZIP transcription factor, Gcn4p. Gnc4p is essential for transcriptional response when S. cerevisiae experiences amino acid starvation. In the filamentous ascomycete fungus, Neurospora crassa, the ortholog of GCN4, called cross pathway control (cpc-1) is required for the ability of N. crassa to induce a number of amino acid biosynthetic genes upon response to amino acid starvation. In this study, we decipher the CPC1 regulog by transcriptional profiling wild-type and a cpc-1 mutant using full genome N. crassa 70-mer oligonucleotide microarrays. We show that 443 genes are either direct or indirect CPC1 targets, including 67 amino acid biosynthetic genes, 16 tRNA synthetase genes and 13 vitamin-related genes. Comparison between the N. crassa CPC1 and S. cerevisiae Gcn4 regulogs showed that regulation of only 18 genes is conserved between these two species, and primarily include amino acid biosynthetic genes. Exploration of regulatory networks in N. crassa and S. cerevisiae sheds light on how gene interaction networks evolve.

143. Development and evaluation of an Affymetrix array for Aspergillus flavus. Andrea Dolezal¹, David Ryan Georgianna¹, Greg OBrian¹, Charles Woloshuk², Nancy Keller³, Jiujiang Yu⁴, Dahlia Nielsen¹, Gary Payne¹. ¹ North Carolina State University, Raleigh, NC. ² Purdue University, West Lafayette, IN. ³ University of Wisconsin, Madison, WI. ⁴USDA/ARS/SRRC, New Orleans, LA.

A multi-species Affymetrix GeneChip array was developed to study development, metabolism, and pathogenicity of A. flavus. This chip, based on the whole genome sequence of A. flavus, contains 13,000 A. flavus genes, 8,000 maize genes, and 25 human and mouse innate immune response genes as well as the fumonisin and trichothecene clusters from Fusarium. These arrays were used to monitor gene expression of A. flavus during aflatoxin biosynthesis in defined media and during infection of developing maize seeds. A parallel study comparing this array with a 5002 element cDNA array showed the same expression profile for the aflatoxin biosynthetic genes when A. flavus was grown in culture on defined media at conducive and non conducive temperatures for aflatoxin production. Gene expression was also monitored in A. flavus during the infection of field grown maize seeds. The profile of aflatoxin gene expression by A. flavus in infected maize kernels was similar to that observed for A. flavus grown in the lab under conductive temperatures for aflatoxin production. In addition, several genes encoding enzymes for the metabolism of complex carbohydrates and for transporters were also elevated during infection of maize seeds. Nonspecific hybridization across species has not been observed in any of our experiments using the Affymetrix GeneChip. These initial observations show that these multi-species arrays will be a powerful tool for studying the complex ecology and metabolism of A. flavus. This research was funded by USDA/NRI/CGP 2006-35604-16666.

144. Gene expression analysis of the basidiomycete wood decay fungus (Heterobasidion parviporum) during adaptive growth on wood and toxic cell wall phenolic (ferulic acid). Fred O. Asiegbu^1,*, Guosheng Li¹ and Jason Osborne². ¹Department of Forest Mycology and Pathology, Swedish University of Agricultural Sciences, Box 7026, 750 07, Uppsala, Sweden ²Department of Statistics, North Carolina State University, Raleigh, USA. Email: Fred.Asiegbu@mykopat.slu.se

The success of many wood rotting fungi lies in their ability to overcome and survive in a phenol-phytoalexin rich toxic environment within litter, wood debris and tissues. Although so much has been learned about the ecology, taxonomy and physiology of several wood decaying basidiomycete fungi, the molecular basis for their survival in a diverse range of substrates and ecological habitats has been very little studied. Using the necrotrophic wood decay fungus (Heterobasidion sp.) as an experimental model, we investigated its transcriptome response during growth on a woody substrate and further compared how the gene expression pattern is affected by exposure to toxic cell wall phenolics (ferulic acid), simple sugars (glucose) and oxalic acid (a component of hyphal exudates). We documented elevated transcription of significantly more genes in glucose starved media (such as in ferulic acid) which probably indicates that the phenolic compound may constitute an important alternative energy source during periods of glucose starvation. The result also revealed similarity in expression profiling pattern of oxalate and glucose media or ferulic acid and wood which suggest that the fungus might use the same genetic programs for responding to growth on certain substrates. The results also indicate that some genes in fungi that determine the outcome of growth on a diverse range of substrates may share common regulatory pattern. The importance of the results in the identification of genes controlling important traits and their regulation during exposure to different substrates as well as how it applies to other related wood rotting fungi is discussed.

The genus Verticillium contains several phytopathogenic species, the most important being V. dahliae, V. albo-atrum, and V. longisporum. The soil-borne habitat of these species, and their capacity to infect a variety of crops, make them chronic economic problems in crop production. Verticillium wilt causes billions of dollars in annual losses worldwide. A comparative genomic approach has been initiated to study the evolutionary processes and genetic mechanisms that underpin the pathogenic development of soilborne fungi among Verticillium spp. For this purpose, we will generate a 7X genome assembly of V. dahliae and a 4X genome assembly of V. albo- atrum, two verticillium species that have distinct phenotypic variation and host ranges. Both of these assemblies, coupled with gene annotation, will provide the tools for comparative analyses to improve annotation, identify SNPs and chromosome structure changes, and create sets of conserved and species-specific genes among these two soilborne pathogens. In addition, 0.5X Fosmid sequence reads of V. longisporum will enable the assessment of whether the diploidization of V. longisporum arose as a result of inter- or intraspecific hybridization events. The synergistic power of comparative genomics will speed Verticillium research toward a greater understanding of pathogenicity. This knowledge will enable new methods for improving disease detection and management.

146. Comparative genomic analysis of Aspergillus fumigatus and sequenced close relatives. William C. Nierman¹, Vinita Joardar¹, Rama Maiti¹, Paolo Amedeo¹, David Denning² Jennifer Wortman¹, and Natalie Fedorova¹. ¹The Institute for Genomic Research, Rockville, MD, USA. ²School of Medicine and Faculty of Life Sciences, The University of Manchester, Manchester, UK

Comparative genomic analyses of pathogenic Aspergillus fumigatus, and closely related species revealed a large pool of variable genes, which may ensure the rapid adaptation to a range of environments. These variable genes are structurally distinct from the rest of the genome and tend to function in secondary metabolite biosynthesis, transport, and detoxification of xenobiotics. Many are up-expressed in a mammalian host suggesting that they may be essential for survival in this environment. In contrast, all previously identified virulence-associated genes are well conserved in all sequenced aspergilli and seem to be evolving under strong purifying selection. The variable genes tend to cluster together in subtelomeric and intrasyntenic chromosomal blocks called accessory zones that share several characteristics with prokaryotic genomic islands. The origin of accessory genes in aspergilli does not appear to involve lateral gene transfer. This analysis also shows that the aspergilli have utilized several mechanisms to ensure a quick diversification and turnover of secondary metabolism gene clusters in the course of evolution.

147. Transcriptome analysis of shiitake mushroom Lentinula edodes development using multiple approaches. W.W.Y. Chum, I.S.W. Kwok, X.L. Bian, K.T.P. Ng, R.S.M. Shih, G.S.W. Leung, T.C.H. Au, and H.S. Kwan. The Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR

Fruiting body development is an important area in mushroom biology and have been analyzed at the molecular level. We aim to analyze the transcriptome during development of Shiitake mushroom Lentinula edodes. First, we used RNA fingerprinting with arbitrarily primed polymerase chain reaction (RAP or Differential Display) to isolate genes differentially expressed. Over 100 genes were isolated and sequenced. Fifteen were studied further. Second, cDNA clones were randomly sequenced to generate about 1000 Expressed Sequence Tags (ESTs). Differential expression of the ESTs were analyzed by dot-blot hybridization and cDNA microarray analysis. Third, Serial Analysis of Gene Expression (SAGE) and LongSAGE were used to determine the proportion of each mRNA among total transcripts in various growth stages. About 30,000 transcripts were counted from seven developmental stages. Expression profiles of the tags were annotated and clustered. About 800 tags matched to our cDNA sequence collection. Forth, over 13000 cDNA sequence contigs were obtained by high-throughput sequence-by-synthesis technology. These cDNA were annotated using programs we developed. Fifth, differential expression of selected genes were confirmed using Northern analysis and real-time RT-PCR. A few genes were localized in various tissues by in situ RNA-RNA hybridization. Gene expression profiles revealed by different approaches were compared and were generally consistent.

148. Isolation and analysis of genes expressed in lignocellulose-grown Lentinula edodes using novel sequencing-by- synthesis approach. Iris S.W. Kwok, Winnie W.Y. Chum, Tommy C.H. Au and H.S. Kwan. Department of Biology, The Chinese University of Hong Kong, Shatin, HKSAR, China

The edible shiitake mushroom Lentinula edodes degrades lignin and cellulose efficiently and completely, so it has been widely used in bioconversion and bioremediation. A cDNA pool prepared from mycelium grown on lignocellulose was sequenced using pico-titered plate based genome sequencer (Roche). 5894 cDNA contigs were obtained. Their sizes ranged from 100bp to 2 kb. More than 2600 contigs could be annotated by BLAST homology search, one third of the matches were highly significant (E-value >10^-5). Using Gene Ontology (GO), these contigs were categorized according to their biological functions and processes involved. We found genes encoding ligninolytic enzymes and cellulolytic enzymes, such as chloroperoxidase, laccase, a and b-glucosidases, and endo-1,3(4)-b-glucanase. We have also obtained genes encoding antioxidative thioredoxins, glutaredoxin, superoxide dismutase (SOD) and catalase. The expression levels of SOD and catalases in mycelia grown in lignocellulose and non-lignocellulose media were compared with quantitative RT-PCR. Surprisingly, their expressions are higher in non-lignocellulose medium. This indicates that growing in lignin, L. edodes is not encountering strong oxidative stress despite that free radicals are produced to degrade lignin. These findings provide new insights on the lignocellulytic system and also the antioxidative activity during lignin degradation of L. edodes.

The e-Compendium is the logical development of earlier printed Compendia (Barratt et al (1954) Adv Gen 6: 1-93; Perkins et al (1982) Microbiol Rev 46: 426-570; Perkins et al, The Neurospora Compendium, Academic Press, 2001), integrating the genetic data accumulated over seven decades with the genome sequence and annotations of the last few years. It presents on line, in searchable form, data on over 2,100 genes structured into fields for gene symbol, name, linkage, product, phenotype, contig, sequence, reference, etc., with web links to sequence at Broad, bibliography at Pubmed, enzyme at Expasy, structure at PDB, pictures of morphological mutants at various locations, etc.. The search function permits keyword searches singly and in combination in different database fields. The e-Compendium also includes compiled chromosome maps based on both genetic and genomic data, and including over 1,700 sequenced genes. These include the products of the author's recent studies of novel genes for tRNA, rRNA, proteases, glycosyl hydrolases etc.. Monitoring software shows that the e-Compendium has been used by over 1,300 individual users in the past 12 months, over half of whom have used the site repeatedly.

150. Searching for the Rosetta stones in the multifunctional proteins of the Phytophthora genomes. Tom Wittenschlaeger ¹, Ryan Austin², Nicholas Provart², Paul F Morris ¹. ¹ Department of Biological Sciences, Bowling Green State University, Bowling Green OH 43403, ²Cell and Systems Biology University of Toronto, Toronto ON M5S 3G5.

Eukaryotic genomes have in common a large number of multifunctional proteins. A global survey of the oomycete Phytophthora sojae genome, identified 274 novel multifunctional proteins using strict criteria that excluded multi- exonic gene models without EST support. These P. sojae proteins have significant BLAST hits to two or more different proteins. Such proteins have been posited as Rosetta stones, since their association in one genome has been used to infer the association of orthologous proteins in other genomes. In our analysis, we adopted the reciprocal smallest distance algorithm (Wall et al 2003) to identify potential orthologs in 34 sequenced genomes. Surprisingly, this approach identified only seven potential Rosetta stones, where each domain of a multifunctional protein had an ortholog in the same organism. A separate phylogenetic analysis has identified several examples where each half of a multifunctional protein, clusters in a node with homologs from separate kingdoms. The evolutionary history of oomycetes involved the endosymbiotic acquisition of a red algae, and subsequent transfer of nuclear and plastid genes to the host nucleus. We postulate that this endosymbiotic event (genome acquisition and recombination) has enabled the ancestral genome to develop metabolic and regulatory pathways that are distinct from those of the animal, fungal and plant genomes. Oomycete pathways that include genes from plant and animal ancestral genomes may have metabolic and regulatory efficiencies that are not present in other organisms. Our observations suggest that the evolutionary strategy of genome acquisition and recombination should also be assessed in other members of the Chromalveolates.

151. Full-Length cDNA Sequences for Genome Annotation in Phytophthora infestans. William Morgan¹, Joe Win¹, Liliana Cano¹, Michael C. Zody², Chad Nusbaum², Sophien Kamoun¹. ¹Department of Plant Pathology. The Ohio State University, Ohio Agriculture Research and Development Center, Wooster, OH 44691, USA. ²The Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.

Phytophthora infestans is a devastating phytopathogenic oomycete that causes late blight on tomato and potato. Recent genome sequencing of P. infestans and other Phytophthora species resulted in vast amounts of sequence data providing opportunities to unlock the complex nature of pathogenesis. However, accurate annotation of Phytophthora genomes is a significant challenge. Full-length cDNA (FLcDNA) sequences are essential for the correct annotation of genomic sequences and for downstream functional analyses. We initiated three separate approaches to generate novel FLcDNA resources for P. infestans. First, bioinformatics analysis of 31592 P. infestans 5’ ESTs identified 4943 putative FLcDNAs predicted to contain a complete open reading frame. Of these, we sequenced the inserts of 480 clones corresponding to unique sequences. Second, we constructed and sequenced normalized FLcDNA libraries using mRNA isolated from various developmental stages of P. infestans strain T30-4 to acquire additional FLcDNA sequences. Finally, we constructed new cDNA libraries that are adapted for high throughput sequencing of 5’ mRNA ends using 454 sequencing. The accurate picture of P. infestans gene structure that emerges will help us develop more precise gene predictions algorithms. In addition, the FLcDNA sequences will allow us to gauge the degree of sequence conservation between P. infestans genes and those of other oomycetes, and identify patterns of gene conservation between P. infestans and various eukaryotes.

152. Proteomic analyses of developmental stage specific proteins in Aspergillus nidulans. Yun-Hee Park¹, Eun Hye Kang¹, Sujin Kim¹, Ja-Young Yun¹, Kwang- Yeop Jang², Keon-Sang Chae², Dong-Min Han³, Suhn-Kee Chae⁴, Pil Jae Maeng¹ and Hee-Moon Park¹. ¹Dept. Microbiol., SBB, Chungnam National University, Daejeon; ²Div. Biol. Sci., Chonbuk University, Chonju; ³Div. Life Sci., Wonkwang University, Iksan; ⁴Dept. Life Sci. & Technol., Paichai University, Daejeon, South Korea.

Aspergillus nidulans which has three different cycles, such as asexual, sexual and parasexual cycle, showed various morphological changes during vegetative and development. For a global analysis of developmental stage specific proteins, we investigated the changes in protein expression profiles during developmental stage. We decided the time points for harvesting cultures which revealed specific change of morphology, such as vegetative growth 9 and 14 hr, asexual development 9 and 18 hr, hypoxia condition 12 hr and sexual development 1, 6, and 24 hr. We extracted total proteins from each stage and performed two-dimensional polyacrylamide gel electrophoresis (2-DE). All 2-DEs were independent and repeating three-times for every stages. The PDQuest image analysis of silver stained 2D-gel revealed about 2400 protein spots. Among those 301 spots with meaningful changes along with the developmental stages, were identified by peptide mass fingerprinting (PMF).

153. Transcriptome analysis during sexual development in Aspergillus nidulans. Yeong Man Yu¹, Yong Jin Kim¹, Dong-Min Han², Kap-Hoon Han³, Hee-Moon Park¹, Suhn-Kee Chae⁴, Kwang-Yeop Jahng⁵, Keon-Sang Chae⁵, Jong-Hwa Kim³, Tae Jeong Oh⁶, and Pil Jae Maeng^1*. ¹Chungnam National Univ., Daejeon; ²Wonkwang Univ., Iksan, Chonbuk; ³Woosuk Univ., Wanju, Chonbuk; ⁴Paichai Univ., Daejeon; ⁵Chonbuk National Univ., Chonju, Chonbuk, ⁶GenomicTree Inc., Daejeon, Korea

In Aspergillus nidulans, although its genome has been fully sequenced, microarray analysis is still in its infancy. Here, we describe analysis of microarrays for A. nidulans using a 70-base-oligomer microarray that assays 10,580 representing ORFs from. To estimate the relative gene expression levels and changes in gene expression during sexual development, we analyzed the microarrays throughout a time course during sexual development using a AnURR (A. nidulans Universal Reference RNA) in all hybridization. In the oligomers designed for annotated genes, we obtained 9,698 reliable genes (92%). The array revealed time-dependent expression of distinct genes set. The most significantly regulated genes (P < 0.01) were grouped in six clusters based on their expression profile. Functional group of the genes contained in each cluster were analyzed by using the MIPS classification. We found several groups of genes that are expected to be involved in the regulation of sexual development at transcriptional level.

154. Microarray profiling of gene expression of Aspergillus nidulans in response to hypoxic stress. Yeong Man Yu¹, Hye Jun Yun¹, Dong-Min Han², Kap-Hoon Han³, Hee-Moon Park¹, Suhn-Kee Chae⁴, Kwang-Yeop Jahng⁵, Keon-Sang Chae⁵, Jong-Hwa Kim³, Tae Jeong Oh⁶, and Pil Jae Maeng^1*. ¹Chungnam National Univ., Daejeon; ²Wonkwang Univ., Iksan, Chonbuk; ³Woosuk Univ., Wanju, Chonbuk; ⁴Paichai Univ., Daejeon; ⁵Chonbuk National Univ., Chonju, Chonbuk, ⁶GenomicTree Inc., Daejeon, Korea

In all aerobic organisms, oxygen is the terminal electron acceptor in the respiratory chain and is required for ATP generation via oxidation phosphorylation. Thus hypoxia is expected to affect the expression of many genes involved in cellular bioenergetics, sterol, heme, and fatty acid synthesis. In order to figure out the change of transcriptome under hypoxic conditions, we analyzed the profiles of mycelia exposed to hypoxic stress by using a 70-base-oligomer microarray. The array revealed hypoxia-specific expression of distinct genes set. The most significantly regulated genes (P < 0.01) were grouped in four clusters based on their expression profile. The clusters analyzed by using the MIPS classification. Our transcriptional profiling data seems to correlate well with the change in biochemical and physiological processes caused by the hypoxic stress. Several groups of genes expected to be involved in the regulation of hypoxic genes were found.

155. Gene clusters for secreted proteins in smut fungi: Determinants for the biotrophic lifestyle. Jan Schirawski, Thomas Brefort, Julia Schöning, Kerstin Schipper and Regine Kahmann. Max Planck Institute for Terrestrial Microbiology, 35043 Marburg, Germany

The two related basidiomycetes Ustilago maydis and Sporisorium reilianum cause smut disease in maize. Upon penetration of the maize cuticle both fungi proliferate extensively within the plant apoplast. While U. maydis induces tumors filled with fungal spores on all aerial parts of its host, spore formation in S. reilianum occurs without prior tumor induction exclusively in the inflorescence. To define determinants of compatibility, we focused on the predicted secretome. In U. maydis a surprising number of secreted protein-encoding genes occur clustered. These clusters seem to encode virulence factors, since deletion of individual clusters in U. maydis in many cases affected pathogenicity. To define conservation/divergence between S. reilianum and U. maydis potentially explaining differences in disease progression, we sequenced the genome of S. reilianum and compared the cluster loci. In U. maydis deletion of the largest cluster (19A) containing 23 genes for potentially secreted proteins resulted in loss of tumor induction and spore formation without affecting preceding developmental stages. A detailed microscopic analysis of the cluster 19A deletion mutant in planta suggested recognition by the plant defense machinery at late stages of development. Intriguingly, interspecies comparison of the cluster loci revealed significantly lower conservation between cluster genes than between neighboring genes. These findings suggest that the secreted cluster proteins are under diversifying selection, potentially arising from the different modes of disease development. Currently we determine localization of secreted proteins of U. maydis cluster 19A in planta and analyze the role of the corresponding genes during biotrophic development of S. reilianum.

156. Functional characterization of acetylglutamate synthase and phosphoribosylamine-glycine ligase genes in Gibberella zeae. Jung-Eun, Kim¹, Kilseon Myong¹, Won-Bo Shim², Sung-Hwan Yun³, and Yin-Won Lee¹. ¹School of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Korea ²Department of Plant Pathology and Microbiology, Texas A&M University, USA ³Department of Biological Resources and Technology, Soonchunhyang University, Asan 336-745, Korea

Gibberella zeae (anamorph, Fusarium graminearum) is an important pathogen of cereal crops in many regions of the world. In this study, we characterized two auxotrophs (designated S4B1279 and S4B3008) that were recovered from a collection of insertional mutants of G. zeae created by the restriction enzyme-mediated integration (REMI) procedure. Both mutants showed pleiotropic phenotypes including reduction in mycelial growth and virulence, and no sexual reproduction. Molecular analyses of the REMI mutations revealed that genes responsible for the arginine and adenine auxotrophy in the strains S4B1279 and S4B3008 are ARG2 (encoding a putative acetylglutamate synthase) and ADE5 (encoding a phosphoribosylamine-glycine ligase), respectively. Subsequent gene deletion and complementation analyses confirmed the functions of ARG2 and ADE5 in G. zeae. In addition, complementation of the auxotrophs with intact copy of ARG2 or ADE5 indicated that both ARG2 and ADE5 can be used as new dominant selectable markers for G. zeae

157. Symbiosis Insights from the Genome of the Mycorrhizal Basidiomycete Laccaria bicolor. Francis Martin, Joint Genome Institute and Laccaria Genome Consortium (fmartin@nancy.inra.fr)

Laccaria bicolor is a ubiquitous fungal symbiont of trees in boreal and temperate forest ecosystems. This biotroph has a beneficial impact on plant growth, yet our understanding of the interactions driving this ectomycorrhizal association are poorly known. To elucidate the genetic basis of this ecologically important behavior, we have sequenced the 65 Mb L. bicolor genome The genome assembly contains about 20,000 predicted protein-encoding genes and a very large number of transposons and repeated sequences (i.e. 21% of the assembled sequence) which contribute to its large size compared to known fungal genomes. Analysis of this gene set yields insights into unexpected aspects of the symbiont biology including the identification of genes potentially associated with saprobic organic matter decay and pathogenesis. This fungus also possesses expanded protein families associated with plant interactions, such as hydrolases, signaling proteins, and small secreted proteins. WD40 repeat- and TPR-containing proteins – key integrators of stress and nutrient availability signals - are very abundant. Comparison of the genomes of pathogenic and saprobic fungi with the L. bicolor genome reveals that the symbiont versatile genome encodes all the features corresponding to the major fungal lifestyles, i.e., saprophytism, pathogenesis and symbiosis. I will close with some thoughts on other strategies, including transcriptomics and proteomics, we are undertaking to fully appreciate the molecular basis of fungal symbiosis.

158. Browsing C. albicans Genomic Information at the Candida Genome Database. Martha B. Arnaud, Maria C. Costanzo, Marek S. Skrzypek, Prachi Shah, Gail Binkley, Stuart R. Miyasato, and Gavin Sherlock. Candida Genome Database, Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305- 5120

Candida Genome Database (CGD, freely accessible at http://www.candidagenome.org/ ) contains genomic sequence data and information about the genes and gene products of the opportunistic human fungal pathogen Candida albicans. CGD supports genome viewing and browsing using GBrowse, an open-source tool developed as part of the Generic Model Organism Database project ( http://www.gmod.org/ ). CGD provides access to the two most current versions of the C. albicans genome sequence, Assemblies 19 and 20. The Assembly 19 contigs and the Assembly 20 chromosomes are independently navigable. The tool displays a user- configurable set of Tracks, which each show a class of features, for example ORFs, tRNAs, or contigs. CGD has generated sequence-based mappings of the contigs from Assemblies 4, 6, and 19 and the ORFs from Assemblies 6 and 19, onto the chromosomes from Assembly 20, and mappings of the older assembly information onto the Assembly 19 contigs. These historical tracks are available in GBrowse, where they serve as a graphical representation of the evolution of the genome assembly. GBrowse may also be used to view the results of any BLAST query. To view the genomic context of the sequence match, select the link from CGD BLAST results page. GBrowse also allows downloading of any selected region of DNA or protein sequence, annotation of restriction sites recognized by a configurable list of restriction endonucleases, and export of publication-quality images. CGD curators welcome comments and suggestions by email, at candida- curator@genome.stanford.edu. CGD is supported by grant RO1 DE15873 from the NIDCR at the NIH.

