Developmental Biology

459. The cyclin Cln3p links G1 progression to hyphal and pseudohyphal development in Candida albicans. Catherine Bachewich and Malcolm Whiteway. BRI, National Research Council of Canada

G1 cyclins coordinate environmental conditions with growth and differentiation in many organisms. In the pathogen Candida albicans, differentiation of hyphae is induced by environmental cues, and was thought to occur independent of the cell cycle. Repressing the G1 cyclin Cln3p under yeast growth conditions caused yeast cells to arrest in G1, increase in size, and then develop into hyphae and pseudohyphae, which subsequently resumed the cell cycle. Differentiation was dependent on Efg1p, Cph1p and Ras1p, but absence of Ras1p was also synthetically lethal with repression of CLN3. In contrast, repressing CLN3 in environment-induced hyphae did not inhibit growth or the cell cycle, suggesting that yeast and hyphal cell cycles may be regulated differently. However, hyphal compartments were longer and morphogenesis was not normal in CLN3-repressed hyphae, suggesting that cell cycle progression and hyphal growth may be co-regulated. We are currently determining the transcription profiles of C. albicans cells depleted of CLN3 to screen for potential factors that mediate the developmental response. Therefore, absence of a G1 cyclin activates developmental pathways in C. albicans and uncouples differentiation from normal environmental controls. Since blocking the cell cycle at other stages produces different responses, the data suggest that G1 phase of the cell cycle may play a critical role in regulating hyphal and pseudohyphal development in C. albicans.

460. Identification of Genes Differentially Expressed Early in the Sexual Development of Schizophyllum commune. Kirk A Bartholomew. Sacred Heart University, Fairfield, CT, USA

The well characterized products of the complex A and B mating-type loci of Schizophyllum commune control activation of sexual development when monokaryotic mycelia of non-self mating type interact. Both the products of the A loci (interacting homeodomain proteins) and the B loci (lipopeptide pheromones and associated G-protein coupled receptors) are proposed to initiate the events of sexual development by controlling the differential expression of target genes in mating mycelia eventually leading to the formation of fully dikaryotic mycelia with binucleate cells and associated clamp connections. In this ongoing project modifications of a membrane culture technique were used to mate colonies of sexually compatible monokaryons by transferring them into close proximity. RNA isolated from the mated mycelia prior to the fully dikaryotic stage of sexual development was compared with RNA from unmated mycelia from the same sexually compatible monokaryons by the reverse transciptase differential display polymerase chain reaction method (RTDDPCR) in order to identify genes differentially expressed during the early stages of sexual development. RTDDPCR analysis with 48 primer pairs identified 15 partial cDNAs that were strong candidates for differential expression. Progress in cloning, sequencing and confirming the differential expression of the candidate cDNAs will be reported.

461. Peroxisomes and sexual differentiation in Podospora anserina: characterization of the peroxisomal protein receptors PEX5 and PEX7. C. Bonnet, Espagne E., Zickler D. and Berteaux-Lecellier V. Institut de Génétique et Microbiologie, UMR8621, Université Paris sud, Bât 400, 91405 Orsay Cedex, France.

Peroxisomes are highly adaptive organelles involved in numerous enzymatic functions and in various developmental decisions. In P. anserina we have shown that they are required at a specific step of the sexual cycle. The peroxisome biogenesis pex2 mutant (Zellweger syndrome in Human) is unable to switch from the mitotic to the meiotic state.

To determine more precisely the link between peroxisomes and differentiation, we characterized the two peroxisomal receptors PEX5 and PEX7. While very few peroxisomal matrix proteins possess the peroxisomal targeting signal recognized by PEX7, PEX5 is responsible for the import of most peroxisomal matrix proteins. The P. anserina pex5 gene encodes a protein of 665 aa composed of 7 TPR repeats while pex7 encodes a protein of 358 aa with 6 WD40 repeats. These two proteins share 90% and 60% similarity with their orthologs in Neurospora crassa and Human, respectively.

Contrary to pex2 mutants, both delpex5 and delpex7 deleted strains are able to achieve the sexual cycle. However, a delay is observed in the transition from the mitotic to the meiotic stage in the delpex5 homozygous crosses. Analysis of the double mutant delpex5delpex7 is under investigation. The properties and cytological characterisation of the known pex mutants will be discussed in the frame of the involvement of peroxisomes in sexual development.

462. Does RIP occur at a specific stage of premeiotic development in Podospora anserina? Khaled Bouhouche, Sylvie Arnaise, and Robert Debuchy. Institut de Genetique et Microbiologie, UMR8621, Orsay, France.

RIP is a homology-dependent gene-mutation mechanism which takes place inside the perithecia before premeiotic replication. Whether RIP occurs during a specific stage of the complex perithecial development preceding the premeiotic replication remains unknown. These stages are investigated in details in Podospora anserina. In this heterothallic fungus, fertilization is followed by the formation of plurinucleate cell containing nuclei of opposite mating type which recognize each other to form dikaryotic hyphae. The tip cell of dikaryotic hyphae differentiates into a specialized cell where karyogamy and meiosis take place. Previous analyses of the four mating-type genes demonstrate that three of them control the internuclear recognition (IR) and that the fourth is essential for the development of dikaryotic hyphae. To determine if RIP occurs before IR, or between IR and dikaryotic hypha formation, or during dikaryotic hypha development, we constructed mating-type gene duplication at the mating-type locus and searched if RIP resulted in an altered progeny. If a gene is RIPed at a stage preceding the step under its control, the altered gene would lead to a mutant phenotype. In contrast, if RIP occurs later than the step controled by the duplicated gene, the strain would not display any defect in crosses, but defective crossing of the progeny containing the RIPed gene would reveal the mutation. Preliminary results with the duplication of a mating-type gene involved in IR will be discussed in the poster.

463. Deletion analysis of genes regulated by cAMP-dependent protein kinase A in Ustilago maydis. Kylie J. Boyce, Luis Larraya, Howard Chang, Cletus D'Souza and James W. Kronstad. The Michael Smith Laboratories, University of British Columbia, Vancouver, B.C. Canada V6T 1Z4

In the corn smut fungus Ustilago maydis, development and virulence are regulated by a cAMP-dependent protein kinase A (PKA) pathway and at least one mitogen-activated protein kinase (MAPK) pathway. To identify additional genes regulating development and virulence in U. maydis we have used serial analysis of gene expression (SAGE) to analyze the transcriptomes of wild-type and mutant strains defective in either the catalytic (adr1) or regulatory (ubc1) subunit of PKA. Mutation of the catalytic unit of PKA results in filamentous cell growth, whereas mutation of the regulatory PKA subunit results in a multi-budded phenotype. Using SAGE we have identified numerous genes whose transcript abundance is altered by a mutation in PKA and is hence up or down regulated during filamentation or budding. In this study we describe the deletion of some of these genes with a focus on the deletion of a gene encoding a Cdc11p, septin homolog. Deletion of the U. maydis Cdc11p homolog, sep1, results in cells with division defects including mis-localization of chitin and failure of cells to separate, as well as morphological and nuclear defects. Strains with the sep1 mutation fail to grow filamentously in response to fatty acids, despite being able to form filaments during mating. Deletion of sep1 does not inhibit pathogenicity but results in cells types with altered morphology in planta. The generation of ubc1 delta sep1 strains has showed that deletion of sep1 prevents the multi-budded phenotype of the ubc1 strain.

464. Analysis of the regulatory multprotein complex COP9 Signalosome in Aspergillus nidulans. Silke Busch, K. Helmstaedt, O. Bayram, O. Valerius, Gerhard H. Braus. Institut für Mikrobiologie und Genetik, Georg-August-Universitaet, Grisebachstrasse 8, D-37077 Goettingen (GER)

The COP9 signalosome (CSN) is a eukaryotic multiprotein complex that connects various signals with multiple downstream molecular pathways controlling cellular differentiation and development. In higher eukaryotes, disfunction of the CSN is embryonic lethal, whereas in Aspergillus nidulans deletion of CSN subunits leads to viable strains with severe developmental defects. We want to understand the molecular networks controlled by CSN action in A. nidulans as a genetically amaneable eukaryotic model system.

In higher eukaryotes, the CSN is composed of eight conserved subunits. We identified proposed open reading frames for all of them in the A. nidulans genome sequence. Using the tandem affinity purification method (TAP) we purified a complex containing several CSN subunits as proven by mass spectrometry. We are currently improving the method to additionally identify CSN associated proteins.

Function of the CSN involves 26S proteasome-dependent regulation of cellular protein levels, mainly by the signalosome's intrinsic deneddylation and associated kinase activities. Accordingly, we observed a different protein expression pattern in a wild-type compared to a csn deletion strain in 2D gel analyses. Differentially expressed proteins were identified by mass spectrometry. These potential downstrem targets of CSN action hint at a CSN function in regulation of metabolism, cytoskeleton composition and stress response.

465. Negative regulation of sexual development by specific interactions of IndB/D proteins with NsdD in Aspergillus nidulans. Nak-Jung Kwon, 1Dong-Min Han, and Suhn-Kee Chae, BioMed RRC and Dept. of Biochem., Paichai University, Daejeon 302-735 Korea, and 1Division of Biological Science, Wonkwang University, Iksan 570-749, Korea.

