Cell Biology

1. Evaluation of a recombinant pH-sensor in Aspergillus niger K Altenbach¹, T Bagar², ND Read¹, M Bencina². ¹Fungal Cell Biology Group, Institute for Cell Biology, University of Edinburgh, Edinburgh EH9 3JH, UK ²Laboratory of Biotechnology, National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia

We have developed a recombinant pH-probe allowing a novel approach to pH-measurement in filamentous fungi. It is known that the absorbance and fluorescence of some GFP mutants depend strongly on the pH. Based on pHluorins, we have designed a novel variant of GFP, called RaVe_C, and expressed it in Aspergillus niger. RaVe_C is a ratiometric dual excitation pH-sensor displaying reversible emission ratio changes in the range of pH 5.5 to pH 7.5. With a pK_a of 7.0 RaVe_C is ideal for monitoring pH-changes in the cytosol. We have analysed the performance of this probe in A. niger using confocal laser scanning microscopy, addressing the following questions: (1) Is the level of expression of RaVe_C in A. niger high enough for imaging? (2) Does the probe respond to changes in intracellular and extracellular pH? (3) How can we best calibrate the pH readout of RaVe_C expressed in A. niger?

2 Molecular events controlling conidial germination in Penicillium marneffei. Kylie J. Boyce, Sophie Zuber, Michael J. Hynes and Alex Andrianopoulos. Department of Genetics, University of Melbourne, Parkville 3010, AUSTRALIA.

Penicillium marneffei is an emerging fungal pathogen endemic to South-east Asia. In response to an extrinsic stimulus (temperature), P. marneffei is capable of alternating between a filamentous and a yeast growth form, a process known as dimorphic switching. P. marneffei grows in the filamentous form at 25̊C and in the yeast form at 37̊C. At 25̊C. Little is known about the molecular events involved in the establishment and maintenance of the various developmental states of P. marneffei and the control of the dimorphic switching process.

The establishment and maintenance of cell polarity is a fundamental aspect of developmental programs and cellular differentiation. Ras and Rho GTPases have been examined in a wide variety of eukaryotes and play varied and often overlapping roles in cell polarisation and development. To investigate the unique functions of these proteins and determine how they interact to co-ordinately regulate morphogenesis during growth and development we undertook a genetic analysis of GTPase function by generating double mutants of the Rho GTPases cflA and cflB and the newly isolated Ras GTPase rasA from P. marneffei. The data shows that small GTPases have both overlapping and distinct roles and genetically interact to co-ordinately regulate development.

3. The role of RacA and the identification of RacA interacting proteins during polarized cell growth in Aspergillus niger. M. Arentshorst¹, R.A. Damveld¹, S.M.J. Langeveld¹, M.S. Roelofs¹, C.A.M.J.J. van den Hondel^1,2 and A.F.J. Ram^1,2. ¹Leiden University, Institute of Biology Leiden, Fungal Genetics Research Group, Leiden, The Netherlands, ²Department of Microbiology, TNO Nutrition and Food Research, Zeist, The Netherlands.

The establishment of cell polarity in filamentous fungi is critical for the control of many cellular and developmental processes, including polarized hyphal growth, intracellular movement of organelles, protein secretion, cell wall biosynthesis and the development of conidiophores. In yeasts, but also in other eukaryotic cells, polarized cell growth is driven by Rho-related GTPases (Rho, Rac and Cdc42). Analysis of the function of the small GTPase RacA in A.niger has implicated a role for RacA during polarized cell growth. Deletion of racA showed abnormal branching at the hyphal tip and delayed sporulation. Overexpression of dominant active RacA resulted in the loss of polarized hyphal growth. To identify RacA interacting proteins Gateway compatible yeast two hybrid cDNA libraries have been constructed. The effects of alteration of expression of these RacA interacting proteins on fungal morphology will be determined.

4. Isolation and characterization of NcSKN7 and NcOCH1 disruptants in Neurospora crassa. Shinpei Banno¹, Makoto Kimura², Isamu Yamaguchi^{2, 3}, Makoto Fujimura¹. ¹Dept of Life Sciences, Toyo Univ, Gunma, Japan. ²Laboratory for Remediation Research, PSC, RIKEN, Wako, Japan. ³Laboratory for Adaptation and Resistance, PSC, RIKEN, Yokohama, Japan

Two-component signal transduction pathway consisting of Os-1p (histidine kinase), Rrg-1p (response regulator) and Os-4p, 5p, 2p (MAP Kinase cascade) plays an important part in osmotic adaptation and fungicide-resistance of Neurospora crassa. Like yeast, N. crassa has two response regulators, Rrg-1p and NcSkn7p. To investigate the function of NcSkn7p branch in histidine kinase pathway, we isolated and characterized the gene disruptants of NcSKN7 and also of NcOCH1. NcSkn7p has HSF-like (heat shock factor) binding domain and response regulator domain. The NcSKN7 gene disruptant by gene replacement didn't affect osmotic sensitivity and fungicide resistance, therefore the NcSkn7p branch might not be directly involved in osmoregulation. The growth and fertility of ncskn7 disruptant were normal, but it was slightly sensitive to oxidative stresses (H₂O₂ and t-butyl hydroperoxide). The insertional disruptant of NcOCH1 gene, which encodes a putative alpha-1, 6-mannosyltransferase, showed slow growth and severe morphological abnormality; coral-like multiple branching hyphae with few conidia. We analyzed electrophoretic mobility of a glycoprotein, acid phosphatase. The ncoch1 disruptant cells produced a smear band with difference mobility by activity staining, suggesting that the function of NcOCH1 was involved in outer chain elongation of oligosaccharides.

5. Identification of Antibiotic Binding Sites in the V-ATPase. Emma Jean Bowman, Marija Draskovic, Molly McCall, and Barry Bowman. Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, CA 95064, USA

The macrolide antibiotics bafilomycin and concanamycin are potent inhibitors of V- ATPases. To identify the binding site of bafilomycin we selected mutant strains of Neurospora crassa (named bfr) that are resistant to this antibiotic. In one class of bfr strains the V-ATPase was resistant to inhibition in vitro. These strains had seven different point mutations in the vma-3 gene, which encodes the hydrophobic c subunit of the vacuolar ATPase. Most of the mutated sites appear to be on the outer face of the “rotor” sector of the enzyme, a region hypothesized to form an interface with the “a” subunit.

Surprisingly, the bfr strains had little resistance to concanamycin, which has a similar structure. By further mutagenizing three bfr strains we obtained 13 new strains that were resistant to both antibiotics. Each of these had two altered residues in the c subunit. Thus, concanamycin does appear to bind to the same region. The positions of four of the mutated residues correspond precisely to the positions of mutated residues in the homologous c subunit of the mitochondrial ATPase that confer resistance to oligomycin. These results suggest that vacuolar and mitochondrial ATPases have an ancient, conserved antibiotic binding site. As the sequences of the polypeptides have diverged new antibiotics that target the same vulnerable site in this family of enzymes have arisen. The data also suggest a model for the tertiary structure of the c subunit of the V-ATPase. To test the model we have developed new procedures for introducing mutations into subunits of the V-ATPase.

6. The role of vacuolar calcium in hyphal morphology and tolerance of high calcium in the growth medium. Barry Bowman, Marija Draskovic, Emilio Margolles-Clark, and Stephen Abreu. Department of Cell, Molecular and Developmental Biology, University of California, Santa Cruz, CA 95064

Calcium, a key signaling molecule in all organisms, has been postulated to play a major role in polar hyphal growth in filamentous fungi. However, we know little about which organelles or transporters are involved in sequestration of calcium within hyphae. Analysis of the Neurospora crassa genome revealed several calcium transport proteins that may function in organelles (Zelter et al. FGB 41:2004). The cax gene encodes a proton/calcium exchange protein, homologous to the Vcx1/Hum1 protein in the vacuole of S. cerevisiae. Nca-2 and nca-3 encode P-type Ca-ATPases homologous to the Pmc protein also reported to be in the vacuole of S. cerevisiae. We generated mutant strains lacking these transporters and used cell fractionation to examine the distribution of calcium. In the wild-type strain only the vacuolar fraction had significant amounts of calcium. The concentration in this organelle was surprisingly high, at least 20 mM. Nca-2 and nca-3 mutant strains were like wild type, but inactivation of the cax gene caused the complete loss of calcium from the vacuole. When strains were inoculated in medium with high concentrations of calcium (50-200 mM) wild-type, nca-3 and cax strains grew normally, but growth of the nca-2 mutant and of the nca-2 cax double mutant was strongly inhibited. In standard growth medium (Vogel's, 1 mM calcium) all the mutant strains had normal hyphal morphology. Thus, the results indicate that the vacuole is a major storage site for calcium; however, loss of vacuolar calcium does not affect polar growth or the ability to tolerate high concentrations of calcium in the medium.

7. Circadian Rhythms in Neurospora crassa: Clock Mutant Effects in the Absence of a frq–based Oscillator. Laura Lombardi, Kevin Schneider, Michelle Tsukamoto, and Stuart Brody

Div. of Biological Sciences, UCSD, La Jolla, CA 92093-0116

 In Neurospora, the circadian rhythm is expressed as rhythmic conidiation due to a feedback loop involving the genes and protein products of frq (frequency), wc-1 (white collar-1), and wc-2, known as the frq/wc (FWC) oscillator. Although null mutations, such as frq10 or wc-2(delta) that lack a functional FWC oscillator, lead to the loss of this rhythm under most conditions, a rhythm can be restored to them by the addition of geraniol or farnesol to the media. Utilizing this restoration as an assay, the effect of other clock mutations in a frq10 or wc-2(delta) null background can then be measured. It was found that existing clock mutants fall into 3 classes:

1) those that showed NO effect in a null background, such as prd-3 frq10 and frq7 wc-2(delta) (proof of principle); 2) those that did have a measurable effect in the frq10 background, such as prd-1, or prd-4; and, 3) those that suppressed the frq10 effect, such as ult-1, i.e. geraniol/farnesol were not required for a visible rhythm. Using analysis of these results along with previous studies, one can classify these double mutants into 3 categories:

I . Both mutations affect the FWC oscillator, such as prd-3 or frq10 or wc-2(delta);

II. Both mutations do not affect the FWC oscillator, such as prd-4, prd-1, cel, chol or ult;

III. One mutation affects the FWC, one does not, such as frq10 or prd-4.

Classifying which clock mutations affect the FWC oscillator and which do not is a first step to understanding a multi-oscillator system.

8. Circadian Rhythms in Neurospora crassa: Vivid has a clock effect in constant light. Stuart Brody and Michelle Shuff, Division of Biological Sciences, UCSD, La Jolla, CA.

Previous studies on vivid (vvd) mutants have shown that vivid over-produces carotenoids in the light, is involved in photoadaptation, and that the bd vvd strain has a normal period in constant darkness (D/D) of 23 hrs., but no observable rhythm in constant light (L/L) under the conditions of race tubes, glucose-arginine media (Shrode et al. , Fungal Genet.Biol., 32,169 (2001), Schwerdtfeger and Linden, Mol. Microbiol., 39, 1080 (2001), Heintzen et al., Cell,104, 453 (2001)). Our studies showed the following: 1) the above-mentioned findings were reproducible; 2) however our use of different media/conditions (maltose/Petri plates) for the bd vvd strain lead to short (7-10 hr.) periods in L/L (fluorescent); 3) the period in D/D was still 23 hrs. ; 4) the bd control strain showed no rhythm in L/L; 5) a bd strain containing the vvd allele (ss) also showed 8-10 hr periods in L/L; 6) the introduction of the csp-1 marker (conidial separation) and the use of a different media, namely acetate/casamino acids, gave the clearest banding, averaging 8-11 hr. periods; 7) the periodicity of the bd csp-1 vvd strains are a function of the intensity of the light; and 8) the rhythm in L/L is temperature-compensated. These findings suggest that the vvd gene plays, under certain conditions, a more significant role in clock timing than previously suggested.

9. Cdk2, a second essential cyclin-dependent kinase in the corn smut fungus Ustilago maydis. Sonia Castillo-Lluva and José Pérez-Martín. Centro Nacional de Biotecnología-CSIC. Campus de Cantoblanco-UAM. 28049 Madrid. Spaain

Cdk2 is a member of the Pho85 family of cyclin-dependent kinases. Unlike other fungal members of this family, Cdk2 appears to be essential for growth in U. maydis. A temperature-sensitive allele of cdk2 was generated, caused cell separation defects, G1 arrest and polarity defects at restrictive temperature. Therefore, the essential function may involve cell cycle control and morphogenesis. To further characterize the roles of Cdk2 in U. maydis, we also analyzed the putative cyclin partners. We found five distinct cyclin genes (pcl1-5) and we started the search for suppressors of the growth defects present in the ts-mutant. Details of this search will be provided.

10. Cytoplasmic flow in Neurospora crassa during the circadian cycle. E. Castro-Longoria¹, S. Brody², and S. Bartnicki-García¹. ¹División de Biología Experimental y Aplicada, CICESE, Ensenada, B. C., México. ²Division of Biology, University of California, San Diego.

The radial growth rate of a colony of Neurospora crassa (bd csp oli strain) undergoes circadian oscillation with a reduction of approximately 42%, coincident with the conidiation process. Conidium-free (interbands) and conidia-laden bands are formed alternatively in each 24h cycle. Since hyphal growth depends on precursors mobilized over long distances, we investigated if the changes in growth rate were correlated with changes in cytoplasmic flow. The flow of cytoplasm in vegetative hyphae was examined at high magnification. At the growing edge of the colony, cytoplasm was found to flow continuously towards the leading hyphae and their branches during formation of either band. Further back, at about 500 m from the edge of the colony, cytoplasm from older branches was observed to drain into leader hyphae, thus contributing to the formation of the growth front. Leader hyphae and branches in the interband, just behind a newly formed conidial band, became progressively vacuolated and the amount of cytoplasm decreased in older sections until a high percentage of hyphae were completely empty. Clearly, the cytoplasm of the interband hyphae is mostly diverted away from the forward extension of the colony towards the formation of aerial hyphae and the subsequent production of conidia. Presumably, the observed circadian decrease in vegetative growth rate is a consequence of the increased demands of cytoplasm to support the conidiation phase.

