112. Pheromone discrimination in Schizophyllum commune and evolutionary aspects. Susanne Gola and Erika Kothe. Friedrich-Schiller-University, Institut für Mikrobiologie, Mikrobielle Phytopathologie, Winzerlaer Str. 10, 07745 Jena
The tetrapolar mating type is defined in part by a multi-specific pheromone/receptor system. The interaction of pheromone receptors and their non-self ligands, encoded by the B loci of Schizophyllum commune, lead to sexual development. There is no induction of sexual development by pheromones of self specificity, of which several are encoded within the locus. Using chimeric receptors composed of Balpha1 and Balpha2 specificity new phenotypes were found. Single point mutations allowed to further characterize the interactions between ligands and receptors in vivo. Our results are in accordance with a multi-state receptor activation for this G protein coupled receptor. The evolution of multiple, independent and multispecific pheromone receptors and several pheromones encoded in the mating type loci is a feature unique to homobasidiomycete fungi. A model for the evolution of new specificities by recombination and selection is proposed which explains evolution of new specificities. The model makes use of the multi-state receptor activation explaining differential responses to different pheromones in one receptor molecule.
113. A yeast-like pheromone/receptor system is involved in the sexual development of mycelial ascomycetes. Stefanie Poeggeler and Ulrich Kuck. Department of Botany, Rhur-University Bochum, Germany
In order to analyze the involvement of pheromones in cell recognition and mating in homothallic and heterothallic ascomycetes, two putative pheromone precursor genes, named ppg1 and ppg2, were isolated from the homothallic fungus Sordaria macrospora. The ppg1 gene is predicted to encode a precursor pheromone that is structurally similar to the alpha-factor of the yeast Saccharomyces cerevisiae. The ppg2 gene encodes a 24-amino-acid polypeptide that contains a putative farnesylated and carboxy-methylated C-terminal cysteine residue. Disruption of both pheromone precursor genes in S. macrospora revealed that pheromones are involved in the sexual development of a homothallic fungus. Detection of pheromone genes in filamentous ascomycetes implicated the presence of pheromone receptor genes. We have identified two pheromone receptor genes, named pre1 and pre2, in each of the genomes Neurospora crassa and of S. macrospora. The deduced pre1 gene products are putative seven-transmembrane proteins, which display a high-level amino acid identity with the S. cerevisiae a-factor receptor Ste3p, and are also homologous to lipopeptide pheromone receptors of basidiomycetes. The deduced pre2 products display significant sequence similarity with the yeast STE2 gene product, the alpha-factor receptor. The two genes are transcriptionally expressed in both N. crassa and S. macrospora. Northern and RT-PCR analyses indicate that in N. crassa, expression of the receptor genes does not occur in a mating type specific manner.
114. SwoHp, A Nucleoside Diphosphate Kinase, Is Essential in Aspergillus nidulans. Xiaorong Lin1, Cory Momany2, and Michelle Momany1, 1Plant Biology, 2Pharmaceutical and Biomedical Sciences, University of Georgia
The ts- swoH-1 mutant of A. nidulans was swollen and lysed during vegetative growth at restrictive temperature. The swoH gene was mapped to chromosome II and cloned by complementation. The sequence showed that the swoH gene encodes a homologue of nucleoside diphosphate kinases (NDKs). Cell extract from the swoH-1mutant is low in NDK activity. Sequence of the mutant allele and structural modeling suggested that the swoH-1 mutation distorts the enzyme active site. The swoH gene fused with 3xHA tag at the N-terminus complemented swoH-1 mutant indicating the functional integrity of the fusion protein. The tagged protein possessed the phosphate transferase activity as analyzed by TLC. Similar to other eukaryotes, SwoHp in A. nidulans forms a hexamer based on results of western blot of a native gel and subunit interactions of the modeled protein structure. Although NDK has been considered a housekeeping enzyme in nucleotide metabolism, recent evidence suggests that it is also an important regulatory protein in many cellular processes. In mammals, NDKs are involved in differentiation and tumor metastasis. In plants, NDK interacts with the phytochrome molecules. Although null mutants of E. coli, S. cerevisiae and S. pombe are viable, deletion of the swoH gene was lethal in A. nidulans suggesting that NDK plays a more important role in this filamentous fungus.
115. Heterologous microarray hybridization identifies genes regulated differentially during sexual development of Sordaria macrospora. Minou Nowrousian, Stefanie Pöggeler, Carol Ringelberg, Jennifer J. Loros, Jay C. Dunlap, Ulrich Kück. Ruhr-University Bochum and Dartmouth Medical School.
The filamentous fungus Sordaria macrospora develops complex fruiting bodies (perithecia) to propagate its sexual spores. Not much is known about gene expression patterns during this developmental process; therefore, we have used microarray hybridization to identify genes that are differentially regulated in the wild type compared to the developmental mutant pro1. We have made use of the fact that S. macrospora is a close relative of Neurospora crassa, for which microarrays have been established previously. Microarrays spotted with N. crassa probes were hybridized with targets derived from S. macrospora RNA. Using this heterologous array hybridization, we were able to identify a number of candidate genes that are more than threefold up- or downregulated in the S. macrospora developmental mutant pro1 compared to the wild type. For several of these genes, verification experiments were performed using Northern blots or real time PCR, and in all cases confirmed the expression patterns observed on the arrays. As the mutant pro1 lacks the zinc finger protein PRO1 which is a putative transcription factor, at least some of the genes that are transcriptionally up- or downregulated in the mutant strain might be target genes directly regulated by the PRO1 protein. Among the genes that are upregulated in the mutant strain is ppg1 (pheromone precursor gene 1), which in S. macrospora and other fungi has been shown to be involved in sexual development.
116. A protein kinase of Phytophthora infestans induced during zoosporogenesis has a novel structure. Flavio A. Blanco, Samuel Roberts and Howard S. Judelson. University of California, Riverside, CA, USA.