159. Determining mode of antifungal activity of phytochemicals using S. cerevisiae and genetic array technologies. Nadereh Hannah Mir-Rashed¹, Imelda J. Galvan², John T. Arnason³, Myron L. Smith¹. ¹ Biology Department, Carleton University, Canada.² Chemistry Department, Universidad Autónoma de Aguascalientes, México. ³ Biology Department, University of Ottawa, Canada.

Host toxicity, a limited spectrum of activity, and propensity to induce or select for resistant fungal pathogens by many current antifungals has created a need for additional agents to treat fungal infections. We have studied how S. cerevisiae responds to sub-lethal exposures of plant-derived compounds, including the alkaloid berberine, cinnamodial, chimaphilin and those in Echinacea extracts. DNA microarray analysis of yeast grown in the presence of berberine revealed significant differential RNA transcript levels for 106 genes which were assigned to seven categories including cell cycle/division, metabolism, cell wall, gene expression, transport/secretion and unknown functions. Based on this transcriptional response, we hypothesize that berberine is an effective antifungal because, at least in part, it is not apparently recognized as a xenobiotic. A yeast Gene Deletion Array (GDA, ~4600 gene deletion mutants) was also used to examine chemical-genetic interactions with yeast exposed to each compound. These experiments provide insight into antifungal mode of action and thus add to the value of these bioactive compounds. For example, a significant pattern to emerge from the GDA analysis was that Echinacea extracts interfere with fungal cell wall functions. Furthermore, the experiments provide a template in medicinal plant research. The future direction of the study is to link the collected data to the antifungal drug discovery and development processes.

160. Telomere instability in Magnaporthe oryzae caused by highly mobile, telomere-targeted tranposons. Mark Farman & John Starnes. Department of Plant Pathology, University of Kentucky, Lexington, KY 40546, USA

 Magnaporthe oryzae isolates that infect perennial ryegrass have unusually unstable telomeres that undergo continual rearrangements in culture and in planta. Sequencing revealed that the chromosome ends of the ryegrass pathogens are organized very differently to those of a rice-infecting strain with stable telomeres. Specifically, the subtelomeres consist of tandem arrays of two types of repetitive elements, with canonical telomere repeats at the ends of the arrays. The elements are found only in subtelomeric locations, so we have named them M. oryzae telomere- exclusive repeats (MoTERs). MoTER1 is 4.6 kb in length and codes for a reverse transcriptase. It lacks terminal repeats and, therefore, appears to be a non-LTR retrotransposon. MoTER2 is only 1.7 kb long and potentially codes for a 204aa protein of unknown function. We have identified two new insertions of MoTER1 at telomeres that originally lacked MoTER elements, thereby demonstrating that MoTER1 is an active transposon. The structures of MoTER1 and MoTER2, and the way they are organized at the chromosome ends, are reminiscent of the retrotransposons TART and HeT-A, which are responsible for maintaining the Drosophila telomeres. Therefore, we hypothesize that M. oryzae strains from ryegrass have dual systems for telomere maintenance – one based on telomerase, and another which uses terminally targeted retrotransposons to repair degraded ends that arise during telomere crisis. Ironically, it appears that the presence of the MoTER elements causes the telomeres to be unstable in the first place – an apparent case of a selfish element working hard to ensure the security of its own job!

We asked if there is any correlation between the pattern of expression and phylogenetic age for Neurospora crassa genes. N. crassa genes were classified into six mutually exclusive groups (Cai et al., J. Mol. Evol 2006): (1) Eukaryote/Prokaryote-core, a group of genes whose orthologs were identified in non-fungal Eukaryotes and/or Prokaryotes, (2) Basidiomycota-core, genes shared with basidiomycetes, (3) Hemiascomycete-core, (4) Euascomycete- specific, (5) N. crassa orphans, which lack sequence similarity to any of the genes in SIMPS database and (6) Remainder. We then examined the mRNA profiles obtained during vegetative growth and conidiation using N. crassa microarray representing all the predicted ORFs (c.a. 11,000). We found an over-representation of Euascomycete-specific genes in very young hyphae (0 to 3 hours old) and in mature hyphae engaged in conidiation (18 to 27 hours old; enrichment test, p <0.01). On the other hand, Eukaryote/Prokaryote-core genes showed peak expression pattern in 6 to 15 h old hyphae. The most notable finding was the expression timing of N. crassa orphan genes. There are c.a. 2,000 orphan genes in the N. crassa genome and 95% of them do not have functional annotation. N. crassa orphan genes were over-represented during development of aerial hyphae and conidiation, which coincided with development of the conspicuous and distinctive asexual morphology characteristic of this fungus.

162. Functional analysis of genes regulated by MAT1-2 in Gibberella zeae using a proteomics approach. Seung-Ho Lee¹, Sung-Hwan Yun², and Yin-Won Lee¹. ¹School of Agricultural Biotechnology, Seoul National University and Center for Agricultural Bio-materials, Seoul 151-921; ²Department of Biological Resources and Technology, Soonchunhyang University, Asan 336-745, Korea

The homothallic ascomycetes Gibberella zeae (anamorph = Fusarium graminearum) is an important pathogen with ubiquitous geographic distribution and causes severe diseases on cereals. Ascospores formed within the sexual fruiting body (perithecium) of G. zeae can overwinter and initiate the primary infection in the next spring. Thus, a greater understanding of sexual development in G. zeae is needed for a comprehensive control strategy of the diseases caused by this fungus. We have focused on identifying the protein specifically controlled by MAT1-2, a master regulator of sexual reproduction in G. zeae. To select differentially expressed proteins under control of MAT1-2 during the perithecial stage, we compared the protein profiles of a self-fertile G. zeae and its isogenic strain deleted for MAT1-2 using a two-dimensional electrophoresis. Total 384 protein spots were visualized by silver staining and 12 spots were differentially expressed during sexual reproduction mode; only two proteins were highly expressed in the MAT1-2 deletion mutant. We identified genes corresponding to these proteins from the genome database and determined their gene expression patterns and functional requirement for sexual development. Of 13 genes examined, 10 genes were essential for either perithecia or ascospore formation in G. zeae. This study is the first report on functional analyses of a set of genes controlled by MAT1-2 at the protein level.

163. Genetic questions that we expect to answer by sequencing the genome of the white-rot basidiomycete Pleurotus ostreatus. Antonio G. Pisabarro, Lucía Ramírez Public University of Navarre, Pamplona, Spain. E-mail: gpisabarro@unavarra.es

The sequencing of the P. ostreatus genome been scheduled for 2007 by the JGI. P. ostreatus has been studied as edible fungus, as model for lignin degradation and as source of poly-aromatic hydrocarbon degrading enzymes. However, the genetic questions that can be addressed using this organism as model are far beyond that. This organism is dikaryotic in all its life cycle except at the basidia. This allows separating the two nuclei present in a mature sexually competent individual to study both parental haplotypes. Electrophoretic analyses of P. ostreatus have revealed prominent length polymorphisms in homologous chromosomes. The sequence of the two sets of homologous chromosomes will make available the two haplotypes of a mature organism for the first time. This will permit to determine the minimal synteny between homologous chromosomes and to study the meiotic behaviour of structurally polymorphic chromosomes. In P. ostreatus various quantitative loci (QTLs) have been identified whose genetic architecture is poorly known. The sequence of the two haplotypes will permit to study this architecture and to evaluate the contribution of other genomic regions interacting with it. Finally, the study of the contributions of the two haplotypes to subcellular structures (nucleus, mitochondria) will be possible, and this will open the door to deeper analyses

164. Phenotypic and metabolomic analyses of the Fusarium graminearum snf1 mutant. Martin Urban, John Antoniw, William Allwood, Andrew Beacham, Jane Ward, Mike Beale and Kim Hammond- Kosack. Rothamsted Research, Herts, AL5 2JQ, UK; Kim.Hammond-Kosack@bbsrc.ac.uk

The Ascomycete fungus Fusarium graminearum (Fg) is a ubiquitous plant pathogenic fungus in all major cereal growing areas of the world. Infections, which occur during plant anthesis, lower grain quality and contaminate grains with trichothecene mycotoxins. The genome was sequenced by the BROAD Institute and completely aligned to the genetic map. Fg has 4 chromosomes and minimal repetitive DNA (http://www.broad.mit.edu/annotation/fungi/fusarium) . In an effort to identify the set of pathogenicity genes in Fg and other fungi, a database of known fungal and Oomycete pathogenicity genes was established at Rothamsted Research ( http://www.phi- base.org) . We have identified all the Homologues of verified pathogenicity and virulence (HvPV) genes in Fg, and created a chromosome visualisation software, called Mycomap to inspect visually the chromosomes for clustering of HvPV genes. This analysis identified a region on chromosome 1, where 5 HvPV genes are clustered together within 24 kb. This includes the S. cerevisiae SNF1 gene homologue, a regulator of catabolite derepression in yeast and a regulator of cell wall degrading enzymes in Cochliobolus carbonum and F. oxysporum. We deleted the snf1 gene in Fg as the first member of the HvPV gene cluster in chromosome 1 to characterise the importance of catabolite derepression during plant pathogenicity and mycotoxin production. The availability of the snf1 mutant, which exhibits highly reduced virulence on wheat ears, has allowed us to use 1H-NMR metabolomic analysis as a tool to identify fungal candidate metabolites with a role in plant pathogenicity. Rothamsted Research receives grant aided support from the Biotechnology and Biological Sciences Research Council.

165. Withdrawn

The sexual development of the heterothallic euascomycete Podospora anserina has been analyzed for many years in terms of signal transduction pathways controlling the switch from vegetative to reproductive state, autophagy requirement for fruiting-body maturation, functions of MAT proteins and the developmental role of peroxisomal proteins. The completion of the P. anserina genome sequence allows all these research areas to benefit now from a microarray approach. Agilent 22K microarrays targeted against the 10,700 putative CDS of P. anserina will be used to compare wild type and the numerous mutants obtained in this model–system. This project is funded by Agence Nationale de la Recherche, grant n° ANR-05-0385-01.

167. Transcriptional Analysis of the Pathogenic Fungus Magnaporthe grisea during rice infection Cécile Ribot*, Arnaud Lagorce***, Aurélie Darchis, Fabien Munier, Jean-Benoit Morel**, Rick De Rose*, Roland Beffa* and Marc-Henri Lebrun*. *Bayer CropScience / CNRS, 69263 Lyon Cedex 09, France **CIRAD, TA73/09, 34398 Montpellier Cedex 05, France ***Institut de Genetique et de Microbiologie, Laboratoire de Genomique des Archaea, Université Paris XI / UMR8621 CNRS, 91405 ORSAY Cedex, France

Magnapothe grisea/Oryza sativa is a relevant model to investigate the molecular mechanisms underlying fungal infection of plants. M. grisea infections are responsible of the main foliar disease of rice and cause severe crop losses world-wide. This fungus is well suitable to molecular biology since most tools (genome sequence, EST databases, DNA chip, forward and reverse genetics) are available. Using Agilent microarrays representative respectively of 14 000 ORF from M. grisea, we performed a genome wide transcriptional analysis to highlight fungal genes expressed during the infection process. Leaves from the rice cultivar IR64 were infected by the virulent M. grisea strain PH14. Infected rice leaves were harvested 5 days after infection at the onset of lesions. This time point corresponds to the active invasion and destruction of plant tissues by M. grisea. Out of the M. grisea 14 000 genes, we could detect hybridization signals for 1800 genes that are expressed during infection. Among these 1800 genes, 240 genes are specifically expressed during infection, as no signal was detected in a mycelium array. The expression of a subset of genes that are either (1) highly expressed during infection or (2) are expressed during infection but not in the mycelium was monitored using real time RT-PCR. This strategy confirmed the expression during infection for 75% of the genes. Interestingly, bioinformatic analyses revealed an over representation of genes encoding for proteins putatively secreted. While only 5 % of the proteins encoded by the whole genome of M. grisea are predicted to be secreted, 43 % (105/240) of the protein specifically expressed during the infection process a signal peptide for protein secretion. These results suggest that M. grisea may produce numerous secreted effector proteins in host tissues that could be involved in the infection process. A systematic analysis of M. grisea proteins secreted during infection has been started to study this process.

168. Relative Protein Quantification through Stable Isotope Labeling by Amino Acids in Aspergillus flavus: Temperature Regulation of Aflatoxin Biosynthesis. D. Ryan Georgianna^1,3, David C. Muddiman², and Gary A. Payne¹. ¹Department of Plant Pathology and Center for Integrated Fungal Research. ²Department of Chemistry. ³Functional Genomics Graduate Program. North Carolina State University,Raleigh, NC 27606 USA.

Aflatoxin biosynthesis is inhibited at 37C, the optimum temperature for growth of Aspergillus flavus. Transcriptional analysis has shown that all aflatoxin biosynthetic genes except the pathway regulatory genes aflR and aflS are down regulated at 37C relative to 28C, suggesting that AFLR may be modified at 37C. To quantify the response of AFLR and other proteins to high temperature we adapted a stable isotope labeling by amino acids (SILAC) strategy for relative protein quantification in A. flavus. SILAC relies on the quantitative incorporation of labeled amino acids into proteins to provide a powerful mass spectrometry based proteomics tool useful for both the rapid quantification of proteins and identification of interactions. This technique has been used in several systems; including yeast, mammalian cells, and Arabidopsis cell culture. Samples were prepared using in-gel trypsin digestion of selected 1D-PAGE gel slices and analyzed on an ESI LTQ linear ion trap mass-spectrometer directly coupled to RP- HPLC. The A. flavus labeling strategy was optimized to provide a homogenously labeled sample with ~90% incorporation of [13C6] arginine. Furthermore, we found that the relative abundance of aflatoxin pathway enzymes compared between 28C and 37C is consistent with the relative abundance of their encoding transcripts at the respective temperatures. This is the first report of SILAC being used to quantify proteins in a filamentous fungus as well as a multi- cellular free-living prototrophic organism.

169. Comparative analysis of the cell wall sub-proteome from genetically-related strains of Cryptococcus neoformans with distinct pathogenic properties. Jamal Stie and Deborah Fox. Research Institute for Children/LSUHSC. Pediatrics and Microbiology, New Orleans, LA

Fungal infections are a frequent and growing cause of morbidity and mortality in the immuno-suppressed patient population, with effective treatment and continued antifungal drug development compromised by the conservation, at the molecular level, of many key physiological processes common to both humans and fungi. However, fungi elaborate one unique structure that is essential for their survival, the fungal cell wall, which acts as a physical barrier, and mediates events necessary for environment sensing, growth and morphogenesis. While the carbohydrate and protein composition of the cell wall has been extensively studied for many fungi, the cell wall of Cryptococcus neoformans, a significant opportunistic fungal pathogen that causes life-threatening meningoencephalitis is largely uncharacterized. Recent efforts to sequence and annotate the genome of this pathogen revealed the 99.5% genetic identity of two related strains of C. neoformans that possess markedly different virulence phenotypes, with the B3501A strain considerably more virulent than JEC21. Because the protein population within the cell wall plays an important role in the mediation of virulence in other pathogenic organisms, we hypothesize that differences in the cell wall protein composition could contribute to differences in virulence. Here we examined the hypothesis that B3501A and JEC21 differentially express proteins within the cell wall sub-proteome, contributing to the differences observed in the virulence properties of the two strains. Using a directed proteomics approach, we have performed a comparative analysis of the cell wall sub-proteomes from each strain, revealing a small subset of proteins that are unique to each, suggesting the presence of strain-specific cell wall-associated virulence determinants. Ongoing studies utilizing LC-MS/MS will permit the identification of both common and unique protein populations within the cell wall sub-proteome for each strain, leading to the elucidation of important surface-associated mediators of host interaction during pathogenesis in C. neoformans.

170. PhESTDB: An Integrated resource for Phytophthora and Soybean EST sequences. Sucheta Tripathy and Brett M. Tyler. Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University, Blacksburg, VA-24061 sutripa@vbi.vt.edu bmtyler@vt.edu

PhESTDB is an unique integrated EST data resource, containing 33,350 P.sojae ESTs, 99,319 P.infestans ESTs and 37,465 public Soybean unigenes. This database is the most complete resource for P.sojae EST sequences to date. The P.sojae sequences were quality trimmed followed by vector/ adaptor, poly A/T trimming. A total of 3769 Soybean EST sequences were separated from the P.sojae ESTs from an infection library using in silico methods. Subsequently, the sequences were clustered and assembled using a wrapper perl script TGICL.The clustering and assembly produced 7863 unigenes of P.sojae, 2292 soybean unigenes from the infection library, and 14754 unigenes of P.infestans. The unigenes were primarily annotated using NCBI- BLASTX against NR database accelerated with a Timelogic board.The unigenes were then passed through a modified algorithm of loglikelihood(McLachlan,1984) to separate the UTRs from the ORFs. The protein sequences were annotated for domains, motifs, profiles and fingerprints using InterproScan. Sequences were submitted to signalp and TMHMM servers to get secretory and trans-membrane domains. The resulting outputs were parsed and stored in the database. The database was designed in mysql and the interfacing was done with PHP and perl CGI. We have created an easy-to-use Perl GD-based browser which is distinct from Gbrowse. In the browser the genome to unigene alignments are shown in greater detail which also indicates any overlaps between the unigenes. Each EST unigene is made clickable to view detailed information about assembly and their primary annotation including various other related information such as NCBI links and links to gene models. Through the gene models this database is linked to the VBI microbial database. This database has a very powerful query page, that can retrieve any kind of information the user may need. Data can also be downloaded from the database in text format. The database is publicly available at http://phytophthora.vbi.vt.edu/EST .

171. Sequencing of the genome of the forest pathogen Heterobasidion annosum s.l. Jan Stenlid¹, Matteo Garbelotto², Ursula Kües³, James B Anderson⁴, Francis Martin ⁵, Halvor Soilheim⁶. ¹Swedish University of Agrcultural Sciences, Uppsala, Sweden, ² University of California, Berkeley, USA, ³ Georg August University of Göttingen, Germany, ⁴University of Toronto, Canada, ⁵ INRA-Nancy, France ⁶ Norwegian University of Life Sciences, Ås Norway

Heterobasidion annosum s.l. causes a devastating root rot in conifer plantations and natural forests throughout the northern hemisphere. The JGI- sponsored genome sequencing for H. annosum will provide the first comprehensive genetic information on a plant pathogenic homobasidiomycete allowing for new insights into plant- microbe interactions with trees, in particular conifers. It is important to broaden the taxonomic base for understanding the mechanisms of plant-microbe interactions, studying genes and proteins involved, and identifying pathogenicity determinants. Comparative genomics of plant pathogens with a gradient of taxonomic relatedness to H. annosum will help to understand the evolution of such factors. Comparisons can also be made in the response of plants to various types of trophic interactions; necrotrophic, biotrophic and mycorrhizal mutualists and the model tree Populus. Moreover, this project will also gain insights into fungal evolutionary history and biology including development, non self recognition, mating, wood degradationand secondary metabolism.

172. High throughput discovery of virulence factors among transcription factors of Alternaria brassicicola. Yangrae Cho, Mauricio La Rota, Kwang-Hyung Kim, Derrick Scott, Amanda Cronin, Meagan Callilhan, Graciela Santopietro, and Christopher Lawrence. Virginia Tech, Blacksburg, VA

Alternaria brassicicola is a ubiquitous necrotrophic fungus causing destructive rots among diverse Brassica family members. We have optimized relatively easy and economical PEG-mediated protoplast transformation methods for this fungus. In order to produce targeted gene knockout (KO) mutants, we have utilized unconventional linear minimal element constructs as well as conventional gene replacement constructs. Using these two methods, we produced knockout mutants for over 100 genes and investigated changes in virulence. Here we present the results related to mutants for 40 protein coding genes. These candidates were comprised of five kinases and four transcription factors (TFs) involved in six known signal transduction pathways. In addition 31 TFs from the partially annotated A. brassicicola genome were also selected (both randomly and based on interesting predicted functional domains) for KO and subsequent phenotypic analyses. Near loss of virulence was observed for mutants of the Amk1, AbSte12, and AbMps1 genes, representing two signal transduction pathways of the mating pheromone responses and hypotonic shock responses. Significant reduction of virulence without any other discernable phenotypes has been observed for mutants of the AbPro1 gene whose homolog is involved in sporulation in diverse filamentous fungi. In addition, we have discovered mutants for two of the 31 selected TF genes showed reduced virulence. The study results thus far show a few common as well as several unique aspects of virulence mechanisms in A. brassicicola.

173. The genome of the necrotrophic fungus Alternaria brassicicola. C. Mauricio La Rota¹, Yangrae Cho¹, Thomas Mitchell², Brett Tyler¹, Sandra Clifton³, Dennis Knudson⁴, Susan Brown⁴, Christian Haudenschild⁵, Blake Meyers⁶ and Christopher Lawrence¹. ¹ Virginia Bioinformatics Institute, Blacksburg, VA. ² Fungal Genomics Lab, North Carolina State University, Raleigh, NC. ³ Washington University Genome Sequencing Center, St Louis, MO. ⁴ Colorado State University, Fort Collins, CO. ⁵ Solexa Inc, Hayward, CA. ⁶ Delaware Biotechnology Institute, Newark, DE.

The genomes of several fungi within the Dothideomycete are in the process of being sequenced at various centers internationally. These fungi are economically important plant pathogens infecting various important crops such as wheat, corn, and Brassicas, to name a few. All of these fungi are capable of leading a saprophytic lifestyle but have a necrotrophic stage in their lifecycle. Comparisons between their gene contents and genome structures are likely to reveal important details about their evolutionary history, production of toxins and the capability to switch from a saprophytic phase to a necrotrophic one in order to infect plant tissues. We present here the first draft assembly of the 30Mb genome of the Brassica pathogen, Alternaria brassicicola with a 6.4x coverage and provide details about the progress in annotating the genome, the manual curation and the functional genomics aspect of the project. Analysis of the genome features and structure will help to further the understanding of necrotrophic pathogen-plant interactions. This project is funded by USDA-CSREES (Proj. # VAR-2004-05551).

Identification of genes involved in aflatoxin biosynthesis through Aspergillus flavus genomics has been actively pursued. A. flavus Expressed Sequence Tags (EST) and whole genome sequencing have been completed. Groups of genes that are potentially involved in aflatoxin production have been profiled using microarrays under different culture conditions and during fungal infection of corn. Preliminary annotation of the sequence revealed that there are about 12,000 genes in the A. flavus genome. Many genes in the genome, which potentially encode for enzymes involved in secondary metabolite production, such as polyketide synthases, non-ribosomal peptide synthases, cytochrome P450 monooxigenases, have been identified. Comparative analysis of A. flavus genome with food grade industrial fermentation organism A. oryzae can help understanding the mechanism of aflatoxin biosynthesis and solving the problem of aflatoxin contamination.

175. Validation of DNA microarrays derived from the Fusarium verticillioides Gene Index. Robert A.E. Butchko, Daren W. Brown and Robert H. Proctor. Mycotoxin Research Unit, National Center for Agricultural Utilization Research, 1815 N. University Street, Peoria, IL 61606

 Fusarium verticillioides is a pathogen of maize and it can produce the toxic polyketide derived secondary metabolites called fumonisins. Fumonisins have been shown to cause animal diseases and are epidemiologically correlated to esophageal cancer and neural tube defects in humans. In an effort to identify genes involved in regulation of fumonisin production and in pathogenicity on maize, a large scale EST sequencing project was conducted in collaboration with The Institute for Genomic Research. The Fusarium verticillioides Gene Index (FvGI) contains up to 11,126 sequences including 7198 tentative consensus (TC) and 3921 singleton EST sequences. DNA microarrays were designed based on the FvGI and were constructed by NimbleGen Systems, Madison WI. Each DNA microarray contains 12 different 24-bp oligonucleotide probes specific to each of the unique sequences in the FvGI. Here, we present data from an experiment where total RNA from a six-point time course of F. verticillioides cultured in a liquid medium conducive to fumonisin production was used to hybridize to the microarrays. Analysis of FUM gene expression patterns over time were used to validate the microarray. The expression of FUM genes determined by the microarray analysis is consistent with expression patterns determined by Northern analysis and RT-PCR. To help determine the orientation of singleton ESTs whose direction could not be determined from sequence data or BLAST analysis, probe sets to both plus and minus strand sequences were included for all of the singletons as well as TCs containing two open reading frames. Microarray analysis successfully resolved the orientation of a number of the singletons for which the orientation was not obvious and demonstrated the existence of two open reading frames in some TCs. Global patterns of gene expression under these conditions are also presented.