A GATA type transcription factor NsdD has been known as a positive regulator of sexual development in A. nidulans. Previously, we isolated two novel proteins, IndB and IndD, that interacted with NsdD by the yeast two-hybrid screening. In vitro and in vivo associations of NsdD with IndB and IndD have been confirmed. Mutated NsdD proteins carrying change in the zinc-finger motif failed to interact with IndB/D, indicating that the intact zinc-finger domain is required for the interactions. Other GATA type proteins of LreA, LreB, and AreA failed to interact with IndB/D, suggesting that NsdD-IndB and -IndD interactions are specific. Northern analysis showed that both of the indB and indD transcripts were highly elevated in veA disruptants, indicating that VeA represses the indB/D gene expression. Overexpression of indB and/or indD strongly inhibited sexual development in veA+ backgrounds. We also identified that IndB/D proteins inhibited DNA bindings of NsdD at putative GATA sites in vitro. Therefore, our results suggest that indB/D function on sexual differentiation negatively via binding to the zinc-finger domain of NsdD to interfere NsdD function, and are regulated by the veA gene, providing a clue of modulating process in sexual development promoted by NsdD and VeA. [Supported by grants from KOSEF]

466. Sumoylation is required for differentiation and DNA repair in Aspergillus nidulans. Nak-Jung Kwon, Jeong-Hwa Park, and Suhn-Kee Chae. BioMed RRC and Division of Life Science, Paichai University, Daejeon 302-735, Korea

SUMO modifies functions of proteins in various ways when attached covalently. The first step of sumoylation is the formation of a thiol ester linkage between the C-terminal glycine of SUMO and an active site cysteine of an SUMO activating enzyme complex, Uba2 and Aos1 (E1). The second step involves the transfer of SUMO to a cysteine in the active site of an SUMO conjugating enzyme of Ubc9 (E2). In the last step, the E2 enzyme may cooperate with an protein ligase (E3) to form an isopeptide bond between the C-terminal glycine of SUMO and a lysine-amino group in the target. Enzymes involved in the sumoylation process are well conserved in A. nidulans based on the analysis of A. nidulans genomic DNA sequence database. The sumO and ubcN genes of A. nidulans encoding homologs of SUMO and Ubc9, respectively, were cloned. Null mutants of sumO and ubcN were not lethal in A. nidulans. Mycelial growth rate was not much affected but conidiation hardly occurred in both null mutants. Furthermore, cleistothecium was never found in various growth conditions, but Hulle cells were still observed. The sumO transcript was expressed to the similar level during asexual and sexual differentiation. delta-sumO and delta-UbcN exhibited high sensitivities to 4-NQO, MMS, CPT, and HU compared to those for wild-type. In conclusion, sumoylation process is required for proper differentiation and DNA repair in A. nidulans. [Supported by KOSEF]

467. Functional study of SMR1, a mating-type gene which does not control self-non self recognition in Podospora anserina. Evelyne Coppin, Sylvie Arnaise, Khaled Bouhouche, Xavier Robellet, and Robert Debuchy. Institut de Genetique et Microbiologie, UMR 8621 CNRS-Universite Paris Sud, Orsay, France

The sexual reproduction of P. anserina requires the coordinate action of 4 mating-type genes. Three of them encode transcriptional factors, which control recognition between mat+ and mat- sexually compatible cells during fertilization, and between nuclei after fertilization, at the transition from a plurinucleate to a dikaryotic stage when ascogenous hyphae are formed. The fourth gene, SMR1, is involved in pre-fertilization or initial steps of perithecial development, and is essential for the dikaryotic stage. By contrast, it is dispensable for the development of uniparental ascogenous hyphae resulting from mat gene mutation affecting nuclear pairing. Orthologs of SMR1 are present in all Sordariomycetes analyzed to date, but the molecular function of these genes remains unknown. Subcellular localization of SMR1 by GFP tagging suggests that it has a cytosolic localization, which does not support previous prediction that it is a transcription factor. A SMR1 disruption results in barren perithecia and suppressors restoring fertility were previously isolated. All of them affect mat genes and nuclear pairing. We have constructed 30 new alleles by alanine scanning and found one mutation which leads to sterility in cross. This point mutation is used to screen suppressors with the aim of characterizing new genetic interactions helping to understand the role of SMR1.

468. The function of the coding sequences for the putative pheromone precursors in Podospora anserina is restricted to fertilization. Evelyne Coppin, Christelle de Renty, and Robert Debuchy. Institut de Genetique et Microbiologie, UMR 8621 CNRS-Universite Paris-Sud, Orsay, France

We addressed the role of the pheromone precursor genes in P. anserina, and explored regulation of their expression by mat+ and mat- mating types. We cloned these genes using their previously isolated homologs in Neurospora crassa and Cryphonectria parasitica. They were named mfp and mfm (mating factor plus and minus) since their transcription is controlled by mat+ and mat-, respectively. The mfp gene encodes a 24-aa peptide ending with the CAAX motif, characteristic of fungal lipopeptide pheromone precursors. The mfm gene encodes a 221-aa polypeptide, which contains two 13-residue repeats assumed to correspond to the mature pheromone. Deletion of each coding sequence impaired male fertility in a mating-type specific manner, without affecting female fertility and vegetative growth. We managed to cross null mutants lacking both mfp and mfm, by complementation or transient expression of the required pheromone gene to trigger fertilization. These crosses were fertile, showing that once fertilization had occured, the pheromones are no longer necessary for the completion of the sexual cycle. Finally, we subtracted both genes from mating-type control by replacing their 5 UTR by the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter and found that expression of both constructs is repressed post-transcriptionally by the non-cognate mating type.

469. Ribonucleoprotein particles that shuttle along microtubules are essential for pathogenic development in Ustilago maydis. Philip Becht, Julian König, Janine Koepke, and Michael Feldbrügge. Max-Planck Institute for Terrestrial Microbiology, Department of Organismic Interactions, Karl-von-Frisch-Strasse, 35043 Marburg, Germany

RNA-binding proteins function as developmental regulators in eukaryotes. To test whether this holds true for pathogenic development of Ustilago maydis, the causative agent of corn smut, we used a reverse genetic approach. We deleted 18 open reading frames encoding putative RNA-binding proteins. One of these designated Rrm4 contained three N terminal RNA recognition motives with an architecture known from ELAV proteins (embryonic lethal abnormal vision). The corresponding deletion strain was impaired in filamentation and pathogenicity. Using time-lapse fluorescence microscopy we were able to demonstrate that Rrm4 assembled into particles that shuttled along microtubules. Thus, Rrm4 is apparently part of ribonucleoprotein complexes that transports RNA from the nucleus to the cell poles. Currently, we are focusing on unravelling the molecular composition of the Rrm4-containing particles to identify the cargo and the mode of transport.

470. Few sequence constraints hinder Schizophyllum pheromone function in yeast. Thomas Fowler, Joseph Pare, and Christopher Baker, Dept of Microbiology and Molecular Genetics, University of Vermont, Burlington, VT 05405 USA

G protein-coupled pheromone receptors of Schizophyllum commune can distinguish among the many dozens of mating pheromones in the species. One group of highly similar lipopeptide pheromones were examined for clues to their activity and specificity using extensive mutagenesis at several amino acid positions within the mature pheromones. An heterologous assay was utilized in which S. commune pheromones and pheromone receptors were functionally expressed in the yeast Saccharomyces cerevisiae. Successful mating of these transgenic yeast strains identified functional mutant pheromones. Many amino acid positions in the mature pheromones are much more mutable than might be predicted from sequence comparisons. Pheromone Bbp2(4) tolerates at least 17 different amino acid substitutions at one amino acid position while more than 10 changes are tolerated at other positions with little effect on specificity. We have begun to test some of the mutant pheromones directly in S. commune to explore the reason for amino acid conservation of the mature pheromones in S. commune in contrast to the results of the heterologous assay. One possibility is that S. commune requires these conserved residues for pheromone processing and export, but not for receptor activation, while yeast employs a very flexible processing and export mechanism for these heterologous pheromones. Regardless of possible differences in pheromone processing and export, the features within the amino acid sequences of mature pheromones that are required for receptor activation are surprisingly minimal.

471. Exploring appressorial formation in the cereal biotroph, powdery mildew. Sally Gilbert1, Andy Bailey2, Bart Fraaije1, Hans Cools1 and John Lucas1. 1Plant-Pathogen Interactions Department, Rothamsted Research, Harpenden, Herts AL5 2JQ England

2School of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 1UG England.

Powdery mildew (Blumeria graminis) is an obligate, fungal biotroph which invades its host using an infection structure, termed appressorium. There is considerable interest in understanding the sequence of events that leads to appressorial formation and ultimately successful penetration of the host plant. A greater understanding of the signal transduction pathway has and continues to facilitate the development of novel fungicides that target specific stages in this pathway.