Zoosporogenesis in P. infestans is a critical step in infecting plants. Proteins involved in this process are good targets for disease control. A gene expressed during zoospore formation was identified that showed high similarity to protein kinases. The predicted sequence of the protein contains all 12 domains diagnostic of Ser/Thr kinases, and the best similarity to the Ca+2/CaM family. However, it lacked the long C terminal regulatory domain typical of such proteins, while having a longer N terminus. The promoter of this gene was cloned upstream of the gus gene and the construct was used to transform P. infestans.Cis elements involved in expression during zoosporogenesis will be discovered by deletion analysis and associated transcription factors will be identified through one-hybrid screening. To study the activity of the protein, it was expressed as a fusion with the maltose binding protein in E. coli. Antibodies were raised to study the accumulation of the kinase during life cycle, its subcellular localization and interacting proteins. At the same time, the kinase was used as bait in a two-hybrid screening of a cDNA library from different stages of zoosporogenesis (from sporangia to swimming zoospores). Several interactors were identified which may represent substrates and regulatory proteins. These studies will reveal the role and regulation of this kinase and identify other components of signal transduction pathways involved in zoosporogenesis.
117. Negative control of asexual sporulation in Aspergillus nidulans: FluG suppressor analyses. Jeong-Ah Seo, Yajun Guan, and Jaehyuk Yu. Department of Food Microbiology and Toxicology, University of Wisconsin, Madison, WI 53706,USA.
Asexual sporulation (conidiation) in Aspergillus nidulans requires an early developmental activator called FluG. Loss of fluG function results in the blockage of both conidiation and production of the mycotoxin sterigmatocystin. Molecular mechanisms of FluG-mediated activation of conidiation are unknown. To investigate molecular events responding to FluG, we carried out an unbiased genetic analysis and have isolated and characterized 40 suppressor mutants that overcome the sporulation defects of a loss of fluG function mutant (suppressor of FluG; SFG). Genetic analyses revealed that an individual suppression is the result of a single second site mutation not linked to fluG, and that most sfg mutations are recessive, only one is dominant. Among many recessive SFGs, we have identified two allelic suppressors (sfgA) that are predicted to encode a novel Zn2Cys6 DNA binding protein of which mRNA is differentially expressed during the lifecycle. Miss-sense mutations in SfgA defined by these allelic suppressors are expected to cause a loss-of-function suggesting that SfgA negatively regulates conidiation downstream of fluG. Moreover, complete loss of sfgA not only suppresses loss of fluG functions, but also induces conidiophore formation in liquid submerged culture within 22 hrs indicating that FluG's main role in activation of conidiation is to remove the negative effects imposed by SfgA. Molecular genetic analysis leading to a finding of the involvement of at least two loci in suppression of fluG is also presented.
118. Polar growth in Aspergillus nidulans requires protein modification: an examination of swoA and swoF. B. D. Shaw, and M. Momany. Department of Plant Biology, Plant Science Building, University of Georgia, Athens, GA, 30602, USA.
Two swollen cell (swo) temp. sensitive A. nidulans mutants that are aberrant in polar growth were previously complemented and their corresponding genes were cloned and sequenced. SwoA is a protein mannosyl transferase responsible for the first step in protein O-glycosylation, i.e. the co-translational addition of a mannose residue to serine or threonine residues in the endoplasmic reticulum . The swoA mutant grows isotropically producing giant >50 micrometer diameter round cells that do not send out a polarized germ tube. SwoF is an N-myristoyl transferase responsible for attaching a 14 carbon fatty acid, myristate, to the N-terminus of a small subset of proteins. This modification is thought to increase the affinity of the target for hydrophobic membranes. The swoF mutant can send out a polarized germ tube but with 10 micrometers of growth the tip ceases extension and begins to swell. Strategies to identify the role of these protein modifications in polar growth will be discussed, including a 2D proteomics approach comparing total protein from wild type and mutant cells. Progress in mutant screens to identify additional swollen cell mutants will be discussed. To date at least six new single locus swo mutants have been identified.
119. The Noxa Gene Encodes a Gp91phox Homologue Necessary for Sexual Development in Aspergillus nidulans. Lara-Ortiz, T. and Jesús Aguirre. Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, 04150 México, D.F.
gp91phox is the catalytic subunit of the respiratory burst oxidase, an NADPH dependent, superoxide generating oxidase from phagocytes. Recently, a new family of gp91phox homologues (Nox) has been described in non-phagocytic mammalian cells, which have been implicated in growth factor signaling, mitogenic responses, apoptosis and oxygen sensing. Although no Nox enzymes were reported in microorganisms, Biochemical evidence from our laboratory suggested the presence of a NADPH oxidase activity in A. nidulans, which led us to request Cereon Genomics LLC for possible gp91phox homologue genes. A 720 bp PCR product was amplified using primers based upon provided partial genomic sequence. Cosmids identified with this fragment were used to obtain entire noxA sequence. Predicted NoxA protein is 47% identical to human gp91phox and highly similar to other Nox enzymes. To characterize noxA function, a disruption construct was generated replacing part of the noxA coding region with selective marker argB. noxA null mutants were unable to develop sexual fruit bodies, indicating that noxA is necessary for sexual development. Accordingly, noxA transcript was induced during sexual development and derepressed in sakA null mutants, which show premature sexual development. Our results indicate that reactive oxygen species are involved in A. nidulans sexual development.
120. Identifying genes involved in asexual sporulation of Phytophthora infestans using cDNA macroarrays. Kyoung Su Kim and Howard S. Judelson, University of California, Riverside, CA USA
Asexual sporangia are the main agents of dispersal of P. infestans, and the main inoculum for the potato and tomato late blight. To understand the factors involved in differentiating sporangia from hyphae, and to identify spore components that participate in germination, 5200 sequenced clones from a sporangial cDNA library were spotted on membranes and hybridized with total cDNA from sporangia, hyphae, germinated spores, starved hyphae, and sporulation-defective strains. This identified 54 genes up-regulated >5-fold in sporangia. For 43 genes, searches against GENBANK predicted functions in regulation (transcription factors, protein phosphatases and kinases, signal transduction, etc.), structure, transport, and metabolism. The most common type of gene induced in spores (>10% of mRNA) were polyol dehydrogenases. These plus selected others were also induced during starvation. The genes could also be grouped based on whether they continued to be expressed at high levels after germination, or in sporulation-defective strains. This identified genes that may participate in early versus later stages of spore biology. The cellular roles of genes with regulatory functions, such as transcriptional activators, are currently being characterized by gene silencing, yeast two-hybrid, and other methods. Fusions between GUS and spore-induced promoters are also being dissected to identify the mechanisms by which the genes are activated.
121. The COP9 signalosome is an essential regulator of development in the filamentous fungus Aspergillus nidulans. S. Busch, S. E. Eckert (1), S. Krappmann, G. H. Braus. Institute of Microbiology and Genetics, Department Molecular Microbiology and Genetics, Georg-August-University, Grisebachstr. 8, D-37077 Goettingen, Germany. (1) Present address: Department of Biosciences, Faculty of Science, Technology and Medical Studies, University of Kent at Canterbury, Canterbury, Kent CT2 7NJ, UK.