11. Biochemical analysis of Dbf4-dependent kinase in Aspergillus nidulans. Bernadette Connors, Jennifer Line, Morgan Campbell, and Steven James. Department of Biology, Gettysburg College, Gettysburg. PA 17325

In the fungus Aspergillus nidulans, nimO and cdc7 encode the regulatory and catalytic subunits of the conserved eukaryotic Dbf4-dependent kinase (DDK). In budding yeast, dbf4p escorts cdc7p to origins of replication, and origin unwinding is triggered through phosphorylation of pre-RC components by cdc7p. Dbf4p is subsequently targeted for destruction by the anaphase promoting complex through D-box motifs found in its amino terminus, thus preventing reinitiation of DNA synthesis. In Aspergillus, the temperature sensitive lethality of the nimO18 mutation can be partially suppressed by mutations in two genes, snoA and snoB (suppressor of nimO). As one approach to investigate DDK function we generated epitope tagged alleles of both cdc7 (HA3::cdc7) and nimO (HM2::nimO). Standard immunoblotting techniques have shown that HA3::cdc7p is a phosphoprotein that is differentially phosphorylated through the cell cycle. Preliminary results indicate this phosphorylation occurs in either a nimO- or snoA-dependent manner and current efforts include a detailed examination of HA3::cdc7p in both nimO18 and snoA mutant backgrounds. HM2::nimOp is also a phosphoprotein and our current efforts include examining nimOp turnover and phosphorylation through the cell cycle in strains that harbor mutations in the two N-terminal D-boxes. Supported by NSF-RUI #01-14446 to SJ.

12. A novel checkpoint system controlling nuclear division during Aspergillus nidulans mitotic exit. Jonathan Davies, Colin De Souza and Stephen Osmani. Department of Molecular Genetics, Ohio State University, Columbus, OH 43210

In order to generate multinucleate cells, filamentous fungi, including Aspergillus nidulans, undergo rounds of nuclear division in the absence of cytokinesis. During these closed mitoses, nuclear division must be driven by forces other than cytokinesis. In contrast, it has been suggested that cytokinesis drives nuclear division in budding yeast by promoting nuclear fission.

In yeasts a "dumbbell" shaped nucleus can be observed during nuclear division. In contrast, we have failed to detect "dumbbell" shaped nuclei during mitotic exit in A. nidulans. Therefore, nuclear fission, during which constriction of the nuclear envelope generates two nuclei from one, may not occur in A. nidulans. However, nuclear division is a regulated process. For instance, dominant versions of tinD promote defective mitoses in which anaphase proceeds but, surprisingly, nuclear division does not occur. The result of this defective mitosis is an intact polyploid nucleus which can progress through further cell cycles.

We propose that a regulatory mechanism exists in A. nidulans which, following detection of mitotic defects, allows completion of DNA segregation but stops nuclear division during mitotic exit. Because mitosis is not followed by cytokinesis, the resulting nucleus is able to contribute to hyphal growth. Through the process of haploidization, normal haploid nuclei can also be subsequently generated. Thus, filamentous fungi can prevent inappropriate segregation of DNA to daughter nuclei after defective mitosis by preventing nuclear division. This survival strategy is not available to unicellular organisms, such as yeasts, because nuclear division is linked to cytokinesis.

13. Mitotic Regulation of the Nuclear Pore Complex in Aspergillus nidulans. Colin De Souza, Shahr Hashmi, Aysha Osmani and Stephen Osmani. Department of Molecular Genetics, Ohio State University, 802 Riffe Building, 496 W 12th Ave Columbus OH 43210.

During Aspergillus nidulans closed mitosis, the nuclear pore complex (NPC) is partially disassembled, facilitating nuclear entry of tubulin and mitotic regulators (De Souza et al. 2004. Current Biology 14:1973-84). Genetic, overexpression and localization studies all implicate the NIMA mitotic kinase in regulating partial NPC disassembly during mitosis. We hypothesize that NIMA triggers the mitotic dispersal of the SONA and SONBn NPC proteins (nucleoporins) from the NPC as the first step in mitotic NPC disassembly. The SONBc^Nup96 structural nucleoporin remains associated with the nuclear envelope throughout mitosis. SONA and SONBn bind each other and are likely tethered to the NPC by binding of SONBn to SONBc^Nup96.

To define how the initial steps in mitotic NPC disassembly are regulated, we have determined the minimal domain within SONBn required for binding to the NPC. We demonstrate that this domain (SONBn^587-1035) disperses from the NPC during mitosis and is mitotically phosphorylated in a NIMA-dependent manner. SONBn^587-1035 contains 8 consensus NIMA phosphorylation sites and we demonstrate that mutation of all eight sites to glutamic acid compromises binding to the NPC. To determine if these sites (or other sites) are phosphorylated in vivo, we have generated an S-tagged version of SONBn^587-1035 allowing single step purification using S-protein agarose. S-tag-SONBn^587-1035 has been purified to homogeneity from mitotic A. nidulans extracts to facilitate the identification of phosphorylated residues utilizing mass spectrometry. Similar strategies are being employed to determine the sites of mitotic phosphorylation in SONBc^Nup96.

14. Functional analysis of ABC transporter genes in Aspergillus nidulans. Maarten A. De Waard¹, Ciska Braam¹, Lute-Harm Zwiers¹, and Alan C. Andrade². ¹Laboratory of Phytopathology, Wageningen University, The Netherlands. ²Embrapa Recursos Genéticos e Biotecnologia, Brasilia-DF, Brazil.

At present, seven ATP-binding cassette (ABC) transporter genes (atrA-atrG) from Aspergillus nidulans are functionally characterized. Previously, atrB and atrD were identified as multidrug transporter and transporter of antibiotics, respectively. Recently, atrG was characterized as a transporter of azole antifungals. Its physiological function may involve transport of sterols, since sterols induce expression of atrG, reduce efflux of azoles from intact cells, and affect growth of atrG knockout mutants in a temperature-dependent manner. A triple knockout mutant of Aspergillus nidulans of atrB, atrD, and atrG is hypersensitive to a wide range of natural toxic products and drugs and its phenotype is the combination of that of the single knockout mutants. The triple knockout mutant can be useful in screening programs to detect new antifungals. The imaB mutation, previously described in relation to pleiotropic drug resistance, enhances transcription of atrD, atrE, atrF, and atrG, suggesting that imaB may be a regulatory gene of ABC transporter genes. Currently, we try to clone imaB by complementation of imaB mutants with a gene-specific cosmid library.

15. het-c mediated heterokaryon incompatibility in Neurospora crassa. Karine Dementhon, Qijun Xiang, Isao Kaneko and N. Louise Glass. Plant and Microbial Biology department, UC Berkeley, California, 94720

Filamentous fungi are capable of undergoing hyphal fusion with each other to form a vegetative heterokaryon (genetically different nuclei in a common cytoplasm). However, if individuals undergoing hyphal fusion differ in allelic specificity at any one of a number of heterokaryon incompatibility loci called het, the heterokaryotic cell is rapidly compartmentalized and destroyed by a programmed cell death reaction. This phenomenon is called heterokaryon incompatibility. This nonself recognition is believed to reduce the risk of transmission of infectious elements such as mycoviruses and debilitated organelles throughout fungal populations, and to restrict resource plundering between individuals.

 In Neurospora crassa, incompatible heterokaryons show growth inhibition, repressed conidiation and hyphal compartmentation and death. Three antagonist het-C alleles are present in populations: het-C^OR, het-C^PA and het-C^GR. HET-C is a plasma membrane protein. het-c incompatibility is suppressed by vib-1, which encodes a putative nuclear- localized transcriptional regulator. Mutations in vib-1 result in strains showing de-repressed conidiation. Here we show that vib-1 over-expression represses conidiation, confirming that VIB-1 is a repressor of conidiation. We show that VIB-1 is a nuclear-localized protein by constructing a GFP fusion protein. VIB-1 is detectable when expressed under its native promotor in vivo. During incompatibility, VIB-1-GFP is also detected in nuclei, with an increased intensity of fluorescence.

Another locus, vib-2, mapped near het-c, is required for het-c incompatibility. vib-2 is predicted to encode a HET domain protein. het-c/vib-2 non-allelic interactions are essential for het-c incompatibility. vib-2^OR and vib-2^PA are highly polymorphic. We cloned the third allele vib-2^GR which also shows a high polymorphism; vib-2^GR is more similar to vib-2^PA then vib-2^OR.

16. No sex in red light - A role for phytochromes in Aspergillus nidulans. Anne Blumenstein¹, Kay Vienken¹, Ronja Tasler², Nicole Frankenberg-Dinkel² and Reinhard Fischer¹, ¹University of Marburg and University of Karlsruhe, ²University of Braunschweig.

Phytochromes are photoreceptors that sense red and far-red light through photo-interconversion between two stable conformations. This distinct feature is mediated by a covalently bound linear tetrapyrrole chromophore. Phytochromes were thought to be confined to photosynthetic organisms including cyanobacteria, but have been recently discovered in heterotrophic bacteria and in fungi, where little is known about their functions¹. PhsA and orthologues from other fungi share chromophore-binding regions, which are phylogenetically closer related to bacteria than to plants, pointing to their evolution from a common bacterial ancestor. In support of that, all fungal phytochromes are multifunctional proteins, where the phytochrome region and histidine kinase domain are combined in a single protein with a C-terminal response regulator domain. We studied the role of the Aspergillus nidulans phytochrome (PhsA). PhsA expressed in E. coli autoassembled with biliverdin and less efficiently with phycocyanobilin. The functionality of the two-component system histidine kinase was shown in an in vitro kinase assay.

We found that A. nidulans PhsA acts as red-light sensor and represses sexual development under red-light conditions. PhsA-GFP was excluded from nuclei, suggesting that red-light perception occurs in the cytoplasm, in contrast to blue-light perception through the nuclear flavoprotein WC-1 in Neurospora crassa ^2,3.

1. Bhoo, S.-H., Davis, S. J., Walker, J., Karniol, B. & Vierstra, R. D., Nature 414, 776-779 (2001).

2. Schwerdtfeger, C. & Linden, H., Eur. J. Biochem. 267, 414-421 (2000).

3. Froehlich, A. C., Liu, Y., Loros, J. J. & Dunlap, J. C., Science 297, 815-819 (2002).

17. Hyphal fusion in Neurospora crassa. Andre Fleissner, Sovan Sarkar and N. Louise Glass. University of California, Berkeley

The mycelial colony of filamentous fungi consists of a network of interconnected multinucleate hyphae. The colony grows by hyphal tip extension, branching and fusion (anastomosis). The ability to form hyphal fusions within one colony, but also between different individuals, enables fungi to establish complex functional units that show coordinated growth and exploration of their environment. To gain a better understanding of the basic, yet not well described, process of hyphal fusion, we are characterizing hyphal fusion mutants in Neurospora crassa.

The soft (so) mutant exhibits lack of anastomosis within a colony or between conidial germlings. We cloned the so gene by complementation. Database analysis showed that this gene is highly conserved in filamentous ascomycetes, but not present in ascomycete yeast species. The encoded protein contains a conserved WW-domain involved in protein-protein interactions. SO-GFP fusion proteins localize to the cytoplasm. Surprisingly the fusion protein localizes to the hyphal plugs in injured or dying cells. Overexpression of the so gene leads to a hyperbranching phenotype.

Using genetic analysis we are investigating the relationship of SO with a MAP-Kinase pathway involved in hyphal fusion. Furthermore we are analyzing the role and function of this signaling pathway by overexpression and introduction of activated alleles. GFP fusions are used to study the localization of these proteins during colony development.

18. Ramosa-1: An evolutionary conserved protein involved in fungal morphogenesis. S.J. Flitter¹, M. Arentshorst¹, C.G. Reynaga-Pena³, S. Bartnicki-Garcia⁴, C.A.M.J.J. van den Hondel^1,2, A.F.J. Ram^1,2. ¹Leiden University, Institute of Biology Leiden, Fungal Genetics Research Group, Leiden, The Netherlands, ²Department of Microbiology, TNO Nutrition and Food Research, Zeist, The Netherlands, ³Dept. de Ing. Genetica Unidad Irapuato, Universidad de Guanajuato, Mexico, ⁴ Centro de Invesigacion Cientifica de Educacion Superior de Endenada, Mexico.

Polarised growth of leading hyphae of many filamentous fungi is characterised by the presence of a Spitzenkörper (Spk) or a Vesicle Supplying Centre (VSC). This structure is thought to allow the efficient delivery of vesicles to the growing apex. To identify proteins involved in this process we have characterised the previously isolated apical branching mutant, ramosa-1 (1), and by complementation of the temperature sensitive phenotype have cloned the ramosa-1 gene. Homology searches indicate that the protein belongs to an evolutionary conserved family of proteins, in all eukaryotic cells. The ramosa-1 homolog gene in S. cerevisiae, YOL078w/AVO1, is an essential gene. Over-expression of the ramosa-1 cDNA using the GAL4 promoter could rescue the S. cerevisiae gene deletion mutant (deltaYol078w), indicating a functional conservation between the two homologs. Ramosa-1 depletion studies in S. cerevisiae indicate that the gene is required for proper morphogeneisis, as is seen in Aspergillus niger. Ramosa-1 homologs have been shown to be involved in several signalling mechanisms in the cell including MAPK signals (S. pombe), cyclic AMP signalling (Dictyostelium) and PI signaling (S. cerevisiae). Current research is aimed at the identification of Ramosa-1 interacting proteins to elucidate its function in fungal cells.

1. C.G. Reynaga-Pena and S. Bartnicki-Garcia. 1997. Apical branching in a temperature sensitive mutant of Apergillus niger, Fungal Genetics and Biology 22, 153-167.