The main aim of this project is to use real-time RT-PCR to identify potential target-encoding gene(s) of a novel fungicide known to inhibit appressorial formation. Six genes that play a role in appressorium formation were chosen. These encode an integral membrane protein (PTH11); a G protein alpha subunit (Bgene); a Mitogen-activated Protein kinase (MAP1); a Protein Kinase C (PKC); a cAMP-dependent Protein Kinase A (CPKA); and an Adenylate Cyclase (BAC1). In addition, a Catalase/Peroxidase encoding gene (CPX) was also chosen as it is involved in scavenging activated oxygen species generated during the plant/pathogen interaction. TaqMan® probes have been designed and used to determine changes in expression of these genes in the presence of fungicides and/or inhibitors as compared to the untreated control.

472. Characterization of components of an apoptotic machinery in the filamentous ascomycete Podospora anserina. Andrea Hamann, Heinz D. Osiewacz. Botanisches Institut, J.W. Goethe-Universitaet, Marie-Curie-Str. 9, D-60439 Frankfurt, Germany

During the last years several analyses demonstrate the existence of apoptotic processes in the ascomycete fungi Saccharomyces cerevisiae and two Aspergillus species. The search for homologues in the genomic sequence of P. anserina ( Projet_GA/GA.html) resulted in the identification of a number of putative members of the apoptotic machinery including two metacaspases, one apoptosis-inducing factor (AIF), and one AIF-homologous mitochondrion-associated inducer of death (AMID) and others. Here we present first data on the characterization of selected components via over-expression and/or deletion of selected genes. During the course of investigations we optimized the gene replacement procedure developed by Chaveroche et al. (2000, Nucleic Acids Res. 28: E97) for use with P. anserina. The features of this modified method are reported. Utilizing this strategy, we generated and initially characterized several strains in which different genes encoding putative components of the apoptotic machinery were disrupted. The deletion of PaMca1, a gene encoding a homologue of a yeast metacaspase, resulted in increased life span and enhanced stress tolerance. The influence of apoptotic components on stress tolerance, life span and on spore degeneration in crosses between so-called killer and non-killer strains will be discussed.

473. The Aspergillus nidulans silG gene functions in repression of sexual development in response to light. Kap Hoon Han1, Bang-Yong Lee, and Dong-Min Han. Div. of Biological Science, Wonkwang University, Korea 1Dept. of Plant Pathology, Seoul National University, Korea

The Aspergillus nidulans nsdD gene encodes a GATA-type transcription factor that is required for sexual development. To further understand the NsdD-mediated regulatory cascade, we isolated various suppressors of nsdD (SND). The silG gene presented in here was identified as a multi-copy suppressor of one of the snd mutations. Multi-copy of silG blocked cleistothecia development in an SND mutant. The silG gene is predicted to encode a 703 aa polypeptide with three C2H2 zinc finger DNA-binding domains at the C-terminus. The silG null mutant produced a high number of cleistothecia even under the visible light, which normally inhibits sexual development. However, high osmolarlity or poor carbon sources blocked sexual development of the silG deletion mutant, suggesting that SilG may play a specific role in negative regulation of sexual development in response to light. Further supporting this hypothesis, over-expression of silG resulted in a great reduction of sexual development in dark, which preferentially enhance sexual development in wild type. Accumulation of silG mRNA undulated throughout the lifecycle in a certain recurring pattern. Interestingly, silG mRNA levels elevated upon exposure to light, and this response requires the functional A. nidulans wcA(lreA) gene. Moreover, mRNA level of silG in veA1 mutant was increased in dark, implying that silG functions downstream of veA and VeA may repress silG expression in dark, which leads to sexual development.

474. Oxidative stress and cell differentiation in Neurospora crassa. Wilhelm Hansberg, Leonardo Peraza, Jesús Aguirre, Karen Delfín, Nallely Cano. Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, México D. F., México.

Morphogenetic transitions of N. crassa life cycles are considered responses to a hyperoxidant state. If so, mutant strains in antioxidant enzymes should favor and in pro-oxidant enzymes should inhibit development. A catalase-3 (cat–3) null mutant strain showed increased protein oxidation and carotene levels in the dark, indicating oxidative stress. The strain formed hyphae aggregates and produced more aerial hyphae and conidia than the WT (1). A catalase-peroxidase (cat–2) null mutant strain was sensitive to peroxides, formed more arthroconidia and more protoperithesia and perithesia than the WT. In contrast, NADPH oxidase-1 (nox–1) null mutant strain formed less aerial hyphae and no protoperithesia and a ?nox–2 formed ascospores that could not germinate. These results indicate that oxidative stress is required for cell differentiation in N. crassa.

1) Michán S, Lledías F, Hansberg W (2003) Asexual development is increased in Neurospora crassa catalase-3 null mutant strains. Eukaryotic Cell 2:798-808.

Acknowledgements: CONACyT C01-40697, DGAPA/UNAM IN228405

475. Functional Comparison of Cdc42 and Rac1 in the Dimorphic Fungus Ustilago maydis. Andrea Hlubek, Kay Schink and Michael Bölker. Fachbereich Biologie, Universität Marburg, Karl-von-Frisch-Str. 8, 35032 Marburg, Germany

Cdc42 and Rac1 are small GTPases which regulate various cellular processes like morphogenesis, vesicle trafficking, cytokinesis and cell polarity. Guanine nucleotide exchange factors (GEFs) convert the GTPases to their active, GTPbound form; GTPase activating proteins (GAPs) stimulate the GTPase activity.

Both genes are not essential for viability in the dimorphic fungus U. maydis, but they share at least one important function. We could show that U. maydis Cdc42, but not Rac1, complements the lethal phenotype of the temperature sensitive cdc42-1 allele in Saccharomyces cerevisiae. To define the regions that are responsible for the specificity of these proteins, we generated a set of chimeric proteins, which were checked for complementation of both U. maydis and S. cerevisiae mutants. These experiments demonstrate that the region between amino acids 41 and 56 is necessary and sufficient to determine the specificity of the GTPases. This could be due either to specific activation of the GTPases by their cognate GEFs or to their interaction with specific downstream effectors. To distinguish between these possibilities we are currently characterizing further GEFs specific for either Cdc42 or Rac1. Preliminary indications suggest that a single amino acid might be important for specific interactions with certain GEFs. Corresponding chimeras of human Cdc42 and Rac1 are currently tested by transient expression in mouse fibroblasts.

476. A putative sugar transporter is involved in the regulation of mushroom development in the basidiomycete Schizophyllum commune. Peter Sage, Josh White, Alaap Shah, Gail Palmer and Stephen Horton. Department of Biological Sciences, Union College, Schenectady, NY 12308 USA

Mushroom development in basidiomycetes is regulated by both genetic and environmental factors. Nutrient depletion is one of the necessary prerequisites for fruiting in these fungi. The gene sts1 encoding a putative sugar transporter has been isolated from Schizophyllum commune by means of a two-hybrid screen using a bait protein implicated in mushroom development. The predicted protein is 550 amino acids in length, and has 12 transmembrane domains typical of these transporters. Co-transformation of a dominant activated mutant version of sts1 into a homokaryotic fruiting recipient strain drastically inhibits mushroom formation in a small number of transformants. Overexpression of this mutant gene using the highly expressed sc3 promoter resulted in a much greater number of affected transformants. Immunofluoresence studies using anti-myosin antibodies showed an accumulation of staining at many of the hyphal septa in non-fruiting sts1 overexpression strains. We hypothesize that the mechanism of the block in fruiting observed in the mutant sts1 overexpression strains may involve disruption of myosin-mediated vesicle transport. Grant support from the NIH-AREA program is gratefully acknowledged.

477. Control of the circadian clock in Aspergillus flavus by LreA and LreB. L. R. Milde1, A. Greene2, D. Bell-Pedersen2, N. P. Keller1. 1Department of Plant Pathology, University of Wisconsin, Madison, WI 53706; 2Program in Biological Clocks, Department of Biology, Texas A&M University, College Station, TX 77843

Endogenous circadian clocks generate and control daily rhythms in biochemical, physiological, and behavioral processes. Circadian rhythms are widespread in nature, occurring in organisms ranging from cyanobacteria to humans. In Aspergillus flavus, fungal clock mechanisms have been shown to control the production of sclerotia, which are important to the development and survival of the fungus. Putative orthologs of Neurospora crassa clock components, WC-1 and WC-2, have been identified in the Aspergilli and are called LreA and LreB, respectively. Using RNA interference technology, both genes were simultaneously silenced in A. flavus, yielding transformants aberrant in rhythmic sclerotial production. Further examination will help to distinguish the specific functions of LreA and LreB in the Aspergilli and to gain a better understanding of circadian-control of the survival structures and virulence determinants of the pathogenic Aspergilli.