We identified the COP9 signalosome (CSN) as an essential regulator of development in the filamentous fungus Aspergillus nidulans. The deduced amino acid sequences of the two genes csnD and csnE show up to 38% and 53% identities to the fourth and fifth CSN subunits of higher eukaryotes. The A. nidulans CSND protein is accumulated in the nucleus and interacts with CSNE in a yeast two-hybrid assay. Deletion of either csn locus resulted in viable strains with identical developmental phenotypes which could be complemented by the according wild-type genes. When grown in an air-medium interface, vegetative cells of A. nidulans csn deletion strains were impaired in cell polarity and showed changes in secondary metabolism resulting in the accumulation of a red pigment. The csn deletion strains showed disturbed light-dependence of developmental initiation processes, including constitutive repression of the onset of the asexual reproductive cycle in developmentally synchronised and competent mycelia. The deltacsn mutants were capable to initiate the sexual cycle and develop primordia of fruit bodies. However, the further maturation and morphogenesis of primordia to sexual fruit bodies was blocked. This developmental arrest could not be overcome by overexpression of the sexual activator velvet (VEA). We conclude that the COP9 signalosome is a key regulator in A. nidulans which is essential for light-dependent signalling and sexual development.
122. Characterization of differentially expressed genes in dimorphism and pathogenesis in Ustilago maydis. María D. García-Pedrajas, David L. Andrews and Scott E. Gold. Department of Plant Pathology, University of Georgia, Athens, GA 30602-7274
Morphogenesis and pathogenesis are closely related in the corn pathogen Ustilago maydis. Upon mating of compatible haploid sporidia U. maydis switches from budding growth to a filamentous dikaryon. This transition in morphology coincides with a change from saprophytic to pathogenic development. Production of teliospores, the only cells competent to undergo meiosis, occurs only inside the plant, therefore the fungus is completely dependant on the plant to complete its life cycle. Previously, we have identified a role for the cAMP signal transduction pathway in dimorphism and pathogenicity. We are now using suppression subtractive hybridization PCR (SSHP) to identify novel genes involved in dimorphism. We have identified a number of genes upregulated during filamentous or budding growth and confirmed differential expression by northern blot analysis. We have produced disruption mutants of select genes to determine their biological roles in morphogenesis and/or pathogenesis. We are also using the same technique, SSHP, to identify genes from both the plant and the fungus involved in gall formation and teliosporogenesis. We have constructed two libraries enriched for genes up and downregulated during gall formation, respectively. Screening of these libraries by reverse northerns has revealed many genes differentially expressed in galls versus infected non-galled tissue. We are now in the process of characterizing individual genes from both libraries.
123. Analysis of the Spore Killer Phenotype of the Ascomycete Podospora anserina. Andrea Hamann, Jens Rabenstein, Heinz D. Osiewacz. J. W. Goethe-Universitaet, Botanisches Institut, Frankfurt/Main, Germany
In some filamentous fungi a special form of meiotic drive, spore killing, can be observed. Crosses between a so-called killer strain and a sensitive strain result in the elimination of spores carrying exclusively the sensitive allele. The ascomycete Podospora anserina normally produces four heterokaryotic – linearily ordered – ascospores. In case of spore killing, only those spores survive that carry at least one killer allele. Spores exclusively containing sensitive alleles degenerate early in development. The nature of the corresponding alleles and the cause for the degeneration of the sensitive spores remain speculative. The degeneration may be triggered by the strictly regulated transposition of mobile elements or by epigenetic factors. To identify components involved in spore killing, two different strategies were followed. Both strategies employ a subtractive suppressive hybridization technique (SSH). First, the genome differences between a killer and a sensitive strain are analysed. Second, genes are identified which become differentially expressed during sexual development and especially during spore killing. Northern and sequence analysis of these genes are presented and discussed.
124. Isolation of the bad42 gene of Coprinus cinereus. Patricia J. Pukkila, Paul Medina, and Suzanne Harrold Dept. Biology U. North Carolina-Chapel Hill
We have taken advantage of several features of the C. cinereus mating system to recover many recessive mutations (induced by UV) that block basidiospore development (bad mutants). To facilitate molecular analysis of these genes, we have identified new badalleles generated by transgene insertions that fail to complement the original mutations. We have used TAIL PCR to obtain sequences flanking the transgene insertions, and recovered cosmid clones that complement the meiotic defects for bad42. Sequencing of a cosmid clone, cDNA clones, and PCR products from the UV-induced allele revealed that the original mutation is a C to T transition generating an in-frame stop codon 392AA upstream from the 3' end of the gene. The non-complementing REMI mutation is a rearranged transgene inserted 215AA upstream from the 3' end. The bad42 gene encodes a basic protein with limited similarities to other gene products that have been previously implicated in meiosis. In C. cinereus, bad42 is a typical class II mutant which exhibits full length axial cores with very little SC. Both the point mutant and the insertion mutant exhibit metaphase I arrest. The cosmid clone integrated at random genomic positions complements the bad42 mutation both when homozygous and when heterozygous. We conclude that either "meiotic silencing by unpaired DNA" does not occur in C. cinereus, or that bad42 sequences escape MSUD, either because of the timing of their expression or because of the particular genomic locations of these ectopic cosmid insertions.
125. External pH Affect Secretion of Pectate Lyase by C. gloeosporioides. H. Kramer- Haimovich1 , N. Drori1 , J. Rollins4, Y. Okon2, O. Pines3 and D. Prusky1. 1Department of Postharvest Science of Fresh Produce, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel. 2Department of Plant Pathology and Microbiology, Hebrew University of Jerusalem, Faculty of Agricultural, Food and Environmental Quality Sciences, Rehovot 76100, Israel, 3Department of Molecular Biology, Hebrew University, Medical School, Jerusalem 91120, Israel. 4Department of Plant Pathology, University of Florida, Gainesville, Florida 32611, USA.
Accumulation of ammonia and associated tissue alkalinization predisposes fruit to attack by C. gloeosporioides. As external pH increases from 4.0 to 6.0, pectate lyase (PL) and other extracellular proteins are secreted and accumulate. At pH 4.0 neither pelB (encoding PL) transcription nor PL secretion were detected; however they were detected as the pH increased. Sequence analysis of the 5’ upstream region of the pelB promoter revealed nine putative consensus-binding sites for the Aspergillus transcription factor PacC. Accordingly transcript levels of pac1 (the C. gloeosporioides pacC homolog) and pelB increased in parallel as a function of pH. Our results suggest that ambient pH is an independent regulatory factors for processes linked to PL secretion and virulence of C. gloeosporioides.