19. Endosome based recycling of the pheromone receptor Pra1 is essential for pheromone perception in mating of the plant pathogen Ustilago maydis. Uta Fuchs and Gero Steinberg. Max-Planck-Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse, 35043 Marburg, Germany

We previously identified the t-SNARE Yup1 as a key receptor for fusion of endocytic vesicles and an early endosomal compartment¹. It was shown that endocytosis is involved in cellular morphogenesis but the relevance for this basic developmental process in pathogenic development of Ustilago maydis was not known. Here we demonstrate that Yup1-dependent endocytosis is essential for early infection stages of U. maydis. Temperature sensitive Yup1 mutants are non-pathogenic, and they are unable to form mating tubes in the presence of synthetic pheromone or the compatible mating partner. This is due to a defect in pheromone perception, while Yup1 only has a minor effect on growth and elongation of existing mating tubes. Pheromone perception in Ustilago maydis is based on the recognition of the pheromone receptor, Pra1/Pra2, and the compatible mfa2/mfa1 pheromone. In unstimulated cells low amounts of Pra1-GFP localize to the plasma membrane and are also found in the the vacuole suggesting that Pra1 is delivered to the vacuole for degradation. Upon pheromone stimulation Pra1 is enriched in the membrane of the growing tip of the mating tube. We show that Pra1 is endocytosed via actin-dependent processes and that it colocalizes with Yup1-tagged early endosomes. In temperature-sensitive Yup1 mutants Pra1 is still internalised but accumulates in the cytoplasm and is not transported to the vacuole. Here Pra1 is depleted from the plasma membrane, suggesting that the pheromone receptor is recycled back to the surface via Yup1-tagged early endosomes. These results argue for an important role of Yup1 mediated endosomal recycling of Pra1 in pheromone signalling and pathogenic development of U. maydis.

¹Wedlich-Söldner, R., Bölker, M., Kahmann, R., Steinberg, G., (2000): A putative endosomal t-SNARE links exo- and endocytosis in the phytopathogenic fungus Ustilago maydis. EMBO J 19: 1974-1986.

20. Microtubules are dispensable for cell fusion but are essential for long distance hyphal growth in the corn smut fungus Ustilago maydis. Uta Fuchs, Isabel Manns and Gero Steinberg. Max-Planck-Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse, 35043 Marburg, Germany

Fungal pathogenicity is often linked to a morphogenic switch between a yeast stage and a filamentous hypha. It is thought that the cytoskeleton underlies this transition, but a detailed understanding of the importance of microtubules and F-actin in morphological switch is missing. In Ustilago maydis, compatible yeast-like cells recognize each other in order to form conjugation hyphae that fuse and give rise to a b-dependent filament. Here we analyze the role of the cytoskeleton in these early steps of pathogenic development. We show that F-actin is essential for growth of yeast-like cells, cell fusion and formation of both conjugation and b-dependent hyphae. In contrast, microtubules have cell cycle-specific roles in both mitosis and cell polarity of yeast-like cells, but were of minor importance for hyphal growth. Conjugation hyphae elongate up to 100 m in length and contain long microtubules that extend their plus-ends towards the growing apex. However, in the absence of microtubules conjugation hyphae still elongate up to 60 m. A similar phenotype was observed in b-dependent hyphae, suggesting that microtubule based transport is dispensable for short distance hyphal growth. A quantitative analysis of fusion of compatible cells revealed that microtubules neither participate in pheromone recognition nor in cell-cell fusion, indicating that short conjugation hyphae are fully functional. These results demonstrate that F-actin has numerous crucial roles in dimorphic transition, while microtubules are essential for extended hyphal growth.

21. Analysis of dynein regulatory complexes using a tandem affinity purification tag for filamentous fungi. Kerstin Helmstaedt, Silke Busch, Ozgur Bayram, and Gerhard H. Braus. Institute of Microbiolgy and Genetics, Georg-August-University, Grisebachstr. 8, D-37077 Goettingen, Germany

In filamentous fungi, migration of nuclei is established through the interplay of the molecular motor dynein in combination with dynactin and microtubuli. Several proteins like NUDF, NUDE, and NUDC had been identified as regulators of dynein-mediated movement, which are not part of the dynein/dynactin motor complex itself (Xiang et al, 1995; Efimov and Morris, 2000). We intend to identifiy interaction partners of these regulatory proteins in Aspergillus nidulans in order to clarify the regulatory pathway leading to dynein activation. For this biochemical approach we used the tandem affinity purification (TAP) tag, which was originally developed for yeast and was optimized for expression in filamentous fungi. The nuclear migration gene nudF was fused to the new N-terminal tag and expressed in A. nidulans from its native promoter for gentle purification of active regulatory complexes at their natural level. Proteins which co-purified with NUDF were identified by mass spectrometry. In first affinity purifications, several hypothetical proteins with homologs in other filamentous fungi and higher eukaryotes, respectively, were found. The method is currently improved and interactions are verified by yeast two-hybrid analysis.

22. White collar homologs sense blue light in basidiomycetes and zygomycetes. Alexander Idnurm and Joseph Heitman. Department of Molecular Genetics and Microbiology, Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710, USA.

Light is the primary energy source for life on earth and as such is a major environmental signal for organisms from all Kingdoms of life, including the fungi. Through candidate gene and insertional mutagenesis approaches we have elucidated the function of two genes whose products perceive light and control mating mating in the basidiomycete human pathogen Cryptococcus neoformans. Light represses mating of C. neoformans, and strains mutated in a homolog of Neurospora crassa white collar 1 (BWC1) mated equally well in the light or the dark. The predicted Bwc1 protein shares identity with N. crassa WC-1, but lacks the zinc finger DNA binding domain. In C. neoformans Bwc1 regulates cell fusion and represses hyphal development after fusion in response to blue light. In addition, bwc1 mutant strains are hypersensitive to ultraviolet light. To identify other light sensing components, a novel self-fertile haploid strain was created and subjected to Agrobacterium-mediated insertional mutagenesis. One UV-sensitive mutant that filaments equally well in the light and the dark was identified and found to have an insertion in the BWC2 gene, whose product is structurally similar to N. crassa WC-2. Finally, we analyzed the zygomycete Phycomyces blakesleeanus, and found two homologs of WC-1. Sequence analysis is underway to determine whether or not these homologs are mutated in the photon insensitive mad strains. These results demonstrate that perception of UV/blue light via the White collar system is an ancient process that predates the divergence of the fungi into the ascomycete, basidiomycete and zygomycete phyla.

23. The cAMP signaling pathway is involved in osmotic tolerance, but not in dicarboximide resistance in Cochliobolus heterostrophus. Kousuke Izumitsu, Akira Yoshimi, and Chihiro Tanaka. Laboratory of Environmental Mycoscience, Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan.

In the fungus Cochliobolus heterostrophus (anamorph: Bipolaris maydis), the mutants resistant to dicarboximide fungicides are sensitive to high osmolarity (Yoshimi et al. 2003). Genetic analysis indicated that three genes were associated with osmosensitivity and dicarboximide resistance: Dic1, Dic2, Dic3. Dic1 encoded the histidine kinase that confered osmotic adaptation and dicarboximide resistance, but the functions of Dic2 and Dic3 are unknown. In Ustilago maydis, it was reported that mutants with disruptions in the ubc1 gene, which encoded the regulatory subunit of PKA (cAMP dependent protein kinase), were resistant to dicarboximide and sensitive to high osmolarity (Marilee et al. 2001). To evaluate the role of cAMP signaling pathway in C. heterostrophus, we isolated and characterized two genes involved in cAMP signaling pathway. These two genes, BmRpk1 and BmCyr1, encoded a PKA regulatory subunit and adenylyl cyclase, respectively. Disruption of BmCyr1 resulted in the significant reduction of conidiation, whereas bmrpk1 mutants showed normal conidiation. Both bmcyr1 mutants and bmrpk1 mutants show sensitivity to dicarboximide, similar to the wild-type strain. This result suggests that, unlike in the case of U. maydis, the cAMP signaling pathway do not mediate resistance to dicarboximide in C. heterostrophus. With regard to the sensitivity to osmotic stress, the growth of the bmrpk1 mutants was significantly reduced on the medium amended with 0.8M KCl, comparing with that of the wild-type strain. This suggests that the cAMP signaling pathway is involved in osmotic adaptation in C. heterostrophus.

24. The absence of meiotic silencing by unpaired DNA (MSUD) in Neurospora tetrasperma. David Jacobson and Namboori Raju. Department of Biological Sciences, Stanford University, California.

Genes that are unpaired during meiosis are silenced in N. crassa. GFP-tagged histone H1 when inserted at the his-3 locus on linkage group I (LGI) allows visualization of MSUD in developing asci by fluorescence microscopy. When homozygous, hH1::GFP is paired during meiosis; it expresses normally and nuclei fluoresce throughout ascus development. However, when heterozygous, it is unpaired and silenced during meiosis until ascospore demilitation. MSUD does not extend into the ascospore maturation stage and hH1::GFP nuclei fluoresce in four of the eight ascospores. N. tetrasperma packages two nuclei of opposite mating type into each of its four ascospores. This is accomplished by blocking recombination in a large region of LGI. LGI also shows a large unpaired region during pachytene. To test whether the genes in this unpaired region are silenced, hH1::GFP at his-3 was introgressed from N. crassa into N. tetrasperma. The initial hybrid cross produced almost all 8-spored asci with a high level of ascospore abortion, but showed no silencing of hH1::GFP. After four backcrosses to N.tetrasperma, all progeny were phenotypically N. tetrasperma: asci were 4-spored and ascospores were heterokarotic for hH1::GFP. All nuclei in the developing asci and in the heterokaryotic ascospores fluoresced brightly. Thus, unpaired hH1::GFP on LGI is not silenced in N. tetrasperma. The absence of MSUD in N. tetrasperma is genome wide, as hH1::GFP constructs inserted on other chromosomes (courtesy of M. Freitag, U. Oregon) were also not silenced. Sad-1 is a suppressor of MSUD in N. crassa, and its role in N. tetrasperma is under investigation.

25. Molecular analysis of snoA (suppressor-of-nimO), a novel regulator of DNA synthesis in Aspergillus nidulans. Elizabeth Wille, Matthew Denholtz, Allison Altenburger, and Steve James. Department of Biology, Gettysburg College, Gettysburg, PA 17325

In Aspergillus nidulans, nimO^Dbf4 and cdc7encode regulatory and catalytic subunits of the conserved Dbf4-dependent kinase (DDK). DDK initiaties DNA synthesis by phosphorylating components of DNA helicase to trigger origin unwinding. The temperature sensitive nimO18 allele can be partially suppressed by mutations in two genes, snoA and snoB. Semi-dominant snoB mutations lie within the cdc7 gene, and suppress by stabilizing the association of cdc7p with nimO18p. Recessive snoA suppressors not only rescue nimO18 ts-lethality, but they also rescue growth of strains hypomorphic for expression of nimO⁺. However, snoA suppressors cannot bypass a complete loss of nimO. Thus, snoA suppression is not allele-specific, and snoA appears to act by influencing the level or stability of nimOp. Bioinformatic and positional cloning approaches were combined to localize snoA within a 340,000 bp interval on Linkage Group VII. snoA was discovered by systematic ORF deletion, in which deletion of one ORF phenocopied the snoA suppressor phenotype by partially rescuing nimO18 ts-lethality. Current efforts include snoA overexpression studies, mapping snoA functional domains, and biochemical analysis of nimOp and cdc7p in snoA mutants. (Supported by NSF-RUI #01-14446)

26. Endocytosis: A Filamentous Fungal Perspective. E.R. Kalkman¹, Robert W. Robertson², and N.D. Read¹ .¹Fungal Cell Biology Group, Institute of Cell Biology, University of Edinburgh, Rutherford Building, Edinburgh EH9 3JH, UK. ²Department of Plant Biology, Arizona State University, Tempe, Arizona, USA

Endocytosis has been well characterized in budding yeast and animal cells, and to a lesser extent in plant cells. In contrast, much less is known about this process in filamentous fungi although it is likely to have important roles in membrane recycling, membrane degradation, and the uptake of signal molecules. This presentation will provide evidence from studies of several different filamentous fungi, most notably Neurospora crassa, which support the occurrence of endocytosis in filamentous fungi. This evidence is: 1) Membrane-selective markers of endocytosis (FM4-64) are internalized; 2) A marker of fluid-phase endocytosis (Lucifer Yellow) is internalized; 3) Internalization of endocytosis markers is an active process and not diffusion since it is reversibly inhibited by azide or cold treatment; 4) Internalization of FM4-64 is actin-mediated because it is inhibited by Latrunculin B; 5) The genomes of several filamentous fungi encode complex endocytic protein machineries

27. Comparison of membranes imaged with fluorescent probes FM4-64 and DiOC6 to GFP-tagged ER, Golgi and microtubules in Aspergillus nidulans. Michelle Hubbard and Susan Kaminskyj, Dept. Biology, Univ Saskatchewan, Saskatoon, SK S7N 5E2, Canada

Fungal tip growth depends on precisely targeted secretion of endomembrane-derived vesicles. The Aspergillus nidulans hypA and hypB morphogenesis loci have roles in ER-Golgi transport via the COPII and COPI pathways, respectively. To facilitate future work in mutant phenotype studies, we imaged A. nidulans endomembranes with the lipophilic dyes FM4-64 and DiOC6 using confocal microscopy. Arrays were compared with GFP-tagged ER, Golgi and microtubule patterns. Effective DiOC6 concentrations were 1000x lower than FM4-64. FM4-64 patterns changed over time, first staining the cell membrane and later being internalized; DiO6 staining of endomembranes was rapid. In growing wildtype cells there was overall agreement between FM4-64 and DiOC6 patterns after 1 h incubation in FM4-64, except that DiOC6 did not label a putative apical vesicle cluster. Time lapse images with both dyes showed endomembrane movements. Although theoretically possible, we were not able to effectively unmix signals from DiOC6 and GFP-tagged cytoplasmic microtubule arrays due to spatial rather than spectral constraints. FM4-64 arrays co-localized well with GFP-ER, but generally not with GFP-Golgi. Comparing endomembrane and cytoplasmic microtubule arrays in developing branches in wildtype and hypA1 mutants, we find that Golgi cluster at branch initiation sites, whereas both Golgi and cytoplasmic microtubules have roles in branch extension.