478. Role of a MAP kinase cascade in the development of an epigenetic cell degeneration. Sébastien Kicka, Fabienne Malagnac, Andrew K. Sobering, Crystel Bonnet, Latha Prabha Ganesan and Philippe Silar. Institut de Génétique et Microbiologie, Université de Paris Sud, 91405 Orsay cedex France

PaASK1, a MAPKKK, and PaNox1, a NADPH oxidase are essential to develop Crippled Growth (CG), an epigenetic cell degeneration of the filamentous fungus Podospora anserina. A model based on the bistable and hysteretic behaviour of the PaASK1 MAP kinase cascade has been proposed to explain the properties of CG, especially its triggering by C, a cytoplasmic and infectious factor. In this model, C is the active form of the cascade. To confirm this model, the downstream MAPKK and MAPK genes have been cloned and null alleles recovered. Inactivation of the MAPKK and MAPK genes triggers the same phenotypes as the inactivation of the MAPKKK gene, confirming the crucial role of the MAP kinase module in the development of CG. Strains harbouring transgenes expressing a MAPK-HA fusion protein have been constructed and used to evaluate the phosphorylation status of the MAPK during Normal and Crippled Growth. Data show that phosphorylation of the MAP kinase perfectly correlates with the presence of the C, as expected from the model. In addition, lack of phosphorylation of the MAP kinase in the PaNox1 mutant confirm that PaNox1 acts upstream of PaASK1. These data are the first steps towards the deciphering of the molecular mechanisms involved in the generation of the Crippled Growth cell degeneration.

479. The nsdC gene encoding a novel C2H2-type zinc finger protein is a key activator of sexual development in Aspergillus nidulans. Hye-Ryoun Kim, Kap Hoon Han1 and Dong-Min Han. Division of Life Science, Wonkwang University, Korea 1Seoul National University, Korea

Sexual development in Aspergillus nidulans is affected by a number of genetic and environmental factors. Previously, we isolated various NSD (never in sexual development) mutants and identified a gene called nsdD encoding a GATA-type transcription factor necessary for sexual development. Here, we report the identification and characterization of the nsdC gene, which is predicted to encode a 643 aa polypeptide with a novel C2H2-type zinc finger DNA binding domain. While deletion of nsdC resulted in the typical NSD phenotype, i.e., lack of sexual development, overexpression of nsdC not only enhanced formation of sexual fruiting bodies (cleistothecia) but also overcame inhibitory effects of certain stresses on cleistothecial development, indicating that NsdC is a key positive regulator of sexual development. The nsdC gene generates two distinct transcripts, 3.2 and 2.8 kb, and its 5' UTR is ~1.5 kb with two relatively long introns (168 bp and 212 bp). Differential accumulation of two nsdC transcripts in various stages of growth and development as well as under the varying environmental conditions indicates that nsdC is subject to complex transcriptional regulation. Carbon source dependent retarded vegetative growth and hyper-active asexual development phenotypes were observed in the nsdC deletion mutant. This result further supports the idea that balanced progression of sexual and asexual development is necessary for the maintenance of genetically programmed lifecycle.

480. A homeobox-domain protein represses sexual development in Aspergillus nidulans. Jee-Hyun Kim, Dong-Beom Lee and Dong-Min Han. Division of Biological Science, Wonkwang University, Korea

Previously we identified NsdD , a crucial activator for sexual development in Aspergillus nidulans. While increased dose of nsdD caused elevated formation of fruiting bodies (cleistothecia) even under the unfavorable environmental conditions, nsdD mRNA levels did not change, suggesting that multiple copies of the nsdD promoter might relieve repressive effects by negative regulators. To dissect complex regulatory networks of sexual development, we identified a multi-copy suppressor (nrsA ) that abolished enhanced cleistothecia formation by increased dose of nsdD. The nrsA gene is predicted to encode a novel 293 aa protein with a homeobox domain at the C terminus. Deletion of nrsA resulted in elevated cleistothecial development even in the presence of stresses, but caused lowered asexual sporulation. Conversely, forced expression of nrsA blocked formation of cleistothecia, while enhancing asexual development, indicating that nrsA act as a key negative regulator of sexual development, which may confer properly balanced progression of sexual and asexual development in response to environmental factors. Steady state mRNA levels of nrsA increased at the later phases of sexual/asexual development as well as in the presence of osmotic stresses during vegetative growth. Taken together, we propose that the homeo-domain protein NrsA administrates coordinated regulation of two distinct developmental programs in A. nidulans.

481. A hydrophobin gene, VDH1, is involved in microsclerotial morphogenesis and spore viability in Verticillium dahliae (Kleb). A. Klimes1 and K.F. Dobinson2. 1,2Department of Biology, University of Western Ontario, London ON N6A 5C1, Canada; 2Southern Crop Protection and Food Research Centre, Agriculture and Agri-food Canada, 1391 Sandford Street, London, ON N5V 4T3, Canada.

The plant pathogen Verticillium dahliae (Kleb) is characterized by the production of microsclerotia (MCS), the desiccation and cold tolerant resting structures that serve as a primary source of disease inoculum in the field. Despite the critical role of these structures in the fungus's life cycle, the molecular mechanisms of microsclerotial development are not well understood. We have identified a class II hydrophobin gene, VDH1, that is important to the formation of these critical structures. A green fluorescent reporter gene under the control of the wild type VDH1 promoter is highly expressed in developing MSC, conidiophores and spores. Targeted disruption of VDH1 severely inhibits MCS production. Although vdh1, mutants do produce conidiophores and spores, the spores are less tolerant to desiccation than are wild type spores. vdh1 disruption mutants are able to cause disease in tomato plants, indicating that the gene is not needed for pathogenicity. While its importance in the development of V. dahliae resting structures is apparent, the precise role of VDH1 in morphogenesis is not yet clear. We are using microarray analysis to determine whether disruption of VDH1 results in changes in expression of other genes which could also be involved in morphogenesis. We are also examining a VDH1 homologue we identified in the plant pathogen V. albo-atrum, to determine if differences in gene sequence or expression may account for the characteristic lack of MSC in this close relative of V. dahliae.

482. Biological function of the genes ras and tpk in the fungus Schizophyllum commune. N. Knabe, E. Kothe. FSU Jena, Jena, Germany

It has been shown that cAMP affects normal development of fruit bodies in S. commune. A certain concentration of cAMP causes many fruit bodies to stop morphogenesis at an early stage. Those fruit bodies that continue development have abnormal or no gills. Therefore, the role of cAMP-dependent signal transduction is investigated in the wood-rotting homobasidiomycete S. commune. Since the components of cAMP signaling cascades are well conserved in eukaryotes, two components are investigated. One is the G-protein Ras which is known to induce adenylate cyclase in Saccharomyces cerevisiae. An activated Ras is therefore expected to lead to high intracellular cAMP concentration and thus to mimic high external cAMP concentrations. A cDNA sequence of ras1 from S. commune could be used to derive PCR primers to amplify a fragment from genomic DNA. The 1.2kb fragment contains the entire ras1 gene and shows 98% amino acid homology to Ras1. Both dominant negative and constitutive Ras will be used to analyze Ras function in sexual development and fruit body formation in S. commune. The second protein under investigation is cAMP dependent protein kinase A which is translating the signal from differing cAMP levels to regulation of transcription factors by phosphorylation. For tpk, encoding the catalytic domain of protein kinase A, a fragment was cloned and sequenced. The fragment of 3.4 kb contains almost the entire tpk gene which shows 60% amino acid identity to the yeast protein Tpk2. A knock-out mutant will be constructed to investigate the role of cAMP-dependent signaling in sexual development and fruit body formation in S. commune.

483. The Aspergillus nidulans F-Box Protein GrrA is required for Ascosporogenesis. Sven Krappmann1, Branka Medic1, Nadja Jung1, Rolf Prade2, and Gerhard H. Braus1. 1 Department of Molecular Microbiology and Genetics, Georg-August-University Goettingen, Germany. 2Department of Microbiology and Molecular Genetics, Oklahoma State University, USA

Aspergillus (Emericella) nidulans is a well-established model organism to study elementary processes of differentiation. A Negative Subtraction Screening procedure was performed with the aim to isolate transcripts that are expressed at elevated levels during cleistothecia formation. Among them, one EST that is specifically up-regulated at the end of fruit body development could be identified. The deduced amino acid sequence of the equivalent full-length cDNA shows similarity to the S. cerevisiae F-box protein Grr1p and the A. nidulans GrrA protein complements the morphology defect of a yeast grr1D mutant strain. The grrA locus was deleted in a wild-type background and the resulting mutant strain was characterised thoroughly: whereas no specific phenotype is obvious with respect to growth capacities, mitosis, asexual sporulation or cleistothecia development, there are no ascospores to be found within the ripe fruiting bodies of the grrAD mutant. As specificity factors of so-called SCF ubiquitin ligase complexes, F-box proteins influence protein stability and turnover. Conclusively, the grrA gene product links ascosporogenesis in A. nidulans to proteasome-executed protein degradation mediated by the SCFGrrA complex.