176. Measuring LOH rates in Candida albicans by fluctuation analysis. Anja Forche ¹, Merima Helić ¹, and Judith Berman¹. ¹University of Minnesota, Minneapolis, MN, USA e-mail: forche@umn.edu

Candida albicans is the most common human fungal pathogen. While it does not undergo meiosis, clinical strains exhibit high levels of genetic and genomic variability that can lead to the development of drug resistance. To explore the mechanisms that give rise to genetic and genomic changes, we have measured basal rates of recombination by following loss of heterozygosity (LOH) in fluctuation assays. We used two counter-selectable genetic markers, GAL1 and URA3, located on opposite alleles at the same locus on chromosome 1. Basal LOH rates were approximately 10^-6 events/cell/generation for each marker, indicating that the assay is effective, that it is independent of the marker used and that both homologues of chromosome 1 undergo equal rates of LOH. SNP microarrays were used to study the extent and the mechanisms of LOH along chromosome 1 in strains that had undergone LOH at GAL1. Eighty-six percent of LOH events were caused by a single cross-over event that extended to the telomere, most likely due to break-induced replication. Shorter gene conversion tracts (9%) and LOH of whole chromosomes (5%) also were observed. We are currently testing if 1) the basal LOH rate is altered in the presence of anti-fungal drugs and other stresses such as temperature and toxic agents, and if 2) LOH rates differ at different chromosomal loci including telomeres, centromeres and mid-arm loci on all eight chromosomes. Preliminary results suggest that LOH is much higher at telomeres than at centromeres or internal chromosomal loci.

177. Creating a Critical Mass of Data for Genome Annotation and Comparative Analysis. Igor Grigoriev^1*, Andrea Aerts¹, Alan Kuo¹, Asaf Salamov¹, Diego Martinez², Jean Challacombe², Thomas Brettin², Erika Lindquist¹, Harris Shapiro¹, Gregory Werner¹, Susan Lucas¹, Jane Grimwood³, Jeremy Schmutz³, Paul Richardson¹, James Bristow¹. ¹US DOE Joint Genome Institute, Walnut Creek, CA 94598 ² US DOE Joint Genome Institute - LANL , Los Alamos, NM 87545 ³ US DOE Joint Genome Institute - Stanford, Palo Alto, CA 94304 ^*ivgrigoriev@lbl.gov

The DOE Joint Genome Institute contributes to the rapidly growing number of fungal genomes available to the research community through high-throughput sequencing and annotation. We sequenced and annotated 9 fungal and 2 oomycete genomes (genome.jgi.doe.gov) and expect to nearly double this number in 2007. The broad and diverse set of genomes covers major branches of the fungal phylogenetic tree and samples some of the branches at higher depth, which allows analysis of differences between clades and within species on the genomic level. In addition to several basidiomycete and ascomycete genomes, we have annotated the genome of the zygomycete Phycomyces blakesleeanus and are currently annotating the chytridiomycete Batrachochytrium dendrobatidis. Many of these genomes are either finished or are in finishing, which enables effective analysis of genome organization and evolution, such as, for example, a conditionally dispensable chromosome in Nectria haematococca MPVI. Genome projects triggered development of additional resources of ESTs, proteomics and microarray data to assist with genome annotation and expression analysis. User Communities with over 100 biologists around the world actively participate in analysis and manual curation of predicted genes and functions using the JGI Genome Portal. Creating a critical mass of data improves genome annotation and enables efficient comparative analysis to address a broad spectrum of biological questions and to find important industrial applications.

In our continuing effort to bring added value to the materials in the FGSC collection, we have endeavored to identify the open reading frame associated with genetically mapped temperature sensitive lesions in Neurospora crassa. There are nearly 60 such ts lesions mapped in the N. crassa genome and because there is no function for most of these, they are known as 'unknown'. We used functional complementation of the temperature sensitivity to identify open reading frames for two such lesions, un-16 and un-4. When we identified un-16, we found that it had been previously identified by Sakai et al (2002) as part of their investigation of the near-by upr-1. un-16 encodes a ribosomal protein and we sequenced the two mutant alleles and found that in both there was a single leucine to arginine substitution. A search of the genome databases revealed that the leucines in question are very highly conserved- from archae to humans. The ability to complement the temperature sensitive lesion in strains carrying the un-16 lesion makes it possible to use this as a selectable marker. The second unknown to be complemented, un-4, encodes a mitochondrial import protein. Our next target is un-7 and currently we have identified 8 candidate ORFs from existing data. The FGSC is supported by grant 0235887 from the US National Science Foundation.

The fungal species Cryptococcus gattii is an environmental saprophyte capable of causing serious disease in people and animals, and is uniformly fatal without treatment. Like most systemic mycoses, cryptococcosis is difficult to treat and this is due to the scarce number of safe and efficacious antifungal drugs, and to our limited knowledge of the intricacies of fungal virulence. Proteome analysis enables the identification and characterization of the protein complement of a cell, tissue or organism at any one time, under specific conditions. The global nature of proteomics means areas of little knowledge, like fungal disease, can be addressed without any prior knowledge or assumptions about the role of differentially expressed proteins. Furthermore, as proteins are targets for drug interventions in microbial infections, proteome approaches are the most rapid, direct and powerful means of identifying novel candidate antimicrobial targets. The major virulence factor of C. gattii is its polysaccharide capsule, composed mainly of glucuronoxylomannan residues and mannoproteins. In our preliminary proteome analysis, an encapsulated C. gattii strain was extracted using the current best practice for two-dimensional electrophoresis of microorganisms. However, this gave very limited extraction of high molecular weight proteins and suggested that the capsule may be trapping the ‘missing’ proteins by ionic interactions. We have developed a novel method of protein extraction using high salt combined with acidic conditions. We present here studies using a range of different salts that greatly increased protein extraction from various encapsulated C. gattii strains.

180. Transcritome analyses of developmental processes associated with hypoxic stress in Aspergillus nidulans and A. fumigatus. Kap-Hoon Han¹, Hyoun-Young Kim¹, Jong Hwa Kim¹, Yeong-Man Yu², Pil-Jae Maeng², Hee-Moon Park², Suhn-Kee Chae³, Keon-Sang Chae⁴, Kwang-Yeop Jahng⁴, and Dong-Min Han⁵. ¹Dept. of Pharmaceutical Engineering, Woosuk University, Wanju, 565-701, ²School of Bioscience & Biotechnology, Chungnam Nat'l University, Daejeon, 305-764, ³Dept. of Life Science & Technology, PaiChai University, Daejeon, 302-735, ⁴Div. of Biological Sciences, Chonbuk Nat'l University, Jeonju, 561-756, ⁵Dept. of Life Science, Wonkwang University, Iksan, 570-749, Korea.

Aspergillus fumigatus is the primary causative agent of an opportunistic fungal disease, aspergillosis. Unlike Aspergillus nidulans, A. fumigatus has no known sexual development process. Furthermore, in A. nidulans, hypoxic condition is the one of most important environmental factor for generating fruiting bodies. This condition is also important to A. fumigatus because of the environment of host cell is usually maintained as hypoxic condition. To study relationship between hypoxic stress, developmental process and virulence, comparative DNA microarray analyses were performed using A. fumigatus and A. nidulans chips. Comparisons of the two transcriptome provided important information of hypoxic stress response and sexual development process. This work was supported by the KOSEF grant (No. R1-2006-000-11204-0) and KRF (KRF-2005-070-C00123).

181. Identification of proteins involved in early phase of conidia germination by quantitative proteomics in Aspergillus nidulans. Young Taek Oh^*, Chun Seob Ahn, Jae Won Kim, Chang-Won Lee**. Department of Microbiology, Research Institute of Life Science, Gyeongsang National University, 900 Gazwa-dong, Jinju 660-701, Korea *presenting author, **corresponding author

The asexual spore, or conidium of A.nidulans become competent for growth and nuclear division in a process called conidial germination, and that begins when the conidium breaks dormancy and growth isotropically. To find out critical proteins involved in early phase of conidia germination, we performed a global proteome analysis of differentially expressed protein between dormant conidia with germinating conidia. More than 1200 protein spots were detected on silver stained 2-D gel with IPG strip pH 4-7. Image analysis revealed that 97 protein spots showed quantitative variations that were significant (P <0.05) and reproducible in the 30minute germinating conidia. And 144 protein spots showed quantitative variations that were significant (P <0.05) and reproducible in the 60minute germinating conidia. Protein identification was carried out peptide mass fingerprinting by MALDI-TOF mass spectrometry. Under 30minute germinating conidia, the protein expression levels of 9 proteins including thioredoxin reductase are up-regulated, and 5 proteins including transketolase are down-regulated. Under 60minute germinating conidia, the protein expression levels of 13 proteins including peptidyl-prolyl cis-trans isomerase are up-regulated, and 12 proteins including mannitol-1- phosphate dehydrogenase are down-regulated. The identified proteins in this report enhance to understanding of the process of conidia germination.

182. Coding tandem repeats generate genetic diversity in Aspergillus fumigatus genes. Emma Levdansky¹, Jacob Romano¹, Kevin J. Verstrepen², Gerald R. Fink² and Nir Osherov¹. Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, MA², Department of Human Microbiology,Sackler School of Medicine¹, Tel-Aviv University, Tel-Aviv, Israel.E-mail: nosherov@post.tau.ac.il

Genes containing multiple coding tandem repeats are highly dynamic components of genomes. In humans, expansion of coding repeats is associated with various diseases, including Huntington’s Disease and Fragile X Syndrome. In fungi, many repeat-containing genes encode for cell-wall proteins containing a leader sequence and glycophosphatidylinositol (GPI)- anchor. Several encode for known adhesins (ie. Saccharomyces cerevisiae FLO11, Candida albicans ALS1, ALS5, ALS7 and ALS9, Candida glabrata EPA1). They undergo frequent recombination-dependent expansion or contraction in size, creating size-variability between different yeast isolates and alterations in adhesion. This variation provides the functional diversity in cell-surface antigens which allows rapid adaptation to the environment. Aspergillus fumigatus is the most common mold pathogen in man, causing both deadly invasive diseases in immunocompromised patients and allergic diseases in patients with atopic immune systems. We analyzed the entire genome of A. fumigatus for open-reading frames (ORFs) containing putative repeats, using the ETANDEM software program, a numerical score for tandem repeats in a nucleotide sequence. Fourteen of 30 top-ETANDEM-scoring genes analyzed showed strain-specific size variation of repeat containing-regions. Four of these, (Afu3g08990, Afu2g05150 (MP-2), Afu4g09600 and Afu6g14090) are putative cell-wall proteins containing a leader sequence and GPI-anchor motif. All four are expressed genes and produce size-variable mRNA encoding a discrete number of repeat amino-acid units. Their expression during development and in response to cell-wall disrupting agents was analyzed. Our findings suggest that a subset of the A. fumigatus cell-surface proteins may be hyper-variable due to recombination events in their internal tandem repeats. We have created knock out strains in A.fumigatus 293 for two putative cell-wall coding genes (Afu3g08990 and Afu6g14090). We are currently studying the effect of the deletion of these genes on the phenotype. Preliminary results indicate that compared to the wild type strain, Afu3g08990-deleted strain exhibits early germination rates and reduced adhesion to ECM (extra cellular matrix) of alveolar lung cells (A549 cells). In addition, we are analyzing Afu3g08990 and Afu6g14090 cellular location by tagging the genes with a myc epitope.

183. Molecular analysis of the response of cultured A549 lung cells to A. fumigatus infection. Haim Sharon and Nir Osherov¹. Department of Human Microbiology, Sackler School of Medicine,¹ Tel-Aviv University, Tel-Aviv, Israel.E-mail: nosherov@post.tau.ac.il

 Aspergillus fumigatus is the most prevalent airborne fungal pathogen which causes fatal invasive aspergillosis in immunocompromised patients. Invasive pulmonary aspergillosis (IPA) is caused by inhalation of A. fumigatus spores and growth of the fungus inside the lungs, often spreading from the initial site of infection in the lungs to attack various organs in the body. Our main goal is to better understand the mechanisms controlling damage in infected lung alveolar epithelial cells. We assumed that lung alveolar cells infected with A. fumigatus undergo a specific and pre- programmed molecular response. Two approaches were used to analyze the response of infected cells: A) Kinome analysis, identification of specific phospho-proteins using a panel of 36 mono- specified antibodies. B) Transcriptional analysis using Affymetrix arrays. A549 lung epithelial cells were infected with A. fumigatus conidia or culture filtrate and compared to uninfected cells. Kinome analysis revealed five main proteins, Adducin gamma, PKR, Erk1/2, JNK and MEK1/2 which showed differences in phosphorylation patterns between infected and uninfected cells. These results suggest that the infected cells mount a strong JNK/MAPK pathway stress response to A. fumigatus infection. Interestingly, adducin, a cytoskeletal protein, was phoshphorylated more intensively in response to conidia than to culture filtrate, whereas PKR, which plays a role in mediating the activation of the MAPK pathways, was phoshphorylated only in response to culture filtrate. Affymetrix transcriptional analysis revealed 388 genes differentially expressed at least 2 fold (p <0.05) in response to infection by germinated conidia, and 478 genes in response to culture filtrate. Interestingly, the response to germinated conidia was characterized by strong (>5 fold) upregulation of 35 genes involved in cell death, immune response, cytokine activity (especially IL8) and response to wounding. A significant number of these genes is controlled by the transcription factor NFk-B. In contrast, the response of A549 cells to culture filtrate was mainly characterized by the down regulation of 277 genes involved in metabolism and biosynthesis. Our results demonstrate that A549 lung cells respond differentially to infection with A. fumigatus conidia or culture filtrate. We propose that during early conidial infection, infected cells mount a vigorous protective response. In contrast, during late infection, the accumulation of secreted culture filtrate elicits a marked inhibition of cellular metabolism. These results help clarify the progression of cellular infection by A. fumigatus at the molecular level, and suggest novel ways to interfere with this destructive process.

184. Metabolic network-driven analysis of genome-wide transcription data from Aspergillus nidulans. Helga Moreira David¹, Gerald Hofmann², Ana Paula Oliveira², Hanne Jarmer³, and Jens Nielsen². ¹Fluxome Sciences A/S, Diplomvej 378, 2800 Lyngby, Denmark ² Center for Microbial Biotechnology, Technical University of Denmark, 2800 Lyngby, Denmark ³ Center for Biological Sequence Analysis, Technical University of Denmark, 2800 Lyngby, Denmark

Aspergillus nidulans is a model organism for aspergilli, an important group of filamentous fungi that encompasses human and plant pathogens, as well as industrial cell factories. Aspergilli have a highly diversified metabolism and, because of their medical, agricultural and biotechnological importance, it is valuable to understand how their metabolism is regulated. We therefore performed genome-wide transcription analysis of A. nidulans grown on glucose, glycerol, and ethanol with the objective of identifying global regulatory structures. We furthermore reconstructed the complete metabolic network of this organism, which resulted in linking 666 genes to metabolic functions, as well as assigning metabolic roles to 472 genes that were previously uncharacterized. Through combination of the reconstructed metabolic network and the transcription data, we identified subnetwork structures that pointed to coordinated regulation of genes involved in many different parts of the metabolism. Thus, for a shift from glucose to ethanol, we identified a coordinated regulation of the complete pathway for oxidation of ethanol, as well as up-regulation of gluconeogenesis and down-regulation of glycolysis and the Pentose Phosphate (PP) pathway. Furthermore, upon a change in the carbon source from glucose to ethanol, the cells shift from using the PP pathway as the major source of NADPH for biosynthesis to use of the malic enzyme. Our analysis indicated that some of the genes are regulated by common transcription factors, making it possible to establish new putative links between known transcription factors and genes, through clustering.

⁵INRA-URGI, 91000 Evry, France; ⁶IGR, Poznan, Poland

Genoscope (National Sequencing Centre), the French sequencing agency is involved in large-scale sequencing of the genome of the Dothideomycete Leptosphaeria maculans. Previous collaborative projects between INRA Versailles and Genoscope consisted in precise sequencing, assembly and finishing of a 1.1 Mb genomic region in isolate v23.1.3. These data enabled characterization of the first known retrotransposons and avirulence genes in L. maculans. These analyses also showed that the genome comprised regions of long A+T-rich regions reminiscent of heterochromatin, alternating with G+C-equilibrated, gene-rich regions. The goal of the genome initiative is to release an annotated assembly with 12-x genome coverage for L. maculans isolate v23.1.3. Additional sequencing of BAC-ends for physical mapping and ESTs from mycelia grown under three different conditions is underway. The first draft 5-x coverage indicated a high content of repeats in the genome. The first 12-x assembly was released Nov. 15, 2006, and comprised of 1277 super contigs of which 142 are more than 100 kb (mean : 39.78 kb; max : 866.65 kb). From these data, a bigger- than-expected genome size of ca. 50 Mb is hypothesised and a ca; 30% of repeats in the genome is suggested. First analysis from the final assembly (expected in January 2007) will be presented. A. Stachowiak was funded by Marie-Curie Fellowship HPMT-CT-2001-00395 ‘FUNGENE’

186. The cyclic AMP induced gene expression of Magnaporthe grisea during appressoria formation. Yeon Yee Oh, Shaowu Meng, Douglas E. Brown, Sean Coughlan¹, Thomas K. Mitchell, Ralph A. Dean Fungal Genomics Lab, North Carolina State University, Raleigh NC. ¹Agilent Technologies, Inc. Wilmington DE.

The cyclic AMP signaling pathway modulates appressoria development in rice blast fungus, Magnaporthe grisea. To identify the cAMP induced cellular processes for appressoria induction, we compared global gene expression patterns in cAMP elaborating appressoria verse germinating spores on non inductive hydrophilic surfaces. After 9 hours incubation, RNA was extracted from tissues of each condition and microarray experiments were performed using the M. grisea v2.0 oligonucleotide array, created in collaboration with Agilent Technology. 8.6 % of M. grisea genes were found to be differentially expressed by cAMP treatments. This included some previously known pathogenecity factors. Functional analysis suggests that melanin biosynthesis, secondary metabolism, protein degradation, protein synthesis and lipid degradation are downstream of cAMP signaling pathway and are necessary for appressoria morphogenesis. This was verified by gene knock out followed by phenotype analysis. We will present the results of gene expression studies, classification of differentially expressed genes and functional characterization of selected genes.

187. Natural history and evolutionary principles of gene duplication in fungi. Ilan Wapinski, Avi Pfeffer, Nir Friedman, Aviv Regev. Broad Institute, MIT, Cambridge, MA USA e-mail: ilan@eecs.harvard.edu

Gene duplication and loss is a powerful source of functional innovation. However, the general principles that govern this process are still largely unknown. With the growing number of sequenced genomes, it is now possible to examine these events in a comprehensive and unbiased manner. Here, we develop a novel procedure that resolves the evolutionary history of all genes in a large group of species. We apply our procedure to seventeen fungal genomes to create a genome- wide catalog of gene trees that determine precise orthology and paralogy relations across these species. We show that gene duplication and loss is highly constrained by the functional properties and interacting partners of genes. In particular, stress-related genes exhibit many duplications and losses while growth-related genes show selection against such changes. This dichotomy is relaxed following whole-genome duplication. Duplicated genes rarely diverge with respect to biochemical function, but typically diverge with respect to regulatory control. Surprisingly, paralogous modules of genes rarely arise, even following whole-genome duplication. Rather, gene duplication drives the modularization of functional networks through specialization, thereby disentangling cellular systems.

188. The effector secretome of Phytophthora infestans: Structure and function. Sophien Kamoun ¹, Joe Win ¹, William Morgan ¹, Sang-Keun Oh ¹, Carolyn Young ¹, Rays Jiang ^{2, 3}, Francine Govers ³, Michael C. Zody ², Chad Nusbaum ². ¹ Department of Plant Pathology. The Ohio State University, OARDC, Wooster, OH, USA. ² The Broad Institute of MIT and Harvard, Cambridge, MA, USA. ³ Laboratory of Phytopathology, Plant Sciences Group, Wageningen University, The Netherlands.

The oomycete Phytophthora infestans causes late blight of potato and tomato and is arguably the most destructive pathogen of solanaceous crops. P. infestans establishes parasitic colonization of plants by modulating host cell defenses through an array of disease effector proteins. The biology of P. infestans effectors is poorly understood, but tremendous progress has been made recently. Two classes of effectors target distinct sites in the host plant: apoplastic effectors are secreted into the plant extracellular space, while cytoplasmic effectors are translocated inside the plant cell, where they target different subcellular compartments. Of particular interest are the RXLR effectors that are characterized by a conserved motif following the signal peptide. The RXLR domain is functionally interchangeable with a malaria host targeting domain and appears to function in delivery into host cells. The recent completion of the genome sequence of P. infestans enables genome-wide cataloguing of the effector secretome. Using computational analyses, we identified up to 700 candidate RXLR effector genes. These were frequently organized in clusters of paralogous genes, many of which exhibit hallmarks of positive selection probably as a result of a coevolutionary arms race with host factors. Predictably, effector genes are typically expressed and often up-regulated during infection. We also utilized the discovered RXLR effectors in high-throughput in planta expression assays to screen for alteration of plant defense response and gain an insight into their function.

189. Withdrawn

190. Withdrawn

The acquisition of iron is known to be an essential step in any microbial infection process due to iron-limiting conditions in the human host. This iron limitation is caused by high-affinity iron-binding proteins like transferrin or lactoferrin in the host. Since iron plays an essential role in key metabolic processes like DNA synthesis, oxidative phosphorylation or electron transport A. fumigatus has to overcome the iron deficiency by the synthesis of siderophores, which chelate iron. It was shown that an A. fumigatus strain unable to synthesize siderophores was attenuated in virulence in a murine infection model. To understand the cellular processes, induced by iron starvation, we analysed the proteome of A. fumigatus strain ATCC 46645 grown under iron-deficiency conditions. Under iron depletion, proteins involved in siderophore biosynthesis are upregulated, e.g. L-ornithine N⁵-oxygenase (SidA), and iron cluster-containing proteins as aconitase or 3-isopropylmalate dehydratase are down-regulated. In addition, proteins involved in the heme biosynthesis are less abundant under iron-deficiency. Further proteins analysed under different non- linear pH-scales will be presented and their putative role will be discussed.

192. Comparative Genomics of plant pathogenic Fusarium species. Li-Jun Ma¹, Won-Bo Shim², Manfred Grabherr¹, Michael Koehrsen¹, Reinhard Engels¹, Sinead O’Leary¹, Matter Pearson¹, Sarah Young¹, David DeCaprio¹, Chinnappa Kodira¹, James Galagan¹, Seogchan Kang³, Charles Woloshuk⁴, Harold Corby Kistler⁵, Bruce Birren¹. ¹Broad Institute of MIT and Harvard. ²Texas A&M University. ³Penn State University. ⁴Purdue University. ⁵University of Minnesota.

The genus Fusarium collectively represents the most important group of fungal plant pathogens, causing various diseases on nearly every economically important plant species. Of equal concern is the health hazard posed to humans and livestock by the plethora of Fusarium mycotoxins. Our goal is to utilize the power of comparative genomics to improve gene annotation, to identify functional non-coding elements and to study the evolution and pathogenicity of this group of economicly important species. F. oxysporum and F. verticillioides have been selected as sequence targets to facilitate the comparative studies with the existing F. graminearum genome. Sequencing, assembly and automated annotation of F. verticillioides is complete and sequencing and assembling for F. oxysporum is ongoing. The released F. verticillioides assembly contains 41.7Mb with 14,206 predicted proteins. More than 99% of the assembly was anchored to the genetic map. A newly constructed optical map for F. oxysporum spans more than 60Mb, which is much bigger than either F. graminearum or F. verticillioides. Preliminary analysis of the sequence indicates that the increased genome size is mainly caused by large segmental duplications and the existence of many transposable elements. One of the focal points of our comparative analysis is to identify gene clusters associated with mycotoxin production and tracing their evolutionary history. We will report our recent progress and discuss the impact of comparative genomics on our understanding of genome dynamics, pathogenicity and mycotoxin production in these genomes. Funding for Fusarium comparative project was provided by USDA/NSF microbial genome sequencing program.

193. Aspergillus Comparative Database: a web-based tool for comparative analysis. Vinita Joardar, Jonathan Crabtree, Rama Maiti, Natalie Fedorova, Paolo Amedeo, Samuel Angiuoli, William Nierman, Owen R. White, and Jennifer R. Wortman. The Institute for Genomic Research, Rockville, MD, USA. vinita@tigr.org

Comparative genome analysis in the genus Aspergillus has been facilitated by the availability of genome sequences for multiple species. Ortholog clusters were computed based on the mutual best blastP hits between the Aspergillus proteomes. Syntenic blocks were identified by searching for collinear orthologs, with allowances for gaps and rearrangements, along the full chromosomes and/or supercontigs. The results of the genome and proteome level computes for the Aspergillus genomes were stored in the Aspergillus Comparative Database (asp), a chado relational database. Sybil, a web-based software package developed at TIGR, was used for visualization and analysis of comparative genomics data. Sybil uses a graphical user interface to present and navigate the information stored in asp. The interactive graphical displays allow the user to navigate from global genome views down to specific protein reports. Protein cluster reports, lists of singletons, comparative sequence displays and publication-quality figures can be customized based on user specifications. The Sybil package also leverages the comparative data for annotation improvement. We present an overview of the Sybil package applied to the comparative analysis of aspergilli ( http://www.tigr.org/sybil/asp/index.html ). The Sybil visualization software is freely available for download from http://sybil.sf.net

194. Using comparative genomics to identify virulence characteristics in pathogenic yeast. David Fitzpatrick and Geraldine Butler. School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Ireland.