28. A Sec7 domain gene has a role in Aspergillus nidulans morphogenesis. Lifeng Chen, Yi Yang, Susan Kaminskyj. Dept Biology, Univ Saskatchewan, Saskatoon, SK S7N 5E2, Canada

A Sec7 domain containing sequence was isolated as an extragenic suppressor of the Aspergillus nidulans hypB5 temperature sensitive morphogenesis defect. A hypB5 mutant strain was transformed with the pRG3-AMA1 library. A complementing plasmid was rescued, and a 5 kb KpnI fragment containing the complementing region was cloned into pBluescript as pYY2. The restrictive phenotype of hypB5 mutant strains is restricted colonial growth, and slow-growing cells with aberrant branching. The pYY2-transformed strains have wildtype colonies at 42C. At 42C these hyphae have shorter basal cells with more nuclei than the wildtype parent strain A28. An intact Sec7domain is required for wildtype colony growth: pYY2 with Tn1000-disruptions in, but not adjacent to, the Sec7 domain failed to rescue the hypB5 temperature sensitive defect. The pYY2 Sec7 domain is 81% identical to Saccharomyces cerevisiae SEC7. In A. nidulans it is located in the predicted protein An6709.2. Structural modeling shows good theoretical agreement with the Sec7 domain of human ARNO (ADP Ribosylation Nucleotide Opener). The Sec7 domain is 10% of the predicted length of An6709.2; the remainder of the predicted gene lacks homologies to characterized sequences. In other Sec7 genes, the conserved F-G loop is critical for function. We are using site-directed mutagenesis to examine effects of single residue replacements on hyphal morphogenesis.

29. Proteins binding to subterminal repeats of transposon Restless. Ilka Braumann and Frank Kempken. Abteilung für botanische Genetik und Molekularbiologie, Botanisches Institut und Botanischer Garten, Christian-Albrechts-Universität Kiel, Am Botanischen Garten 1-9, 24118 Kiel, Germany

The fungal transposon Restless is a member of the hAT family of eukaryotic transposable elements (1,2). In previous studies we have demonstrated the activity of Restless and its use for gene tagging in its host Tolypocladium inflatum (3). In addition we observed alternative splicing (4), excision, and the generation of deleted transposon copies (5) in the foreign host Neurospora crassa.

To further analyse the function and transposition of Restless we set out to identify cis-acting elements involved in transposition. ³²P-labeled PCR fragments of Restless terminal and subterminal regions were used for gel shift analysis. UV cross linking experiments were employed to identify Restless-binding proteins. Results of these experiments will be shown and discussed. In addition we present data regarding the expression of the Restless transposase.

(1) Kempken F, Windhofer F (2001) Chromosoma 110:1-9, (2) Kempken F (2003) In: Arora DK, Khachatourians GG (eds) Applied Mycology and Biotechnology, Vol. 3 Fungal Genomics, Elsevier Science Annual Review Series, pp83-99, (3) Kempken F, Kück U (2000) Mol Gen Genet 263:302-308, (4) Kempken F, Windhofer F (2004) Current Genetics 46:59-65, (5) Windhofer F, Hauck K, Catcheside DEA, Kück U, Kempken F (2002) Fungal Genet Biol 35:171-182

30 Ambient pH signaling influences phosphate acquisition in Neurospora crassa. Patrick W. Kennedy¹, Robert L. Metzenberg², Maria J. Harrison³ and Wayne K. Versaw¹. ¹Dept of Biology, Texas A&M University, College Station, TX ²Dept of Chemistry and Biochemistry, University of California, Los Angeles, CA ³Boyce Thompson Institute for Plant Research, Cornell University, Ithica,

N. crassa exhibits two modes of phosphate uptake – constitutive, low-affinity transport and phosphate-repressible, high-affinity transport. The two transport systems can be operationally distinguished not only by available phosphate concentration but also by ambient pH. At pH values above neutrality, the Km of the low-affinity system rises sharply so that this system is unable to support growth under modest phosphate concentrations without the assistance of at least one component of the high-affinity system. Thus a mutant lacking the high-affinity transporters PHO-4 and PHO-5 is unable to grow under the restrictive conditions. The identity of the transporter(s) that comprise the low-affinity system has not been reported but a mutant isolated as a suppressor of the pho-4; pho-5 double mutant's growth defect displays altered phosphate transport activity. Complementation of this mutant revealed that the affected gene encodes a protein homologous to PalF of Aspergillus nidulans, which is one component of a highly conserved, ambient pH signaling pathway. Deletion of the pacC homolog, another component of the same pathway, gives rise to an identical phenotype indicating that pH signaling has a role in modulating low-affinity phosphate transport.

31. Pheromone receptor genes are essential for mating type-specific trichogyne chemotropism and female fertility, and are also involved in perithecial development in Neurospora crassa. Hyojeong Kim and Katherine A. Borkovich. UC Riverside.

In the heterothallic ascomycete Neurospora crassa, mating involves differentiation of protoperithecia and chemotropic growth of trichogynes towards opposite mating type cells in a pheromone-mediated process. The pheromone receptor genes, pre-1 and pre-2, are predicted to encode G-protein-coupled receptors with sequence similarity to other fungal pheromone receptors. Northern analysis indicates that pre-1 and pre-2 are preferentially expressed in mat A and mat a strains, respectively, under mating conditions. Deletion of pre-1, pre-2 or both does not greatly affect vegetative growth, heterokaryon formation or male fertility in either mating type. During sexual development, delta pre-1, delta pre-2 and delta pre-1 delta pre-2 strains form normal protoperithecia and are fertile as males. However, protoperithecia from pre-1 mat A, pre-2 mat a and both mating types of pre-1 pre-2 double mutants do not undergo fertilization, due to inability of their trichogynes to recognize and fuse with opposite mating-type cells. Consistent with a requirement for GNA-1 (G alpha subunit) and GNB-1 (G beta protein) for female fertility in N. crassa, trichogynes of gna-1 and gnb-1 mutants display defects in growth towards and fusion with male cells. Results from studies involving forced heterokaryons of double mutants or inappropriate expression of the receptors suggest that one compatible pheromone/pheromone receptor combination is sufficient for perithecial development in N. crassa.

32 GPR-1, a G-protein coupled receptor, regulates sexual development in Neurospora crassa. Svetlana Krystofova and Katherine A. Borkovich, Dept. Plant Pathology, University of California Riverside, Riverside

The analysis of the genome sequence revealed that Neurospora crassa has at least 10 G-protein coupled receptors (GPCRs), including a novel gene family consisting of the GPCRs GPR-1, GPR-2 and GPR-3. Predicted protein sequences of these receptors share similarity to GCR1 from Arabidopsis and CrlA, CrlB and CrlC from Dictyostelium. The gpr-1, gpr-2 and gpr-3 genes are expressed at various developmental stages of the N. crassa life cycle. The highest expression of gpr-1 was detected in sexual tissues. Deletion of gpr-1 leads to pleiotropic defects during sexual development. Protoperithecia are pale and significant number are small and buried in solid medium. Perithecia have deformed beaks and often lack ostioles. Studies are in progress to determine possible downstream effectors of GPR-1, as well as genes that might be regulated by GPR-1.

33. The Colletotrichum lagenarium Genes ClaKEL1 and ClaKEL2 are Involved in Appressorium Morphogenesis. Yasuyuki Kubo, Kyoto Prefectural University, Laboratory of Plant Pathology, Shimogamo Japan

Colletotrichum lagenarium is the causal agent of cucumber anthracnose disease. The infection process involves a series of change in fungal morphology. We have isolated two novel genes, ClaKEL1 and ClaKEL2, from C. lagenarium. ClaKEL1 was disrupted in the pathogenicity mutant P24 generated by restriction enzyme-mediated integration. ClaKEL1 encodes a putative protein of 555 amino acids containing kelch motif at the C teminal region. To define the function of ClaKEL1, we isolated clakel1 knock-out mutants by homologous recombination. The clakel1 disruptant produced less conidia than the wild type on PDA medium, conidia formed malformed appressoria on glass slides, and showed weak pathogenicity on cucumber leaves. A second gene containing the kelch motif, ClaKEL2, was isolated by PCR using degenerate oligonucleotide primers based on the kelch motif. ClaKEL2 encoded a putative protein of 1577 amino acids containing the kelch motif at the N terminal region, and had significant homology with Saccharomyces cerevisiae KEL1 which has a role in cell morphogenesis and cell fusion, and Schizosaccharomyces pombe TEA1, which is required for microtubule dynamics and proper rod like cell morphology. The phenotype of clakel2 knock-out mutants was impaired maintenance of conidial dormancy. On glass slides, conidia formed normal melanized appressoria but most of them underwent lateral germination, and failed to penetrate cellulose membranes. These results indicate that ClaKEL1 and ClaKEL2 are involved in appressorium morphogenesis.

34. Cytoplasmic dynein is targeted to the spindle poles during mitosis. Shihe Li*, C. Elizabeth Oakley#, Guifang Chen*, Xiaoyan Han*##, Berl R. Oakley# and Xin Xiang*. *Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814. #Department of Molecular Genetics, Ohio State University, Columbus, Ohio 43210. ## Richard Montgomery High School, Rockville, MD 20852.

In Aspergillus nidulans, the spindle-pole localization of cytoplasmic dynein and its regulator NUDF/LIS1 is regulated in an interesting manner during mitosis. While NUDF localizes to the poles of spindles of various lengths, cytoplasmic dynein's spindle pole localization is more apparent on longer spindles, and is dependent upon the function of BIME/APC1, a component of the anaphase-promoting complex (APC). Moreover, while NUDF's localization to the spindle poles is not affected by a loss-of-function mutation of the cytoplasmic dynein heavy chain, the spindle-pole localization of cytoplasmic dynein is greatly diminished in a nudF loss-of-function mutant, suggesting that the function of NUDF is required for cytoplasmic dynein's targeting to the spindle poles. The localization of either NUDF or cytoplasmic dynein is not sensitive to transient microtubule depolymerization, suggesting that cytoplasmic dynein and NUDF bind directly to the spindle pole body (SPB) during mitosis. Interestingly, a gamma-tubulin mutation, mipAR63A, significantly eliminates the localization of cytoplasmic dynein to the spindle poles, while it has no apparent effect on NUDF's spindle-pole localization. These results suggest that the targeting of dynein, and possibly its cargoes, to the SPB is regulated during mitosis.

35. Characterization of Aspergillus nidulans mutants that display altered responses to a novel bacterial anti-fungal compound. Shaojie Li, Gary Y. Yuen, and Steven D. Harris, University of Nebraska, Lincoln, NE 68583

HSAF is a novel antifungal lipopeptide produced by a bacterial biocontrol agent, Lysobacter enzymogenes strain C3. HSAF can confer protection against plant pathogens though its mode of action remains unknown. Here, we show that HSAF induces dramatic morphological changes in germinating Aspergillus nidulans spores and growing hyphae. These include formation of multiple branches, aberrant accumulation of cell wall material and abnormal patterns of action organization. Genetic analysis shows that dominant mutations affecting at least two genes, barA and barB, cause resistance to HSAF, and a recessive mutation affecting the basA gene cause hypersensitivity to HSAF. The barA mutant also displays recessive temperature sensitive growth and morphological defects consistent with failure to maintain hyphal polarity. Molecular characterization of barA revealed that it is the A. nidulans homologue of yeast Lag1, which is essential for Acyl-CoA-dependent ceramide synthesis. Supplementation experiments strongly suggest that BarA is functionally analogous to Lag1p. The basA mutant fails to maintain hyphal polarity and displays aberrant accumulation of cell wall material at restrictive temperature. BasA is the A. nidulans homologue of yeast Sur2, sphinganine C4-hydroxylase, which catalyses the conversion of sphinganine to phytosphingosine. Supplementation experiments strongly suggest that BasA is functionally analogous to Sur2p. Collectively, these results suggest that the mode of HSAF action is related to sphingolipid synthesis.

36. A putative carbon sensing G protein coupled receptor. Liande Li and Katherine A Borkovich. Plant Pathology, University of California Riverside

At least 10 seven-transmembrane helix GPCRs have been predicted in the N. crassa genome (Borkovich et al, 2004). One of the GPCRs is named GPR-4 which is most similar to the putative sugar sensory receptors in yeasts. The gpr-4 gene structure was verified by RT-PCR, which contains two exons and one intron. Expression analysis indicated that gpr-4 expression level is very low. Quantitative RT-PCR showed that gpr-4 has the highest level of expression in 3 day solid cultures. gpr-4 mutants have different response to different carbon sources, suggesting that GPR-4 may act as a carbon source sensor. The complemented gpr-4 strain could totally or partially rescue the phenotype of delta gpr-4 to wild type level. Epistasis analyses showed that GPR-4 is upstream of GNA-1, not GNA-2 nor GNA-3, which indicate that GPR-4 is probably coupled to GNA-1, but not GNA-3 nor GNA-2. gpr-4 mutants also share characteristics with gna-1 mutants on such aspects as premature conidiation, H₂O₂ resistance and induced thermotolerance in germlings, and so on. Exogenous cAMP could partially rescue the phenotypes of delta gpr-4 . Taken together, this research showed that GPR-4 may act as a carbon sensor, may couple to GNA-1 and is likely to be in a carbon-sensing GPCR, Galpha protein-adenylyl cyclase-cAMP-PKA pathway. More experiments are in progress to further prove this hypothesis.

37. A single septin gene from Aspergillus nidulans induces filamentous growth in Saccharomyces cerevisiae. Rebecca Lindsey, Youngsil Ha and Michelle Momany* Department of Plant Biology, University of Georgia, Athens, Georgia 30602 USA

Septins were first discovered in S. cerevisiae where they are thought to form a scaffold that organizes the bud site and they are a component of the morphogenesis checkpoint that monitors bud shape and coordinates budding with cytokinesis. Here we show that one of the A. nidulans septins, AspC, induces filaments and elongated asci containing up to twelve spores in S. cerevisiae. AspC-induced filaments are noninvasive, form in haploid and diploid strains and require the presence of a functional yeast septin Cdc10. Based on comparisons of amino acid sequence, AspC is most similar to the S. cerevisiae septin Cdc12. The A. nidulans aspC gene complements cdc12 and cdc3 yeast mutants. When GFP-Cdc12 and AspC are co-expressed Cdc12 localizes to the necks of buds, but it does not localize to the necks of AspC-induced filaments. In budding yeast, the Bni1 formin and Cdc12 are known to interact. When aspC is introduced into an S. cerevisiae bni1 delete strain, a novel bent filament phenotype is seen. This phenotype is suppressed by extra copies of CDC12. Our results suggest that the A. nidulans septin AspC competes with Cdc12 for incorporation into the septin scaffold at the S. cerevisiae neck and that when AspC is present it perturbs normal yeast bud morphology causing it to take on the more elongated shape common to filamentous fungi.