484. A potential role of basidiolipids as physiological ligands of galectins during sexual development of the mushroom Coprinopsis cinerea. Martin Waelti1, Piers Walser2, Rinaldo Bertossa3, Cristina Villalba1, Ursel Kuees4, Markus Aebi1 and Markus Kuenzler1. 1Institute of Microbiology, ETH Zuerich, Switzerland; 2Present address: Institute for Molecular Bioscience, University of Queensland, Australia; 3Present address: Department of Genetics, University of Groningen, The Netherlands; 4Institute of Forest Botany, Georg-August Universitaet Goettingen, Germany

Galectins, a family of beta-galactoside-binding lectins with a characteristic carbohydrate-recognition-domain (CRD) signature, have been implicated in cell differentiation, cell adhesion, cancer, apoptosis, and pre-mRNA-splicing in animals. Interestingly, the protein family is absent from archaea, bacteria, plants and most fungi but is found in mushrooms. The temporal and spatial expression pattern of two isogalectins, CGL1 and CGL2, in the ink cap mushroom Coprinopsis cinerea suggests a role for these galectins in fruiting body formation. We are aiming to identify the physiological ligand(s) of the C. cinerea galectins in order to understand their molecular function in this developmental process. Our preliminary results point to a subfamily of glycosyl-inositol-phosphoryl-ceramides (GIPCs) containing beta-galactosides as candidate ligands. These so-called “basidiolipids” have been described in a number of different mushrooms thereby revealing a remarkable species-to-species variability in their sugar headgroup. At present, we are in the process of isolating basidiolipids from C. cinerea and characterizing them both structurally and with regard to their galectin-binding activity. We hypothesize that the binding of developmentally regulated galectins to basidiolipids may play a crucial role in mushroom development.

485. Classical genetics in fruiting body development of Coprinopsis cinerea. P. Srivilai, W. Chaisaena and U. Kües. Georg-August-University Göttingen, Institute for Forest Botany, Göttingen, Germany

Coprinopsis cinerea is an excellent model to study fruiting body development in the basidiomycetes. The self-compatible homokaryon AmutBmut with mutations in the A and B mating type genes serves us to isolate mutants in fruiting body formation. Genetic analysis of such mutants turned out to be difficult by lack of compatible co-isogenic monokaryons. Progenies from crosses of both homokaryon AmutBmut or AmutBmut mutants with foreign strains often show an extreme variety in fruiting behaviour (lack of initiation, block of fruiting at various developmental stages, defects in fruiting body maturation, defects in sporulation), indicating that in most monokaryotic strains the natural sets of fruiting genes are suboptimal. Therefore, by repeated back-crossing, we generated wildtype monokaryons with different mating types that are co-isogenic to homokaryon AmutBmut. In crosses with mutants, these strains give inheritance patterns that are possible to interpret. Genetic analysis of some interesting AmutBmut fruiting mutants will be presented. Moreover, we will present observations on influences of mating types on fungal growth.

Our laboratory is supported by Deutsche Bundesstiftung Umwelt (DBU) and scholarships by the Mahasarakham University (to PS) and the Rajamangala Institute of Technology (to WC).

486. Monstrosities under the Inkcap mushrooms. M. Navarro-González, A. Domingo-Martínez, S. Navarro-Gonzáles, P. Beutelmann* and U. Kües. Georg-August-University Göttingen, Inst. Forest Botany, Göttingen and *Johannes Gutenberg-University of Mainz, Inst. General Botany, Mainz, Germany

Inkcaps are a group of basidiomycetes whose mushrooms usually deliquesce shortly after maturation for spore liberation. Until recently, inkcaps were compiled under one single genus Coprinus. However, molecular data divided the fungi into four new genera: Coprinus, Coprinopsis, Coprinellus and Parasola (Redhead et al. 2001). Species within these genera are often difficult to recognize beyond doubt and, most likely, many species are still undescribed. Rhacophyllus lilacinus is a mushroom-like anamorph found on horse dung. It carries lysomeres or bulbils (large, round propagules) on lamella and on the upper surface of the cap. Occasional formation of basidiospores in these structures classified the fungus as Coprinus clastophyllus, respectively Coprinopsis clastophylla (Maniotis 1964). Environmental conditions appear to decide whether the monstrous anamorphs are formed or fruiting bodies that carry four meiospores at the basidia on the lamella. Molecular analysis of ITS sequences confirmed assignment to the genus Coprinopsis. The species is closest related to Coprinopsis stercorea. Another strain isolated from horse dung forms crippled mushrooms of various shapes (“hunchbacks”, “picked helmets”, “split umbrellas”, “cedar-shape”). So far, this strain could not be identified on the species level. ITS sequencing groups it close to Coprinellus curtus. Supported by the DBU and CONACYT.

487. The Role of Protein Myristoylation in Fungal Development. Soochan Lee and Brian D. Shaw. Program for the Biology of Filamentous Fungi, Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas, 77803, USA.

In a previous study the A. nidulans polarity mutant swoF1 was found to be mutated in a gene encoded a protein N-myristoyl transferase (NMT). NMTs transfer the 14 carbon fatty acid myristate to the N-terminus of a small group of proteins. This modification allows otherwise cytoplasmic proteins to associate with membranes. We hypothesize that a myrisotylated protein downstream of SwoF plays an important role in growth polarity. Six suppressor of swoF1 (ssf) mutants have been identified. Genetic analysis has shown that all six mutations are extragenic to swoF. At least two distinct, reduced growth phenotypes are observed when each ssf mutant is released from the swoF background. Interestingly, ssfA, ssfC and ssfD secrete a red pigment into the medium similar in color to ascoquinone, as made during ascosporogenesis. This is of particular note, since the G alpha proteins GanA, GanB, and FadA are likely targets for myristoylation. The distinct reduced growth phenotype of each mutant allows for cloning by complementation. To date ssfD has been complemented using a plasmid based genomic library, but the complementing clone has not been analyzed. Continued analysis of these mutants will be discussed. Progress using an in vivo labeling approach to ascertain myrisotylated targets of SwoF will be discussed.

488. Microarray analysis of the Gibberella zeae cDNA clones obtained by subtractive hybridization against an isogenic mat1-2 deletion strain. Seung-Ho Lee1, Sanghyeob Lee2, Doil Choi2, Sung-Hwan Yun3, and Yin-Won Lee1. 1School of Agricultural Biotechnology, Seoul National University, Seoul 151-921; 2 Korea Research Institute of Bioscience and Biotechnology, Taejeon 305-600; 3Division of Life Sciences, Soonchunhyang University, Asan, 336-745, Korea

Gibberella zeae is a homothallic ascomycete causing head blight on several cereal crops. Ascospores of this fungus can overwinter within the sexual fruiting body (perithecium) and initiate the primary infection in the next spring. Thus, a greater understanding of sexual development in G. zeae is needed for a comprehensive disease control strategy. We have focused on identifying the genes specifically controlled by MAT gene, a master regulator of sexual reproduction in G. zeae. To do that, we employed suppression subtractive hybridization between self-fertile G. zeae Z3643 and an isogenic strain deleted for MAT1-2 (delmat1-2). In total, 1,000 expressed sequence tags (ESTs) were generated from the cDNA subtraction library and 378 EST unigenes were identified. To select the genes expressed under control of MAT1-2, we performed a cDNA microarray analysis using the unigenes. Among them, 228 (61.1%) clones were highly expressed in strain Z3643, when grown on mating plates, but not in delmat1-2. These included the genes similar to a Ste12-like transcription factor, Grg1 protein involved in glucose-repression, a glutamate carboxypeptidase-like protein1, a NADPH-cytochrome P450 reductase, and to several hypothetical proteins. Differential expression of the 15 genes from this collection was confirmed by Northern blot analysis.

489. Identification of a sclerotial-specific storage protein gene (ssp1) and transcript profiling of sclerotial development by microarray in Sclerotinia sclerotiorum. Moyi Li, Andrew R. Hutchens and Jeffrey A. Rollins. University of Florida, Dept. of Plant Pathology, Gainsville, FL

Sclerotial development plays a key role in the life cycle of Sclerotinia sclerotiorum. To gain an understanding of this regulatory process we are isolating and characterizing genes differentially expressed during the transition from vegetative growth to sclerotial development. The first recipient of this analysis is a gene encoding a sclerotial-specific storage protein (ssp1), initially identified by peptide sequencing and translated EST comparison. SSP1 makes up 35%-40% of the total protein in mature sclerotia and is also abundant in apothecial tissue. Northern analysis revealed that the ssp1 transcript accumulated in all stages of sclerotial developmental but was absent in both apothecial tissue and vegetative hyphae. These results suggest that ssp1 transcript accumulation is exclusive to the sclerotial stages of the life cycle, but that the SSP1 protein is translocated to the apothecium during carpogenic germination. We have hypothesized that SSP1 functions as a carbon and nitrogen source during apothecial development. Degradation of SSP1, however, is not detected in sclerotia or apothecia. An ssp1 gene deletion mutant will be constructed to further investigate its function during sclerotial development. To identify other genes differentially regulated during sclerotial development, we have constructed a small scale, 500 element, cDNA microarray. Hybridization analysis indicated that ~60 genes were up-regulated and ~50 genes were down-regulated greater than 2-fold during sclerotial initiation relative to vegetative mycelial growth. We are investigating the functions of these sclerotia-related genes by sequence analysis and gene deletion.