126. Factors Controlling Expression of Mep2p in Saccharomyces cerevisiae: interplay of phosphorylation state and the cAMP-dependent PKA pathway. Casey Shawler, David Smith, Michael H. Perlin. University of Louisville, Louisville, Kentucky, USA
The Mep2 protein acts as both a high-affinity transporter of ammonium and as a sensor tied to a signal transduction pathway that, under conditions of nitrogen limitation, shifts budding yeast cells into psuedohyphal growth. Genetic evidence ties Mep2p to the cAMP-dependent PKA pathway, since mep2 deletion mutants defective in the pseudohyphal response are corrected by addition of exogenous cAMP or by bypass of cyclase mutations (bcy) that allow constitutive PKA. Interestingly, we have identified in Mep2p (see poster by Smith, Shawler, Lovely, and Perlin) a putative target for phosphorylation by PKA. We have made several site-directed mutants in this position, one of which mimicks phosphorylation. In order to examine possible interactions that regulate Mep2p expression, we have constructed fusions between the MEP2 promoter and the lacZ gene. This construct was placed into wildtype S. cerevisiae as well as strains with mutations in different genes within the PKA pathway. Moreover, expression was also examined for strains containing a second construct in which the Mep2p produced contained a mutation that made the protein appear phosphorylated. Like the wildtype protein, this mutant was able to complement the pseudohyphal defect in a strain deleted for the three MEP genes. However, expression of the mutant Mep2p in atpk2/tpk2 deletion strain, defective in PKA activity, allowed this strain to overcome its pseudohyphal defect on low ammonium, suggesting that over-expression of the "modified" Mep2p is epistatic to PKA. When these results are combined with those on transcriptional effects of mutations in the PKA pathway, an interesting picture will begin to emerge of the relationships between this conserved ammonium transporter, the even more highly-conserved PKA pathway, and their respective roles in dimorphism of fungi.
127. Role of inositol triphosphate and calcium signalling in gene expression during zoosporogenesis in Phytophthora infestans. Shuji Tani, Flavio A. Blanco, and Howard S. Judelson. Department of Plant Pathology, University of California, Riverside, California 92521, USA.
Most infections by the oomycete Phytophthora infestans, the late blight pathogen, are initiated by zoospores released from asexual sporangia. This release occurs at cool temperatures in a process known to involve Ca2+ or other cations. At higher temperatures, germ tubes emerge directly from sporangia. To better understand these pathways, we tested the effects of inhibitors on the release of zoospores, direct germination, and gene expression during zoosporogenesis. Inhibitors of ion channels, kinases, and inositol triphosphate (IP3) pathways impaired zoospore release, but not direct germination. To identify genes induced during zoosporogenesis, candidates from EST database mining approaches were tested by Northern blotting. This identified 12 genes induced in sporangia undergoing cleavage into zoospores, but not in directly germinating sporangia. Most genes were repressed by one or more inhibitors, although a broad spectrum of quantitatively distinct responses were observed and some genes were insensitive to all inhibitors tested. The majority of genes were repressed drastically by 2APB, an inhibitor of IP3-induced Ca2+ release, and by the phospholipase C inhibitor U73122. The functions and transcriptional regulation mechanisms of the cleavage-induced genes are now being examined. Emphasis is being placed on studying a family of transcription factor-binding proteins including the analysis of interactors identified by yeast two-hybrid analysis.
128. The role of the transcriptional regulator RfxA during growth and development of the dimorphic pathogen Penicillium marneffei. Hayley Smith, Michael J. Hynes and Alex Andrianopoulos. Department of Genetics, The University of Melbourne, Parkville 3010, Australia.
Penicillium marneffei is a thermally dimorphic ascomycete capable of causing infection in immunocompromised individuals. At 25̊C, P. marneffei exhibits filamentous growth and is also capable of undergoing asexual development to produce uninucleate conidia. Upon transfer to 37̊C a process known as arthroconidiation occurs where cellular and nuclear division become coupled, double septae are deposited and fragmentation of the hyphal filaments leads to the production of uninucleate yeast cells that divide by fission. We are interested in examining the regulatory networks controlling cell-type specificity, development and pathogenesis in P. marneffei. The RFX proteins (Regulatory Factor X) comprise a family of transcriptional regulators containing a novel DNA binding domain, the winged helix motif, which is highly conserved from humans to yeast. Genes encoding RFX-type proteins have been isolated from a number of fungi. In S. cerevisiae Crt1p is required for regulating the expression of DNA damage-inducible genes, while the essential sak1 protein of S. pombefunctions downstream of PKA and is required for the completion of mitosis and entry into either stationary phase or the sexual differentiation pathway. The cpcR1 gene of A. chrysogenum, initially identified as a key regulator of cephalosporin C biosynthetic genes, is also required for the hyphal fragmentation of mycelium in liquid culture leading to the production of short, swollen yeast-like arthrospores. Given that this process is highly analogous to arthroconidiation in P. marneffei, common mechanisms may be acting to regulate this morphological transition. A P. marneffei cpcR1 homologue, rfxA, has been cloned using degenerate PCR and displays significant homology to other fungal RFX family members. The role of rfxA during development and yeast-hyphal morphogenesis is being investigated through the analysis of both rfxA deletion and overexpression mutants.
129. Development, oxidative and general stress signal transduction in Aspergillus nidulans. Laura Kawasaki, Olivia Sanchez, Kasuhiro Shiozaki2, Teresa Lara-Ortiz and Jesus Aguirre, Instituto de Fisiolgia Celular-UNAM, Apartado Postal 70-242, 04510 Mexico, D.F. and 2University of California-Davis.