38. Novel regulators of septation in Aspergillus nidulans. Ling Lu, Rongzhong Shao, Jung-Mi Kim, and Bo Liu. Section of Plant Biology, University of California, Davis, CA 95616, USA

The kinase cascade of the septation initiation network (SIN)/ mitotic exit network (MEN) plays a regulatory role for septation in fungi. The evolutionarily conserved MOB1 protein is a novel protein that is associated with the terminal kinase of this cascade. The exact role of MOB1 is unclear. In the filamentous fungus A. nidulans, AnMOB1 is required for septation and conidiation, but is not essential for hyphal extension and colony formation. Because the A. nidulans mycelium contains multinucleate cells, novel mechanisms may exist to regulate septation in this organism. To identify novel septation regulators in A. nidulans, by UV mutagenesis we have isolated suppressor (smo) mutations that restored conidiation when AnMOB1 was down-regulated/not expressed. Microscopic examination indicated that the restored conidiation was concomitant with restored septation in the absence of the AnMOB1 protein. Among 110 independent smo mutants, five in two complementation groups demonstrated reduced hyphal growth, colony formation, and conidiation in the presence of AnMOB1. These five smo mutations also rendered hypersensitivity to low doses of the microtubule-depolymerizing agent benomyl. However, none of these smo mutations altered the localization of AnMOB1. Therefore, regulators antagonizing the SIN/MEN pathway likely exist in A. nidulans. Isolation of smo genes will shed light on regulatory mechanisms underlying septation in filamentous fungi. Progresses on characterization of the smo mutants, and identification of the smo genes will be presented. This work was supported by the NSF.

39. The csnD-E signalosome genes are involved in the Aspergillus nidulans DNA damage response. Iran Malavazi¹, Joel F. Lima¹, Marcia R.V.Z.K. Fagundes¹, Marcela Savoldi¹, Maria H. S. Goldman², Gerhard Braus³ and Gustavo H. Goldman¹. ¹Faculdade de Ciências Farmacêuticas de Ribeirão Preto and ²Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Brazil. ³Department of Microbiology and Genetics, Institute for Microbiology and Genetics, Georg-August-University Gottingen, Germany.

The signalosome (CSN) is a conserved complex involved in protein turnover and eukaryotic development, and is also required to activate ribonucleotide reductase for DNA synthesis. In A. nidulans, csnD and csnE, are key regulators of sexual development. We investigated if these genes are involved in the DNA damage response. The growth of the csnD-E deletion mutants is reduced by sub-inhibitory concentrations of camptothecin (CPT) and 4-nitroquinoline oxide (4-NQO). In A. nidulans, septum formation is inhibited by DNA damage in a checkpoint-dependent manner. Nevertheless, septation is not inhibited in the csnD-E deletion mutants upon DNA damage caused by methyl methane sulfonate (MMS). The csnD mRNA expression was induced by CPT, MMS, bleomycin (BLEO) (about 3-fold), and 4-NQO (9-fold). The csnE gene encoding the deneddylase activity which is required to modify E3 SCF ubiquitin ligases, has high levels of mRNA expression induced by all drugs (from 10-fold, CPT to 37-fold, 4-NQO). Germinating conidia of csnD-E deletion strains are more sensitive to ultraviolet light than the corresponding wild type strain. We constructed double mutants with csnD-E deletion, npkA and uvsB^ATR. The NpkA is a cdc2-related kinase that is involved in the S-phase. The double csnD npkA and csnE npkA mutants are much more sensitive to DNA damaging agents than the respective single mutants. However, the intra-S-phase and DNA replication checkpoints are intact in these mutants. Our results suggest that csnD-E genes are involved in the DNA damage response and that NpkA genetically interacts with the CSN.

Financial support: FAPESP and CNPq, Brazil

40. Studies of the Neurospora crassa adenylyl cyclase, CR-1. Sara Martinez and Katherine Borkovich. University of California, Riverside.

Heterotrimeric G proteins are involved in the ability of eukaryotic cells to react to various stimuli. In pathogenic filamentous fungi, G proteins are essential for virulence. In the non-pathogenic filamentous fungus Neurospora crassa, there are three G alpha subunits (GNA-1, GNA-2, GNA-3), one G beta subunit (GNB-1) and one G gamma subunit (GNG-1). One of the proteins acting downstream of G proteins is the adenylyl cyclase, CR-1. The G alpha subunits have been shown to regulate CR-1 level and function. CR-1 activity in gna-1 deletion mutants is decreased compared to wild type, but protein levels are equal, suggesting GNA-1 affects CR-1 function. In gna-3 deletion mutants, both activity and level of CR-1 is significantly lower than wild type, indicating that a loss of GNA-3 causes a decrease in CR-1 protein levels. GNA-2 is thought to function as a compensatory protein to GNA-1 and GNA-3, and levels and activity of adenylyl cyclase are normal in gna-2 deletion mutants. Furthermore, the cr-1 mutant has more severe forms of phenotypes observed in gna-1 mutants. This data suggests that GNA-1 could be directly interacting with CR-1. Results will be presented from experiments testing for interactions between the CR-1 and G proteins. The understanding of how CR-1 functions, its role in G protein signaling, and its downstream effectors will give insight as to how eukaryotes respond to stimuli. This data can be applied to pathogenic fungi as well as mammalian studies to aid in the fight of plant and animal disease.

41. The snxA1 and nimA5 mutations of Aspergillus nidulans Interact to Affect Mitotic Spindle Structure. R. Day, B. Fontenelle, S. Chandna, and S.L. McGuire, Millsaps College, Jackson, MS, USA

The snxA gene interacts with NIMX^cdc2 to affect mitosis, and its mutation causes abnormal nuclear morphology at 17C, while the nimA gene affects the nuclear import of NIMX^cdc2 and when mutated blocks mitotic entry at 42C. To characterize the effects of the snxA1 mutation on microtubule and nuclear structure, strains expressing a GFP-tubA (alpha-tubulin) gene were generated with either snxA1 or nimA5 mutations alone or with a snxA1/nimA5 double mutant. At 17C 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 yet others were bifurcated or trifurcated and had fragmented, variably condensed nuclei along their lengths. Similar abnormal nuclei and spindle structures were observed when snxA1/nimA5 strains were germinated at 32C and upshifted to 42C for 3 hours. Additionally, snxA1 was shown to suppress a checkpoint defect observed in nimA5 cells: Double mutants were able to grow in the presence of 20mM hydroxyurea, while nimA5 cells were sensitive at 10mM hydroxyurea. Cloning and further characterization of the snxA gene will provide clues to the interactions of NIMXCDC2, NIMA, SNXA, and the regulation of mitotic spindle formation. Supported by NIHGM55885-03 and the Mississippi Functional Genomics Network.

42. The isolation and identification of a Cephalosporin transporter homologue in Penicillium chrysogenum. Nijland JG, Evers ME, Driessen AJM. Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.

The pathway of the biosynthesis of penicillin and the cellular localisation of the critical enzymatic steps has been elucidated. Little is known about the transport processes that play a role in beta-lactam excretion by Penicillium chrysogenum. Overexpression of the CefT gene in Acremonium chrysogenum resulted in a two-fold increase in cephalosporin C production in this organism. Therefore, we have used a PCR-based cloning strategy to identify transporters involved in antibiotics transport in P. chrysogenum. By means of sets of degenerate primers, a MFS transporter homologue was identified that is expressed under conditions of beta-lactam production. The full length CefT gene was obtained from a phage DNA library and showed homology (53%) to the CefT in A. chrysogenum. Expression studies revealed the increased level of mRNA under penicillin inducing conditions of the CefT homologue.

Expression of the CefT gene of A. chrysogenum and the homologues gene of P. chrysogenum, both fused to GFP were overexpressed in P. chrysogenum. Both genes show localisation at the plasma membrane.

To determine the substrate specificity of the CefT of A. chrysogenum it was also expressed in E.coli and in insect cells. By the use radiolabeled derivatives of cephalosporin, the transport activity will be determined.

43. Disordered cell integrity signaling caused by disruption of the kexB gene in Aspergillus oryzae. Osamu Mizutani, Tomonori Fujioka, Youhei Yamagata, Keietsu Abe, and Tasuku Nakajima. Molecular and Cell Biology, Tohoku University, Sendai, Japan

To examine the physiological role of kexB, which encodes a subtilisin-like processing enzyme, in A. oryzae, we constructed a kexB disruptant (delta-kexB), which formed shrunken colonies with poor conidia generation on Czapek-Dox (CD) agar plates. The phenotypes of the delta-kexB strain were restored under high-osmolarity in solid culture conditions. We found that transcription of the mpkA, which encodes a putative mitogen-activated protein kinase involved in cell integrity signaling, was significantly higher in delta-kexB cells than in wild-type cells. The delta-kexB cells also contained higher levels of transcripts for cell wall-related genes encoding ss-1,3-glucanosyltransferase and chitin synthases. As expected, constitutively increased levels of phosphorylated MpkA were observed in delta-kexB cells on the CD plate culture. High osmotic stress greatly downregulated the increased levels of both transcripts of mpkA and phosphorylated form of MpkA in delta-kexB cells, concomitantly suppressing the morphological defects. These results suggest that the upregulation of transcription levels of mpkA and cell wall biogenesis genes in the delta-kexB strain is autoregulated by phosphorylated MpkA as the active form through cell integrity signaling. We think that KexB is required for precise proteolytic processing of sensor proteins in the cell integrity pathway or of cell wall-related enzymes under transcriptional control by the pathway and that the KexB defect thus induces disordered cell integrity signaling.

44. Vegetative incompatiblity, TOR pathway and autophagy in Podospora anserina. Bérangère Pinan-Lucarré and Corinne Clavé, IBGC UMR 5095 CNRS, laboratoire de génétique moléculaire des champignons, 33077 Bordeaux France.

Vegetative incompatibility is an ubiquitous phenomenon in filamentous fungi. It prevents the formation of viable heterokaryons between strains non-isogenic at het loci. The heterokaryon resulting from cell fusion is rapidly destroyed by a cell death reaction. A main feature of the incompatibility reaction is the intense vacuolization of the cytoplasm. Autophagy is a pathway allowing degradation of macromolecules and organelles in vacuoles. It is also associated with type II programmed cell death. We have shown that autophagy is induced during incompatibility. We propose to determine if autophagy is responsible for cell death by incompatibility. Therefore we constructed a mutant strain disrupted for the autophagy gene atg1-Pa. We have got cytological evidence that autophagy is blocked in this mutant. Surprisingly, this mutant is not impaired in rapid and intense vacuolization of the cytoplasm and the incompatibility reaction still occurs. In eukaryotes, autophagy is negatively regulated by TOR kinase, a central growth controller. Treatment of Podospora with rapamycin, a highly specific TOR inhibitor in most eukaryotes, induces a lot of features of incompatibility including cell death. We proposed that interaction between incompatible het genes results in Pa-tor inactivation.

In order to ensure that rapamycin has also a direct effect on TOR in Podospora, we constructed a mutant allele by site directed mutagenesis in the conserved rapamycin binding domain. Transformation of Podospora with this allele confers rapamycin resistance. This indicates that the toxicity of rapamycin is mediated via the TOR pathway in Podospora.

45. Ceramide Signals Death in the Heat-Stress Response. Nora Plesofsky and Robert Brambl, Department of Plant Biology, University of Minnesota, St. Paul, MN 55108.

Neurospora crassa displays a typical heat shock response at 45ºC, synthesizing the major heat shock proteins strongly, but transiently. This response allows Neurospora to survive an otherwise lethal exposure (2 hr) to 50ºC. More recently we have found conditions that are lethal for Neurospora when it is exposed to 44.5ºC, by adding inhibitory glucose analogs, thereby reducing availability of carbohydrate and energy stores. Surprisingly, the lethality of this combined heat and carbon stress depends on biotin being present in the culture medium; without biotin the cells survive the combined stresses. The response of relevant mutant strains of Neurospora indicates that fatty acid synthesis is required for lethality. To identify the type of lipid molecule responsible for the stress-caused death, we added various fatty acids and sphingolipids to the biotin-lacking cells during stress. These experiments strongly suggest that ceramide (the mammalian type) is the cause of death. We are in the process of determining what sphingolipids are induced in vivo by the combined heat and carbon stress that might be responsible for signaling cellular death in Neurospora. Our first approach has been to radiolabel lipids being synthesized under various normal and stress conditions and to separate the component lipids by thin layer chromatography. We have also added inhibitors of de novo sphingolipid biosynthesis to the cells to help identify these molecules.

46. Molecular and functional characterization of spk1 encoding a putative sphingosine kinase of Fusarium oxysporum. Rafael C. Prados-Rosales and Antonio Di Pietro. Departamento de Genetica, Universidad de Cordoba, Campus de Rabanales Ed. C5, 14071 Cordoba, Spain

Sphingolipid long chain bases are important eukaryotic signalling molecules that control vital cellular processes such as proliferation, differentiation and death. In mammalian systems, sphingosine kinase, the enzyme that converts sphingosine to the phosphorylated derivative sphingosine 1-phosphate (S1P), has been implicated in the activation of the extracellular signal-regulated kinase (ERK) MAPK cascade. The orthologous Pmk1-type MAPK cascade in fungi is required for plant infection in a large number of biologically diverse pathogens. At present, the signals and upstream components that activate the Pmk1 pathway during infection are largely unknown. We have initiated a study on the possible role of sphingosine kinase in the activation of Fmk1, a Pmk1-type MAPK from the vascular wilt fungus Fusarium oxysporum that is required for root penetration and invasive growth on plant tissue. To this end, we have cloned the F. oxysporum spk1 gene, encoding a putative polypeptide with homology to sphingosine kinases of yeast and humans, and have constructed an spk1 allele disrupted by the hygromycin B resistance marker. Knockout mutants are being generated to study the role of Spk1 in signalling and pathogenicity of F. oxysporum.