490. Sexual reproduction between partners of the same mating-type in Cryptococcus neoformans. Xiaorong Lin1, Christina M. Hull15, and Joseph Heitman1, 2, 3, 4. Departments of Molecular Genetics and Microbiology1, Medicine2, Pharmacology and Cancer Biology3, the Howard Hughes Medical Institute4, Duke University Medical Center, Durham, North Carolina 27710 Current address5: Depts. of Biomolecular Chemistry and Medical Microbiology & Immunology, University of Wisconsin - Madison Medical School, Madison, WI 53706-1532

C. neoformans is a ubiquitous bipolar basidiomyceteous fungus with a defined sexual cycle involving haploid cells of alpha and a mating-type, yet the vast majority of clinical and environmental isolates are alpha cells. Sexual reproduction plays an important role in evolution, yet given the preponderance of alpha strains in the environment it has been a mystery how C. neoformans mates to produce meiotic progeny. C. neoformans grows as a budding yeast in culture and in the infected host, but it undergoes a filamentous dimorphic transition during the sexual cycle to produce spores, the suspected infectious agent. Alpha strains can also filament and sporulate by a process known as monokaryotic fruiting, which resembles mating but was previously thought to be asexual with no ploidy change. We discovered that key hallmarks of mating occur during monokaryotic fruiting, including a change in ploidy (diploidization) and meiosis (ploidy reduction, frequent recombination). Efficient monokaryotic fruiting requires mating machinery and the key meiotic factor Dmc1. These studies reveal how sexual reproduction can occur in nature between isolates of the same mating-type.

491. A mutation in Aspergillus nidulans chaperonin subunit, cctA results in a tip splitting, polarity maintenance phenotype. Melissa Long and Brian D. Shaw. Program for the Biology of Filamentous Fungi, Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas, 77803, USA.

The A. nidulans cctA1 temperature sensitive mutant is defective in polarity maintenance when grown at restrictive temperature (39C). When germinated at 39C cctA1 extends initially a germ tube more or less normally but the conidium continues to expand isotropicaly, resulting in a swollen basal cell twice the size of wild type. The most profound phenotype of the cctA1 mutant at 39C is dichotomous branching at the growing tip of the germ tube. The mutant is complemented by a plasmid containing An5713.2 as designated in The Broad Institute A. nidulans genomic database. Transposon insertion within An5713.2 disrupts the ability of this clone to complement the cctA1 mutant. The An5713.2 predicted protein encodes a chaperonin subunit with 65% identity (e value 0) to S. cerevisiae protein Cct7. The chaperonin is a complex made up of two each of eight subunits in a double ring that assists in folding of approximately 10% of newly synthesized proteins. Though the exact make up of the substrates of this complex is not fully understood, the best studied to date are the cytoskeletal proteins, actin and tubulin. Homologs of all eight S. cerevisiae chaperonin subunits are found in the A. nidulans genome. It is hypothesized that a cytoskeletal defect leads to the cctA1 tip splitting phenotype. Analysis of this hypothesis will be discussed.

492. Role of Cdc42 and Rac1 for cytokinesis and morphology in Ustilago maydis. Michael Mahlert, Verena Welteke and Michael Bölker. University of Marburg, Dept. of Biology, Karl-von-Frisch Str. 8, D-35032 Marburg

We are studying signalling cascades regulating morphology and cytokinesis in Ustilago maydis. Small GTPases of the Rho/Rac family act as molecular switches. The GTP bound form stimulates downstream effectors.

We were able to construct a viable cdc42 deletion mutant which only displayed a cell separation defect. Another small GTPase, Rac1, shows high sequence similarity to Cdc42. Deletion of the rac1 gene resulted in a phenotype, which is clearly different from that of cdc42 deletion strains: rac1 mutants are unable to bud, instead they multiply by fission at a centrally located septum. This indicates, that Rac1 plays a role in the regulation of cell polarity and bud formation. A double mutant, in which cdc42 is deleted and rac1 is expressed under the control of an inducible promoter displays a lethal phenotype under repressing conditions. Therefore Cdc42 and Rac1 share at least one essential function.

Interestingly, overexpression of wildtype Rac1 induced the formation of long filaments similar to that formed by dikaryotic cells. Expression of constitutive active Rac1Q61L resulted in a lethal phenotype which is characterized by the formation of large vacuolated cells. Currently we are screening for synthetic lethal mutants which depend on either Rac1 or Cdc42. We aim to identify novel components of the cascades containing Rac1 and Cdc42.

493. Differential Regulation of Fruitbody Development and Meiosis by the Unlinked Aspergillus nidulans Mating Type Loci. Karen Y. Miller, Autumn Nowell and Bruce L. Miller. University of Idaho.

The haploide genome of the homothallic fungus Aspergillus nidulans possesses both mating type genes previously described for other heterothallic and homothallic filamentous fungi. matA encodes the HMG box protein and homolog of N.crassa MAT-a (general MAT-2). matB encodes the alpha box protein and homolog of N.crassa MAT-A-1 (general MAT-1-1). Homologs of MAT-1-2 and MAT-1-3 do not exist in A.nidulans. Unlike other fertile filamentous fungi, the A. nidulans mating type genes are located on separate chromosomes and freely segregate as independent loci. The A.nidulans mating type loci are not idiomorphs and suggests that homothallism in A. nidulans may not have arisen via a recombination event in a heterothallic predecessor. Vegetative growth and conidiation are not affected in single or double matA/matB mutant strains. A matA deletion strain differentiates only reproductive foci (proto-cleistothecia) and Hulle cells. A few mature fruiting bodies are formed after three weeks (~1% of wild type levels). However, the internal hyphae of these cleistothecia undergo greatly delayed and limited differentiation of ascospores. By contrast, developmental timing and numbers of cleistothecia in a matB deletion strain are similar to wild type. However, there is a complete failure of meiosis, the internal tissue eventually disintegrates and the cleistothecia lyse. matA/steA and matB/steA double mutant strains have phenotypes similar to a steA mutant. This suggests that steA functions upstream of the mating type genes, or is essential for amplification of mating type gene expression. The medA gene (medusa) is also required for self-fertility in A.nidulans. medA mutants abort at Hulle cell and foci formation. medA encodes a transcription factor that directly regulates matA expression and directly, or indirectly, regulates matB expression.

494. The JAMM motif of the COP9 signalosome is essential for Aspergillus nidulans sexual development. Krystyna Nahlik1, Silke Busch, Elke U. Schwier, Gerhard H. Braus2. Institut of Microbiology and Genetics, Georg-August-Universitaet, 37077 Goettingen, Germany

The COP9 signalosome (CSN) is a conserved multiprotein complex, playing an essential role in development of many eukaryotes, including the filamentous fungus Aspergillus nidulans. The deduced amino acid sequences of four recently identified A. nidulans genes: csnA, csnB, csnD and csnE, show high identities to the respective subunits of higher eukaryotes. Considering the conservation of the proteasome-related machinery in A. nidulans, it provides an easily amenable model for studying the broad array of CSN developmental functions.

Deletion of either csnD or csnE results in pleiotropic phenotypes affecting formation of fruit bodies during sexual development, light-dependent signalling and secondary metabolism. We are trying to determine how exactly the observed phenotypes arise: are they associated with disturbed deneddylation activity of COP9 (the removal of NEDD8 tag from cullin, a subunit of ubiquitin ligase), are kinases associated with CSN responsible for these effects, or is it a combination of both?

We have addressed the first part of this question by point mutagenesis of the csnE, containing the JAMM metalloprotease motif, which has been shown to convey the deneddylation activity. Mutant strains with csnE bearing point mutations in the codons for H134, H136 and D145 of the JAMM motif were constructed. The JAMM mutant phenotype is identical with that of the DcsnE strain, indicating that the COP9 deneddylation activity alone is essential for sexual development in A. nidulans. Analysis of cullin neddylation state in these mutants is underway.

495. Characterization of transcription factors from the filamentous ascomycete Sordaria macrospora and their implications on fruiting-body development. Nicole Nolting, Stefanie Poeggeler. Ruhr-University, 44780 Bochum, Germany

Fungal morphogenesis is regulated by a complex network of various transcription factors. In this study we analysed the mating-type HMG-domain gene Smta-1 and the putative MADS-box gene mcm1 of the homothallic ascomycete Sordaria macrospora. We constructed deletion- and overexpression strains.

The mating-type gene Smta-1, encoding an HMG-domain protein, was shown to be involved in fruiting-body morphogenesis, since deletion of Smta-1 resulted in mutant strains that are impaired in perithecia development.

The S. macrospora MCM1 protein is a member of the MADS-box family of transcription factors. MADS-box proteins are combinatorial transcription factors that often derive their regulatory specificity from other DNA binding factors. So far, little is known about the role of MADS-box proteins in fruiting-body development. A yeast two hybrid-analysis and in-vitro analyses revealed that the S. macrospora MCM1 protein has the capability to interact with the mating-type protein SMTA-1. Overexpression of mcm1 led to an increased production of perithecia. Thus, our results suggest an involvement of the S. macrospora transcription factors SMTa-1 and MCM1 in fruiting-body development.