Due to their increasing clinical importance, nine Candida and closely related species have been sequenced. The wealth of genomic data now available, allows us to investigate the differences that account for increased pathogenicity in particular Candida species. In order to identify specific metabolic activity associated with pathogenesis, we have assigned KEGG orthology identifiers to all available Candida genome data, using a bidirectional best BLAST strategy against the manually curated KEGG GENES database. All annotated Candida data has subsequently been mapped onto reference metabolic pathways from the KEGG PATHWAY database. Using the information gleaned from our metabolic pathway analysis we have attempted to correlate species-specific components with virulence, by noting which are specific to pathogenic Candida such as Candida albicans, Candida parapsilosis and Candida tropicalis but absent in closely related non-pathogenic species such as Debaryomyces hansenii and Lodderomyces elongisporus. Pathways that are present in numerous pathogenic Candia species are of special interest as they have the potential to yield broad-spectrum targets for novel antifungal drugs. To help visualize genotypic and metabolic differences between species, we have also developed the Candida Gene Order Browser, an online tool that displays the syntenic context of genes from all available Candida genomes.

195. The grass endophyte Epichloe festucae: genome, unigenes, and gene expression. U. Hesse, P. Maynard, S. Macmil, G. Wiley, K. Andreeva, W. E. Beech, C.D. Van Horn, V.C. Bumgardner, V. Gopal- Puram, J. Wiseman, J. Webb, L. Gill, S. Alluri, E. Arnaoudova, M.L. Farman, J. W. Jaromczyk, B.A. Roe, C.L. Schardl. University of Kentucky, Plant Pathology Dept.

The ascomycete, Epichloe festucae, is a model endophyte that 1) switches between mutualistic and antagonistic states, 2) is seed transmissible, 3) has a sexual state amenable to genetic analysis, and 4) is rich in bioprotective alkaloids. This fungus grows systemically and intercellularily throughout the life of its host plant. On each reproductive tiller the fungus either infects benignly and transmits clonally in seeds, or produces its sexual state (stroma) and chokes inflorescence development. The E. festucae genome was estimated at 29 Mb in 6 chromosomes. The genome sequence was assembled from cloned insert end reads (4.2 x coverage) and preassembled pyrosequencing reads (454-sequencing: 20 x raw, 1.7 x assembled), giving 3967 supercontigs, of which 1004 larger than 2 kb covered ca 92% of the genome. Gene prediction (FGENESH) identified 9912 putative genes. We sequenced 25,000 ESTs from each of two normalized libraries — one of choked inflorescences, the other of benignly infected inflorescences — yielding 5077 E. festucae unigenes annotated by BLAST and InterPro. Additionally, ca 700,000 pyrosequencing reads were obtained from non-normalized cDNA derived from four biological replicates each of asymptomatic and choked inflorescences. Of these, 118,865 were identified in the E. festucae genome sequence, 23,046 in the unigene set, and 21,703 in the predicted gene set, allowing analysis of gene expression and in symbio regulation. Sequences are BLASTable at http://www.genome.ou.edu/blast/ef_blastall.html, Sequence data and annotations are stored in a MySQL database, soon to be published in GBrowse.

196. Analysis of TOX2, the HC-toxin biosynthetic gene cluster in Cochliobolus carbonum. Kouhei Ohtani, John S. Scott-Craig, and Jonathan D. Walton. MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA.

HC-toxin is a cyclic tetrapeptide host-selective toxin produced by the filamentous fungus Cochliobolus carbonum, a pathogen of maize. HC-toxin production in C. carbonum is controlled by a single locus, Tox2, which is composed of at least seven duplicated and coregulated genes involved in HC-toxin biosynthesis, export, and regulation. The Tox2 locus was earlier partially characterized by mapping, sequencing, and gene disruption but we still didn't know its overall structure nor how many additional genes are required for HC-toxin biosynthesis. To understand more thoroughly the structure of Tox2 and to find new genes involved in HC-toxin biosynthesis, we constructed a bacterial artificial chromosome (BAC) library from a C. carbonum toxin-producing strain and screened it with known Tox2 genes. More than six BAC clones were identified and shotgun sequenced to at least 4x coverage. To date we have identified several new putative Tox2 genes. One encodes an alpha subunit of fatty acid synthase, which is predicted to complex with the product of TOXC (encoding a beta subunit of fatty acid synthase) to synthesize the backbone of Aeo (2-amino-9,10-epoxi-8-oxodecanoic acid). Other new genes encode three P450s and an isoamyl alcohol oxidase. Like the known Tox2 genes, all of these new genes are absent in toxin non-producing isolates of C. carbonum. All have plausible roles in HC-toxin biosynthesis, which is being tested by targeted gene disruption.

197. The Genome Sequence of the Brown Rot Fungus Postia placenta. Jean F. Challacombe¹, Diego Martinez¹, Thomas S. Brettin¹, Susan Lucas², Paul Richardson², Eddy Rubin², and Dan Cullen³. ¹Los Alamos National Laboratory and DOE Joint Genome Institute, Los Alamos, NM. ²DOE Joint Genome Institute, Walnut Creek, CA. ³ USDA, Forest Products Laboratory, Madison, WI.

The brown rot fungi, a group of filamentous basidiomycetes, rapidly depolymerize the cellulose in wood without significant lignin removal. This type of decay differs from white rot fungi, such as Phanerochaete chrysosporium, which simultaneously degrade lignin and cellulose. Both white and brown rot fungi are common inhabitants of forest litter where they play an important role in carbon cycling. Brown rot fungi are most commonly responsible for the destructive decay of wood in buildings and other structures, and it has been estimated that 10% of the U.S. timber harvest decays in service each year. Irrespective of their economic importance, the mechanism of brown rot wood decay is poorly understood. To better understand the biology of this type of wood degradation, the Department of Energy’s Joint Genome Institute has sequenced the entire 33Mb genome of the model brown rot, Postia placenta. Using genomic DNA from dikaryotic strain MAD-698, the JGI generated 571,000 reads that assembled into 1243 haplotype scaffolds. Our investigation into the genome will provide an invaluable mechanistic perspective on cellulose depolymerization, and the unique haplotype assembly will allow us to gain insights into the complexities of genome evolution.

198. Withdrawn

199. Whole genome sequencing of the fungal plant pathogens Botrytis cinerea and Sclerotinia sclerotiorum. Sabine Fillinger¹,François Artiguenave², Alain Billault³, Mathias Choquer¹, Arnaud Couloux², Christina Cuomo⁴, David DeCaprio⁴, Martin Dickman⁵, Elisabeth Fournier¹, James Galagan⁴, Corinne Giraud¹, Chinnappa Kodira⁴, Linda Kohn⁶, Fabrice Legeai², Caroline Levis¹, Evan Mauceli⁴, Cyril Pommier², Jean-Marc Pradier¹, Emmanuel Quevillon^2,8, Jeffrey Rollins, Béatrice Ségurens³, Adeline Simon¹, Muriel Viaud¹, Jean Weissenbach³, Patrick Wincker³, and Marc-Henri Lebrun⁸. ¹UMR BIOGER, INRA, Versailles, France; ²URGI,INRA, Evry, France;³Génoscope- CNS, Evry, France; ⁴ The Broad Institute of Genome Research, Cambridge MA, USA; ⁵ Inst. for Plant Genomics and Biotech, College Station TX, USA; ⁶ University of Toronto, Mississauga, Canada; ⁷Department of Plant Pathology,University of Florida, Gainesville, FL, USA ⁸ Plant and Fungal Physiology, CNRS-BayerCropScience, Lyon, France

Botrytis cinerea and Sclerotinia sclerotiorum are destructive polyphageous pathogens of many economically important crops provoking grey and white mould respectively. Both fungi are leotiomycetes. In 2005, the Broad Institute released the assembly of a 4-5 x genomic sequence from B. cinerea strain B05-10 (TMRI/Syngenta) as well as the 7-8 x genomic sequence of S. sclerotiorum. At the same time, the French national sequencing center (Genoscope) started the genome sequencing of grapevine and two of its pathogens: Stolbur phytoplasm and B. cinerea strain T4. In addition to WGS 50.000-60.000 ESTs have been sequenced for both species. Automatic gene prediction with ab initio and similarity softwares has been applied to the three genomes suggesting 14.000 to 16.000 potential gene calls for both species. A manual annotation process involving an international consortium of 20 research groups will validate and correct the data. Comparative analyses of both B. cinerea strains on one hand and B.cinerea > vs. S. sclerotiorum on the other hand revealed a common set of 8400 gene calls, 1200 of which are absent from other fungal genomes. In addition both species present a high degree of sequence similarity (76 % average sequence identity in orthologous proteins), and of synteny at the contig level. S. sclerotiorum has twice as much repetitive elements (7.7 %) than B. cinerea (3 - 4 %) with an enrichment of all transposable elements and tandem repeats. RIPping also occurred at higher levels in S. sclerotiorum. The comparison between B. cinerea and S. sclerotiorum offers the first opportunity to compare the genomes of two closely related necrotrophic plant pathogens helping understanding evolutionary trends that have shaped their genomes.

200. Global Gene Expression Profiles of Phytophthora ramorum strain Pr102 in response to plant host and tissue differentiation. Caroline Press and Niklaus Grunwald, USDA, 3420 NW Orchard Ave., Corvallis, OR 97330. pressc@science.oregonstate.edu

The release of the draft genome sequence of P. ramorum strain Pr102, enabled the construction of an oligonucleotide microarray of the entire genome of Pr102. The array contains 344,680 features (oligos) that represent the transcriptome of Pr102. P. ramorum RNA was extracted from mycelium and sporangia and used to compare gene expression across tissue types and in the presence of the host (Rhododendron sp.). The purpose of the experiment was to identify genes where expression was responsive to tissue types and upon exposure to the host plant for further study. Gene expression studies were performed using a Nimblegen microarray. Genes were determined to be differentially expressed between tissue types if they were statistically significant (P = 0.05 after FDR) and resulted in a greater than 20-fold change in gene expression. In the comparison between mycelia and sporangial tissues, 263 genes demonstrated a greater than 20 fold change in gene expression. Of those genes, 52 genes were significantly downregulated in sporangia as compared to mycelium and 214 genes were significantly upregulated in sporangia as compared to mycelium. Several of the differentially expressed genes appear to be of the same type as those in a similar study in Phytophthora infestans by Kim and Judelson (2003) and include genes involved in cell wall restructuring, cell division and signaling functions. Not surprisingly, several genes involved in energy production, electron transport chains and growth are reduced in sporangia. 50% of the genes with differential expression have not yet been classified as to function in the current annotation. Further experimentation and analysis is ongoing.

201. Computational Resources for Fungal Genomics. Gangman Yi¹, Jaehee Jung¹, Serenella A. Sukno², and Michael R. Thon^1,2. Texas A&M University, ¹Department of Computer Science, and ²Department of Plant Pathology & Microbiology, College Station, TX USA

As the number of sequenced fungal genomes continues to grow, there is an increasing need for effective algorithms for whole genome analyses. We present two applications that are aimed at improving our knowledge of gene function and genome evolution. First, we present C-Hunter, an algorithm and software application designed to identify clusters of functionally related genes in genomes. C-Hunter utilizes functional categories defined in graph-based vocabularies such as the Gene Ontology (GO). Clusters identified in this manner need only have a common function and are not constrained by gene expression patterns or other properties. C-Hunter source code and example report files are freely available from our web site. We also present AAPFC (Automated Annotation of Protein Functional Class). AAPFC addresses the need for high quality, automated functional annotation of proteins in fungal genomes. The method utilizes data mining and statistical pattern recognition techniques to learn models for assigning Gene Ontology functional categories to unannotated proteins. In its current implementation, AAPFC is trained using fungal GO-annotated proteins obtained from the UniProt database. The AAPFC web site provides users with GO annotations and protein features in graphical and textual formats. Users can annotate small sets of proteins via a web interface and large sets of proteins by contacting the authors.

202. Transcriptional patterns in the extramatrical mycelium of the ectomycorrhizal fungus Paxillus involotus in response to various sources of nitrogen. Derek P. Wright, Dag Ahren, Anders Tunlid & Tomas Johansson Microbial Ecology, Ecology Building, Lund University, Sweden

The positive effects of ectomycorrhizal (ECM) fungi on plant nutrition have traditionally been attributed to the quantitative effect of the extramatrical mycelium on uptake of dissolved nutrients, such as nitrogen. In forest soils nitrogen is present either in inorganic (mainly as ammonium) or organic (amino acids, peptides and proteins) forms and nitrogen mobilization by hyphae from soil is directly linked to hyphae uptake capacities. Transcriptional programs for assimilation of various sources of inorganic and organic forms of nitrogen were investigated in the ECM association between Paxillus involotus and birch. Nitrogen sources of various degrees of complexity, ammonium phosphate, ammonium sulphate, glutamine, chitin, and bovine serum albumin were provided as patches in peat microcosms for fungal in growth. After fungal establishment in these patches total RNA was isolated and used for global transcriptional analyses. We used a custom cDNA microarray containing approx. 4,900 gene representatives. By bioinformatic means and using EST sequence information we could among those identify 462 reporters representing putative enzymes. From a metabolic reconstruction of these 462 enzymes we found them potentially involved in 901 enzymatic reactions and 173 metabolic pathways. By combining this information with transcriptional data we could observe major shifts in a number of metabolic pathways as responses to the nitrogen source provided.

203. Comparative Genomic Hybridization in the Genus Neurospora: Assessing the Ability to Recover Evolutionary Relationships. Luz B. Gilbert*, Lee Chae, Takao Kasuga, Louise Glass, and John W. Taylor Department of Plant and Microbial Biology, UC Berkeley, *lgilbert@berkeley.edu

Comparative Genomic Hybridization (CGH) using DNA microarrays is becoming a popular method of determining phylogenetic relationships among individuals and even species. Although researchers praise the large-scale genome information output as an advantage of this technology for use in phylogenetic analysis, few have questioned the reliability of CGH to correctly determine evolutionary relationships. With the tools at my disposal for the filamentous fungus Neurospora: a genome sequence, a DNA microarray, and a well supported phylogeny for this genus, I had the ability to rigorously address the utility of CGH for phylogenetic studies using both experimental and simulated data. The results suggest constructing phylogenetic trees from this type of data requires careful consideration of which tree-building method is used and which taxon is printed on the array.

Magnaporthe grisea, a fungal plant pathogen, is a major threat to rice production globally. The M. grisea genome encodes many secreted proteins and in response to various growth conditions it secretes extracellular proteins (ECPs) which play indispensable roles in host pathogen interactions. For many years, plant pathologists have concentrated on elucidating the relation between environmental factors and field development of the disease, developing fungicides, or breeding resistant cultivars. However, molecular basis of the resistance mechanisms in rice plants against M. grisea or of host specificity for M. grisea has not been clearly elucidated. A more extensive catalog of the secretome would therefore represent a valuable resource to elucidate processes such as pathogenesis and disease resistance. The aim of this study is to gain new insights into the dynamics and complexity of the proteins that are secreted by both Magnaporthe grisea and its rice host (Oryza sativa L.) during infection. To date, most of the proteomics studies in plant pathogenic fungi have been limited to 2-D gel analysis. Here we present a simple and effective “non-gel”-based fractionation method to separate the secreted proteins from M. grisea and rice plant, followed by LC-MS/MS analysis for protein identification and statistical validation with the PROVALT algorithm."

205. Preliminary GO annotation of a blast fungus Magnaporthe grisea. Shaowu Meng, Douglas E. Brown, Thomas K. Mitchell, Ralph Dean*. Center for Integrated Fungal Research, North Carolina State University, Campus Box 7251, 851 Main Campus Drive, Raleigh, NC 27695

The Magnaporthe grisea genome assembly Version 5 was released in the spring of 2006 along with automatic annotations. The next step is a curated community annotation effort. Gene ontology (GO) based systems are evolving into a reliable and rapid means of assigning annotations. Here, we report our preliminary GO annotation of M. grisea genome. This was done in two steps. Firstly, we identified orthologs between M. grisea and Saccharomyces cerevisiae through cataloging reciprocal best hits between translated ORFs of the two organisms using blastp. The cataloging was iteratively performed on the remaining un-annotated genes until the last reciprocal best hit was found. The result was 4,464 unique M. grisea ORFs annotated using 2,581 unique S. cerevisiae GO terms totally at 26,966 times. Next, for those M. grisea ORFs which did not have orthologs in the S. cerevisiae database, we applied the cataloging process using the GO proteins databases, which included multiple organisms. In this step, 1,760 unique M. grisea ORFs were annotated using 1,263 unique GO terms totally at 5,381 times. The GO annotation of M. grisea covered 50.9% of predicted genes. Our GO annotation provides a base for further functional genomics analysis of M. grisea, and for assigning newly created terms from the PAMGO community that are specific to plant pathogens. ^*Corresponding author.

206. Conserved Synteny in Trichoderma ressei with Related Filamentous Ascomycetes. Diego Martinez^1,2, Mary Anne Nelson² and Margaret Werner-Washburne². ¹Los Alamos National Laboratory/Joint Genome Institute, Los Alamos, NM 87018 ²Department of Biology, University of New Mexico, Albuquerque, NM 87131

The current pace of whole genome sequencing has made it almost trivial to produce nearly complete fungal sequences. This now gives us the ability to perform whole genome comparisons of fungi to investigate biological questions and genome evolution. To this end, syntenic regions between Trichoderma reesei and four other filamentous fungi (Fusarium graminearum, Neurospora crassa, Magnaporthe grisea and Aspergillus nidulans) were identified. An algorithm was designed that takes homologs from two fungi and places them side by side, optimizing homolog density, and minimizing gaps between homologs and the number of homologs in a syntenic region. As would be expected, the percent coverage of synteny between T. reesei and the other four ascomycetes declines with time since the last common ancestor, with F. graminearum having the highest synteny and A. nidulans the lowest. This approach enabled the identification of regions in the T. reesei genome that were conserved in multiple genomes. Finally, for genes within these highly conserved regions, GO terms and Enzyme Commission codes were assigned. These results were used to assess the possibility that there is selective pressure to force genes that are in the same biochemical pathway, have similar function or are involved in similar processes to maintain proximity.

207. Comparative proteomics of the in vitro and in planta secretomes of Fusarium graminearum. Janet M. Paper, John S. Scott-Craig and Jonathan D. Walton. DOE-Plant Research Laboratory, Michigan State University, E. Lansing MI 48824

F. graminearum (Fg) is the cause of head blight and stalk rot of wheat and maize. Its sequenced genome is predicted to encode ~12,000 proteins. Secreted fungal proteins have several known functions related to pathogenesis, including phytotoxicity, degradation of cell wall polymers (penetration, ramification, and nutrition), and inhibition of plant defense proteins. We are using peptide mass fingerprinting to analyze the secretome of Fg in vitro and in planta. Secreted proteins from Fg grown in vitro on different media or extracted from infected wheat heads by vacuum infiltration were digested en masse with trypsin and analyzed by LC-MS/MS. From culture filtrates of Fg grown on sucrose, carrot or maize cell walls, collagen, xylan, pectin, or oat bran, ~230 proteins could be identified with high statistical probability. Because many secreted proteins are catabolite-repressed and/or substrate-induced, the spectrum of proteins was influenced by the medium, e.g., collagen induced the most proteases and sucrose yielded the fewest hydrolases. Despite the preponderance of host proteins in infected wheat heads, we could identify 120 fungal proteins. Many of these (25) were predicted to interact with host cell walls (e.g., hydrolases, sugar oxidases, cellulose-binding proteins, acetylxylan esterases). Some (42) in planta proteins were not found any of the in vitro analyses. About 27% of the in planta proteins were of unknown function or predicted to be non-enzymatic. Although >85% of the in vitro proteins were predicted to have signal peptides, only 61% of the in planta proteins were. Among the many predicted non-secreted proteins found in planta were 13 housekeeping enzymes including enolase, triose phosphate isomerase, phosphoglucomutase, calmodulin, aconitase, and malate dehydrogenase. The presence of these proteins in the in planta secretome might be due to fungal lysis in planta, but several of them have been reported to be immunogens secreted by animal pathogenic fungi.

208. Simple Sequence Repeats in Neurospora crassa : distribution, polymorphism, and marker potential. Tae-Sung Kim¹, James Booth², Hugh G. Gauch, Jr.³, Qi Sun⁴, Jongsun Park⁵, Kwangwon Lee¹. ¹Department of Plant Pathology, ²Department of Biological Statistics and Computational Biology, ³Department of Crop and Soil Sciences, Cornell University, Ithaca, NY 14850 USA ⁴Cornell Theory Center, Ithaca NY 14850 USA ⁵Fungal Bioinformatics Laboratory and Department of Agricultural Biotechnology Seoul National University, Seoul 151-921, Korea.

Simple sequence repeats (SSRs) have been successfully used as molecular markers for various genetic studies in eukaryotic systems. Despite the growing number of sequenced genomes, a thorough statistical examination on the distribution of the SSRs and experimental verification of the usefulness of SSRs in filamentous fungi for genetic and genomics studies are lacking. We identified and characterized the 2749 SSRs of 963 SSR types in the whole genome of the model fungal system Neurospora crassa. The tri-nucleotide (nt) repeats were the most common SSRs in N. crassa. The distribution of the tri-nt SSRs was significantly biased in exons. We further characterized the distribution of 18 abundant SSR types, which account for 71% of total SSRs in the N. crassa genome. We tested whether the occurrence rate of SSRs is random across chromosomes using Poisson log linear model. Based on our analyses, we concluded that there are systematic differences in SSR occurrence rates between chromosomes as well as between SSR types. We attempted to explain the non-randomness of the SSR occurrence between the chromosomes using factors such as chromosome size and gene density. We also characterized the size variation of SSRs among accessions using Polymorphic Index Content (PIC). We characterized the PIC scores of abundant SSR types using randomly selected 162 SSR loci in seven N. crassa accessions. Finally, utilizing the polymorphic SSRs, we built three linkage maps from three intra-species specific populations. The genetic orders of SSR markers along the chromosomes from the three linkage maps were highly consistent with the position in the physical map suggesting that the genetic architectures of the six natural accessions are similar to each others.

209. Annotation and analysis of the genome of Phycomyces blakesleeanus, a model photoresponsive zygomycete. Alan Kuo^1*, Asaf Salamov¹, Jasmyn Pangilinan¹, Erika Lindquist¹, Harris Shapiro¹, Scott Baker², Luis Corrochano³, and Igor Grigoriev¹. ¹DOE Joint Genome Institute, Walnut Creek, CA, USA. ²Pacific Northwest National Laboratory, Richland, WA, USA. ³Departamento de Genetica, Universidad de Sevilla, Spain. ^*akuo@lbl.gov

Light induces in P. blakesleeanus multiple developmental and biochemical responses (sporangiophore growth and development, beta-carotene synthesis). P. blakesleeanus is an intensively studied, experimentally tractable model organism, and whole-genome analysis is expected to further elucidate the signaling pathways underlying its photoregulation. To this end the genome was sequenced to 7.49X depth and assembled into 475 scaffolds totaling 56Mbp, and 47847 ESTs were assembled from cDNAs of light and dark cultures. We combined into a single annotation pipeline a variety of gene modeling methods (homology-based, EST-based, and ab initio), and predicted 14792 protein-coding genes. Many of these gene predictions are supported by homology in nr (68%), by Pfam domains (44%), or by ESTs (35%). We next assigned GO terms to 41% of the proteins and EC numbers to 16%. We then distributed these annotations to the Phycomyces consortium, along with tools to curate them manually. We expect that the annotation will provide a solid platform for expression analysis. In addition to its value as a model organism, P. blakesleeanus is the second zygomycete with a sequenced genome, after the related Rhizopus oryzae. We therefore will present preliminary results of comparative analysis between the two zygomycetes. LBNL-62091 ABS This work was performed under the auspices of the US Department of Energy's Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Livermore National Laboratory under Contract No. W-7405-Eng-48, Lawrence Berkeley National Laboratory under contract No. DE-AC02-05CH11231 and Los Alamos National Laboratory under contract No. DE-AC02-06NA25396.