47. Web-based illustrations of the Neurospora sexual biology. Namboori B. Raju, Stanford University.

The newly crafted Perkins lab website at Stanford University provides useful resources for Neurospora biologists, as well as general mycologists and students. The resources include a photo collection covering: a) meiosis and ascospore development in wild type and mutants of Neurospora crassa and N. tetrasperma, b) Spore killers, c) chromosome rearrangements, d) meiotic silencing, e) visualization of gene expression using GFP-tagged genes, and f) programmed ascospore death in Coniochaeta tetraspora. The photo series are organized into various chapters, each with a summary description and captioned illustrations. The photo archive is accessible through the internet (http://stanford.edu/group/neurospora), and high-resolution versions of these photos are available for educational purposes (not for profit) upon request from the author. The website also includes a complete list of publications from the Perkins lab since 1949, with PDF files of many recent papers and reviews. David Jacobson's help in creating the web site is greatly appreciated. (Support: MCB 0417282 from NSF.)

48. Visualization of meiotic silencing by unpaired DNA (MSUD) using GFP-tagged histone H1 and ß-tubulin in Neurospora crassa. Namboori B. Raju¹, Michael Freitag², and Robert L. Metzenberg³. ¹Stanford Univ., ²Univ. Oregon, and ³UCLA.

Shiu et al. (2001, Cell 107:905) discovered that genes (e.g. actin, ß-tubulin, mei-3) inserted into N. crassa genome at an ectopic location are not expressed during meiosis in heterozygous crosses where they are unpaired; they coined the term "meiotic silencing by unpaired DNA" (MSUD). With GFP-tagged genes, we can not only localize the gene product, but also observe the expression of MSUD by examining developing asci (Freitag et al. 2004, FGB 41:897). We used histone H1-GFP and ß-tubulin-GFP gene inserts at his-3. -- When heterozygous, hH1-GFP was silenced during ascus development, but only until the time of ascospore delimitation, when it again became active, and the nuclei in four of the eight ascospores fluoresced during development and maturation. In contrast, hH1-GFP was not silenced in homozygous asci, where the nuclei glowed throughout ascus development. ß-tubulin-GFP was also silenced when heterozygous, but not when homozygous. -- RNA-dependent RNA polymerase is required for meiotic silencing. Mutations in sad-1, which lacks the enzyme, suppress meiotic silencing in a semidominant manner (Shiu and Metzenberg 2002, Genetics 161:1483). In crosses of hH1-GFP and ß-tubulin-GFP with Sad-1, both hH1-GFP and ß-tubulin-GFP are expressed throughout ascus development. Spore killers Sk-2 and Sk-3 also act as suppressors of MSUD, similar to Sad-1. (Support: MCB 0417282 and MCB 0131383 from NSF.)

49. The suppression of meiotic silencing by Spore killers Sk-2 and Sk-3 in Neurospora crassa. N. B. Raju¹, P. K.T. Shiu² and R. L. Metzenberg². ¹Stanford Univ., ²UCLA.

When genes that are required for meiosis are inserted at an ectopic location, they are not expressed in heterozygous crosses because of meiotic silencing by unpaired DNA (MSUD); consequently, ascus development is abnormal. RNA-dependent RNA polymerase is required for MSUD, and mutations in sad-1, which lack the enzyme, suppress MSUD in a semidominant manner (Shiu et al. 2001; Shiu and Metzenberg 2002). – Spore killers (Sk-2^K or Sk-3^K), when crossed with wild type (Sk^S), cause the death of four of the eight ascospores that do not contain the killer (Turner and Perkins 1979). We report here that Sk-2 and Sk-3 resemble Sad-1, a suppressor of MSUD, in suppressing the silencing of various ectopically inserted genes (Actin, ß-tubulin, Mei-3, hH1-GFP and ß-tubulin-GFP). Meiotic silencing of Asm-1 is also suppressed but only partially, and Round spore is not suppressed at all. We have demonstrated the suppression of meiotic silencing visually by observing developing asci in Sk^K x hH1-GFP and Sk^K x ß-tubulin-GFP. Both hH1-GFP and ß-tubulin-GFP were expressed during meiosis, and all eight young ascospores showed expression of GFP. As the asci matured, the four Sk^S ascospores aborted and degenerated. The remaining four Sk^K ascospores developed normally, with or without the GFP glow, depending upon the segregation of Sk and GFP-tagged genes, which are unlinked. Suppression of MSUD by Sk-2 or Sk-3 does not depend upon spore killing, however. Although both Sad-1 and the two Spore killers suppress MSUD, they differ significantly from one another: Sad-1 is homozygous barren, where as Sk-2 and Sk-3 are both homozygous fertile. (Support: MCB 0417282.)

50. A new strategy for identifying cell wall biosynthetic genes in Aspergillus niger. R.A. Damveld¹, M. Arentshorst¹, P.A. vanKuyk¹, F.M. Klis², C.A.M.J.J. van den Hondel^1,3, A.F.J. Ram^1,3*. ¹Leiden University, Institute of Biology Leiden, Fungal Genetics Research Group, Leiden, The Netherlands, ²University of Amsterdam, Swammerdam Institute for Life Sciences, Amsterdam, The Netherlands. ³Department of Microbiology, TNO Nutrition and Food Research, Zeist, The Netherlands.

The fungal cell wall is an essential organelle. We are interested in identifying new target genes involved in cell wall formation for the development of new antifungal compounds. We found that the expression levels of several genes involved in cell wall biosynthesis were heavily up-regulated when the cell wall integrity of germlings was compromised by the addition of Calcofluor White (CFW). Northern analysis indicated that the mRNA levels of agsA, encoding an alpha-1,3-glucan synthase, and gfaA, encoding an glutamine:fructose-6-phosphate amino-transferase involved in de biosynthesis of UDP-N-acetyl-glucosamine, were induced 20-fold and 4-fold, respectively. Both genes including at least 2-kb of their respective promoter sequences were cloned. Subsequently, we designed a genetic screen for the isolation of cell wall mutants by cloning the agsA promoter region in front of a selectable marker (AmdS). The rationale of this screen is that a mutation in a cell wall biosynthetic gene is expected to result in cell wall weakening and as a result will trigger the expression of the amdS gene from the agsA promoter, allowing growth on acetamide as the sole N-source. Two hundred and forty mutants were isolated that were able to grow on acetamide. Thirteen of these showed an osmotic-remediable, temperature-sensitive growth defect at 37̊C. Complementation of the mutants and identification of the genes involved is expected to lead to the discovery of new antifungal targets related to cell wall biosynthesis.

51. Characterisation of CwpA, a putative glycosylphosphatidylinositol anchored cell wall mannoprotein in the filamentous fungus Aspergillus niger. R.A. Damveld¹, M. Arentshorst¹, P.A. vanKuyk¹, F.M. Klis², C.A.M.J.J. van den Hondel^1,3, A.F.J. Ram^1,3*. ¹Leiden University, Institute of Biology Leiden, Fungal Genetics Research Group, Leiden, The Netherlands, ²University of Amsterdam, Swammerdam Institute for Life Sciences, Amsterdam, The Netherlands. ³Department of Microbiology, TNO Nutrition and Food Research, Zeist, The Netherlands.

In fungi, glycosylphosphatidylinositol (GPI)–anchored proteins are found at the cell surface, either attached to the plasma membrane (GPI-PMPs) or attached via a remnant of the GPI-anchor to the cell wall. In the yeasts S. cerevisiae and C. albicans, it has been shown that GPI-anchored cell wall mannoproteins (GPI-CWPs) become attached to the beta-1,3-glucan or chitin part of the cell wall through a flexible beta-1,6-glucan moiety. GPI-CWPs can be extracted from the fungal cell wall by treatment with hydrofluoric acid (HF), which cleaves the phosphodiester bonds which is present in the remnant of the GPI-anchor. We show that A. niger contains at least five HF-extractable cell wall mannoproteins. A gene encoding an HF-extractable cell wall mannoproteins was cloned and named cwpA. The protein sequence of CwpA indicated the presence of two hydrophobic signal sequences at the N-terminus and C-terminus of the protein, for entering the ER and the addition a GPI-anchor, respectively. A CwpA specific antiserum was raised and in combination with simple fractionation experiments, we confirmed that this protein was hardly present in the membrane fraction and abundantly present as a HF-extractable protein in the cell walls. Deletion of cwpA did not show an apparent difference in growth under normal growth conditions. The cwpA deletion strain displayed however a more sensitive phenotype towards the cell wall disturbing compound Calcofluor White. The latter result indicate the CwpA might have a structural role in maintaining the integrity of the cell wall and that the absence of CwpA leads to weakening of the cell wall which makes the fungal more sensitive to Calcofluor White.

52. A Rho-type GTPase, rho-4, is required for septation in Neurospora crassa. Carolyn G. Rasmussen and N. Louise Glass. Department of Plant and Microbial Biology. 111 Koshland Hall, University of California, Berkeley, CA 94720-3102

Proteins in the Rho family are small monomeric GTPases primarily involved in polarization, control of cell division and reorganization of cytoskeletal elements. Phylogenetic analysis of predicted fungal Rho proteins suggests that a new Rho-type GTPase family, whose founding member is Rho4 from the archiascomycete Schizosaccharomyces pombe, is involved in septation. S. pombe delta-rho4 mutants have multiple, abnormal septa. In contrast to S. pombe delta-rho4 mutants, we show that rho-4 loss-of-function mutants in the filamentous fungus Neurospora crassa lead to a loss of septation. Both epitope and GFP-tagged RHO-4 were targeted to septa and to the plasma membrane. RHO-4 formed a ring at incipient septation sites that appeared to constrict with the formation of the septum. RHO-4 remained at mature septa and formed a ring around the septal pore. In other fungi, the steps required for septation include formin, septin and actin localization followed by cell wall synthesis and the completion of septation. rho-4 mutants were unable to form actin rings, suggesting that RHO-4 acts upstream of actin localization. Further, the maintenance of RHO-4 localization was independent of an intact actin cytoskeleton or microtubule cytoskeleton. Characterization of activated alleles of rho-4 showed that RHO-4-GTP is likely to initiate new septum formation.

53 Cell biology of conidial anastomosis tubes in Neurospora crassa. M.G. Roca, J. Arlt, C.E. Jeffree and N.D. Read. Institute of Cell Biology/COSMIC, University of Edinburgh, Edinburgh EH9 3JH, UK

Although hyphal fusion has been well documented in mature colonies of filamentous fungi, it has been little studied during colony establishment. Here we show that specialized hyphae, called conidial anastomosis tubes (CATs), are produced by all types of conidia and by conidial germ tubes of Neurospora crassa. The CAT is shown to be a cellular element that is morphologically and physiologically distinct from a germ tube, and under separate genetic control. In contrast to germ tubes, CATs are thinner, shorter, lack branches, exhibit determinate growth and home towards each other. Evidence for an extracellular CAT inducer derived from conidia was obtained because CAT formation was reduced at low conidial concentrations. A cr-1 mutant lacking cAMP produced CATs indicating that the inducer is not cAMP. Evidence that the transduction of the CAT inducer signal involves a putative transmembrane protein (HAM-2) and the MAK-2 and NRC-1 proteins of a MAP kinase signaling pathway was obtained because ham-2, mak-2 and nrc-1 mutants lacked CATs. Optical tweezers were used as a novel experimental assay to micromanipulate whole conidia and germlings to analyze chemoattraction between CATs during homing. Strains of the same and opposite mating type were shown to home towards each other. The cr-1 mutant also underwent normal homing indicating that cAMP is not the chemoattractant. Fusion between CATs of opposite mating type was partially inhibited providing evidence of non-self recognition prior to fusion.

54. Live imaging of the secretory pathway in hyphae of Neurospora crassa. Meritxell Riquelme¹, Michael Freitag², Eddy Sánchez León-Hing¹ and Barry Bowman³. ¹Department of Microbiology. Center for Scientific Research and Higher Education of Ensenada. Baja California. México; ²Institute of Molecular Biology, University of Oregon. Eugene; and ³Department of Molecular, Cell & Developmental Biology. University of California, Santa Cruz.

Fungal hyphae elongate by apical growth, a complex process that involves the polarized traffic of organelles to the cell apex, and the exocytosis of vesicles in areas of active cell growth. One of the unanswered questions about the establishment of fungal polarity is how vesicles are formed and sorted from sites of synthesis to sites of exocytosis at the plasma membrane. Knowing the correct localization and traffic routes of membrane proteins is essential to better understand how the secretory machinery operates in polarized hyphae. As part of an ongoing project to characterize the organization of the secretory pathway in filamentous fungi, we have set out to localize the plasma membrane H⁺-translocating ATPase, a protein carried by secretory vesicles from their point of synthesis in the ER to their destination, along hyphae of Neurospora crassa.

We have fused the H⁺-ATPase encoding gene pma-1 from N. crassa, with gfp and studied its expression in living hyphae of N. crassa. Transformants showing positive fluorescence were selected and analyzed by confocal microscopy. The GFP labeled H⁺-ATPase was found at the septa and the cell surface of distal parts of the hyphae but not at the apex. Our results confirm earlier predictions (C. L. Slayman, Yale Univ.) that the H⁺-ATPase is deficient or inactive at the apex but abundant distally, and provide insight into one of the different pathways for the delivery of proteins from Golgi to the cell surface.

55. Characterization of the Neurospora crassa DNA-repair mutants, mus-43 and mus-44. Masahito Sato, Takeru Toko, Takaharu Niki, Yasunori Kato, Masashi Tani, Shintaro Amano, Akihiko Ichiishi. Dept of Life Sciences, TOYO University, Itakura, Gunma, Japan

We previously identified the mus-43 gene, which was the homolog of human XPA and Saccharomyces cerevisiae RAD14, and the mus-44 gene, which was the homolog of human ERCC1 and S. cerevisiae RAD10. Using the repeat-induced point mutation (RIP), we isolated the mutants of those genes. Those mutants were sensitive both to UV and 4-NQO, but not sensitive to MMS. The mus-44 mutant showed the same sensitivity to UV as the mus-38, the previously isolated NER mutant. While the sensitivity of the mus-43 mutant to UV were milder than the mus-38 or mus-44. To confirm thatthose genes were exclusively involved in NER, epistasis were tested for other repair genes. The four genes, the NER gene mus-38, the post-replication repair gene uvs-2, the recombination repair gene mei-3, and the second excision repair gene mus-18, were selected for this purpose. The mus-38 mus-43 and the mus-38 mus-44 double mutants showed the same UV-sensitivity as the mus-38 mutant, while the other double mutants were more sensitive to UV than the parental single mutants.¡¡These results indicate that the mus-43 and the mus-44 are components of NER and not involved in other repair systems.