We have proposed that reactive oxygen species play a central role in microbial development. During the past years, our group has characterized several catalase genes and studied their regulation, as an effort to understand the antioxidant response and its relation to development in Aspergillus nidulans. Recently, we have found that the sakA gene encodes a member of the Hog1/Spc1/p38 stress MAPK family. SakA MAPK is activated in response to oxidative and osmotic stress and mediates part of catalase gene regulation. On the other hand, asakA null mutant shows development and cell-specific phenotypes. First, it displays premature steA-dependent sexual development. Second, it produces asexual spores that are highly sensitive to oxidative and heat shock stress and lose viability upon storage. Indeed, SakA is transiently activated early after induction of conidiation. Our results indicate that SakA MAPK is involved in stress signal transduction, catalase gene regulation, repression of sexual development, and is required for spore stress resistance and survival. At least part of the effects on sexual development is related to regulation of the NADPH oxidase ecoding gene noxA, which is essential for this process. We thank CONACyT and PAPIIT-UNAM, Mexico for financial Support and Cereon Genomics LLC for partial noxA DNA sequence.
130. PiCDC14 expression is specific to asexual sporulation in the oomycete Phytophthora infestans. Audrey M V Ah Fong and Howard S Judelson. Department of Plant Pathology, University of California Riverside, CA 92521.
CDC14 protein phosphatases have been shown to control mitotic exit and/or cytokinesis in a range of ascomycetes and metazoans. Their genes are normally constitutively transcribed and regulated post-translationally. We have identified a CDC14 homologue from the oomycete P. infestans, designated PiCDC14, which interestingly was transcribed only during sporulation. The gene was identified using an in silico data mining strategy for developmentally regulated phosphatases. The gene is single-copy in Phytophthora and complemented the S. cerevisiae cdc14-ts mutant. Northern blot and promoter::GUS fusion analyses showed that PiCDC14 is only expressed during asexual sporulation, and not in non-sporulating hyphae or cultures forming sexual spores. PiCDC14::GUS activity was detected in sporangiophore initials and along the length of sporangiophores bearing immature sporangia, and upon maturation was restricted to sporangia. PiCDC14 mRNA was present in zoospores and cysts, but disappeared from germlings a few hours before the onset of mitosis. The analysis of transformants from homology-dependent silencing experiments supported the role of PiCDC14 in sporulation. It may synchronize nuclear division during sporulation, and then help maintain sporangia in a state of dormancy by inhibiting cell division and growth. The absence of PiCDC14 during vegetative growth may explain why nuclear division is normally asynchronous in hyphae.
131. Genomic analysis of the transcription factors controlling pseudohyphal growth in S. cerevisiae. Anthony R. Borneman and Michael Snyder. Department of Molecular, Cellular and Developmental Biology. Yale University, New Haven, CT, USA.
When exposed to an environment low in nitrogen, diploid cells of the yeast S. cerevisiae undergo a transition from an ellipsoidal, unicellular, vegetative state to a pseudohyphal growth form. Pseudohyphae are characterised as being composed of elongated cells which fail to detach from their mother cell. These cells therefore form branching chains of elongated, conjoined cells that radiate out from the colony, presumably aiding in dispersal to areas of higher nitrogen availability. Several transcription factors, including Tec1p, Ste12p, Sok2p and Phd1p, have been shown to affect pseudohyphal growth through the modulation of downstream effector gene expression. While some of these targets, such as the cell wall flocculin, Flo11p, have been identified, it is likely that there are many other genes which remained to be discovered. Chromatin immunoprecipitation microarrays (chIP chip's) were therefore used to elucidate, on a genomic scale, the binding targets for several transcription factors that have been shown to be involved in pseudohyphal growth. This data has therefore allowed for the formulation of a transcriptional network for pseudohyphal growth which encompasses these transcription factors and their downstream genes. This provides the basis for future experiments into the function of previously unidentified/uncharacterised genes in cellular development in S. cerevisiae.
132. Oxidative stress and conidiation in Neurospora crassa. Wilhelm Hansberg, Shaday Michán, Fernando Lledías, Adelaida Díaz, Leonardo Peraza, Pablo Rangel. Instituto de Fisiología Celular, Universidad Nacional Autónoma de México
Morphogenetic transitions of N. crassa asexual life cycle are responses to a hyperoxidant state. Catalase activity induction and catalase oxidation by singlet oxygen are consequences of this hyperoxidant state. The two large monofunctional catalases (Cat–1 and Cat–3) and catalase-peroxidase (Cat–2) are resistant to molar concentrations of hydrogen peroxide. They are oxidized by singlet oxygen at the heme, without significantly affecting enzyme activity, but are degraded rapidly. Cat–1 is expressed in non-growing cells, such as hyphae in stationary growth and conidia. Cat-1 is accumulated to high levels in conidia. Crystallographic structure of Cat-1 showed an oxidized heme and an inusual covalent bond at the active site. Cat–2 is associated with lysing cells, such as hyphae in late stationary growth, in conidiating substrate mycelium, and base of aerial hyphae. Cat–3 is associated with growing hyphae and is expressed during late exponential and pre-stationary growth. Cat–3 has a signal peptide and is secreted. Light and oxidative stress induces Cat-3. A Cat–3 null mutant strain showed increased protein oxidation and carotene levels in the dark, indicating oxidative stress. It formed hyphae aggregates and produced six fold the amount of wild type strain aerial hyphae and conidia. Aerial hyphae developed earlier and in higher number per area than wild type. Results support our hypothesis of cell differentiation as response to oxidative stress. Acknowledgements: CONACyT 33148N, DGAPA/UNAM IN225402-2
133. Catalase-peroxidase of presumable bacterial origin. Leonardo Peraza and Wilhelm Hansberg. Instituto de Fisiología Celular, Universidad Nacional Autónoma de México
Neurospora crassa Catalase-2 (Cat-2) was purified and characterized. It is a homodimer of 83.4 kDa subunits. Absorption spectrum and HPLC analysis indicated that its prosthetic group is a two hydroxyl/two carboxylic porphyrin that is different from protoporphyrin IX. The enzyme has catalase and peroxidase activity with similar kinetic constants to other catalase-peroxidases (CP). An internal peptide from the purified enzyme was sequenced and the entire cat-2 cDNA sequence determined. Comparison between cDNA and the genomic sequence (Whitehead Institute/MIT) revealed an ORF with no introns encoding a 753 amino acid CP. Phylogenetic analysis indicates that Cat-2 corresponds to a typical CP that, together with another fungal CP, diverged from the Burkholderia (b-proteobacteria) branch. We suggest a bacterial origin for fungal CPs. Cat-2 transcript and activity were found in late stationary-phase mycelia, in growing hyphae treated with heat shock or H2O2 or growing on fatty acids. Cat-2 was developmentally regulated: after 30 min induction of conidiation by air exposure of a mycelial mat, cat-2 mRNA accumulated and Cat-2 activity was detected after 4 h when aerial hyphae were formed. Both, mRNA and Cat-2 increase further and were present in aerial hyphae and conidia. In general, Cat-2 was associated with cells undergoing lysis. Acknowledgements: CONACyT 33148N