210. Development of wheat leaf rust, Puccinia triticina, ESTs from various spore stages. Guus Bakkeren¹, Junhuan Xu¹, Rob Linning¹, Yehoshua Anikster², Brent McCallum³, Travis Banks³, Sarah Munro⁴, Michael Mayo⁴, Brian Wynhoven⁴, Johar Ali⁴, Richard Moore⁴. ¹Agriculture & Agri-Food Canada, Pacific Agri-Food Research Centre, Summerland, BC, V0H 1Z0, Canada; ²Tel Aviv University, Israel; ³ Agriculture & Agri-Food Canada, Cereal Research Centre, Winnipeg, MB, R3T 2M9; ⁴ Michael Smith Genome Sciences Centre, Vancouver, BC, V5Z 4S6, Canada. bakkereng@agr.gc.ca

We are in the process of increasing genomic resources for the obligate wheat pathogen, the leaf rust fungus P. triticina. Approximately 17,000 EST reads covering a teliospore stage from senescent wheat and pycnio- and aeciospore stages from an alternate host infection on Thalictrum speciosissimum (meadow rue), were added to our previously established, 30,000 reads-large EST database (holding ESTs from urediniospore, appressorium and other wheat infection stages). These new spore stages contributed approximately 65%, 80% and 45% new unigenes to the existing database, respectively. From initial analyses it appears that aeciospores might express gene complements more similar to the urediniospore stages from wheat we sampled previously; this might illustrate the possible resemblance of function of these spores, i.e., infecting wheat. The pycniospores stage, which represents the sexual stage, displayed the largest pool of new genes. Other trends and comparisons will be presented.

211. The secretome of Coprinopsis cinerea. Andrzej Majcherczyk, Dorothea Fragner, Ravi Chandra Dwivedi, Mojtaba Zomorrodi, and Ursula Kües. Institute of Forest Botany, Georg-August-University, Göttingen, Germany

Basidiomycetes secrete many different proteins for degradation of complex substrates. In the complex fungal secretomes, only few proteins can be studied by direct detection of enzymatic activities and changes in substrate composition. To understand and to follow degradation processes, we use mass spectrometry for the analysis of tryptic digests of proteins with a Mascot database containing known protein sequences, annotated genomic sequences and ESTs from Coprinopsis cinerea, Trametes versicolor and Pleurotus ostreatus, respectively. Analysis of the C. cinerea secretome showed an increasing complexity with rising age of cultures. Several different glucosidases, oxidative enzymes (e.g., laccases, manganese-dependent peroxidases, glyoxal and glucose oxidases), lectin-like proteins and multiple proteolytic enzymes have been identified in liquid culture. Other enzymes effective in substrate degradation localize to the fungal cell wall and methods are developed to identify their individual nature. Part of this work is supported in frame of a Common Lower-Saxony-Israel Project (VW-Vorab). The Deutsche Bundesstiftung Umwelt financially supported our laboratory.

212. Identification of pathogenicity factors among secondary metabolite-related genes in Alternaria brassicicola. Kwang-Hyung Kim¹, Yangrae Cho¹, Mauricio La Rota¹, Robert Cramer², and Christopher Lawrence¹. ¹ Virginia Bioinformatics Institute and Department of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061. ² Department of Molecular Genetics and Microbiology, Duke University Medical Center, Duke University, Durham, NC 27708-9902

Very little information is currently available concerning the pathogenic determinants produced by Alternaria brassicicola, the causal agent of black spot disease of Brassica plants. Production of secondary metabolites with host and non-host specific phytotoxicity has been proposed to play a role during the infection process of plants by fungi, especially in necrotrophic fungal pathogens. Gene clusters containing Polyketide Synthase (PKS) and Non-Ribosomal Peptide Synthetase (NPS) coding genes have been shown to be a major source of toxic secondary metabolites in fungi. To test the hypothesis that secondary metabolites produced by A. brassicicola contribute to pathogenicity, 8 NPS and 9 PKS genes were first identified by mining the A. brassicicola genome sequence database in our lab. Knockout (KO) mutants, constructed by gene disruption technology developed in our lab, were obtained for all 17 genes and evaluated for changes in fungal development and pathogenicity. Results of studies so far indicate some of the KO mutants of PKS and NPS genes showed interesting phenotypes and/or reduced virulence. Here we present functional characterizations of two interesting NPS genes, AbNPS2 and AbNPS4. As a result of NCBI BLAST searches, AbNPS2 and AbNPS4 showed high similarity (E-value 0.0) to NPS4 and NPS12 of Cochliobolus heterostrophus, respectively. To further characterize the abnps2 and abnps4 transformants, we are conducting chromatographic and histomorphological analyses. Future studies include further characterization of other PKS and NPS genes, KO mutagenesis and plant pathogenicity analyses, and structural analyses (LC-MS and NMR) of the in vitro-produced metabolites identified thus far.

213. Contributions of aneuploidy to fluconazole resistance in Candida albicans. Anna Selmecki, Anja Forche, Judy Berman. University of Minnesota, Minneapolis, MN selme003@umn.edu

Genomic instability and aneuploidy are common in Candida albicans. Using comparative genome hybridization (CGH) microarrays, we found a strong correlation between aneuploidy and azole resistance¹. Furthermore, we identified a segmental aneuploidy of chromosome 5L (Ch5L) DNA that was due to the formation of an isochromosome (a chromosome with two identical arms), in many fluconazole resistant (Flu^R) strains. Isochromosome 5L (i(5L)) formation is often accompanied by loss of heterozygosity (LOH) along Ch5L and while i(5L) is relatively stable under rich culture conditions, loss of i(5L) does occur and always correlates with a loss of Flu^R. Microarray analysis of i(5L) strains show that most genes on Ch5L exhibit increased expression and may contribute to Flu^R. Several well characterized genes involved in drug transport, ERG11 and TAC1, are present within the isochromosome and thus present in increased gene copies. We found that deletion of one or two extra copies of these genes in an i(5L) strain background has only partial effects on Flu^R, suggesting that increased copies of these genes alone are not responsible for the high level of resistance seen in the i(5L) strain. We are currently using telomere-mediated chromosome truncations of Ch5L to determine the degree to which different gene regions convey resistance. The effects of aneuploidy on gene copy number, gene expression level, LOH, and acquired azole resistance will be discussed. 1. Selmecki et al. (2006) Science 313: 367-370.

214. Initial analyses of the transposable elements of Rhizopus oryzae. Margi Butler¹, Manfred Grabherr², Tim Goodwin¹, Russell Poulter¹, Michael Koehrsen², Reinhard Engels², Sinead O’Leary², Chinnappa Kodira², Bruce Birren² and Li-Jun Ma². ¹ University of Otago, NZ. ²Broad Institute of MIT and Harvard, Cambridge MA. margi.butler@otago.ac.nz

Rhizopus oryzae is the first Zygomycete fungus to be sequenced and differs from all previously sequenced fungal genomes in being highly repetitive. Repeat sequences account for approximately 35% of the genome. Such a heavily repetitive genome provides the unique opportunity to study the origin of the repeats and their impact on genome evolution. Analyses show that more than half of the identified repeats are derived from transposable elements. Representatives of all the mobile elements, including class I retrotransposons (LTR retrotransposons, non-LTR retrotransposons and DIRS-like elements) and class II DNA transposons (DDE transposons, Helitrons and Cryptons) are found in the genome assembly. Remarkably, many of the mobile elements are apparently functional. No evidence of RIP (repeat induced point mutation) was detected among copies of transposable elements. The presence of abundant, apparently functional, elements will provide a unique resource for analysing transposon and retrotransposon replication. We will describe the frequency, diversity and distribution of these elements in the genome and will discuss the their impact on the genome dynamics of organism. Funding for the Rhizopus oryzae genome sequencing was provided by NHGRI

215. Identifying Divergent Genes in the Genus Neurospora and Related Species Using Comparative Genomic Hybridizations. Luz B. Gilbert*¹, Takao Kasuga¹, Jeff Townsend ², Louise Glass¹, John W. Taylor¹. ¹Department of Plant and Microbial Biology, UC Berkeley, ²Department of Ecology and Evolutionary Biology, Yale University, *lgilbert@berkeley.edu

Array based Comparative Genomic Hybrization or CGH is a useful tool to identify candidate genes that differ between species. Using the 70mer expression array designed for Neurospora crassa we compared seven species of Neurospora, as well as Podospora anserina and Sordaria macrospora in an effort to detect divergent genes. A second whole-genome tiling array was constructed by Nimblegen Systems for Neurospora crassa for a more comprehensive CGH analysis in both genic and intergenic regions of the genome. Two of the nine species used in the 70mer study were competitively hybridized to the Nimblegen tiling array. Genes found divergent using both arrays were classified according to functional category and a subset were selected for sequencing analysis.

216. Whole transcriptome analysis of Coprinus cinereus meiotic development: a nosey look at mushroom sex. Claire Burns¹; Jason Stajich²; Jason Lieb³; Walt Lilly⁴; Allen Gathman⁴; Mimi Zolan¹; Pat Pukkila³. ¹Dept. of Biology, Indiana University, Bloomington, IN, USA; ²Dept. of Plant Biology, University of California, Berkeley, CA, USA; ³Dept. of Biology, University of North Carolina, Chapel Hill, NC, USA; ⁴SE Missouri State University, Cape Girardeau, MO, USA

Coprinus cinereus, a basidiomycete mushroom fungus ideally suited to meiotic studies due to its synchronous development, has a near-completed genome. EST information is available for ca. 2500 genes, and gene prediction algorithms indicate a total of ca. 12,500 genes. We have designed and fabricated a 13,230 element 70-mer oligonucleotide microarray using ArrayOligoSelector. Design criteria and array formulation will be presented. These arrays are to be made available to the wider Coprinus community. The tightly-regulated light and temperature dependent life cycle of the mushroom allows sampling of gill tissue in which cells are at a specific meiotic stage at a certain time. Meiosis occurs synchronously and takes ca. 12 hours. Our initial analyses using the new Coprinus arrays concern a broad 15-hour timecourse through the meiotic process, describing events prior to fusion of dikaryotic nuclei, nuclear fusion, condensation and alignment of meiotic chromosomes, first meiotic division, and completion of the second division. Analysis of resultant data using several algorithms, including ArrayLod and SAM, will be presented. This work is supported by NIH, and by the Indiana METACyt Initiative of Indiana University, which is funded in part through a major grant from the Lilly Endowment, Inc.

217. Genome sequence of Batrachochytrium dendroabatidis. Christina Cuomo¹, Manfred Grabherr¹, Lucia Alvarado¹, David DeCaprio¹, Chinnappa Kodira¹, James Galagan¹, Timothy James², Joyce Longcore³, and Bruce Birren¹. ¹Broad Institute of MIT and Harvard, Cambridge, MA ²Uppsala University, Uppsala, SWE ³University of Maine, Orono, ME

Batrachochytrium is a pathogen of amphibians implicated as a primary causative agent of amphibian declines. This recently emerging pathogen was identified in 1998 as the cause of amphibian deaths in Australian and Central America. More recently, B. dendrobatidis has been implicated in population declines of frog species in North America, South America, Europe and Africa. We chose to sequence strain JEL423, isolated from a sick Phylomedusa lemur frog from Panama. We produced a 7.4X whole genome shotgun assembly, which contains 23.4Mb of sequence in 348 contigs, which are linked into 69 scaffolds. The sequence is highly repetitive

 (~24%), but no significant similarity is found to previously characterized transposons. Progress in automated gene prediction will be described. This is the first representative of the Chytrid order to have its genome sequenced, and will provide the opportunity for comparisons across the fungal clade along with the sister animal clade.

218. Withdrawn

219. Fungal Comparative Genomics – Progress under the Fungal Genome Initiative. Christina Cuomo, Matthew Henn, Li-Jun Ma, Kurt Labutti, Sean Sykes, Manfred Grabherr, Evan Mauceli, Sante Gnerre, Matthew Crawford, Michael Koehrsen, Reinhard Engels, Lisa Larson, Phil Montgomery, Matthew Crawford, Jared White, Matthew Pearson, Clint Howarth, Qiandong Zeng, Chandri Yandava, Sinead O’Leary, Lucia Alvarado-Balderrama, Sarah Young, Dave DeCaprio, Chinnappa Kodira, David Jaffe, Chad Nusbaum, James Galagan, and Bruce Birren. The Broad Institute of MIT and Harvard, Cambridge MA, bwb@broad.mit.edu

The Fungal Genome Initiative was launched to generate genomic resources to promote research on organisms across the fungal kingdom. The FGI has been creating fungal genome sequences, annotations, and analytic tools to support studies of individual organisms and their evolutionary origins. To date the FGI has released genome sequence for 28 fungi, including fungi from all four major branches of the fungal tree. In addition, 35 approved fungi are currently in the sequencing queue. A primary focus of the FGI has been to promote comparative studies by sequencing clusters of related fungi and phenotypic variants of species to help interpret the biology of key models or pathogens. Recently initiated projects include major sequencing efforts around human pathogenic fungi, Candida albicans (5 species), dermatophytes (5 species), Coccidioides (14 new strains), and other dimorphic pathogens (9 strains of 4 species). Comparative sequencing projects around plant pathogenic Fusarium and Verticillium species are also underway. We will describe recent progress on sequencing as well as new tools and web sites to help make these comparative data most useful to the community.

220. Sequencing and Comparative Analysis of Candida Genomes. Christina Cuomo¹, Matthew Rasmussen², Manfred Grabherr¹, David DeCaprio¹, Lohith Kini², Chinnappa Kodira¹, Esther Rheinbay², Radek Szklarczyk², Geraldine Butler³, Neil Gow⁴, Joseph Heitman⁵, Michael Lorenz⁶, Andre Nantel⁷, Manolis Kellis^1,2, and Bruce Birren¹. ¹Broad Institute of MIT and Harvard, Cambridge MA ²MIT Computer Science and Artificial Intelligence Laboratory, Cambridge, MA ³ University College Dublin, Dublin, Ireland ⁴ University of Aberdeen, Aberdeen, United Kingdom ⁵Duke University Medical Center, Durham, NC ⁶University of Texas Health Science Center, Houston, TX ⁷National Research Council of Canada, Montreal, PQ, Canada

Analysis of an individual fungal genome such as Candida albicans can be greatly enhanced by comparison to genomes of related species. Comparative genomics highlights sequences conserved between species and can inform such basic questions as how genomes evolve, what genes make a species unique, and what genes and noncoding sequences are shared among species. As part of the Fungal Genome Initiative, we have sequenced five related Candida species: C. albicans (strain WO-1), C. tropicalis, Lodderomyces elongisporus, C. guilliermondii, and C. lusitaniae. We have released assemblies and preliminary gene sets from each genome. Additionally we have generated comparative genomic resources including whole genome alignments, protein families, species specific genes, and Candida specific genes. These resources are being utilized by a community based comparative analysis project which is currently underway. The growing set of Candida genome sequences allows comparisons across a range of evolutionary distances, enabling many different approaches to study the conservation of genes and regulatory elements as well as the evolution of these elements and genomic architecture within Candida species.

221. DelsGate, a robust and rapid gene deletion construction method for functional genomics. María D. García-Pedrajas^1†, Laura B. Kapa¹, Michael H. Perlin², and Scott E. Gold¹. ¹Department of Plant Pathology, University of Georgia, Athens, GA ², Department of Biology, University of Louisville, Louisville, KY ^†Estación Experimental del Zaidín, CSIC, Granada, Spain

With increasing availability of fungal genome sequences there is great demand for fast, simple methods to generate gene deletion constructs. We describe a method, named DelsGate, that combines PCR and Gateway cloning technology together with the I-SceI homing endonuclease to generate precise deletion constructs in a simple, universal and robust manner in just two days. These constructs may then be used to produce deletion mutants in the organism of interest. The initial version (DelsGate?) involved PCR of the entire ORF and 1 kb of 5’ and 3’ flanks, followed by self-ligation, inverse PCR and Gateway cloning to generate the final construct. A simplified improved version (DelsGate) was later devised consisting of PCR of only the 5’ and 3’ 1 kb gene flanks followed directly by in vitro Gateway cloning and final circular deletion construct generation by in vivo recombination in E. coli. For use in DeslGate we have produced vectors for transformation of Ascomycetes and the Basidiomycete fungus Ustilago maydis. We have tested the feasibility and reproducibility of the DelsGate approach by generating deletion constructs for 12 genes in U. maydis. Although not tested here DelsGate should be well suited for high-throughput approaches to gene deletion construction in fungal species.

222. High throughput gene knockouts in Neurospora. Patrick D. Collopy¹, Hildur V. Colot¹, Gyungsoon Park², Carol Ringelberg¹, Liubov Litvinkova², Susan Curilla¹, Lorena Altamirano², Norma Gorrochotegui-Escalante¹, John Jones², Katherine A. Borkovich², and Jay C. Dunlap¹. ¹Department of Genetics, Dartmouth Medical School, Hanover, NH, USA ²Department of Plant Pathology, University of California, Riverside, CA

Gene disruptions have been made in predicted open reading frames in the annotated Neurospora crassa genome using a high throughput procedure as part of an NIH-funded Program Project (P01)*. Knock-out (KO) cassettes were created by yeast recombinational cloning techniques and electroporation of N. crassa conidia can be performed in a 96-well format. In order to expedite molecular confirmation of KO mutant strains, we have developed a program ( http://borkovichlims.ucr.edu/southern/ ) that allows automated identification of the appropriate restriction enzyme to use during Southern analysis. Additionally, we have developed and implemented a Laboratory Information Management System (LIMS; http://borkovichlims.ucr.edu/php/sLIMS.php ) to keep track of the various steps of our gene KO processes. All plates and tubes used during the knockout procedure are labeled with barcodes and managed systematically. Completed N. crassa KO mutant strains are submitted to the Fungal Genetics Stock Center after targeted gene replacements are confirmed by Southern analysis. The list of submitted strains is available at the Neurospora genome project website ( http://www.dartmouth.edu/~neurosporagenome/knockouts_completed.html ). Use of these tools and our current progress in creating KO mutants will be presented. *Colot and Park et al., 2006. A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors. PNAS 103:10352-10357

223. High-throughput mutation procedure for Neurospora genes. Lorena Altamirano¹, Luibov Litvinkova,¹, Gyungsoon Park¹, John Jones¹, Hildur V. Colot², Patrick D. Collopy², Norma Gorrochotegui- Escalante², Carol Ringelberg², Jay C. Dunlap² and Katherine Borkovich¹. ¹ Department of Plant Pathology, University of California, Riverside, CA ² Department of Genetics, Dartmouth Medical School, Hanover, NH, USA

Gene deletions have been made in predicted open reading frames and genes in the annotated Neurospora crassa genome using a high throughput procedure as part of an NIH-funded Program Project (P01). Many steps of the process utilize a 96-well format, including construction of knock-out (KO) cassettes (using yeast recombinational cloning techniques), electroporation of N. crassa conidia and subsequent analysis of transformants. In order to expedite molecular confirmation of KO mutant strains, we have developed a program ( http://borkovichlims.ucr.edu/southern/ ) that allows automated identification of the appropriate restriction enzyme to use during Southern analysis. Additionally, we have created and implemented a Laboratory Information Management System (LIMS; www.borkovichlims.ucr.edu/php/sLIMS.php ) to track the various steps in the gene KO process. All plates and tubes used during the knockout procedure are labeled with barcodes and managed using the LIMS. Completed N. crassa KO mutant strains are submitted to the Fungal Genetics Stock Center after the gene replacements are confirmed by Southern analysis. The list of submitted strains is available at the Neurospora genome project website ( http://www.dartmouth.edu/%7Eneurosporagenome/knockouts_completed.html ). To date, we have constructed knockout mutants for1480 Neurospora genes and a total of 2233 mutant strains have been submitted to the FGSC. Another 960 genes are in various stages of the knockout procedure.

224. The Comparative Fungal Genome Websites of the Broad Institute. M. Crawford, M. Koehrsen, R. Engels, L. Larson, P. Montgomery, J. White, M. Pearson, C. Howarth, T. Ledlie, D. Park, D. DeCaprio, J. Galagan, B. Birren. Broad Institute, Genome Biology, Cambridge, MA

Efforts at the Broad's Fungal Genome Initiative focus on the sequencing of clusters of related organisms. To make these data as useful as possible we are developing a set of comparative group web sites to analyze multiple genomes. These sites feature redesigned statistics pages that highlight differences and similarities between different assemblies, genomes, and gene predictions. BLAST and feature searches can be performed simultaneously on the genomes of all the organisms in the group, with the results tabulated for meaningful comparison. The process for downloading has also been adjusted: sequences and other files generated for several organisms can be conveniently downloaded together. For group sites where the corresponding genes between organisms have been mapped, a set of views and tools facilate the analysis of related genes, including a Tree View and an Alignment view. The Argo browser now incorporates a comparative view that can assist in analyzing two or more genomes simultaneously. To make these tools as widely useful as possible we are integrating published genomes generated by others to give users a consistent analysis platform.

225. Tagging target genes of the MAT1-2-1 transcription factor in Fusarium verticillioides (Gibberella fujikuroi MP-A). Anita Keszthelyi¹, Apor Jeney¹, Zoltán¹, Odette Mendes², László Hornok¹ and Cees Waalwijk². ¹Agricultural Biotechnology Center, Szent-Györgyi A. u. 4, H-2100 Gödöllõ, Hungary ²Business Unit Biointeractions and Plant Health, Plant Research International BV, P.O. Box 16, 6700 AA Wageningen, The Netherlands cees.waalwijk@wur.nl

Mating type in filamentous ascomycetes is controlled by idiomorphic alleles, named MAT1-1 and MAT1-2, which contain one to three genes. Of these genes MAT1-1-1 and MAT1-2-1 encode putative transcription factors and are thus considered to be the major regulators of sexual communication and mating. Fungi with no known sexual stage may also have fully functional mating type genes therefore it was plausible to hypothesize that the MAT products may also regulate other types of genes not involved directly in the mating process. To identify putative target genes of these transcription factors in Fusarium verticillioides, DMAT1-2-1 knock out mutants were produced and transcript profiles of mutant and wild type were compared by means of differential cDNA hybridization. Clones, either up- or down- regulated in the DMAT1-2-1 mutant were sequenced and a total of 248 sequences were blasted against the NCBI database as well as the G. zeae and G. moniliformis genomes. Fifty-five % of the clones were down-regulated in the mutant, indicating that the MAT1-2-1 product positively affected these tagged sequences. On the other hand, 94 sequences (~45%) were found to be up-regulated in the mutant, suggesting that the MAT1-2-1 product also exerted a negative regulatory function on this set of genes. Sequences involved in protein synthesis and metabolism occurred more frequently among the clones, up-regulated in the mutant, whereas genes belonging to cell signalling and communication were especially frequently tagged among the sequences, down-regulated in the mutant.

226. Computational Predictions of Gene Structure in the Filamentous Fungus. Aspergillus niger. Christine Chee¹, Diego Martinez^1,2 and Mary Anne Nelson¹. ¹Department of Biology, University of New Mexico, Albuquerque, NM 87131; cchee@unm.edu, manelson@unm.edu ²Los Alamos National Laboratory/Joint Genome Institute, Los Alamos, NM 87018; admar@lanl.gov

The Department of Energy‘s Joint Genome Institute has recently sequenced the genome of the filamentous fungus Aspergillus niger. Like all eukaryotic organisms, A. niger has many genes with complex structure. To assist in the discovery of this large number of genes, the JGI utilizes a gene prediction pipeline to automatically predict the intron – exon structure of genes. All information is displayed on an interactive website, The JGI Genome Portal (http://genome.jgi-psf.org/index.html). Due to the complexities of gene structure, it is necessary to manually verify and correct about 25% of the predicted gene models; the most common errors are truncated 5’ and/or 3’ ends. The interactive Genome Portal allows for comparison of A. niger predicted proteins against the proteins of many other species simultaneously, as well as known partial cDNAs and mRNA information. If editing is required, the curator can adjust the gene model structure or create an entirely new gene model. The combination of sequence comparisons and editing ability allows the researcher to create the most accurate gene models currently possible.

227. An update on the Neurospora crassa community genome annotation project. Heather M. Hood¹, Matthew R. Henn², Dave DeCaprio², James E. Galagan², Alan Radford³, Bruce W. Birren², Jay C. Dunlap⁴, & Matthew S. Sachs¹. ¹Oregon Health & Science University, Beaverton, OR 97006; ²Broad Institute of MIT & Harvard, Cambridge, MA 02141; ³University of Leeds, Leeds, UK; ⁴Dartmouth Medical School, Hanover, NH 03755.

To produce a more accurate Neurospora protein-coding gene catalog, we need to include the wealth of information about gene structure and function that is contained within the scientific community. A web-based community annotation resource was developed and released in March 2006 to help accomplish this (http://www.broad.mit.edu/annotation/genome/neurospora/CAHome.html). Community annotators can use this site to add a variety of information to genome features, including both genetic and functional data. They can also correct gene models and add alternative transcripts. Immediately following submission, community annotations are appended to the gene detail page and are publicly available. Each submitted annotation is inspected for accuracy by curators; once curated, the annotation is scheduled to be incorporated into the official gene detail page as part of an updated genome release. A new release of Neurospora gene predictions (Version 3) included community input. As of December 2006, the community annotation project has 57 registered participants that have contributed over 500 annotations, including 130 citations. Over 1100 E-compendium entries were also incorporated as community annotations, further enriching the data set. Finally, a Textpresso database was implemented for Neurospora literature. This database contains over 2500 searchable full-text articles related to Neurospora and fungal biology. Taken together these new community resources extend the utility of the genome sequence data.