 The mus-43 and mus-44 mutants were analyzed whether it had the ability to remove CPDs using the Micrococcus luteus endonuclease specific to CPDs. These mutants did not show the ability to remove CPDs from the damaged DNA. The two mutants were also analyzed by ELISA using the antibodies specific to CPDs or TC(6-4) photoproducts.

56. Geosiphon pyriformis as a model organism for the arbuscular mykorrhiza. Arthur Schuessler and Holger Martin. Darmstadt University of Technology, Institute of Botany, Darmstadt, Germany.

Geosiphon pyriformis is the only known fungus forming endosymbiosis with cyanobacteria. Together with the arbuscular mycorrhizal (AM) fungi, forming AM with the majority of land plants, Geosiphon belongs to the Glomeromycota. The Geosiphon-Nostoc association therefore represents a symbiosis of an ‘AM fungus' with a photoautotrophic prokaryote. Due to this unique life style, the investigations of Geosiphon offers some fundamental advantages when compared to AM. E.g., for gene expression studies, the fungal poly-A mRNA can be easily isolated. Therefore, we use Geosiphon for the identification of 'AM fungal' genes. In the Geosiphon/Nostoc and AM symbioses, the symbiotic interface is the site of nutrient exchange. Via the perisymbiotic membrane the mycobiont receives large amounts of carbohydrates from the photobiont and specific fungal nutrient-transporters are expected to exist.

Methods were established to reproducibly isolate fungal mRNA from small samples. After RT, cDNA was amplified by SMART-LD-PCR, size fractionated, ligated in a yeast expression vector, and E. coli cells were transformed to construct an expression library.

Since we try to characterize transporter genes involved in nutrient exchange, we used the library for functional complementation of yeast mutants deficient for uptake of amino acids or hexoses. A yeast mutant deficient in hexose uptake was functionally complemented and results about the characterization of the respective gene will be shown.

57. Induction of Apoptosis in Aspergillus Nidulans by the Candida Albicans Quorum- sensing Molecule Farnesol. Camile P. Semighini, Kenneth W. Nickerson ¹ and Steven D. Harris. Plant Science Initiative, ¹ School of Biological Sciences, University of Nebraska Lincoln

Candida albicans produces farnesol as an extracellular quorum-sensing molecule. After accumulation above a threshold level, farnesol prevents the yeast to mycelium transition and also blocks formation of biofilm. To our surprise, at concentrations ranging from 20 mM and 200 mM, farnesol has no apparent effect on hyphal morphogenesis in Aspergillus nidulans. Instead, it triggered rapid DNA condensation independent of mitosis, intense DNA fragmentation and exposure of phosphatidylserine. These are morphological features that are characteristic of apoptosis. Moreover, farnesol treatment also induced the accumulation of reactive oxygen species. Farnesol is not produced by A. nidulans hyphae during growth in liquid culture media, suggesting that it is not acting as a quorum-sensing molecule to regulate colony morphology. Instead, we propose that A. nidulans responds to farnesol produced by other fungal species. Future efforts will address the mechanism of farnesol-induced apoptosis, with the goal of identifying potentially novel targets for the development of anti-fungal compounds.

58. Six localisations for six hydrophobins: differential targeting in C. fulvum. Hélène LACROIX and Pietro D SPANU, Imperial College London

Cladosporium fulvum has at least 6 hydrophobins named HCf-1 to -6. We have shown that expression of these genes is differentially regulated. We have now investigated the localisation of the hydrophobins during development. To do this we have created transgenic strains of the fungus that carry hydrophobins with the V5 epitope tag. Antibodies that recognise this tag do not cross react with any other C. fulvum epitope, the tag is retained in the mature protein if attached close to the C-terminus and does not interfere with the activity of the hydrophobin for which we observe a phenotype. The V5 tag therefore allows us to localise specifically all the hydrophobins identified in this fungus. Our findings to date show that in axenic culture, not only are the hydrophobins produced at different times, but, most unexpectedly, that they were located in different portions of the mycelium. Thus HCf-1 was found in large patches on the conidia, and on the aerial hyphae that produce the conidia. HCf-2 was in a similar position to HCf-1 but the patches were much less frequent. HCf-4 appeared in small areas both in the mycelium that is submerged in the agar and in the aerial hyphae. HCf-5 was visible only on the basal portion of the aerial hyphae at the time of sporulation, but was not found on the conidia. HCf-6 was secreted by the young hyphae 24 hours after germination and was evident as a layer on the surface of the growth medium, not associated with the fungal structures. We will also present data on the localisation of the hydrophobins as C. fulvum infects its host, tomato and then discuss the significance of these findings in relation to the possible structure and function of these ubiquitous fungal proteins.

59. Myosin V and conventional kinesin cooperate in hyphal growth of the corn smut fungus Ustilago maydis. Isabel Manns, Daniela Aßmann, Anne Straube¹, Uta Fuchs and Gero Steinberg. Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Straße, D-35043 Marburg, Germany; ¹Wellcome Trust Centre for Cell Biology, University of Edinburgh, Scotland

Long-distance membrane transport is crucial for function and survival of polarized growing cells, such as neurons and fungal hyphae. While evidence exists for a role of microtubules-based kinesins and F-actin associated myosins in polar cell expansion, their functional interplay is only poorly understood. Here we set out to elucidate the role of motors in polarized hyphal growth of fungus Ustilago maydis. In hyphae ~90% of all microtubules plus-ends are directed to the expanding tip. However, out of 8 kinesins only Kin3, a Kif1A-like kinesin, and the conventional kinesin Kin2 are crucial for hyphal growth. Dkin3, Dkin2 and kin2/kin3 hyphae were irregular and much shorter, but were still able to grow polarized. A similar phenotype was found in hyphae deleted in myo5, a class V myosin. Interestingly, cell polarity is lost in Dkin2Dmyo5 double mutant, suggesting that both motors cooperate independently to support polar hyphal growth. However, in Dkin2 and Dmyo5 cells, as well as in Dkin2Dmyo5 double mutants phosphatase secretion was reduced to a similar extend, indicating that Myo5 and Kin2 deliver the same class of secretory vesicles. In addition, the tip localization of Kin2-GFP3 requires F-actin and Myo5, and a YFP-Kin2tail fusion protein moves independently of microtubules in a Myo5/actin-dependent manner, suggesting that conventional kinesin is a cargo of myosin V.

60. Blue light negatively regulates the sexual filamentation via the Cwc1 and Cwc2 proteins in C. neoformans. Ying-Ku Lu¹, Kai-Hui Sun¹, and Wei-Chiang Shen ¹. ¹Department of Plant Pathology and Microbiology, National Taiwan University, Taipei 106, Taiwan.

Most organisms are capable of sensing light and make appropriate physiological adjustments via complex mechanisms. We use Cryptococcus neoformans, a heterothallic basidiomycetous fungus, as a model system to dissect the molecular mechanisms regulating the light responses. In our studies, we demonstrate that the production of sexual dikaryotic filaments is inhibited by blue light. To reveal the molecular mechanisms of blue light photoresponses in C. neoformans, we have identified and characterized two genes, CWC1 and CWC2, which are homologs of the Neurospora crassa wc-1 and wc-2 genes. Analyses of the features and organization of the putative domains indicate that the functions of Cwc1 and Cwc2 proteins may be evolutionally conserved. Through mutation analyses, both cwc1 and cwc2 mutant strains have shown to be insensitive to light. Furthermore, overexpression of the CWC1 or CWC2 gene requires light activation to inhibit sexual filamentation. Taken together, our findings illustrate that blue light negatively regulates the sexual filamentation via the Cwc1 and Cwc2 proteins in C. neoformans. Models are proposed to describe how blue light responses are regulated by the Cwc proteins in C. neoformans, and research is ongoing to test these hypotheses.

61. The ClaCWH41 gene is involved in appressorial formation and pathogenicity of Colletotrichum lagenarium. Rie Matsui¹, Toshihiko Miyaji¹, Gento Tsuji¹, Tomonori Shiraishi², Richard O'Connell¹ and Yasuyuki Kubo¹. ¹Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto Prefectural University, Kyoto, Japan. ¹Laboratory of Plant Pathology, Faculty of Agriculture, Okayama University, Okayama, Japan.

Colletotrichum lagenarium is the causal agent of anthracnose of cucumber. This fungus forms a heavily melanized infection structure called an appressorium. Recently we isolated the mutant p51 by Restriction Enzyme-Mediated Integration mutagenesis, which showed weak pathogenicity. We rescued a fragment from the disrupted gene in the mutant p51. The rescued fragment showed homology with the CWH41 gene of Saccharomyces cerevisiae. The CWH41 gene encodes alpha-glucosidase and is involved in cell wall alpha-1,6-glucan biosynthesis. We designated the disrupted gene as ClaCWH41. To define the character of ClaCWH41, we constructed ClaCWH41 gene disruption mutants. The clacwh41 mutants were weakly pathogenic on host plants and showed decreased ability to penetrate artificial cellulose membranes. The colony morphology of clacwh41 mutants was normal, but hyphal growth rates were slightly reduced compared to the wild-type. The most clear difference was observed in appressorium development on glass slide. The clacwh41 mutants formed abnormally shaped appressoria. These results indicate that the ClaCWH41 gene plays a key role in appressorium development.

62. Live-cell imaging of the secretory pathway in Trichoderma reesei. Mari Valkonen^1,2, Merja Penttilä¹, Markku Saloheimo¹ and Nick D. Read². ¹VTT Biotechnology, P.O. Box 1500, FIN-02044 VTT, Finland. ²Fungal Cell Biology Group, Institute of Cell Biology, University of Edinburgh, Edinburgh EH9 3JH, UK

Available data suggest that the basic principles of protein secretion obtained from studies on S. cerevisiae and animal cells also apply to filamentous fungi. Although considerable research has been performed on the secretion process in filamentous fungi, little is known about their secretory machinery.

The transport of proteins between the cellular compartments occurs in membrane-bounded vesicles that bud from the donor and fuse with the acceptor compartment. Membrane bound proteins called SNAREs (Soluble N-ethylaleimide-sensitive factor Attachment protein REceptors) play a key role in membrane fusion. SNAREs have been found to be involved in most membrane fusion events in the cell.

This study describes the cloning and characterization of a putative exocytotic t-SNARE (sso1) from Trichoderma reesei. We cloned the T. reesei homologue of Neurospora crassa nsyn2. This protein was labeled with the fluorescent protein YFP and the fusion protein expressed in T. reesei. Transformants expressing the fusion protein exhibit a clear labeling of the plasma membrane. The fusion protein is uniformly distributed along the hyphae with no tip-focused gradient.

We are precently labeling the v-SNARE SNCI with mRFP with the aim of analyzing its interaction with the t-SNARE SSOI using fluorescence resonance energy transfer (FRET) imaging

63. Study of cell wall integrity and disturbing antifungal compounds with a novel GFP-based reporter method. Robert Damveld¹, John van Dam ¹, Frans Klis², C.A.M.J.J. van den Hondel^1,3 and A.F.J. Ram^1,3. Institute of Biology,Wassenaarseweg 64, 2333 AL Leiden, The Netherlands, ² Swammerdaam Institute for Life Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam ³Department of Applied Microbiology and Gene Technology, TNO-Nutrition, 3700 AJ Zeist, The Netherlands.

Activation of the fungal cell wall integrity pathway is a mechanism used by fungi to escape from cell wall threatening conditions. In Aspergillus niger we have previously shown that the agsA gene, encoding an á-1,3-glucan synthase submit is transcriptionally activated in response to cell wall stress, and that this response is mediated via a RLMA dependent signal transduction pathway. We have used the agsA promoter region to set up a GFP-based reporter system to identify compounds that activate the agsA expression by fusing the PagsA to the cytosolic GFP or to a nuclear targeted version of GFP. The reporter strains confirmed earlier observation that the agsA gene is activated in response to various cell wall disturbing compounds such as CFW, caspofungin and tunicamycin. Other forms of stress (osmotic or oxidative stress) did not activate agsA expression, indicating that the induction of agsA is cell wall stress specific and not a general stress response. We have used the cell wall stress reporter system to study the effect of 20 known antifungal compounds in relation to cell wall remodeling.

64. Isolation of septal pore caps from basidiomycetous fungi. Kenneth G.A. van Driel¹, A.F. van Peer, H.A.B. Wösten, A.J. Verkleij, W.H. Müller, T. Boekhout. ¹Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands.

The septal pore cap (SPC) structure covers the dolipore, a septal pore surrounded by a donut-like swelling, in many basidiomycetous fungi. The SPC is a membranous structure associated with endoplasmic reticulum. The morphology of the SPC is diverse within the different phylogenetic groups of basidiomycetes and can be divided in several main categories: vesiculate or tubulate, imperforate, and perforate. Though electron microscopical (EM) studies revealed the SPC in great detail, the function of the SPC is only poorly understood. Our aim was to isolate and enrich SPCs to characterize its proteins and genes that are involved in the formation of the SPC. This will lead to a better understanding of the role of SPCs in basidiomycetous cells .

We successfully enriched SPCs from Rhizoctonia solani cell fractions. After EM studies we observed that the plug material at the orifice of the septal pore channel stayed attached via fibrillar material to SPCs. Protein electrophoresis showed that a 18 kDa glycoprotein was enriched in the SPC fraction. This protein was N-terminally sequenced. We raised antibodies against this protein to perform immunolabeling studies. From our observations we think that the SPC may be involved in the production of plugging material. Alternatively, the SPC may function as a repository of the plugging material that can be released upon plugging the septal pore during i.e. stress situations.

65. Complementation of a Calcofluor-hypersensitive mutant in Aspergillus nidulans by a novel transmembrane protein. Stanley Vance, Lisa Harsch, Darlene Loprete and Terry Hill. Departments of Biology and Chemistry, Rhodes College, Memphis, TN.