496. Identification of a fruiting body-specific protein from pyrenomycetes. Minou Nowrousian1, Markus Piotrowski2, Ulrich Kück1. 1Lehrstuhl für Allgemeine und Molekulare Botanik and 2Lehrstuhl für Pflanzenphysiologie, Ruhr-Universität Bochum, 44780 Bochum, Germany

Native polyacrylamide gel electrophoresis of soluble proteins from several Neurospora species and related ascomycetes identifies an acidic protein that is preferentially present in perithecial extracts (Nasrallah and Srb, Proc Nat Acad Sci USA 70: 1891-1893, 74: 3831-3834). We have now isolated a protein from Sordaria macrospora showing similar biochemical features: The protein appears as a strong band on native gels of perithecial extracts but is absent from mycelial protein preparations. Its molecular weight was determined by SDS gel electrophoresis to be ~24 kDa which is similar to the corresponding N. crassa protein. We have obtained peptide sequences from the S. macrospora protein by mass spectroscopy and were able to identify the corresponding gene. The protein does not have significant homology to any characterized proteins; but interestingly, the corresponding gene was identified previously by microarray hybridization as being strongly downregulated in three developmental mutants from S. macrospora that carry mutations in signal transduction proteins or transcription factors and do not form any mature fruiting bodies (Masloff et al. Genetics 152: 191-199, Pöggeler and Kück Eukaryot Cell 3: 232-240). Taken together, these data indicate that the isolated protein might play a role in fruiting body development of pyrenomycetes.

497. Spatial and temporal expression of laccase in Coprinopsis cinerea using galectin promoters. J. K. Pemmasani, R. Velagapudi, S. Kilaru, K. Kaur, P. Hoegger, U. Kües. Georg-August-University of Göttingen, Institute for Forest Botany, Göttingen, Germany

Fruiting body development in homobasidiomycete Coprinopsis cinerea is a complex, poorly understood cellular process. Galectins are beta-galactoside binding lectins expressed during initiation and development of fruiting bodies in C. cinerea. The proteins are encoded by two genes, cgl1 and cgl2. cgl2 is expressed in the mycelium under dark conditions at the initiation of fruiting body development till the end of primordia formation, whereas expression of cgl1 starts later with the light-induced formation of secondary knots, compact hyphal aggregates (Boulianne et al. 2000, Microbiology 146: 1841; Bertossa et al. 2004, FGB 41: 1120). In this study, we establish the C. cinerea laccase gene lcc1 as a reporter gene to study spatial and temporal regulation of the cgl1 and cgl2 promoters. Within agar plates, promoter activity is locally detected by conversion of the laccase substrate ABTS into a colored substance. Within fruiting bodies, promoter activity is detected by enzyme tests with isolated tissues. Enzymatic tests for specific detection of promoter activities within tissues of intact structures are to be developed. The methods will be useful for later analysis of other promoter activities during fruiting body development.

Our laboratory is financially supported by the Deutsche Bundesstiftung Umwelt (DBU).

498. Pheromones and pheromone receptors of the homothallic ascomycete Sordaria macrospora. Severine Mayrhofer, Stefanie Poeggeler. Ruhr-University, 44780 Bochum, Germany

The genome of the homothallic filamentous ascomycete Sordaria macrospora is predicted to encode two pheromone precursors and two seven-transmembrane pheromone receptors. The deduced proteins of the pheromone precursor genes ppg1 and ppg2 are structurally similar to the alpha-factor precursors and a-factor precursors of the yeast Saccharomyces cerevisiae, respectively. The products of the pheromone receptor genes pre2 and pre1 display significant sequence similarity with the S. cerevisiae Ste2p alpha-factor receptor and Ste3p a-factor receptor, respectively. Deletion of pheromone genes ppg1 and ppg2 does not affect vegetative growth or fertility of S. macrospora. However, deletion of pheromone receptor genes resulted in delayed fruiting body and ascospore development. Double mutants e.g. deltapre2/deltappg2 produce only a drastically reduced number of perithecia and ascospores, thus implying an involvement of pheromones in fruiting body development of homothallic ascomycetes.

In order to analyse the functionality of pheromone /receptor pair of the homothallic S. macrospora, we used a heterologous yeast assay. The S. macrospora alpha-factor-like pheromone precursor PPG1 was shown to be processed to an active pheromone by yeast MAT alpha cells. The S. macrospora PRE2 receptor was demonstrated to be a G-protein coupled receptor that can substitute for the endogenous Ste2p receptor in yeast MATa cells.

499. Ras Gap 1 of the homobasidiomycete Schizophyllum commune regulates growth rate, hyphal growth orientation and fruitbody formation. Daniela Schubert and Erika Kothe. Institute of Microbiology, Friedrich-Schiller-University, Jena, Germany.

The white rot fungus Schizophyllum commune is used for the analysis of mating and sexual development in homobasidiomycete fungi. We isolated the gene gap1 encoding a GTPase-activating protein for Ras. Disruption of gap1 should therefore lead to strains accumulating Ras in its activated, GTP-bound state and to constitutive Ras signaling. Haploid delta gap1 monokaryons of different mating types did not show alterations in mating behaviour in the four different mating interactions in a tetrapolar mating system. Instead, growth rate in delta gap1 monokaryons was reduced about 25 % and about 50 % in homozygous delta gap1/delta gap1 dikaryons. Monokaryons, as well as homozygous dikaryons carrying the disrupted gap1 alleles exhibited a disorientated growth pattern. This failure of maintenance of growth direction was especially obvious in dikaryons during clamp formation as hook cells failed to fuse with the peg beside them. Instead, the dikaryotic character of the hyphae was rescued by fusion of the hooks with nearby developing branches. delta gap1/delta gap1 dikaryons formed increased numbers of fruit body primordia, whereas the amount of fruit bodies was not raised. Mature fruit bodies formed no or abnormal gills. No production of spores could be observed.

500. Characterization of subunit one and two of the A. nidulans COP9 Signalosome. Elke U. Schwier, Oliver Draht, Silke Busch, Krystyna W. Nahlik, Gerhard H. Braus. Institut für Mikrobiologie und Genetik, Georg-August-Universitaet Goettingen (GER)

The COP9 Signalosome (CSN) is a highly conserved protein complex critical to the accurate development of numerous multicellular organisms. It shares structural similarities with the lid subcomplex of the proteasome and the translation initiation factor 3 (eIF3). Recently two subunits of the COP9 Signalosome were isolated in the filamentous ascomycete A. nidulans. Deletion of either csnD or csnE resulted in pleiotrophic phenotypes affecting the sexual cycle, secondary metabolism and light signaling.

We report the isolation and characterization of csnA and csnB from A. nidulans. The csnA coding sequence of 1497 bp is interrupted by three introns resulting in a protein of approximately 55,7 kDa. The approximately 54,2 kDa Protein CSNB is encoded by a DNA sequence of 1670 bp. Both deduced amino acid sequences contain a PCI domain and show high identity to the analogous CSN subunits of higher eukaryotes. Deletion of either csnA or csnB resulted in viable strains with hyphae appearing partly red. The sexual cycle is initiated but fruit body formation is blocked at the stage of primordia. These defects are identical to those found for the csnD and csnE deletion strains. We conclude that csnA and csnB code for the first two subunits as defined by database searches of the COP9-Signalosome in A. nidulans. Strains carrying a csnA::gfp fusion are constructed to verify where the protein is localized at different developmental stages.

501. Multiple roles of a heterotrimeric G protein gamma subunit in governing growth and development of Aspergillus nidulans. Jeong-Ah Seo, Kap-Hoon Han, and Jae-Hyuk Yu

Dept. of Food Microbiology and Toxicology, & Food Research Institute, University of Wisconsin-Madison, 1925 Willow Drive, Madison, 53706 USA

Vegetative growth signaling in the filamentous fungus Aspergillus nidulans is primarily mediated by the heterotrimeric G protein composed of FadA (Galpha), SfaD (Gbeta) and a presumed Ggamma. Analysis of the A. nidulans genome identified a single gene named gpgA encoding a putative Ggamma subunit. The predicted GpgA protein consists of 90 amino acids showing 72% similarity with yeast Ste18p. Deletion (D) of gpgA resulted in reduced vegetative growth and lowered asexual sporulation. Moreover, despite highly elevated Hülle cell production, the DgpgA mutant was unable to produce sexual fruiting bodies (cleistothecia) in self-fertilization and was severely impaired with cleistothecial development in outcross, indicating that GpgA is required for sexual development. Developmental and morphological defects caused by deletion of flbA encoding an RGS protein controlling FadA-signaling were suppressed by DgpgA, suggesting that GpgA primarily functions in vegetative growth signaling. However, deletion of gpgA could not bypass the need for fluG in asexual sporulation, indicating that GpgA indirectly affects conidiation. We propose that GpgA defines the Ggamma subunit constituting the FadA-SfaD::GpgA heterotrimer, and is required for normal vegetative growth and proper asexual/sexual developmental progression. Effects of deletion of gpgA, sfaD and/or fadA on mRNA levels of remaining G protein subunits are also presented.