134. An unusual Cys-Tyr covalent bond present in the crystall structure of Neurospora crassa catalase-1. Hansberg, W., Díaz, A., Horjales, E., Rudiño-Piñera, E. and Arreola, R. Instituto de Fisiología Celular and Instituto de Biotecnología, Universidad Nacional Autónoma de México
Catalase-1 (Cat-1) is one of the two large monofunctional catalases from N. crassa. Cat-1 is associated with non-growing cells and is accumulated in conidia. Cat-1 heme is modified by singlet oxygen during cell differentiation and under stress conditions. Here we present the crystallographic structure of Cat-1 determined at 1.75 Å resolution. The structure of Cat-1 was determined by molecular replacement using Escherichia coliHPII catalase as a starting model. Cat-1 has a C-terminal domain with a flavodoxin topology similar to E. coli HPII and a catalase from Penicillium vitale. Cat-1 dimer is formed by mutual insertion of the N-terminal into the loop of the wrapping domain between the Q-related subunits. The heme group, not covalently bound to the protein, is buried inside the Cat-1 tetramer at 20 Å from the molecular surface. Substrate arrives to the active site through a hydrophobic channel filled with water. Cat-1 has protoheme IX (heme b) (57%) and an oxidized heme (heme d) (43%), originated by dihydroxylation at ring D and subsequent formation of a spirolactone with the propionyl group of this ring. E. coli HPII and a catalase from P. vitale also have heme d. Cat-1 has an unusual covalent bond between the sulfur of Cys 356 and Cb of Tyr 379 that coordinates the FeIII of the heme. The distance of Cys-Tyr bond is 1.9 Å. HPII has a similar covalent bond between the essential Tyr 415 and His 392. This bond could be related to Cat-1 resistance to inactivation by substrate. Acknowledgements: CONACyT 33148N
135. Molecular regulation of sclerotial development and sclerotial-specific gene expression in Sclerotinia sclerotiorum. Jeffrey A. Rollins, Wayne M. Jurick II, and Ulla K. Benny. Department of Plant Pathology, University of Florida, Gainesville FL.
Sclerotia play a key role in the life cycle of Sclerotinia sclerotiorum. These compact, melanized, multihyphal resting structures serve as long term survival structures that myceliogenically and carpogenetically germinate to propagate the fungus. Functional analysis of the pH-responsive transcription factor Pac1 has revealed a key role for Pac1 in sclerotial development and maturation. pac1 transcript accumulation is low in vegetative hyphae but dramatically elevated during sclerotial development. pac1 null mutants produced by gene-replacement form coalescing, multihyphal structures without distinct melanized rind layers. Other genes with sclerotial-specific transcript accumulation have been identified from a small EST collection. Amino acid sequence data obtained from tryptic fragments of a major sclerotial-specific storage protein (Ssp) were used to identify a cDNA clone encoding the Ssp protein. ssp transcript accumulation is high and tightly restricted to the sclerotium. A second unrelated gene with no known function exhibits an identical pattern of transcript accumulation. The most highly represented clone (ca. 6.5% of the total) in the EST collection, encodes a profilin homolog. Transcripts of this gene are abundant during vegetative growth, apothecial development, and sclerotial initiation but greatly reduced during sclerotial development and exposure to alkaline pH. The functions of these genes in sclerotial development and the role of Pac1 in their regulation are currently being evaluated.
136. To "P" or not to "P"?:The action of methylammonium permeases in fungal dimorphism depends on their ability to be phosphorylated. David Smith1, Casey Shawler1, C. Ben Lovely1, Joe Heitman2, and Michael H. Perlin1. 1 University of Louisville, Louisville, Kentucky, USA and 2 Duke University, Durham, North Carolina, USA
While first cloned from yeast and Arabidopsis, the genes for methyalammonium permease homologues (MEPs) are found in organisms ranging from bacteria to humans. InSaccharomyces cerevisiae, one of the three MEPs, Mep2p, seems to be a nitrogen sensor and plays a direct role in the switch from yeast cells to pseudohyphal growth in this organism. InUstilago maydis, the causative agent of galls on maize, the corresponding homologue, Ump2, is required for the ability of haploid yeast-like cells to grow filamentously on low nitrogen. Moreover, Ump2 can substitute for Mep2p in S. cerevisiae mep1 mep2 mep3mutants normally incapable of pseudohyphal growth on low nitrogen. The effects of disruption ofmep2 could be reversed by supplying exogenous cAMP. Such results suggest a connection to the respective cAMP-dependent PKA pathways in these two fungi. Comparison of the amino acid sequences of various MEPs revealed a putative phosphorylation site in MEPs of fungi that was absent in MEPs of other organisms. Site-directed mutagenesis of this site inmep2 and ump2, revealed that replacement of the putative target residue(s) had no effect on growth of S. cerevisiae on low ammonium, but eliminated filamentation under these conditions. In U. maydis, this mutant still provided filamentation on low ammonium. But, a second mutant in which the amino acid change mimicked phosphorylation failed to complement the filamentation defect of ump2 knock-outs. Together these results provide a model in which the potential phosphorylation state of MEPs controls their roles in filamentous growth for two disparate fungi.
137. Filamentous growth and ammonium uptake in Ustilago maydis andSaccharomyces cerevisiae: the methylammonium permease, Ump2, performs both functions. David G. Smith1, Maria D. Garcia-Pedrajas2, Scott E. Gold2, and Michael H. Perlin1. 1 University of Louisville, Louisville, Kentucky, USA and 2 University of Georgia, Athens, Georgia, USA
Nutrient sensing plays important roles in fungal development in general, and specifically in critical aspects of pathogenicity and virulence. Many dimorphic pathogens must switch from a yeast-like to a filamentous form in order to cause disease. This switch is also required for the phytopathogenic smut fungi, U. maydis and Microbotryum violaceum. Three genes encoding methylammonium permeases were identified from these fungi and all the encoded proteins were most similar to Mep2p, the high-affinity permease from S. cerevisiae that plays a direct role in pseudohyphal or filamentous growth for that organism. Each of the smut genes was expressed in diploid S. cerevisiae mutants deleted for all three mep genes (mep1mep2mep3). Each could complement the severe growth defect of the S. cerevisiae mutant on low ammonium. Moreover, the U. maydis ump2 gene was also able to complement the pseudohyphal defect characteristic of the mutant yeast. This is the first report of a heterologous mep gene capable of restoring pseudohyphal growth in yeast. In U. maydis, disruption of ump2 eliminated the filamentous phenotype of haploid cells on low ammonium. The ump1ump2 double mutant produced a branched budding phenotype and when grown in liquid medium, appeared to flocculate. Of particular note was that the double mutant regained the ability to filament on low nitrogen; however, the filaments of the double mutant appeared more entangled than wildtype. Although all three types of mutants showed reduced uptake of methylamine, the most significant drop in uptake was seen for the ump2 mutant and the ump1ump2 double mutants. These findings allow us to present a model of how ammonium transporters play a role in regulating dimorphic growth in fungi.