228. Almost finished: the complete genome sequence of Mycosphaerella graminicola. Gert H.J. Kema¹, Igor Grigoriev², Andrea Aerts², Asaf Salamov², Hank Tu², Harris Shapiro², Jim Bristow², Jane Grimwood³, and Stephen B. Goodwin⁴. ¹ Plant Research International B.V., P.O. Box 16, 6700 AA Wageningen, The Netherlands. ² Department of Energy, Joint Genome Institute, 2800 Mitchell Drive, Walnut Creek, CA, 94598, USA. ³ Stanford Human Genome Center, 975 California Avenue, Palo Alto, CA 94304, USA. ⁴ USDA-ARS, Purdue University, 915 West State Street, West Lafayette, Indiana, 47907-2054, USA.

Mycosphaerella graminicola is a haploid ascomycete causing septoria tritici blotch of wheat. An 8.9x shotgun sequence of bread wheat strain IPO323 was generated through the Community Sequencing Program of the U.S. Department of Energy’s Joint Genome Institute (JGI), and was finished at the Stanford Human Genome Center. The finished genome of M. graminicola contains 39.6 Mb. All available ESTs (37,747) were placed on the finished assembly to assess completeness. In addition, 1793 Diversity Array Technology markers were sequenced at JGI and aligned with the physical map. The genetic and physical maps showed near-perfect colinearity, demonstrating the high quality of both the genetic linkage map and the genome assembly. Fifteen scaffolds are complete and represent entire chromosomes with sizes from 548 kb to 6 Mb. In addition, six scaffolds (ranging from 21 kb to 2.4 Mb) contain a single telomere and could connect in any combination. Two additional scaffolds do not contain telomeres and only 17 gaps remain to be closed. Our sequence data suggest that isolate IPO323 contains 18-20 chromosomes, which represents the highest number of chromosomes reported among ascomycetes. This conclusion is corroborated by high-density genetic linkage maps and detailed pulsed-field gel analyses. The complete mitochondrial sequence is a circular genome of 43,947 bp. The machine annotation predicted 13,413 genes, of which 1126 have been annotated manually (http://www.jgi.doe.gov/Mgraminicola). Gene annotations generally indicate that M. graminicola has fewer genes per family than other plant-pathogenic filamentous fungi, which may reflect its initially extracellular biotrophic and subsequently necrotrophic lifestyle. This genome represents the first nearly finished genome of any filamentous plant pathogen and is of great importance for comparative genomics involving other Dothideales, such as the destructive banana Black Sigatoka pathogen Mycosphaerella fijiensis that currently is sequenced at the ~7.1x level.

229. Improvements in Aspergillus fumigatus annotation. Paolo Amedeo, Natalie Fedorova, Rama Maiti, Vinita Joardar, Jonathan Crabtree, Samuel Angiuoli, William Nierman, Owen White, Jennifer Russo Wortman. The Institute for Genomic Research 9712 Medical Center Dr., Rockville, MD 20850

Aspergillus fumigatus was among the first fungal genomes to be fully sequenced and annotated. At the time, few characterized genes in closely-related organisms were available in the public databases, which had a negative effect on the quality of annotation. Since then annotation tools have improved and several other Aspergilli have been fully sequenced and annotated. We have revised the annotation of Aspergillus fumigatus leveraging comparative analysis techniques and the newly-available data from related organisms. Here we describe these processes and discuss improvements made to the annotation.

230. Withdrawn

231. Mitochondrial Genomics in the Genera Pythium and Phytophthora. Frank N. Martin¹ and Paul Richardson². ¹USDA-ARS, Salinas, CA, ² Joint Genomics Institute, Walnut Creek, CA.

Mitochondrial genomics can be useful for investigating the processes contributing to evolutionary divergence at a mtDNA sequence level and hence, provide insight to the phylogenetic relationships of the organisms under study. While the mitochondrial genomes of the related genera Pythium and Phytophthora encode a similar suite of genes, they differ from each other by the presence of a large inverted repeat (IR) that is found in Pythium (it can represent approximately 80% of the genome size). In an effort to gain a better understanding of the evolutionary forces responsible for sequence divergence in genomes with and without an IR, as well as to clarify the phylogenetic relationships within the individual genera, the mitochondrial genomes of 15 Pythium and 10 Phytophthora species were sequenced. Comparative genomics among species within a genus indicated that certain regions of the genome were more polymorphic than others. In Pythium, the most polymorphic region was the small unique region and adjacent IR sequences. In Phytophthora genomic inversions were observed with many of the rearrangements corresponding to phylogenetic groupings. Two closely related species (P. ramorum and P. hibernalis) also were found to have small IRs (1.1-1.5 kb in size). While the IR in Pythium appeared to stabilize the genome from rearrangements, the data suggests that the rate of evolutionary divergence was more dependent on the specific gene rather than its location within the IR.

232. Withdrawn

233. Transcriptome of conidium and ascospore development in Fusarium graminearum. Kye-Yong Seong¹, Matias Pasquali¹, Jin-Rong Xu², and H. Corby Kistler^1,3. ¹University of Minnesota, St. Paul, MN, USA; ²Purdue University, West Lafayette, IN, USA; ³USDA ARS Cereal Disease Laboratory, St.Paul, MN, USA.

To understand the infection cycle of the head blight pathogen F. graminearum, gene expression profiles were monitored during both ageing and germination for conidia and ascospores. Ascospores and conidia were treated in a similar manner, either aged under desiccating conditions or suspended in liquid germination medium. RNA was extracted from cultures and used to query the 18K feature F. graminearum Affymetrix GeneChip. Overall, a slightly greater number of probe sets corresponding to genes were detected in ascospores (9,207) than in conidia (8,815; detection p value <0.001) but the majority of probe sets (8,068) were shared between conidia and ascospores. While a similar number of genes were detected at most stages of development, the biggest difference among spore types was upon desiccation where the number of probe sets detected in ascospores (6,801) was more than twice the number detected in conidia (2,916). These results indicate that ascospores remain more metabolically active than conidia upon ageing. Peroxisomal proteins and genes involved in lipid beta-oxidation are strongly up-regulated both in fresh conidia and in ascospores. After suspending conidia or ascospores in liquid germination medium, numerous genes involved in transcription, RNA splicing, protein synthesis, and amino acid and nucleotide metabolism were highly induced. Up-regulation of proteasome components and secretory proteins were observed as spores established polarized growth after 8h of incubation. Comparing gene expression in spores with expression in hyphae under a variety of environment regimes indicates that a total of 328 probe sets were specific for ascospores and another 150 were specific for conidia. Spore-specific gene expression may be used to develop hypotheses concerning spore maturation, dormancy and initiation of germination. This work is supported by the National Research Initiative of the USDA CSREES, award 2004-35604-14327.

234. Restriction-site Associated DNA (RAD) mapping can be used to rapidly map mutations using Neurospora microarrays. Zachary A. Lewis, Anthony Shiver, Michael Miller, Eric A. Johnson, and Eric U. Selker. University of Oregon, Eugene, Oregon

Many important insights into fundamental biological processes have come from the study of filamentous fungi. The wealth of sequence information currently available for this group has facilitated evolutionary analysis, genome level expression analysis, and reverse genetic studies. However, the ability to carry out forward genetic screens and selections continues to drive discovery in the fungi. Whereas isolation of mutants is relatively easy, mapping and identification of the mutant genes is often the "rate limiting" step in genetic studies. We have performed a selection for genes that are defective in DNA methylation (dim). In order to identify the mutant genes, we have adapted microarray-based RAD mapping for use with Neurospora oligo arrays. We demonstrate that this technique can be used to detect genome wide restriction site polymorphisms from two polymorphic Neurospora crassa strains, Mauriceville and Oak Ridge. Moreover, we demonstrate that this technique can be used to rapidly map a mutation. RAD mapping can be used to identify restriction site polymorphisms in any two polymorphic strains without a priori knowledge of the polymorphisms. Therefore, in addition to forward genetics, we propose that RAD mapping will useful for analysis of phenotypic variation in natural populations (e.g. QTL analysis).

Industrial Biology and Biotechnology

235. Targeted Introduction of Point Mutations in Epichloë festucae. Kurtis Knapp, Richard Johnson, Christine Voisey, and Gregory Bryan. AgResearch Ltd.

AgResearch has a number of proprietary endophytes that increase plant yield and provide resistance to abiotic stress. However, these strains can not currently be commercialized because they produce mammalian toxic alkaloids. We will eliminate production of these alkaloids using a point mutagenesis technique called Rapid Trait Development System (RTDS). In RTDS conversions, one transforms an oligonucleotide that hybridizes with the gene of interest but contains a single base mismatch. The cell then alters the mismatched base in the genome. This technique is attractive to agricultural biotechnology because it avoids the untoward changes of random mutagenesis and the regulatory issues of transgenics. We will present progress toward implementation of RTDS to fungal endophytes of grasses. Initial work will be on mutations with a selectable phenotype in the Epichloë festucae strain FL1.

236. New screening approaches for fungal strain development. Peter J. Punt¹, Xavier O. Weenink², Marc van der Maarel¹, Jan Jore¹, Arthur Ram², Cees van den Hondel². ¹TNO Quality of Life, Zeist, the Netherlands,²Leiden University, Leiden, the Netherlands

Since the development of recombinant DNA technologies for yeast and filamentous fungi, a considerable part of the strain development programs was diverted to the use of molecular genetic tools. Whereas these approaches have exerted considerable success, recent developments in our laboratory have shown that new developments in classical biological screening approaches, or a combination of both, can still be very useful. A first purely classical approach is based on the discovery of a so-called suicide (SUI) substrate, which we have successfully used for the selection of protease deficient fungal host strains. These protease deficient strains show an increased resistance to the SUI substrate allowing their selection. The advantage of this non-GMO approach is that it can be applied to new and already established production strains. A combination of a molecular and classical approach is based on the use of the so-called glucoamylase carrier approach. Combining this approach with fungal strains unable to use starch as a carbon source allowed us to select for hyper secretive fungal strains generated by classical mutagenesis (Weenink et al., 2006). Moreover, the same approach also allows for selection of the highest producers in a collection of primary transformant strains expressing a glucoamylase-fusion gene.

237. Oxidative protein folding in Aspergillus niger. Anna Harvey¹, Andrew Plumridge¹, Mick Ward², Huaming Wang² & David B Archer¹. ¹School of Biology, University of Nottingham, University Park, Nottingham, NG7 2RD ²Genencor Intl., 925 Page Mill Road. Palo Alto, CA 94304, USA plxarh@nottingham.ac.uk

The formation of protein disulphide bonds is one of several processes that occur within the endoplasmic reticulum (ER) of eukaryotes, which can be vital for the correct folding of secreted proteins. The pathway for the formation of protein disulphide bonds has been characterised in the yeast model organism, Saccharomyces cerevisiae. In S. cerevisiae, two proteins, Ero1p and Pdi1p are essential for the correct formation of protein disulphide bonds, and their expression is regulated by ER stress through the unfolded protein response. In the filamentous fungus, Aspergillus niger, the presence of a Pdi1p homologue, PDIA, has already been demonstrated. We have identified a homologue of the Ero1p protein and we show that its expression is transcriptionally regulated by ER stress. The functions of eroA and the product EROA are being further characterised by investigating the effects of deletion and over- expression.

238. Chrysonilia sitophila: a Case Study for Ascomycete Biodegradation and Bioremediation Potential. Carvalho, MB^1,a, Martins, I^1,a, Garcia, H², Spencer-Phillips, P³, Gil, A², San Romão, MV ^1,4 and Silva Pereira, C ^{1,* 1}ITQB-UNL/IBET, Instituto de Tecnologia Química e Biológica - Universidade Nova de Lisboa / Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal. ²Universidade de Aveiro, Departamento de Química,Aveiro, Portugal ³University of the West of England, Bristol, UK ⁴EVN, Estação Vitivinícola Nacional, Dois Portos, Portugal. * Corresponding author, spereira@itqb.unl.pt ^a Equal contributing authors

Persistent organic pollutants, such as pentachlorophenol (PCP), are globally recognized as a major environmental concern. PCP constitutes an acute risk to health considered, by the WHO (2004), as one of the most hazardous substances. PCP use is nowadays wide restricted but is still frequently detected in all environments. Whole fungal cell biocatalysis represents an elegant way for elimination of extreme toxic products. However, fungi exploitation for biodegradation is often sub-optimal and rarely reliable. One of the underlying reasons for the fragility of many approaches is the lack of scientific knowledge concerning the diversity, activity and dynamics of the microbial communities responsible for the biodegradation processes. There is a close relation between fungi biodegradation and bioremediation abilities because the aromatic matrix of lignin resembles the basic molecular skeleton of PCPs. During cork stopper manufacturing process, cork slabs are initially colonised by Chrysonilia sitophila and latter by other fungal species, such as Penicillium glabrum and Trichoderma longibrachiatum. These fungal species are able to perforate the complete thickness of the cork cell wall (lignin 20%, suberin 40%), suggesting their active biodegradation ability and were able to degrade a less, relative to PCP, chlorinated chlorophenol. Fungi biodegradation ability was screened following cork decay by spectroscopy (FITR). C. sitophila degrades lignin and suberin, tolerates concentrations of PCP above 20 mg/L and can apparently grow solely in the presence of PCP. Growth media supplementation with lignin/suberin enriched composite apparently reduces PCP toxicity. PCP degradation pathway by C. sitophila is being analysed following PCP degradation intermediates formed and the fungi proteome during growth on different substrates (presence and absence of PCP).

239. Bottlenecks in Bacterial Transglutaminase Production in Trichoderma reesei. Marja Paloheimo¹, Susanna Mäkinen¹, Kim Langfelder² and Jari Vehmaanperä¹. ¹Roal Oy, Rajamäki, Finland. ²AB Enzymes GmbH, Darmstadt, Germany.

Transglutaminases catalyse cross-linking in proteins via an acyl transfer reaction between peptide-bound glutamine and lysine. TGases are widespread in mammals and they have also been found in microorganisms, plants, invertebrates, fish and birds. The cross-linking activity of TGases is exploited in several food applications, e.g. in improving the texture of processed meat and fish products. There is a growing interest to use TGases in technical applications, such as use in the textile industry. TGases could be used in modification of silk and wool fibres. For technical applications a cheap source of TGase must be available. For this reason a preliminary study was performed to determine whether Trichoderma reesei is a suitable host for production of a bacterial transglutaminase. The S. mobaraensis tg gene was used as a test gene. The TGase is activated after secretion by two-step cleavage of the pro-peptide from the precursor. Constructs encoding the full-length and mature and the corresponding inactive TGase forms were expressed in T. reesei. In addition the pro-sequence was expressed separately in strains already expressing the mature form of TGase. The TGase encoding sequences were expressed in T. reesei as 3´-fusions to a sequence encoding a carrier polypeptide. The results obtained will be shown and discussed.

240. Targeting cellular stress response systems with natural compounds to enhance antifungal activity. Jong Kim¹, John Beck¹, Shen-Chieh Chou², Noreen Mahoney¹, Kathleen Chan¹, Russell Molyneux¹ and Bruce Campbell^1*. ¹ Plant Mycotoxin Research, USDA, Albany, CA 94710 USA. bcc@pw.usda.gov. ² School of Pharmacy, University of Colorado Health Science Center, Denver, CO 80262.

A series of natural phenolic compounds (i.e., benzoic/cinnamic acid derivatives, etc.) were applied to various gene deletion mutants of Saccharomyces cerevisiae in order to identify gene targets for fungal control. Four groups of molecular targets were identified, as follows: transcription regulators of pH response or glutathione transferase; vacuolar H(+)- ATPase; mitogen-activated protein kinase kinase (MAPKK); and antioxidative enzymes. Treatments with 2,3- dihydroxybenzoic acid disrupted fungal glutathione homeostasis. Several compounds targeted mitochondrial superoxide dismutase (Mn-SOD). Functional complementation analysis using Aspergillus flavus sodA, encoding Mn-SOD verified increasing oxidative stress improved antifungal activity of commercial fungicides. Activity of strobilurin was greatly elevated on A. fumigatus and A. nidulans by co-application with berberine or veratraldehyde. These compounds also prevented A. fumigatus MAPK mutants from escaping toxicity of fludioxonil. Esterification of dihydroferulic acid (DFA) with methyl, ethyl, or propyl side chains greatly enhanced antifungal acitivity. We conclude that targeting fungal antioxidative stress systems with phenolic agents improves fungal control.

241. Withdrawn

242. Ceramidase CerA of Aspergillu oryzae promotes degradation of biodegradable plastic containing urethane bonds. Shinsaku Ohtaki¹, Hiroshi Maeda², Toru Takahashi¹, Youhei Yamagata¹, Katsuya Gomi¹, Fumihiko Hasegawa², and Keietsu Abe^{1, 2*}. ¹Grad. Sch. of Agricul. Science, ²New Industry Creation Hatchery Center, Tohoku University, Japan.

When the industrial fungus Aspergillus oryzae is grown in a medium containing a biodegradable plastic polybutylene succinate-co-adipate (PBSA) as a sole carbon source, the fungal cells secret several proteins including cutinase CutL1 as a PBSA degrading enzyme and several biosurfactant proteins that bind to the surface of PBSA and recruit CutL1 to promote PBSA degradation. From the culture broth, we found and purified another novel protein having a molecular mass of 100 kDa. Because the purified protein is predicted to be a neutral ceramidase by using A. oryzae genome information, the gene encoding the 100 kDa protein is designated cerA. We cloned the cerA gene from A. oryzae and constructed a recombinant A. oryzae strain highly expressing cerA. Purified recombinant CerA hydrolyzed C12-NBD-ceramide. Although CerA scarcely cleaved ester bonds between succinate and 1,4-butanediol in PBSA, CerA promoted degradation of PBSA in combination with CutL1. Since PBSA contains urethane bonds in every 200-300 butylene-succinate ester units and the urethane bonds are similar to the amide bonds in ceramides, CerA seems to degrade PBSA endogenously at the urethane bonds and to be applicable degradation of ester polymers containing urethane bonds.

243. Characterization of novel thermostable fungal cellobiohydrolases. Terhi Puranen¹, Sanni Voutilainen², Matti Siika-aho², Jarno Kallio¹, Satu Hooman², Liisa Viikari², Anu Koivula², and Jari Vehmaanperä¹. ¹Roal Oy, Rajamäki, Finland, ²VTT Technical Research Centre of Finland, Espoo, Finland

Cellulose degradation requires sequential or simultaneous synergic action of three types of hydrolytic enzymes: cellobiohydrolases, endoglucanases and beta-glucosidases. Various filamentous fungi produce these enzymes in order to hydrolyze insoluble cellulose into glucose. Cellulases are currently extensively studied in enzyme industry for cellulosic biomass conversion to ethanol. Here, molecular cloning of three different cellobiohydrolase genes from thermophilic ascomycetes is presented together with their heterologous expression in Trichoderma reesei. The recombinant cellobiohydrolases that belong to the glycosyl hydrolase (GH) family 7 were purified and characterized in terms of pH optimum, thermal stability and kinetic parameters. Thermostable cellulases such as described here have been proposed to improve the overall process economy of the biomass conversion with favourable impact on enzyme need, hydrolysis performance and flexibility of the process.

Trichoderma reesei is an industrially significant organism, particularly in the cellulase production industry. The industrial hyper cellulase producing strain RUT-C30 was found to contain a mutation within the coding region of the T. reesei homologue of the carbon catabolite repressor, CreA. The carbon catabolite repression gene (CCR) creB has been previously implicated in the regulation of genes encoding enzymes involved in cellulose utilisation. In Aspergillus nidulans, disruption of the creB gene results in deregulation of cellulase encoding genes normally subject to strong CCR, without the severe reduction in mycelial growth that is present in creA disruptions. The T. reesei homologue of creB is an ideal candidate for disruption for the development of a commercially exploitable strain, since no extreme effects on morphology are predicted. A homologue, labelled cre2, was identified within the T. reesei genome. In order to test for complementation of an A. nidulans creB mutant strain, the T. reesei cre2 gene was amplified and used to transform an A. nidulans strain containing a creB null mutation. The T. reesei cre2 gene complemented the effect of the creB mutation on utilisation of quinate and proline, and sensitivity to allyl alcohol in the presence of glucose. A disruption of cre2 was generated through the introduction of a selectable marker within the coding region. The disrupted strain was found to deregulate genes normally subject to CCR.

245. Withdrawn

246. Optimization of cellulase expression in Trichoderma reesei for the production of cellulose ethanol. Rebecca D. Taylor¹ (taylorr@agr.gc.ca), Anne Johnston¹, Daneille Schneiderman¹, Jiro Hattori¹, Nick Tinker¹, Linda Harris¹. ¹ Agriculture and Agri-food Canada, Eastern Cereal and Oilseeds Research Center, Ottawa, Ontario Canada K1A 0C6. Alex Sobko², Wei Ling Tan², Jason Edwards², Chris Hindle², Theresa White². ² IOGEN Corporation, 310 Hunt Club Road East, Ottawa, Ontario, Canada K1V 1C1.

The fungus Trichoderma reesei is used by IOGEN Corporation to produce cellulase enzymes for the production of cellulose ethanol from farm silage (wheat straw, corn stalks) and other biomass. The goal of this project was to analyze gene expression changes in T. reesei upon overexpression of cellulase proteins. To this end, we acquired 9298 high quality expressed sequence tags (ESTs) from 10 cDNA libraries constructed from fungal cultures grown under varied conditions. We also directly PCR amplified additional genes of interest not present in our EST population. In total, we have 2979 unique sequences including 899 contig and 2080 singleton sequences representing approximately one third of predicted T. reesei genes. cDNA microarrays were constructed using these unique sequences. Biological samples for array hybridizations were harvested from 14L T. reesei batch fermentation cultures in the presence of soluble cellulase inducer. The results from these experiments will be discussed.

247. Engineering of a novel protein secretion pathway in Aspergillus niger. Robbert Damveld¹, Miranda Hartog¹, Peter ten Haaft¹, Inge Minneboo¹, Cees Sagt¹, Han de Winde¹, Thibaut Wenzel¹. ¹DSM Food Specialties, P.O. Box 1, 2600 MA Delft, The Netherlands.

Production of enzymes on an industrial scale is often limited to extracellularly enzymes. When enzymes are produced intracellular, the downstream processing is costly and process robustness is not guaranteed. We have developed a technology which enables the secretion of enzymes which are normally localized in the cell. The folding conditions of intracellular proteins differ from those of secreted proteins. In the cytosol proteins fold under relative reducing conditions compared to the oxidizing environment in the ER. Moreover, many secreted proteins undergo extensive N- glycosylation and disulphide bridge formation. The set of folding enzymes and chaperones in the secretory pathway and in the cytosol are different as well. Therefore the simple solution of forcing intracellular proteins through the secretory pathway does in many cases fail to result in biologically active secreted enzymes. We have developed a method to secrete intracellular enzymes in an active form. This technology allows the intracellular enzymes to be folded in their native environment in the cytosol using cytosolic folding enzymes and chaperones. After folding the intracellular proteins are translocated to a modified cell compartment. After translocation in this modified cell compartment the content is released into the medium by a specific process. Using controlled fermentations we have established that the physiology of cells containing this novel secretory system is not dramatically different form non- modified cells. We will show the recently obtained proof of principle of this concept with the Green Fluorescent Protein (GFP). In addition we have demonstrated the concept for intracellular enzymes, which are secreted in an active form using this novel approach. Moreover we also have preliminary evidence that this technology can be used to secrete metabolites, which are normally localized intracellular. This is the first report that describes the introduction of a completely novel secretory pathway in eukaryotic cells, which allows the production of intracellular enzymes in a secreted active form enabling industrial application on an economically feasible scale.

248. A transiently disrupted non-homologous end joining pathway in Aspergillus nidulans allows simple and efficient gene targeting. Jakob Blæsbjerg Nielsen, Michael Lynge Nielsen, and Uffe Hasbro Mortensen. Center for Microbial Biotechnology, BioCentrum-DTU, Technical University of Denmark, Denmark jbn@biocentrum.dtu.dk

Gene targeting was developed more than a decade ago for high-throughput gene deletions in the unicellular Saccharomyces cerevisiae owing to the fact that DNA double strand breaks are effectively repaired by the homologous recombination (HR) pathway. In multicellular eukaryotes, from filamentous fungi to man, DNA double strand breaks are preferentially repaired by the non-homologous end joining (NHEJ) repair pathway. When performing gene targeting in such multicellular organisms, the influence of NHEJ compromises the efficiency by causing random integration of the gene targeting DNA. Elimination of NHEJ by deleting one of the main genes in the pathway, e.g. ku70, ku80 or lig4 has been done in several fungal species including Aspergillus fumigatus, A. nidulans, Sordaria macrospora and Neurospora crassa. In such strains, the gene targeting efficiency is often as high as 100%. The phenotype of NHEJ strains includes altered colony morphology and an increase in sensitivity to various genotoxins. This raises the concern that phenotype of NHEJ deficient strains may produce synthetic effects with other mutations introduced in this background. This may hamper direct interpretation of the phenotype of this new mutation. In line with this, authors often recommend as a safety measure to reconstitute the NHEJ pathway, e.g. by retransforming with the functional gene or sexual crossing with wild-type strains, before analyzing the effects of novel mutations. In both cases, strain reconstruction constitutes a bottle neck in large scale gene-targeting experiments. To bypass these problems, we have developed an effective alternative in A. nidulans. The system employs a strain with a transiently disrupted ku70 homolog as the starting point for efficient gene targeting. The ku70 mutation in this strain can via a simple selection scheme be reverted to wild type after the desired genetic manipulation(s) have been carried out. The system can easily be adapted to other filamentous fungi.