502. Aspergillus nidulans Morphogenesis Mutant, swoK1, is defective in a gene that encodes an RNA binding protein. Srijana Upadhyay and Brian D. Shaw. Program for the Biology of Filamentous Fungi, Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas, 77803, USA.

The Aspergillus nidulans swoK1 mutant is defective in polarity maintenance when grown at restrictive temperature (38C). Upon germination, the swoK1 mutant extends a primary germ tube that swells to an enlarged, non-uniform cell with pronounced wall thickenings. The mutant is fully restored to wild type growth when transformed with a plasmid containing the gene An5802.2 as designated in The Broad Institute A. nidulans genomic database. The locus in the swoK1 mutant contains a single base deletion (G) 545 bp downstream from the predicted start codon that results in a frame-shift that is predicted to terminate in a stop codon 18 amino acids down stream of the lesion. The SwoK predicted protein contains an N-terminal RRM (RNA Recognition motif) and a highly repetitive C-terminus with SR repeats and RD repeats. The SwoK protein is 43% identical to the S. pombe protein Srp1. The proteins each contain the N-terminal RRM and in the SR repeats, but SRP1 does not contain the RD repeats. In S. pombe Srp1 is both an RNA splicing factor and responsible for RNA translocation out of the nucleus. Full length homologs of this protein are found in the filamentous fungal genomes of N. crassa and M. grisea. The possible role of this protein in polarized development will be discussed.

503. How many hydrophobins does a mushroom need? R. Velagapudi, S. Peddireddi*, P.J. Hoegger, A. Majcherczyk, A. Polle and U. Kües. Georg-August-University Göttingen, Institute for Forest Botany and NHN*, Göttingen, Germany

Within the genome of the dung fungus Coprinopsis cinerea ( we identified 34 potential type I hydrophobin genes by multiple BLAST searching, using known hydrophobin sequences from the NCBI data base. Compared to other fungi, this is the largest hydrophobin group known so far. For example, there are 20 different genes in the white-rot fungus Phanerochaete chrysosporium (, two in the smut Ustilago maydis and none in the human pathogen Cryptococcus neoformans ( In C. cinerea, the closely related genes Coh1 to Coh7 are clustered together in the genome as the closely related genes Coh29 to Coh34, suggesting them to have been arisen via duplications. Other less related genes are spread within the genome either singly or in groups of two or three. Within vegetative mycelium and fruiting bodies, we detected multiple stage-specific hydrophobins of about 10 kDa and different proteins were identified by LC-MS. Within phylogenetic trees, there is no defined correlation of C. cinerea proteins with fruiting- and mycelium-specific hydrophobins from other basidiomycetes. Further protein analysis and gene expression studies will verify how many of the multiple hydrophobin genes are functional at the different stages of development.

Financial support by the Deutsche Bundesstiftung Umwelt, the European Regional Development Fund and the country of Lower Saxony is acknowledged.

504. Two putative Zn(II)2Cys6 transcription factors, RosA (repressor of sexual development) and NosA (no sexual development) trigger early developmental decisions in the filamentous fungus Aspergillus nidulans. Kay Vienken1, Reinhard Fischer2. 1Biochemistry; Max-Planck-Institute for terrestrial Microbiology; Marburg; 2Applied Microbiology, TU Karlsruhe; Germany

Morphologic differentiation processes such as spore production in fungi are largely controlled by stage-specific expression of regulatory proteins. We have analyzed two putative Zn(II)2Cys6 transcription factors in the filamentous fungus A. nidulans. One gene, rosA, encodes a 713 amino acid long protein transiently expressed during asexual rather than sexual development.

Expression was also induced after carbon starvation. Deletion of the gene caused the fungus to initiate sexual spore production under non-appropriate conditions, such as carbon-starvation or in liquid culture. Subcellular localization of a RosA-GFP fusion protein suggested that shuttling of the factor between the cytoplasm and the nucleus may be important for the function. In summary, RosA is involved in the transduction of environmental conditions into developmental decisions. The second regulator, NosA, is in contrast to the negative regulator RosA, essential to induce sexual development. The expression of the gene appeared to be constitutive suggesting

posttranscriptional regulation of NosA activity. To identify putative target genes for the two important regulators we performed DIGE-2D-gel electrophoresis with subsequent peptide mass fingerprinting for protein identification.

505. Withdrawn

506. COT1, POD6 and LRG1 are required for hyphal elongation in Neurospora crassa. N. Vogt, N. Rabanizada, G. H. Braus, and S. Seiler. Inst. of Microbiology & Genetics, Goettingen, Germany

In a screen for mutants defective in hyphal morphogenesis, we have identified temperature-sensitive mutants in cot-1, pod-6 and lrg-1 that result in the cessation of hyphal elongation and induction of hyperbranching at restrictive temperature. COT1 is related to Rho kinases. POD6 is a novel protein with similarity to the STE20 superfamily, and the GTPase activating domain of LRG1 suggest an involvement of Rho proteins in hyphal elongation. cot-1;pod-6 double mutants are identical to the parental strains, while cot-1;lrg-1 and pod-6;lrg-1 are synthetic lethal. Mutations in the dynein complex and in the RNA binding protein GUL1 were identified as common suppressors for all three mutants. Also, enhanced sensitivity to oxidative stress and partial suppression of the growth defect through osmotic stabilizers was observed for all mutants. COT1 and POD6 colocalize in a vesicular network throughout the hypha. This distribution is dependent on the activity of both dynein and conventional kinesin. Domain analysis of LRG1 indicates that the LIM and GAP domains are both necessary for wild type growth. Overexpression of Rho1 result in full suppression of the lrg-1 growth defect. In vitro GAP assays are in progress to characterise the function of the GAP domain at a molecular level. Taken together, these results suggest that COT1/POD6 are acting together in a functional complex, but that this complex needs the activity of an independent LRG1/RHO1 dependent pathway to coordinate hyphal morphogenesis.

507. Growth and developmental mutants of Aspergillus fumigatus: Comparative forward genetics. Jae-Hyung Mah, Kap-Hoon Han, Greg Flygt and Jae-Hyuk Yu. Food Microbiology and Toxicology, University of Wisconsin-Madison

Aspergillus fumigatus (AF) is the most prevalent airborne pathogen, which causes severe invasive aspergillosis in immunocompromised patients. While the available genome sequences of AF and the model fungus Aspergillus nidulans (AN) permits comparative reverse genetic studies, gene-deletion or overexpression may not be sufficient to unveil the molecular mechanisms underlying species-specific physiological outcomes. In fact, disruption of two major developmental regulators flbA and fluG in AF provided evidence that, although AF and AN share conserved G protein-mediated growth signaling, AF has distinct sporulation processes. To further dissect upstream regulation of growth and development in AF, we carried out forward genetic studies and isolated four groups of AF mutants exhibiting: 1) enhanced colony growth 2) moderately fluffy-autolytic 3) wet-melting spores and 4) non-conidial fluffy phenotypes. Mutations responsible for the mutant groups 1 and 2 were mapped within AF-GpaA (Galpha) or AF-flbA. In conjunction with deletion analyses of AF-fluG and AF-flbA, identification of these mutations further supports the hypothesis that AF-GpaA and AF-FlbA coordinate balanced growth and sporulation, but AF has distinct and persistent sporulation mechanisms. These findings suggest that genes required for AF sporulation may only be identified via forward genetic approach. Transformation-based gene identification is underway. Specific mutations, associated phenotypic changes and speculated genetic model are presented.

508. GzRum1, a putative transcription regulator required for ascospore development in Gibberella zeae. Hee-Hyung Kim1, Theresa Lee2, Yin-Won Lee3, and Sung-Hwan Yun1. 1Div. of Life Sciences, Soonchunhyang Univ., Asan, 336-745; 2Gene expression team, NIAB, RDA, Suwon 441-707; 3School of Agricultural Biotechnology, Seoul Nat¡̅l Univ., Seoul 151-921, Korea

Gibberlla zeae is a homothallic ascomycete with ubiquitous geographic distribution. This fungus infects several cereal crops and causes disease symptoms such as scab and head blight. The importance of G. zeae ascospores in disease development had been recognized since the sexual spores that can overwinter in a sexual fruiting body (perithecium) cause early infection in the next spring. Our goal is to identify and characterize the genetic traits involved in sexual development by G. zeae. In this report, we show the function of a cDNA clone (GzRum1) obtained by cDNA subtraction analysis of the wild-type G. zeae Z3643 against an isogenic mat1-2 deletion strain. GzRum1 exhibited a high sequence similarity to a transcriptional repressor encoded by Rum1 in Ustilago maydis, which is essential for sexual spore formation in U. maydis. Targeted deletion of GzRum1 caused no phenotypic changes in the GzRum1-deletion mutants except ascospore formation. The mutant produced normal-looking perithecia but no ascospores. RNA blot analysis revealed that expression of GzRum1 strongly increased only during the perithecial stage and that the GzRum1 transcript was not detected in the mat1-2 deletion strain, indicating that transcription of GzRum1 is regulated by MAT1-2 in G. zeae.