In a search for as-yet-unidentified genes whose function bears upon cell wall integrity, this laboratory is screening strains from the Harris et al. collection (Genetics 136:516-532, 1994) of Aspergillus nidulans temperature-sensitive morphology mutants, as well as mutant strains of our own creation, using hypersensitivity to the cell wall compromising agent Calcofluor White (CFW) as the primary screening criterion. One such strain, ts1-49, shows hypersensitivity not just to CFW, but also to the unrelated wall-compromising agent Congo Red and to the detergent SDS, when compared to the respective wild type. The strain does not, however, manifest a morphological phenotype at 42C. We have complemented the hypersensitive aspects of the phenotype with two separate plasmids from the Osherov and May (Genetics 155: 647-656, 2000) "AMA NotI" genomic DNA library. The overlapping regions of the two plasmids contain a single predicted gene, designated AN2880.2 (Aspergillus Sequencing Project, Center for Genome Research). The ca. 114 kDa hypothetical translated product contains a ca. 538 amino acid long internal sequence which is homologous to DUF221 domains found in a variety of multipass transmembrane proteins of unknown function. On-line domain-predicting programs predict eleven membrane-spanning helices, with the N-terminus oriented towards the cell interior. Work is underway to sequence the corresponding region of the mutant (ts1-49) genomic DNA, to determine whether AN2880.2 is the gene mutated in strain ts1-49 or a high-copy suppressor.

66. The role of a spindle pole body associated protein in cytoplasmic microtubule organization. Daniel Veith and Reinhard Fischer. University of Karlsruhe and University of Marburg.

Polarized growth and nuclear migration are key features of both uninucleate yeasts and syncytial filamentous fungi. Microtubules and microtubule-dependent motor proteins are key players, whose arrangement, interaction and contribution to nuclear distribution are in the focus of interest. We have studied an A. nidulans mutant, apsB, in which nuclear positioning is disturbed and identified ApsB (GFP-ApsB) as a novel spindle-pole body (SPB) associated protein. Interestingly, ApsB not only localises to SPB́s of interphase nuclei and the poles of the mitotic spindle, but unexpectedly, it was also detected at septa. Even more surprisingly, ApsB was transported very fast along microtubule tracks towards the plus- or the minus-end of microtubules. Recently, it was shown that microtubule-organizing centres were localized at nuclei, in the cytoplasm, and at septa. Thus, ApsB appeared to play a role in microtubule formation. In agreement with this, in apsB mutants the network of cytoplasmic microtubules as well as the number of astral microtubules was dramatically reduced, explaining the disturbed nuclear distribution pattern.

67. Flo11p, an S. cerevisiae flocculin, mediates adhesion to extracellular matrix proteins. S. Venkatraman, D.C. Bartelt and A.M. Dranginis. St. John's University, Dept. Biology, Queens, NY 11439.

The cell wall protein Flo11p is required for a variety of processes that involve adhesion in S. cerevisiae, including flocculation, adhesion to plastic and agar substrates, development of pseudohyphae and formation of biofilms. Flo11p is not expressed in the standard laboratory strain of S.cerevisiae, S288C, due to a mutation in a transcriptional activator required for its expression. Unlike many fungi, S288C-background yeast is not known to adhere specifically to mammalian extracellular matrix (ECM). We tested the hypothesis that Flo11p is the molecule responsible for ECM binding, by coating wells of microtiter plates with various ECM proteins and measuring the binding of yeast to the wells. FLO11-expressing yeast bound to wells coated with the ECM proteins fibronectin and laminin, whereas cells with a mutation in FLO11 failed to bind under these conditions. Flo11p does not mediate nonspecific binding to all glycoproteins, since no binding was observed to wells coated with carboxypeptidase Y, fetuin or asialofetuin. The binding of Flo11-bearing cells to fibronectin was saturable and dose-dependent. Pre-incubation of the yeast cells with fibronectin resulted in greatly reduced binding, as did pre-incubation of the coated wells with antibodies to fibronectin. The binding of FLO11-expressing yeast to ECM proteins thus appears to be specific, and provides a model for the study of adhesion of fungi to human tissue.

Supported by NIH grant 1R15A143927-01 and NSF grant MCB-9973776.

68. Unique roles for Aspergillus nidulans Spa2 and Bud6 homologues in polarized growth and septation of hyphae . Aleksandra Virag, Claire Pearson, Steven Harris. Plant Science Initiative, University of Nebraska, Lincoln, NE

Although a growing number of studies address hyphal development in filamentous fungi, how cellular components organize to initiate, maintain and regulate highly polarized hyphal growth is not fully understood. In yeast, a complex termed the polarisome containing Spa2, Pea2 and Bud6, is present at sites of polarized growth, with Bni1 (formin) interacting with both Spa2 and Bud6. These gene products are involved in directing components that are to be incorporated into sites of cell surface expansion to their final destinations. In filamentous fungi, homologues of some polarisome components, such as Bni1, Spa2 and Bud6 are present, while others, such as Pea2, are absent, suggesting a unique role for these genes and gene products in polarized growth of filamentous fungi. In Aspergillus nidulans the Bni1 homologue, SepA, is present at sites of hyphal tip growth, as well as sites of septation. We constructed GFP fusions with the A. nidulans Bud6 and Spa2 homologues (BudA and SpaA) and looked at the distribution of the fusion proteins. SpaA-GFP localized to the hyphal tip, while the BudA-GFP localized to sites of septum formation. Under repressing conditions, BudA::GFP expressed under the regulation of the alcA promoter showed reduced branching and a delay in septation. Our results suggest that both SpaA and BudA function at hyphal tips and branching sites, and that BudA may have an additional function at sites of septum formation. We also present a method that identifies gene products involved in reconstituting a lost polarization axis by assessing the number of hyphae that resume their original direction of growth after depolarizing the tip with cytochalasin A.

69. Initial characterization of a mutation isolated as a suppressor of the col-16 mutation of N. crassa. Michael K. Watters and Margaret Munich. Valparaiso University, Valparaiso, IN.

Growth in filamentous fungi occurs at a tip which branches as it extends. The col-16 mutant of N. crassa is one of many which displays a greater branching frequency than the wild type and therefore grows more densely. Following UV mutagenesis, colonies were screened for those in which more wild type growth had been restored with the goal of finding suppressors. One of the these suppressed strains (col-16, sup28) was chosen for further characterization. Strains containing the suppressor sup28 separated from col-16 were obtained using unordered tetrads. Examination of the morphology of a sup28 mutant strain shows that while the rate of growth is severely reduced, the basic branching pattern (type of branches as well as distance between branch points) is unaltered. Further characterization of the sup28 mutation suggests that it is a leaky auxotroph.

70. In vivo time lapse microscopy of organelle distribution shows that the WASP family member protein Wal1p is required for vacuolar motility but not for mitochondrial distribution in the filamentous ascomycete Ashbya gossypii. Andrea Walther and Jürgen Wendland. Dept. of Microbiology, Friedrich-Schiller University and Leibniz Institute for Natural Products Research and Infection Biology - Hans-Knoell-Institute, Jena, Germany

During polarized hyphal growth in the Ashbya gossypii wild type cytoplasmic streaming can be visualized as the tipward movement of large vacuoles using differential interference contrast (DIC) imaging methods. Interestingly, the direction of cytoplasmic streaming can be reversed due to either contact inhibition of the hyphal tip that blocks further elongation or cessation of growth at the tip. This suggests that hyphal tips act as a sink for cytoplasmic transport . However, septation blocked the passage of vacuoles and thus restricted the cytoplasm in hyphal compartments.

We have analyzed the requirements of the actin cytoskeleton for these processes and describe the role of the Wiskott-Aldrich Syndrome Protein (WASP)-homolog of A. gossypii encoded by WAL1 involved in endocytosis and vacuolar movement.

Wal1 is required for the maintenance of polarized hyphal growth and wal1 mutants exhibit a drastically decreased hyphal growth rate. In the wal1 mutant strain cortical actin patches are not localized to the hyphal tip but accumulate in subapical regions. This positioning of patches directs fluid phase endocytosis away from the hyphal tip to subapical regions. Loss of WAL1 results in defects in endocytosis, vacuole motility and vacuole movement. In contrast, the tipward localization and redistribution of mitochondria is not dependent on Wal1p.

Our results suggest that Wal1p dependent actin assembly is required for endocytosis and vacuolar movement but that other processes, e.g. formin mediated actin assembly may be involved in mitochondrial distribution.

71. Role of Neurospora crassa kinesins. Renate Dombi, Florian Fuchs, Sarah Adio, Benedikt Westermann, Manfred Schliwa, Guenther Woehlke. Adolf-Butenandt-Institute, University Munich, Germany

The genome of Neurospora crassa contains genes for 10 kinesin-related proteins, among them 3 putative microtubule-dependent vesicle motors. The conventional kinesin NcKin has been known for several years, and extensively been characterized in vivo and in vitro. Two unconventional kinesins of the kinesin-3 (fromerly Unc104 or Kif1) family are also present, one of which shows similarity to other kinesin-3 motors (NcKin2). The other kinesin (NcKin3) is unusually short and seems to be specific for filamentous fungi.

We cloned the cDNA of NcKin3, and expressed truncated variants of the motor in bacteria. The in vitro ATPase and motility behavior shows that NcKin3 is a dimeric microtubule motor protein that moves at a speed of 0.6 micro-m/s, but apparently by a different mechanism than conventional kinesins.

To investigate the cellular function of both kinesin-3 familiy motors, we knocked out NcKin2 and NcKin3. None of the knock-out strains shows any apparent phenotype, suggesting that Neurospora is able to suppress the phenotype under laboratory conditions. Unlike in the NcKin knock-out, the growth rate is normal, and the distribution of organelles is unchanged. To test whether both motors act redundantly, we are in the process of crosssing the knock-out strains, and to generate dominant negative mutants. So far, we were able to detect an up-regulation of NcKin3 in the NcKin2 knock-out strain, but no obvious suppressor for the NcKin3 knock-out using a proteomic approach.

We acknowledge financial support from the Deutsche Forschungsgemeinschaft.

72. Optical Tweezer Micromanipulation of Filamentous Fungi. Graham Wright^1,2, Jochen Arlt², M. Gabriela Roca^1,2, Wilson Poon^2,3 & Nick Read^1,2. ¹Institute of Cell Biology, University of Edinburgh, Edinburgh EH9 3JH, UK. ²COSMIC, University of Edinburgh, Edinburgh, EH9 3JZ, UK. ³School of Physics, University of Edinburgh, Edinburgh, EH9 3JZ, UK

Optical tweezers allow the non-invasive micromanipulation of living cells by utilising the forces generated from the interaction of laser light with the sample. We have designed and built a simple, user friendly, steerable optical tweezer system (using a 785 nm diode laser) to manipulate spores, spore germlings and mature hyphae of the model filamentous fungus, Neurospora crassa. More specifically we are: (1) manipulating the so called Spitzenkorper, the behaviour of which is intimately associated with the pattern and direction of tip-growth; (2) using beads to assess and quantify the growing forces of hyphal tips and germlings; (4) disrupting the chemotropic gradients between spore germlings which are growing towards each other by moving one of the participating germlings; and (5) manipulating the Woronin body, an organelle involved in occlusion of septal pores after hyphal damage.

73. The histidine kinase Dic1p is a positive regulator of the Hog1-type MAP kinase in Cochliobolus heterostrophus. Akira Yoshimi¹, Kaihei Kojima^{2, 3}, Yoshitaka Takano², and Chihiro Tanaka¹. ¹Laboratory of Environmental Mycoscience, Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan, and ²Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan. ³Present address: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA.

In Southern corn leaf bright fungus Cochliobolus heterostrophus, the histidine kinase Dic1p is involved in resistance to dicarboximide and phenylpyrrole fungicides and osmotic adaptation. We previously reported that the phenylpyrrole fungicide fludioxonil led to improper activation of Hog1-type MAPKs in some phytopathogenic fungi including C. heterostrophus. To elucidate the relationship of C. heterostrophus BmHog1p (Hog1-type MAPK) and the histidine kinase Dic1p, the phosphorylations of BmHog1p were analyzed in the wild-type and the dic1 deficient strains by western blotting. In the wild-type strain, the phosphorylated BmHog1p was detected after exposure to both dicarboximide iprodione and phenylpyrrole fludioxonil, even at concentration of 1 µg/ml. In the dic1 strain, no phosphorylated BmHog1p was detected after exposure to 1 µg/ml and 10 µg/ml of the fungicides. Similarly, in response to osmotic stress (0.4 M KCl), a little amount of phosphorylated BmHog1p was found in the dic1 strain, whereas the band representing the active BmHog1p was clearly detected in the wild-type strain. Similar results were obtained in the case of Os2p MAPK phosphorylation in the histidine kinase gene os-1 mutants of Neurospora crassa. These results suggest that in general the Dic1/OS1-related histidine kinase is a positive regulator of Hog1-type MAPKs in filamentous fungi. Importantly, the activations of Hog1-type MAPKs were observed at much higher levels of the fungicides (100 µg/ml) and osmotic stress (0.8 M KCl) in these histidine kinase mutants of C. heterostrophus and N. crassa. This suggests that another signaling pathway activate Hog1-type MAPKs, especially when cells are exposed to high-levels of the fungicides and osmotic stress.

74. Use of RNAi for partial inactivation of the essential COT1 kinase in Neurospora crassa. Carmit Ziv, Efrat Dvash and Oded Yarden. Department of Plant Pathology and Microbiology and Otto Warburg Center for Agricultural Biotechnology, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel

Neurospora crassa is a useful model organism for studies of cell polarity. The N. crassa cot-1 gene encodes a serine/threonine protein kinase involved in apical hyphal cell elongation. Disfunction of this kinase leads to a dramatic and pleotropic change in hyphal morphology; however, its actual role has yet to be determined. We adapted RNAi as a silencing mechanism to reduce cot-1 gene expression to learn its role in the hyphal elongation process. A hairpin construct of the cot-1 gene was assembled using the first 1800bp of the genomic fragment that includes the cot-1 promoter and the first two exons. Transformants were screened for altered morphology and were subjected to morphological and molecular characterization. Our results demonstrate that RNAi can be employed as a partial gene silencing mechanism, resulting in a range of different mutants. This wide-range silencing system may enable us to study the different functions of cot-1 by disassociating some of the multiple defects from each other and reducing the pleiotropy resulting from highly impaired COT1 activity.