249. Applied genome-scale modelling of Aspergillus niger. Mikael Rørdam Andersen, Michael Lynge Nielsen, Jens Nielsen. Center for Microbial Biotechnology, Build. 223, Technical University of Denmark, DK-2800 Lyngby, Denmark, jn@biocentrum.dtu.dk

The filamentous fungus Aspergillus niger has through the years fascinated academic and industrial researchers alike due to its innate ability to produce a large number of enzymes and high concentrations of acids. However, the regulatory mechanisms governing these processes are not thoroughly understood. We are combining genome-scale stoichiometric modeling, transcriptomics and novel tools for graphical analysis to elucidate these mechanisms. A genome-scale model was constructed based on literature of A. niger and related aspergilli. Completion of pathways was performed using pathway databases. Genomic information was added based on the sequencing and annotation provided by DSM Food Specialities (Pel et al, manuscript accepted). The model comprises 1230 biochemically unique reactions (isoenzymes not included). 850 are supported by literature and 873 are backed by the genomic evidence of 1024 open reading frames. The remaining reactions are transport reactions and other reactions added for connectivity. Using a digital pathway map of the model, the computed metabolic fluxes were analysed for a large number of substrates. The results are in accordance with the available literature. Using a fast PCR-based cloning-free approach (Nielsen et al, 2006) adapted for A. niger, deletion mutants of carbon metabolism regulators of A. niger have been constructed. These are to be analysed using steady-state cultures and DNA arrays for A. niger.

250. Withdrawn

251. Coprinopsis cinerea as a host for recombinant laccase production. Sreedhar Kilaru^1,2, Martin Rühl¹, Patrik J. Hoegger¹, Aletta Grimrath¹, Karin Lange¹, Mojtaba Zomorrodi¹, Andzrej Majcherczyk¹, and Ursula Kües¹. ¹Institute of Forest Botany, Georg-August-University, Göttingen, Germany ² School of Biological Sciences, University of Bristol, Bristol, UK

Laccases (EC 1.10.3.2) from basidiomycetes are used for various industrial applications, e.g. in paper pulp bleaching, bioremediation, textile dye decolourization, and wood composite production. For overproduction of laccases, we developed a recombinant expression system using the basidiomycete Coprinopsis cinerea as a host. Various homologous and heterologous laccase genes have by now been expressed in C. cinerea under control of the Agaricus bisporus gpdII promoter. Yields of enzyme activities differ from gene to gene. Alteration of growth conditions (media, temperature, aeration) improved yields with 20fold increases in the best cases. At our current best cultivation conditions and our best gene, laccase activities of up to 30 U/ml are obtained. Individual enzymes are characterized in their properties and found to differ from each other, making them attractive for different biotechnological applications. Our laboratory is financially supported by the Deutsche Bundesstiftung Umwelt (DBU).

252. Multivalent Influenza antigens produced in N. crassa, using a novel application of heterokaryon fusion technology. Edward B. Cambareri, Yuliya Brudnaya and W. Dorsey Stuart Neugenesis Corporation, 849 Mitten Rd, Burlingame, CA 94010

Neugenesis Corporation has adapted patented technologies, developed to produce multimeric subunit proteins such as human IgGs, for use in the production of multivalent vaccines. Individual glycoprotein antigens or antigen variants can be expressed in different strains of the fungus, and then fused to create a heterokaryon strain that can produce either a mixture of secreted soluble antigens or high molecular mass particles that display the antigens. New production strains can be generated in as little as six weeks, and different antigens can be mixed and matched using the inherent combinatorial properties of fungal strain fusion. Commercial levels of tailored antigen mixtures can be rapidly isolated from simple, defined growth medium. Data will be presented demonstrating production from heterokaryons of a pandemic influenza vaccine candidate; a virus-like particle (VLP) displaying active H5 Hemagglutinin and N1 Neuraminidase glycoproteins.

253. Isolation of isoosmotic up-regulated genes in Aspergillus oryzae and use of its promoters for protein expression system. Ken Oda¹, Kazutoshi Sakamoto², Toshihide Arima³, Yuka Okita², Dararat Kakizono², Osamu Yamada², Shinichi Ohashi¹, Osamu Akita⁵, Kazuhiro Iwashita^2,4. Kanazawa Institute of Technology¹,3-1 Yatsukaho, Hakusan, Ishikawa, Japan tel:D+81(76)274-7500, fax+81(76)274-7511, e-mail:Fodaken@neptune.kanazawa-it.ac.jp National Research Institute of Brewing², Prefecture Univ. of Hiroshima³, Hiroshima Univ.⁴, Jissen Women's Univ.⁵, Japan

The mechanism of response to osmotic pressure exists in eukaryotes. In A. orzyae, response to osmotic pressure is significant factor for environment recognition in koji-making (solid-state culture), and in fact it was suggested that osmotic pressure regulation component, such as AtfA, or HogA, is important. We analyzed the genes which respond to isoosmotic pressure (0.8M NaCl and 1.2M sorbitol) by microarray analysis. In isoosmotic condition 96 genes in 3000 spots array were 2-fold up- regulated from 30 to 90 min after induction. Some genes that are needed to respond to osmotic stress, such as atfA, , gpd, , and srk1, were isolated. 19 genes were over 5-fold up-regulated, and named as Isoosmotic Up-regulated Genes (IUG). 5'-upstream region of 10 genes in IUG were cloned and applied to GUS reporter assay to evaluate induction ability in isoosmotic condition. The promoters of two genes (IUG2 and IUG9) were strictly regulated. To construct protein expression system the promoter region of high expression vector pNGA142 was substituted with IUG2 and IUG9 promoters. EGFP as a model of heterologous protein was expressed after 2hr only in induction conditions, suggesting that response speed of this system is fast and control of expression is strict. We constructed new protein expression system which protein expression was induced by osmotic pressure and their level of expression are controlled by NaCl concentration.

254. Chemostat cultivation of Aspergillus niger for C-source mediated induction of the unfolded protein response and transcriptome analysis. Theo Goosen^1,2, Thomas Jørgensen^1,3, Jens Iversen^1,3, Arthur Ram¹ and Kees van den Hondel¹. ¹Leiden University, Institute of Biology Leiden, Fungal Genetics Research Group, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands, ²BioCentre, HAN University, Laan van Scheut 2, 6525 EM Nijmegen, The Netherlands, ³Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, 5230 Odense, Denmark.

Folding of nascent proteins in the endoplasmic reticulum (ER) has been identified as a major bottleneck in production of heterologous proteins. Accumulation of unfolded proteins in the ER elicits a stress response called the unfolded protein response (UPR). We have used C-limited chemostat cultures of Aspergillus niger to investigate UPR resulting from expression of a gene encoding a mammalian antibody fragment (scFv). Chemostat cultivation provides well-defined growth conditions and reduces biological sample variation among replicates, which make it an excellent method for both physiological and molecular studies, like transcriptomics. Two strains were cultivated for comparison. Strain ABGT1026 expresses lama scFv under transcriptional control of the glaA promoter from a single targeted gene. Strain AB94-85 is an isogenic control strain lacking the scFv gene-fusion. Both strains were cultured in chemostat cultures, at a dilution rate of 0.16 h-1, initially with xylose-limitation. After five retention times the medium was changed to maltose-limitation (inducing conditions for the glaA promoter). Cultures were performed in triplicate. There was no apparent physiological difference between the two strains. Culture profiles and yield coefficients of all six cultures were almost identical. Mycelium obtained from fifth retention time on xylose and maltose was subjected to expression analysis using Affymetrix arrays with probe sets based on the A. niger genome. Among 14554 probe sets, glaA had highest expression-level on maltose. It was also expressed on xylose; but at a level four times lower than on maltose. ANOVA of triplicate expression results of four conditions (two strains and two C-sources) revealed that transcription of around 290 genes were >2-fold up- or down-regulated (p 4-fold). A single gene was identified as differentially expressed, when comparing the two strains. Interaction analysis showed this was independent of C-source. Expression of scFv did not result in increased UPR at steady state on maltose, however expression of some genes associated with UPR and protein secretion was enhanced (1.6-2-fold) on maltose compared to xylose. The investigation has yielded one new lead regarding the low yield production of heterologous proteins, and a number of clues on the differences in expression of genes involved metabolism of xylose and maltose.

Biochemistry and Secondary Metabolism

255. Chemogenomics of antioxidant inhibition of aflatoxin biosynthesis. Jong Kim¹, Jiujiang Yu^2,3, Noreen Mahoney¹, Kathleen Chan¹, Deepak Bhatnagar², Thomas Cleveland², Russell Molyneux¹, William Nierman^3,4 and Bruce Campbell^1*. ¹Plant Mycotoxin Research, USDA, Albany, CA 94710 USA. bcc@pw.usda.gov. ²Food and Feed Safety Research, USDA, New Orleans, LA 70124 USA. ³The Institute of Genomic Research, Rockville, MD 20850 USA. ⁴The George Washington University, Washington, DC 20006 USA.

Natural compounds were used to probe the functional genomics (chemogenomics) of aflatoxin biosynthesis. Caffeic acid (12mM) treatment of Aspergillus flavus inhibited aflatoxin biosynthesis without affecting growth. Microarray analysis showed genes in the aflatoxin biosynthetic cluster were completely down-regulated (log2 ratio –0.04 to –3.13). However, aflatoxin pathway regulator genes, aflJ or laeA, and sugar utilization cluster genes showed only minor changes in expression (log2 ratio 0.08 to –0.58). Genes in amino acid biosynthetic and aromatic compound metabolism were up-regulated (log2 ratio > 1.5). Most notable was up-regulation (log2 ratio 1.08 to 2.65, qRT-PCR) of four peroxiredoxin genes orthologous to the AHP1 gene (alkyl hydroperoxide reductase) of Saccharomyces cerevisiae. Antioxidants trigger induction of ahp genes in A. flavus to protect the fungus from oxidizing agents, e.g., lipoperoxides, reactive oxygen species, etc. This detoxification attenuates upstream oxidative stress-response pathway signals, and suppresses aflatoxigenesis.

256. BDM-1, a phosducin-like protein required for G-protein signaling in Cryphonectria parasitica, is a phosphoprotein targeted by casein kinase II. Joanna Salamon-Kozubowska¹ and Angus L. Dawe ^1,2. ¹Department of Biology and ²Molecular Biology Program, New Mexico State University, Las Cruces, NM 88003

G-protein signaling modulates many responses in fungi, including virulence of plant and animal pathogens. Previous studies with the plant pathogen C. parasitica have identified components that include a phosducin-like protein (BDM-1). Perturbation of the G-protein signaling pathways has been shown to affect virulence, pigmentation and sporulation. Deletion of the beta subunit CPGB-1 or BDM-1 affected these characteristics in an identical manner and caused similar alterations in transcriptional profile. G-proteins levels were reduced in accumulation following infection with virulence-attenuating hypoviruses. In this study we have shown by western blotting that CPGB-1 protein levels are undetectable in the absence of BDM-1 and by phosphatase treatment that BDM-1 exists in a phosphorylated form. Re- phosphylation of BDM-1 was accomplished in the presence of total protein lysates, but this activity was inhibited by a casein kinase II inhibitor, indicating that BDM-1 is a substrate for this kinase. With these tools it is now possible to explore the effects of phosphorylation on BDM-1 function and determine if this modification is altered by the presence of hypoviruses, thereby providing a model for exploring the molecular events associated with viral-mediated changes of cellular signaling pathways.

257. Functional characterization of two cytochrome P450 monooxygenases, PaxP and PaxQ, involved in the biosynthesis of the indole-diterpene, paxilline. Sanjay Saikia¹, Emily Parker^2,3 and Barry Scott¹. ¹Institute of Molecular Biosciences and ²Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand. ³Current address: Department of Chemistry, Canterbury University, Christchurch, New Zealand.

Indole-diterpenes are a large, structurally and functionally diverse group of secondary metabolites produced by filamentous fungi. Biosynthetic schemes have been proposed for these metabolites but until recently none of the proposed steps had been validated by biochemical or genetic studies. Using Penicillium paxilli as a model experimental system to study indole-diterpene biosynthesis we previously showed by deletion analysis that a cluster of seven genes is required for paxilline biosynthesis. Two of these pax genes, paxP and paxQ (cytochrome P450 monooxygenases), are required in the later steps in this pathway. Here we describe the function of paxP and paxQ gene products by feeding proposed paxilline intermediates to strains lacking the pax cluster but containing ectopically integrated copies of paxP or paxQ. Transformants containing paxP converted paspaline into 13-desoxypaxilline as the major product and beta-PC-M6 as the minor product. beta-PC-M6, but not alpha-PC-M6, was also a substrate for PaxP and was converted to 13-desoxypaxilline. paxQ-containing transformants converted 13-desoxypaxilline into paxilline. These results confirm that paspaline, beta-PC-M6 and 13- desoxypaxilline are paxilline intermediates and that paspaline and beta-PC-M6 are substrates for PaxP, and 13- desoxypaxilline a substrate for PaxQ. PaxP and PaxQ also utilized beta-paxitriol and alpha-PC-M6 as substrates converting them to paxilline and alpha-paxitriol, respectively.

258. LaeA, a methyltransferase regulating secondary metabolite production in Aspergillus spp. Perrin, Robyn M; Bok, Jin Woo; Keller, Nancy P. 1630 Linden Dr., University of Wisconsin, Madison, WI USA 53706

Filamentous fungi are unique organisms – rivaled only by Actinomycetes and plants - in producing a wide range of secondary metabolites. These compounds often have obscure or unknown functions in the producing organism but have tremendous importance to humankind, displaying a broad range of useful pharmaceutical activities as well as less desirable phyto- and mycotoxic activities. The biosynthetic genes of most fungal secondary metabolites are clustered in genetic loci. LaeA is a nuclear methyltransferase that transcriptionally regulates secondary metabolite gene clusters in the filamentous fungal genus Aspergillus. Deletion and over- expression of laeA either silences or increases, respectively, the production of multiple secondary metabolites and expression of genes for their biosynthesis. Mutation of a canonical amino acid motif found in all methyltransferases results in a loss of function phenotype, indicating that methytransferase activity is critical for LaeA regulatory activity (Bok et al 2006 Mol Microbiol 61:1636). In order to understand the mechanism of LaeA function, we are using an A. nidulans strain expressing epitope-tagged LaeA and anti-LaeA antibodies to identify LaeA methylation targets and interacting proteins. Further analysis of biochemical function is being conducted using a series of mutated and truncated versions of recombinant LaeA. Elucidation of LaeA biochemical function is critical to understanding its role as a global regulator of secondary metabolism.

259. Which “alphabet” do zygomycetes use for their chemical language? Mareike Richter, Doreen Schachtschabel, Ute Neugebauer and Wilhelm Boland. Max Planck Institute for Chemical Ecology, Jena, Germany, mrichter@ice.mpg.de

During sexual interaction, zygomycete fungi communicate via carotene-derived compounds, namely trisporic acid and its precursors. The first sexually determined structures of Mucor mucedo are specialised aerial hyphae, called zygophores. Proteins are distributed uniformly in these hyphae. Carotenoids, in contrast, accumulate in the hyphal tip, shown by 2D Raman spectroscopy. The same technique also depicts the possible presence of trisporoids in the zygophore tips. The formation of zygophores is an important parameter to assess the bioactivity of trisporoids. Various synthetic trisporoid analogues exhibit a differential influence on zygophore formation in Mucor mucedo. Moreover, trisporoid production and carotene biosynthesis are linked via feedback regulation. The early precursor beta- carotene can be readily quantified by HPLC. After culture stimulation the correlation of morphological responses with beta-carotene concentration is allowed. The mechanistic basis of the induced regulation mechanisms remains to be established. The impact of trisporoids on gene transcription necessary for carotene synthesis and degradation is studied by real-time PCR. The early carotene biosynthesis is not under the control of trisporoids. But later enzymatic transformations, e.g. by phytoene desaturase and carotene dioxygenase respond to stimulation with synthetic trisporoids. In the future we want to investigate the consequences of different stimulating trisporoid analogues.

260. Agrobacterium tumefaciens-mediated disruption of a polyketide synthase gene in Cryphonectria parasitica. Keiichi Sudo¹, Heather L. McLane², Stuart B. Krasnoff⁴, Sandra L. Anagnostakis³, Donna M. Gibson⁴, and Alice C.L. Churchill¹(acc7@cornell.edu). ¹Department of Plant Pathology, Cornell University, Ithaca, NY, ²Boyce Thompson Institute, Ithaca, NY, ³Connecticut Agricultural Experiment Station, New Haven, CT, ⁴USDA-ARS, Plant Protection Research Unit, Ithaca, NY.

Cryphonectria parasitica is the causal agent of chestnut blight, which decimated American chestnut populations during the first half of the 20th century. Infection of the fungus with naturally occurring Hypovirus can reduce virulence, sporulation, and the production of fungal pigments, as well as perturbing signal transduction pathways. We cloned fragments of eight C. parasitica genes predicted to be involved in the synthesis of small molecule natural products. Most are polyketide synthase (PKS) genes, which encode key enzymes for the synthesis of linear and aromatic polyketides. We identified and sequenced a ~6.0 kb gene, PKS1, which is a member of a predicted secondary metabolite gene cluster containing at least 13 other putative regulatory, modifying and efflux genes within a ~37 kb genomic region. The PKS1 predicted protein is highly similar to several other uncharacterized fungal PKSs (E value of 0.0; greater than 60% identity). We disrupted PKS1 in C. parasitica by Agrobacterium tumefaciens-mediated transformation and have begun characterization of the targeted gene knockout (KO) strains. There are no significant differences between the KO and ectopic control strains in colony growth rate on an agar medium or in Golden Delicious apple fruits, which are used as an alternate virulence assay. Virulence and mating assays on dormant chestnut stems are in progress, as are efforts to identify potential differences in development or nutritional requirements.

The polyketide virulence determinant, T-toxin, is produced by race T of the filamentous ascomycete Cochliobolus heterostrophus, the cause of Southern Corn Leaf Blight. The genetics, biosynthesis, and evolution of the ability to produce T-toxin differ significantly from those of other polyketides (PK). Unlike genes required for biosynthesis of the PKs, lovastatin and aflatoxin, for example, T-toxin genes are not clustered but are distributed at two loci encompassing ca. 1.2 Mb of AT rich, highly repetitive DNA present in race T but absent in non-T-toxin producing C. heterostrophus race O. Prior to this study, three genes were known to be required for T-toxin synthesis: two polyketide synthases (PKS) and the decarboxylase DEC1. We have identified six additional genes, all unique to race T. These include LAM1, OXI1 and HYD1, encoding proteins similar to a 3- hydroxyacyl-CoA dehydrogenase, an oxidoreductase, and a serine hydrolase, respectively, and three genes encoding putative reductases, RED1, RED2, and RED3. Regarding function, some of the PKS reducing domains are degenerate and we speculate that enzymes encoded by the new genes assume these roles. Regarding gene distribution, T-toxin genes are not present in race O, in other Cochliobolus species, or in neighboring genera. An ortholog of one of the PKSs genes, however, is found in the corn pathogen, Didymella zeae-maydis and in the distantly related dung fungus, Delitschia winteri. This discontinuous distribution of the T-toxin genes, together with their physical location within AT rich, repetitive DNA at chromosomal breakpoints, suggests a complex evolutionary history.

262. Identification of a compound that stimulates perithecial formation in Giberella zeae. Seunghoon Lee¹, Jianming Jin¹, Sung-Hwan Yun², and Yin-Won Lee¹. ¹School of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Korea. ²Dpartment of Biological Resources and Technology, Soonchunhyang University, Asan 336-745, Korea

Giberella zeae, a homothallic ascomycete, develops a fruiting body called perithecium during sexual stage. Many studies suggested that fungal sexual development is induced by specific changes in both gene expressions and metabolism. G. zeae is well known as a producer of many secondary metabolites such as mycotoxins and pigments. Recently, several genes required for production of metabolites such as polyketides and non-ribosomal peptides have been reported in this fungus; some are specifically related to perithecial development. To investigate the enhancement of sexual development by specific fungal metabolites, we searched and identify such compound from carrot agar culture of G. zeae. Exogenous supply of the purified compound to fungal culture caused increase in both the number of perithecia and black pigment production accumulated on the agar surface where peritheica were formed. NMR and MS analyses estimated that it is a long unsaturated compound with a formula C₃₈H₆₆O₅, Mw 604.57. Detailed characterization of the compound is in progress. Further studies will be performed to identify the genes responsible for the biosynthesis of this compound.

263. Identification of AatB, a new component of the penicillin biosynthesis pathway of Aspergillus nidulans. Petra Sproete ¹, Michael J. Hynes ², and Axel A. Brakhage ¹. ¹ Leibniz Institute for Natural Product Research and Infection Biology (HKI) / Friedrich-Schiller- University Jena, Germany ² Department of Genetics, University of Melbourne, Australia

The acyl coenzyme A:isopenicillin N acyltransferase (IAT) of A. nidulans, which is encoded by the aatA gene, catalyzes the final step of the penicillin biosynthesis, i.e., the exchange of the hydrophilic L-alpha-aminoadipic acid side chain of isopenicillin N for a hydrophobic acyl group. By analyzing a GFP-IAT protein fusion it could be shown that in A. nidulans – as in Penicillium chrysogenum – the IAT and therefore the enzymatic reaction is located in the peroxisomes. Further studies indicated a PTS1 dependent transport of the enzyme since the deletion of the rather untypical putative peroxisomal targeting sequence 1 (PTS1) Ala-Asn-Ile at the C terminus of the protein led to cytoplasmic localization of the IAT. Nevertheless, unlike the IAT of P. chrysogenum, such a mislocated enzyme seems to be functional because both, an A. nidulans strain lacking the PTS1 transporter and a strain possessing a mislocated IAT still produced about 50% and 80% of penicillin, respectively, compared to wild-type levels. Because an aatA disruption strain still was able to produce small amounts of an inhibitory substance, the A. nidulans database was searched for a putative redundant protein. A gene displaying a very similar exon distribution and a 58% similarity with the aatA gene but lacking the PTS1 encoding sequence was named aatB. First analyses of an aatB disruption strain indicated a participitation of AatB in penicillin biosynthesis of A. nidulans.

264. Identification of all chitin synthase genes in Rhizopus oryzae genome. Ayumi Abe and Teruo Sone. Research Faculty of Agriculture, Hokkaido University, Sapporo, JAPAN

Chitin and chitosan are main components of cell wall of zygomycetes. Chitin synthases in zygomycetes are thought to be the most important factor for the cell morphogenesis, but they have not been studied well. We aimed to identify all chitin syanthase genes in R. oryzae genome sequence data and to collect the information about the expression of those genes under the various culture conditions. We identified 26 chitin synthase genes from R. oryzae genome sequence, using conserved amino acid sequences by BLASTP analysis.

Phylogentic analysis resulted in the clustering of chitin synthases in zygomycetes independent from those of ascomycetes or basidiomycetes on which current classification is based. This indicates that a new classification of chitin synthases including the data of zygomycetes'

enzyme is appropriate. Notably, two genes in R. oryzae made a distinct clade, thus a new class of the enzyme, class VIII was proposed. Almost all of R. oryzae chitin synthase genes were expressed during at least one culture condition. Seven genes were specifically expressed during later period in solid culture, indicating the relationship to sporangiospore formation. These results may reflect the fact that zygomycetes' cell wall consists mainly of chitin and chitosan, and thus they conserved many isozymes of chitin synthases with different roles, in the genome.

The enzyme cyanase has been found in several organisms such as plants, bacteria and archae. However, so far this enzyme that catalyzes the reaction of cyanate with bicarbonate to produce ammonia and carbon dioxide has not been characterized in fungi. In nature the substrate cyanate is formed from the spontaneous dissociation of urea in aqueous solution. Structural analysis of the Escherichia coli cyanase revealed that the enzyme is a homodecamer of 17 kDa subunits. Although cyanate at high concentrations can be fairly toxic to E. coli, cyanate can serve as the sole source of nitrogen for growth of E. coli as a result of cyanase-catalyzed decomposition to ammonia. In this study, we investigated the role of the cyanase-like gene cyn1 from the filamentous ascomycete Sordaria macrospora. BLAST searches of a S. macrospora genomic sequence revealed a high degree of homology to cyanase encoding genes from various bacteria. The S. macrospora cyn1 gene encodes a putative protein of 164 amino acids with a predicted molecular mass of 18.7 kD. Homologues of the S. macrospora cyn1 gene were identified within the genomes of filamentous ascomycetes and basidiomycetes but not in the genomes of the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. To better understand the function of the S. macrospora cyanase, we generated a deltacyn1 gene disruption mutant. Analysis of the mutant strain, cellular localization of the cyanase and enzyme activity will be presented.