138. Switching fungal reproductive mode by manipulation of mating-type gene. Jungkwan Lee1, Theresa Lee1, Yin-Won Lee1, Sung-Hwan Yun2, and B. Gillian Turgeon3.1School of Agricultural Biotechnology and Research Center for New Bio-materials in Agriculture, Seoul National University, Suwon 441-744, Korea. 2Division of Life Sciences, Soonchunhyang University, Asan 336-745, Korea. 3Department of Plant Pathology, Cornell University, 334 Plant Science Building, Ithaca, NY 14853.
Fungi capable of sexual reproduction use heterothallic (self-sterile) or homothallic (self-fertile) mating strategies. In most ascomycetes, a single mating type locus, MAT, with two alternate forms (MAT1-1 and MAT1-2) called idiomorphs, controls mating ability. In heterothallic ascomycetes these alternate idiomorphs reside in different nuclei. In contrast, most homothallic ascomycetes carry both MAT1-1 and MAT1-2 in a single nucleus, usually closely linked. An economically important example of the latter isGibberella zeae, a devastating cereal pathogen of ubiquitous geographic distribution and also a producer of deadly mycotoxins that threaten human and animal health. Because analysis of traits of interest is difficult in homothallic species, we asked if G. zeae could be made strictly heterothallic by manipulation of MAT. Targeted gene replacement was used to differentially delete MAT1-1 or MAT1-2 from a wild typeMAT1-1/MAT1-2 strain, resulting in MAT1-1/MAT1-0,MAT1-0/MAT1-2 strains that were self-sterile, yet able to cross to wild type testers and more importantly, to each other. These results indicate that differential deletion of MATidiomorphs eliminates selfing ability of G. zeae, but ability to outcross is retained. They also indicated that both MAT idiomorphs are required for self fertility. Furthermore, to our knowledge, this is the first report of complete conversion of fungal reproductive strategy from homothallic to heterothallic by targeted manipulation of MAT. Practically, this approach opens the door to simple and efficient procedures for obtaining sexual recombinants ofG. zeae that will be useful for genetic analyses of pathogenicity and other traits, such as production of threatening mycotoxins.
139. NpgAp encoding the homolog of 4'-phosphopantetheinyl transferase is required for cellular growth and conidiation in Aspergillus nidulans. Jung-Mi Kim, Dong-Min Han1, Keon-Sang Chae, Hwan-Gyu Kim and Kwang-Yeop Jahng. Division of Biological Sciences, Chonbuk National University, Jeonju, South Korea; 1Division of Life Sciences, Wonkwang University, Iksan, South Korea
In Aspergillus nidulans, the npgA gene has been known that it plays an important role in pigmentation. We previously isolated and sequenced the DNA fragment that complemented npgA1 mutation from genomic cosmid library of A. nidulans. The npgA gene encoded a putative protein of 344 amino acids that has 42% of similarity with phosphopantetheinyl transferase encoded by the LYS5 gene of Saccharomyces cerevisae, which transfers the 4'-phosphopantetheinyl (P-pant) moiety of coenzyme A. The cDNA of npgA of A. nidulans recover the growth defect of the lys5 strain of S. cerevisae, indicating that npgA could functionally complement lys5 deletion of S. cerevisae. The deletion mutant of npgA neither grew nor produced secondary metabolites such as the sterigmatocystin and the siderophore. The defect of growth and conidiation in npgA knock-out mutant was cured when provided with the culture filtrate of wild type. These results suggested that the NpgAp might be involved in the posttranslational modification of enzymes required for primary growth or the synthesis of secondary metabolites such as pigment and antibiotics in A. nidulans.
140. Altering sexual reproductive mode by interspecific exchange of MATloci. Shun-Wen Lu1, Sung-Hwan Yun2, Theresa Lee3 and B. Gillian Turgeon1. 1Department of Plant Pathology, Cornell University, Ithaca, NY 14853 2Division of Life Science, Soonchunhyang University, Asan 336-745, Korea 3School of Agricultural Biotechnology, Seoul National University, Suwon 441-744, Korea
Sexual fungi can be self-sterile (heterothallic, requiring genetically distinct partners) or self-fertile (homothallic, requiring no partner). Our goal is to understand the molecular basis for these distinct reproductive modes. The model fungi we have chosen are two closely related filamentous ascomycetes, Cochliobolus heterostrophus (heterothallic) and C. luttrellii (homothallic). We previously demonstrated that the C. luttrellii MAT gene alone conferred homothallism when expressed in a MAT-deleted strain of heterothallicC. heterostrophus. Here we report a reciprocal study in which the C. heterostrophus MAT genes were expressed, separately, in a MAT-deleted C. luttrellii strain, which is sterile. A C. luttrellii MAT-deleted strain carrying ChMAT-1 produced fertile pseudothecia when mated with a C. luttrellii MAT-deleted strain carrying ChMAT-2. Fertility of pseudothecia was similar to that of wild type. Tetrad analysis confirmed that progeny segregated for parental markers. Surprisingly, each transgenic strain was also able to self although all pseudothecia produced were smaller than those of wild type and the fertility was low (number of asci was about 5% of the number of wild type asci). These data support the argument that the primary determinant of reproductive mode is MAT itself.