266. An efficient gene targeting system to generate knockout mutants in the penicillin producer Penicillium chrysogenum. Birgit Hoff and Ulrich Kück. Department of General and Molecular Botany, Ruhr-University Bochum, Universitaetsstr. 150, 44801 Bochum, Germany, birgit.hoff@rub.de

Penicillium chrysogenum is the main producer of the pharmaceutical relevant beta-lactam antibiotic penicillin. DNA-mediated transformation of this filamentous fungus results mostly in ectopic integrations of the molecules. For a functional analysis, it is however desirable to obtain gene substitutions by homologous recombination, a process which occurs with a rather low frequency in this fungus. A cellular feature that decreases homologous recombination is the non- homologous end-joining (NHEJ) pathway, a mechanism that involves the binding of the highly conserved Ku heterodimer at the ends of a DNA double-strand break. Integration events mediated by NHEJ do not rely on homologous sites and therefore attenuate homologous recombination, which results in decreased frequencies of a gene knockout in a given transformation experiment. To improve gene targeting efficiency in P. chrysogenum, we successfully deleted the P. chrysogenum Pcku70 gene. No impairment in vegetative growth, sporulation and penicillin production could be assessed for the corresponding mutant strain but relative frequencies of homologous recombination were drastically increased to about 50- 80 % as deduced from targeting several different genes. To test the applicability of the Pcku70 knockout strain, genes for global regulators were substituted by the phleomycin resistance gene. From the sum of our investigations, we can conclude that the Pcku70 knockout strain is an ideal recipient for targeting of genes involved in regulation of secondary metabolism and fungal morphogenesis, and can therefore be useful in strain improvement programs.

267. Regulation of genes coding for synthesis of beta-lactam antibiotics in Acremonium chrysogenum and Penicillium chrysogenum. Birgit Hoff, Jacqueline Dreyer, and Ulrich Kück. Department of General and Molecular Botany, Ruhr-University Bochum, Universitaetsstr. 150, 44801 Bochum, Germany, birgit.hoff@rub.de

The filamentous fungi Acremonium chrysogenum and Penicillium chrysogenum are the main producer of the pharmaceutical relevant beta-lactam antibiotics cephalosporin C and penicillin. Expression of genes coding for the biosynthesis enzymes is known to be controlled by a complex network of global regulators such as CRE1, PACC, and CPCR1 etc. Using gene disruption approaches, we have generated knockout strains of both fungi to study the homologue of the Aspergillus nidulans velvet gene (veA). VeA co-ordinates asexual and sexual development in this homothallic fungus and was also found to control secondary metabolism. Expression and HPLC analyses have clearly indicated that the veA homologue acts as a regulator of beta-lactam biosynthesis in both fungi. In addition, detailed microscopic analyses have shown that deletion of veA caused alterations in hyphal morphology and fragmentation. From the sum of our investigations, we can conclude that the veA homologue in the two fungi controls both antibiotic biosynthesis and fungal morphogenesis.

268. Use of the DsRed reporter gene to establish an RNAi system for the beta-lactam-antibiotic producer Acremonium chrysogenum. Danielle Janus, Birgit Hoff, and Ulrich Kück. Department of General and Molecular Botany, Ruhr-University Bochum, Universitaetsstr. 150, 44801 Bochum, Germany, danielle.janus@rub.de

In filamentous fungi, RNA-silencing is an attractive alternative to disruption experiments for the functional analysis of genes. We have adapted the gene encoding the autofluorescent DsRed protein as a reporter to monitor the silencing process in fungal transformants. Using the cephalosporin C producer Acremonium chrysogenum, strains showing a high level expression of the DsRed gene were constructed, resulting in red-colored fungal colonies. Transfer of a hairpin-expressing vector carrying fragments of the DsRed gene allowed efficient silencing of DsRed expression. Monitoring of this process by northern hybridizations, real-time PCR, and spectrofluorometric measurements of the DsRed protein confirmed that downregulation of gene expression can be observed at different expression levels. The usefulness of this system was demonstrated by investigating co-silencing of DsRed together with pcbC, encoding the isopenicillin N synthase (IPNS), an enzyme involved in cephalosporin C biosynthesis. Downregulation of pcbC can easily be detected by a bioassay, measuring the antibiotic activity of individual strains. In addition, the presence of IPNS was investigated by western blot hybridization. All transformants having a colorless phenotype show a simultaneous downregulation of the pcbC gene. The RNA-silencing system presented here will be a powerful tool for strain improvement and genome-wide analysis of this biotechnically important filamentous fungus.

We have identified a cluster of eight genes (gene loci fg08077 – fg08084) that is concomitantly up-regulated (Northern and qPCR analysis) under growth conditions that promote mycotoxin production. Proteomics experiments (iTRAQ analysis) have confirmed the up-regulation of proteins encoded by two of these genes under toxin induction conditions. Gene disruption and add-back experiments followed by metabolite analysis of the transformants indicated that one of the genes, fg08079, is directly involved in the synthesis of butenolide, a mycotoxin derived from glutamic acid. As expression of these genes can be detected very early during plant infection, butenolide may play a role in plant infection. However, greenhouse testing for FHB (Fusarium Head Blight) using disruption mutants of fg08079 showed that this gene did not contribute significantly to virulence in wheat heads. The mycotoxin butenolide is produced by several Fusarium species and has been suggested to be associated with tall fescue toxicoses in grazing cattle. We are exploring interspecies conservation of this gene cluster by PCR screening and sequencing in other Fusarium genomes.

270. Identification of a biosynthetic gene cluster for a secreted glycolipid with antifungal activity in Ustilago maydis. Beate Teichmann, Lidan Liu and Michael Bölker. Department of Biology, University of Marburg, Karl-von-Frisch-Str. 8, D-35032 Marburg, Germany. E-mail: teichma3@staff.uni-marburg.de

Under conditions of nitrogen starvation, U. maydis secretes large amounts of the cellobiose lipid ustilagic acid. This amphiphatic glycolipid acts as biosurfactant and displays antibacterial and antifungal activity. Ustilagic acid consists of a cellobiose moiety O-glycosidically linked to the terminal hydroxyl group of di- or trihydroxypalmitic acid, respectively. We have identified a large gene cluster comprising 12 open reading frames that is responsible for UA biosynthesis. By mutational analysis and mass spectrometry we could characterize several enzymes that are involved in hydroxylation, glycosylation and secretion of UA. This allows us to propose a biosynthetic pathway for ustilagic acid. The cluster contains a putative Zn-finger DNA-binding protein, which is involved in regulation of cluster genes. We could show that expression of this transcriptional factor is necessary for UA production. Further we could demonstrate, that U. maydis has biocontrol activities against the plant pathogenic fungus Botrytis cinerea, which causes gray mold on tomato leaves. Coinoculation of haploid U. maydis cells with B. cinerea spores suppress all symptoms of disease. UA production is critical for the antagonistic activity of U. maydis cells against B. cinerea spores, since mutants defective for UA biosynthesis are unable to interfere with B. cinerea infection.

271. Secondary metabolites: fundamental and niche-specific roles in fungal development and cell-cell interactions. B. Gillian Turgeon and Shinichi Oide. Cornell University, New York

An exhaustive characterization of non-ribosomal peptide synthetase (NPS) genes of the corn pathogen, Cochliobolus heterostrophus, and the small peptides produced by the enzymes they encode, has been undertaken to ascertain what peptide secondary metabolite products are doing for the fungal cell. To date, the NRPS method of peptide biosynthesis has been described for filamentous ascomycetes (and to a limited extent, for basidiomycetes) and bacteria, only. NRPs have a broad spectrum of biological activities, many are useful in medicine, agriculture, industry, and biological research. Despite the fact that activities of the peptide products with respect to interactions with other organisms are well-documented, to suggest that this is their primary function is likely incorrect. For example, only one of the 12 C. heterostrophus NPSs is required for virulence. In fact, the physiological significance of these small peptides to the producing fungi is largely unknown. We document that NRPS enzymes are purveyors of small molecules for both basal metabolism and for specialized environmental niches. Certain NPSs are required for sexual reproduction, asexual reproduction, virulence, as nutrient ‘gatherers’ (such as iron-gathering siderophores), for responses to oxidative, nitrosative, nutrient, pH stress etc, for growth, and for hydrophobicity. Eight of the 12 C. heterostrophus NPSs are not conserved, while four are conserved in other ascomycetes. We have examined functional conservation of conserved genes in the wheat pathogen, Fusarium graminearum, and the Arabidopsis pathogen, Alternaria brassicicola. Comprehension of secondary metabolite function, from the perspective of the fungal cell itself, impacts our understanding of evolution of fungal pathogenicity mechanisms by addressing how pathogens become pathogens.

272. CoA-Transferase from Aspergillus nidulans is required to forward the CoA-moiety from propionyl-CoA to acetate. Christian Fleck, Matthias Brock. Leibniz Institute for Natural Product Research and Infection Biology e.V. – Hans-Knöll-Institute (HKI) christian.fleck@hki-jena.de

Metabolism of amino acids and propionate leads to the toxic intermediate propionyl-CoA. Efficient removal via the methylcitrate cycle is guaranteed by a functional methylcitrate synthase. Deletion of the coding region of this enzyme leads to the inability to grow on propionate or ethanol/propionate but not acetate/propionate. This led to the assumption that the level of propionyl-CoA may become reduced by direct transfer of the CoA-moiety to acetate. Therefore, we purified a CoA-transferase from Aspergillus nidulans and characterised the enzyme biochemically. The CoA-transferase was specific for the CoA-esters succinyl-CoA, propionyl-CoA, and acetyl-CoA as CoA-donors and the corresponding acids as acceptors. No other donors or acceptors suited as substrates. To elucidate the role of the enzyme under in vivo conditions the corresponding gene was deleted. The deletion mutant only showed mild phenotypes when tested for growth and development on propionate containing media. Therefore, a double mutant with a methylcitrate synthase deletion strain was created by sexual crossing. In contrast to the methylcitrate synthase deletion strain, the double mutant was neither able to grow on ethanol/propionate nor on acetate/propionate media. Thus, CoA-transferase is essential for removal of toxic propionyl-CoA in the presence of acetate as a CoA-acceptor.

273. The aflatrem biosynthetic genes in Aspergillus flavus and A. oryzae are clustered in two separate regions of the genome. Matthew J. Nicholson¹, Brendon J. Monahan¹, Bethan Pritchard², Gary A. Payne², and Barry Scott¹. ¹Massey University, New Zealand. ²North Carolina State University, USA.

Aflatrem is a potent tremorgenic toxin produced by Aspergillus flavus and is a member of a structurally diverse group of fungal secondary metabolites known as indole-diterpenes. Using Penicillium paxilli as a model experimental system for investigating indole-diterpene biosynthesis, we have shown that the products of four genes (paxG, paxM, paxB and paxC) are necessary for the production of paspaline, the first stable indole- diterpene intermediate, and that two P450 monooxygenases (encoded by paxP and paxQ) are required for the subsequent conversion of paspaline to paxilline. We previously identified a cluster of genes in A. flavus NRRL6541 proposed to be involved in aflatrem (atm) biosynthesis, that included homologues of paxG (atmG), paxM (atmM) and paxC (atmC) (Zhang et al., 2004). Here we report the identification of a homologous atm gene cluster in the sequenced genomes of A. flavus NRRL3357 and A. oryzae RIB40, and the identification of a second gene cluster, containing homologues of paxB, paxP and paxQ in these genomes. Comparative analysis of sequences from all three genomes revealed total syntenic conservation and high nucleotide identity both within and between genes. In both species the aflatrem biosynthetic genes are clustered in two discrete genomic regions. In A. oryzae, the first cluster (containing homologues of paxG, paxC, and paxM) is located subtelomerically on chromosome 7, whilst the second cluster (containing homologues of paxB, paxP and paxQ) is located on chromosome 5. Comparison with the paxilline gene cluster of P. paxilli provides important insights into the evolutionary history of these aflatrem gene clusters.

274. Epigenetic regulation of the Aspergillus chemical arsenal. Elliot Schwab and Nancy Keller. University of Wisconsin- Madison, Plant Pathology. Madison, WI.

Bioactive small molecules are critical in Aspergillus species during their development and interaction with other organisms. Genes dedicated to their production are encoded in clusters that can be located throughout the genome. We show that deletion of hdaA, an Aspergillus nidulans histone deacetylase (HDAC), causes transcriptional activation of two subtelomeric gene clusters - and subsequent increased production of the corresponding molecules - but not of a telomere distal cluster. Furthermore, treatment of other fungal genera with HDAC inhibitors results in overproduction of several metabolites. The regulatory role of gene clustering and subtelomeric positioning of clusters will also be examined. Chromatin regulation of small molecule gene clusters may enable filamentous fungi to successfully exploit environmental resources by modifying chemical diversity.

275. Characterization of a putative fusarin mycotoxin biosynthetic gene cluster in Fusarium. Robert H. Proctor, Robert A.E. Butchko and Daren W. Brown. USDA-ARS National Center for Agricultural Utilization Research, Peoria, Illinois.

Fusarium verticillioides is a stalk and ear rot pathogen of maize and can produce the polyketide mycotoxins fumonisins in infected kernels. Although the genetics and biochemistry of fumonisin biosynthesis are relatively well understood in F. verticillioides, little is known about the biosynthesis of other secondary metabolites produced by this fungus and whether the regulatory mechanisms that affect fumonisin biosynthesis also affect biosynthesis of other secondary metabolites. To begin to address such issues, we are using microarray analysis to determine whether 15 previously identified F. verticillioides polyketide synthase (PKS) genes (Kroken et al, 2003 PNAS 100:15670- 15675) are located in clusters of coordinately regulated genes. To date, the analysis indicates that three of the PKS genes are located in clusters. For example, PKS10, which is required for the biosynthesis of fusarin mycotoxins, and eight contiguous genes adjacent to it exhibit similar changes in expression over time in liquid GYAM medium. These same nine genes also exhibit relatively high expression on whole maize kernels and low expression on embryo tissue. The predicted functions of some of these genes are consistent with enzymatic activities predicted to be required for fusarin biosynthesis. Similar arrangements of PKS10 and at least some of the adjacent genes are present in the genomes of F. graminearum and Nectria haematococca. Together, the data suggest that PKS10 and some of the genes next to it constitute a fusarin biosynthetic gene cluster.

276. Characterization of a Fumonisin Biosynthetic Gene Cluster in Fusarium oxysporum strain O-1890. Robert H. Proctor¹, Jeong-Ah Seo², Mark Busman¹, Yin-Won Lee² and Ronald Plattner¹. ¹ USDA-ARS National Center for Agricultural Utilization Research, Peoria, Illinois, USA. ² Seoul National University, Seoul, Korea.

Fumonisins are carcinogenic mycotoxins produced by some Fusarium species. Most species, including F. verticillioides, produce predominantly B fumonisins (FBs), but F. oxysporum strain O-1890 produces predominantly C fumonisins (FCs). FBs have an alanine-derived methyl function that is absent in FCs. In this study, we determined the nucleotide sequence of the fumonisin biosynthetic gene (FUM) cluster in strain O-1890. Genes in the cluster were present in the same order and orientations as in the previously described F. verticillioides cluster. The FUM gene coding and intergenic regions were 89 – 92 % and 40 – 77% identical, respectively, in F. oxysporum and F. verticillioides. In contrast, the sequences flanking the cluster were completely different in the two species. As previously reported, the F. verticillioides FUM cluster gene FUM8 encodes an oxoamine synthase and fum8 mutants cannot produce fumonisins. In the current study, transformation of a fum8 mutant with a wild-type F. verticillioides FUM8 restored FB production while transformation with a F. oxysporum O-1890 FUM8 resulted in FC production. These data indicate that the ability to produce FBs versus FCs is determined by different FUM8 homologues.

277. Functional analysis of the Hypocrea jecorina lxr1 demonstrates that Lxr1 encodes a D-mannitol dehydrogenase and is not involved in L-arabinose catabolism. Benjamin Metz, Stefan Polak, Christian P. Kubicek and Bernhard Seiboth. Research Area Gene Technology and Applied Biochemistry, Institute of Chemical Engineering, TU Wien, Austria.

The pentose L-arabinose is one of the main compounds of plant hemicelluloses and pectins. Most genes of the fungal L- arabinose pathway have been cloned for the industrial important fungi H. jecorina (anamorph: Trichoderma reesei). Cloning of an H. jecorinai L-xylulose reductase gene (lxr1) was reported previously by heterologous expression in Saccharomyces cerevisiae which enabled this yeast to grow on L-arabinose. A functional analysis of lxr1 in our lab has now questioned the role of Lxr1 in L-arabinose catabolism: Growth of a Δlxr1 strain was not affected on L-arabinose and total L-xylulose reductase activity remained unchanged. In addition Northern analysis showed that lxr1 was not L-arabinose inducible. In summary, our results clearly show that Lxr1 cannot be the enzyme which is active in the L-arabinose catabolic pathway. Our data suggest now that Lxr1 is a D-mannitol 2-dehydrogenase which is involved in developmental processes such as sporulation and germination. The polyol D-mannitol is accumumlated intracellularly by many fungal species and during spore germination D-mannitol is metabolized rapidly by D-mannitol 2-dehydrogenase. This is supported by our data that D-mannitol dehydrogenase activity was reduced in Δlxr1 strains and transcription levels were up-regulated during sporulation or germination.

278. Search for genes involved in the neoechinulin A biosynthetic pathway in Aspergillus spp. Masahiro Takeno, Momoko Horiuchi, Mayu Ohishi, Koji Kuramochi, Fumio Sugawara, and Takashi Kamakura. Tokyo University of Science, Noda, Japan.

Neoechinulin A is a member of the family of indole alkaloids comprised of tryptophane and alanine. It was isolated in 1973 from Aspergillus rubber by Yamazaki and co-workers. It was reported that the neoechinulin A was an antioxidant compound which suppresses lipid peroxidation in dried bonito flakes, Katsuobushi, and results of many studies suggested that Neoechinulin A might be useful as a protectant against neuronal cell death in neurodegenerative diseases. Although the chemical synthesis method of neoechinulin A is already established, the biosynthetic pathway and genes involved in the biosynthesis of neoechinulin A are not determined yet. Therefore we have tried to find the genes by phage display method. Phage display method is one of the ways to find the binding protein(s) or peptide(s) to a particular compounds and at least some proteins involved in the neoechinulin A biosynthesis must have affinity with the neoechinulin A molecule. Since we expect that genes for biosysthesis of neoechinulin A may form gene cluster(s) in the fungal genome similar to other gene clusters for secondary metabolites, the gene isolated by phage display could be the guide for us to access the gene cluster for neoechinulin A biosynthesis. As a result, we found a candidate of biosynthesis gene of neoechinulin A. The candidate had homologous sequence with non-ribosomal peptide synthetase (NRPS).

279. Sweet 'n' greasy: glycolipid biosynthetic gene clusters from Ustilago maydis. Sandra Hewald, Beate Teichmann, Lidan Liu, Uwe Linne and Michael Bölker. University of Marburg, Dept. of Biology, Karl-von Frisch-Strasse 8, D-35032 Marburg, Germany. e-mail: boelker@staff.uni-marburg.de

Under conditions of nitrogen starvation, Ustilago maydis produces two classes of extracellular glycolipids that act as biosurfactants. Ustilagic acid (UA) consists of a cellobiose moiety glycosidically linked to the omega-hydroxyl group of 15,16-dihydroxypalmitic acid. In addition, U. maydis cells secrete mannosylerythritol lipids (MELs), which consist of the disaccharide mannosylerythritol esterified with acyl and acetyl groups at the mannosyl group. We have identified two large gene clusters, which are responsible for extracellular glycolipid production. Database comparisons and mutational analysis allowed us to propose biosynthetic pathways for both of these unusual secondary metabolites. Since U. maydis is very amenable to genetic analysis this organism can be used for metabolic engineering of these extracellular glycolipids. Deletion of a MEL-specific acetyl transferase resulted in the production of deacetylated derivatives of this glycolipid. By targeted disruption of UA-specific hydroxylases we were able to create strains that secrete novel variants of ustilagic acid.

280. Integrated database for functional analysis in Aspergillus flavus. C. P. Smith¹, C. P. Woloshuk², N. P. Keller³, J. Yu⁴, and G. A. Payne⁵. ¹North Carolina State University, Raleigh, USA. chris@statgen.ncsu.edu. ² Purdue University, West Lafayette, IN, USA. woloshuk@purdue.edu. ³University of Wisconsin, Madison, WI, USA. npk@plantpath.wisc.edu. ⁴USDA/ARS/SRRC, New Orleans, LA, USA. jiuyu@srrc.ars.usda.gov. ⁵North Carolina State University, Raleigh, USA. gary_payne@ncsu.edu.

Aspergillus flavus is a plant and animal pathogen that also produces the carcinogen, aflatoxin. Because of its economic importance and well characterized pathway of aflatoxin biosynthesis, several labs are studying the development, metabolism, ecology, and pathogenicity of this fungus. To facilitate the research efforts in these areas and to identify potential genes and pathways for functional analysis, we are developing a database to integrate multiple categories of data. This database resource will serve two important functions: 1) it will provide a platform for the deposition of data from individual experiments; and 2) it will permit the ready analysis of composite data from all experiments enabling researchers to mine a larger data set. It will include phenotypic measurements, gene expression data from microarrays, and metabolic profile information and will be flexible enough to allow the addition of new types of measurement in the future. Users will interact with the database through a web based interface and will be able to: describe experiments; upload data gathered during those experiments; run analyses on the data; select and download raw data; select and download the results of analyses. Recently acquired lab and field data will be used to highlight the structure and utility of this database. These measurements will include fungal growth, aflatoxin concentrations, and gene expression data acquired from lab studies and infected developing maize seeds. This research was funded by USDA/NRI/CGP 2006-35604-16666.

281. Analysis of Methylene Tetrahydrofolate Reductases in filamentous fungi. Rasmus J.N. Frandsen and Henriette Giese, Department of Ecology, Section for Gen and Micro, Copenhagen University, Denmark. raf@kvl.dk and heg@kvl.dk

A. tumefaciens mediated random mutagenesis of Fusarium pseudograminearum led to the isolation of a mutant which accumulated rubrofusarin instead of the normal mycelium pigment, aurofusarin. Determination of the T- DNA integration site and a blast analysis against F. graminearum showed that a gene (Fg-met13) with homology to methylene tetrahydrofolate reductases (MTHR) was affected. Analysis of all fully sequenced fungi showed that they all had two MTHR genes. Multiple sequence alignment of the identified Met12 and Met13 sequences shows that the sequences clustered in two groups, suggesting distinct evolutionary histories and functions. Deletion of met12 and met13 in S. cerevisiae have earlier showed that Sc-met13 is required for methionine biosynthesis, while no function or phenotype have been assigned to Sc-met12. Targeted replacement of Fg-met12 and Fg- met13 showed that Fg-met12, and not as expected Fg-met13, is required for methionine biosynthesis in Fusarium. Complementation of the Sc-met13 deletion strain with Fg-met12 and Fg-met13 showed that both are functional MTHRs. We are currently testing the expression patterns to see if this can account for the differences between the two species. The situation in other fungi is also under investigation. We are currently exploring the possibility that Fg-met13 is indirectly involved in aurofusarin formation, possibly by synthesising a co-factor required by one of the enzymes known to participate in the pathway.

282. FvVE1 Differentially Regulates the Biosynthesis of Fumonisins and Bikaverin in Fusarium verticillioides. Kyung Myung ¹, Shaojie Li¹, Hamed K. Abbas² and Ana M. Calvo¹. ¹Department of Biological Sciences, Northern Illinois University, 1425 W Lincoln Hwy., Dekalb, Illinois. 60115, USA. ²Crop Genetics and Production Research Unit, USDA-ARS, Stoneville, Mississippi 38766, USA.

The veA homologous genes positively regulate sterigmatocystin and aflatoxin production in Aspergillus nidulans and in Aspergillus parasiticus and Aspergillus flavus, respectively. Whether veA homologs have a role in regulating secondary metabolism in other fungal genera is unknown. In this study, we examined the role of the veA homologous gene FvVE1 on the production of two different types of toxins in the plant pathogen Fusarium verticillioides. We found that deletion of FvVE1 completely suppressed fumonisin production on two natural substrates, corn and rice, but did not completely eliminate fumonisin production in fumonisin-inducing complex liquid medium GYAM. In contrast, deletion of FvVE1 reduced the yield of bikaverin in GYAM while it increased bikaverin production in corn. The effects of FvVE1 deletion on toxin production were found to be the same in the two different mating types. Complemented strains restored the production of both fumonisins and bikaverin to wild-type levels. Our results strongly suggest that FvVE1 differentially regulates fumonisin and bikaverin biosynthesis in a medium-dependent manner.