142. Isolation and characterization of mutants that can sexually develop in the presence of visible light. Jong-Hak Kim, Min-Su Kim, Yoon-Hee Cheon, Keon-Sang Chae1, Dong-Min Han. Dept., Biol., Wonkwang University, Iksan, Chonbuk 570-749, Korea1Div., Biol., Sci., Chonbuk National University, Chonju, Chonbuk, 561-756, Korea
When a homothallic ascomycete, Aspergillus nidulance, is exposed to visible light, cleistothecial development is inhibited and instead plenty of asexual spores developed. The ratio of sexual/asexual development increases as light intensity decreases and the sexual development preferentially takes place in dark. This light responsiveness of development implies the existence of delicate regulation process including reception and translocation of light signaling and determination of development. In order to study the genes involved in this regulatory network, we first attempted to isolate mutants that could develop cleistothecia even in the presence of relatively intense visible light. More than 200 mutants were isolated and analyzed. They were divided into two groups. One included those that had defects in the response specific to light and the other those that developed cleistothecia in regardless of the presence of any other inhibitory environmental stress such as high osmolarity. We selected 8 mutants that belonged to the former group. Six mutants were revealed to have single gene mutation and grouped into different complementation groups(silA-F). The mutant alleles were all recessive to that of wild type. The silA gene was cloned and sequence-analyzed. The putative SilA has a Zn2Cys6 zinc finger motif at N terminus and shows high amino acid sequence similarity to Aro80p of Saccharomyces cerevisiae.
143. Sexual development in the euascomycete Podospora anserina: the role of pheromones. Evelyne Coppin1, Deborah Bell-Pedersen2, Dan Ebbole2 and Robert Debuchy1. 1 Institut de Genetique et Microbiologie, UMR8621, 91405 Orsay, France. 2 Texas A&M University, College Station, TX 77843-2132.
The role of pheromones during fertilization has been investigated in several Euascomycetes, but little is known about their function after fertilization. We have cloned the pheromone genes of P. anserina to investigate their possible function after fertilization. Degenerate primers have been designed from the alignement of N. crassa mfa and M. grisea MF1-1and low stringency PCR with these primers allowed us to isolate mfp. Low stringency hybridization with ccg4 of N. crassa on P. anserina genomic DNA led to the isolation of mfm. The deletion of mfp results in male sterility in mat+strains but does not affect female fertility and has no effect in mat- strains. Reciprocally, the deletion of mfm results only in male sterility in mat- strains. Specific transcription of mfm and mfp in mat- and mat+ strains indicates that these genes are under the control of the mating-type genes. The genetic analysis of strains containing transcriptionally deregulated pheromone genes suggests that the mating types also control the post-transcriptional events required for the production of active pheromones. Experiments aimed at testing the function of pheromones after fertilization are underway. This work is supported by the ACI Biophys 2001 (project n̊ 185).
144. Temporal localization of RIP and rearrangements in Podospora anserina. Khaled Bouhouche, Sylvie Arnaise and Robert Debuchy. Institut de Génétique et Microbiologie, UMR8621, 91405 Orsay, France.
Genetic analyses in P. anserina suggest that RIP and sequence rearrangements (RIP/R) occur between fertilization and premeiotic replication. A few events have been identified during this period in P. anserina, offering the possibility to specify the time of RIP/R. Fertilization is followed by the division of male and female nuclei inside a syncytium. The mating-type genes FPR1, FMR1 and SMR2 control the recognition between male and female nuclei. This step is associated with a developmental arrest which is overcome by the action of SMR1. Then pairs of male and female nuclei form dikaryotic hyphae in which premeiotic replication, karyogamy, meiosis and ascospore formation take place. We examined if RIP/R occur before or after the action of each one of the mating-type genes, based on the construction of strains disrupted for each mating-type gene and carrying a cis-duplication of this gene. If RIP/R occur before the action of the duplicated gene, the strain will give a chacteristic progeny indicating that the gene has been altered. If RIP/R occur after the action of the duplicated gene, the strain will not display any phenotype in crosses, but the progeny will produce an affected progeny upon crossing. Preliminary results indicate that RIP/R occur mainly before the action of SMR1. Studies are underway to determine if RIP/R occur before or after the action of FPR1, FMR1 and SMR2, namely before internuclear recognition or during the developmental arrest.
145. Laccases in the dung fungus Coprinus cinereus. M. Navarro-Gonzalez, P. Hoegger, M. Hoffmann, S. Kilaru, R. Dviwedi, M. Zommorodi, A. Majcherczyk, U. Kües. Georg-August-University Göttingen, Institute of Forest Botany, Göttingen, Germany
Laccases (EC 220.127.116.11) belong to the family of multi-copper oxidases and are classified as oxidoreductases acting on polyphenolic substrates. In fungi, they function in lignin and phenol degradation and likely in development and pigment synthesis. Fungal laccases may be secreted into the medium or remain attached to the fungal cell wall. In the basidiomycete Coprinus cinereus, laccase activity in the monokaryon can be induced by copper. Moreover, phenol oxidase activity has also been detected when the fungus is grown on wood (oak, poplar) and straw (wheat). C. cinereus has at least six different genes for laccases. We cloned these genes from homokaryon AmutBmut and established and compared their sequences. In this strain, being self-compatible due to defects in the mating-type loci, enzymatic laccase function correlates with fruiting body initiation. Inducers of laccases stimulate premature fruiting and enhance initiation frequencies. Laccase activity associates with the mycelium and is not or only poorly detected in the surrounding medium. Work in our laboratory is supported by the DBU (Deutsche Bundesstiftung Umwelt).
146. Mutants in initiation of fruiting body development of the basidiomycete Coprinus cinereus. P.-H. Clergeot1, G. Ruprich-Robert1, Y. Liu1, S. Loos1, P. Srivilai2, R. Velagapudi2, S. Goebel2, M. Künzler1, M. Aebi1, U. Kües2. 1ETH Zurich, Institute for Microbiology, Zurich, Switzerland. 2Georg-August-University Göttingen, Institute of Forest Botany, Göttingen, Germany
Homokaryon AmutBmut is self-compatible due to mutations in the mating-type loci and fruits without the need to mate to another strain. Therefore, we used the strain to produce mutants in fruiting body initiation. Fruiting body initiation can be divided into a dark-dependent step, primary hyphal knot formation, and a light-dependent step, secondary hyphal knot formation. The primary hyphal knot is a loose hyphal mesh that arises on one or a few carrier hyphae through intense localized formation of branches of restricted tip growth. In the dark, it will mature into a sclerotium, a compact globular resting structure with a melanized rind. In contrast, upon light illumination, the loose hyphal mesh of the primary hyphal knot develops by aggregation and further branching into a small mycelial ball of originally uniform structure, the secondary hyphal knot in which stipe and cap tissues of the mushroom will promptly differentiate. In both steps, we identified gene functions by mutant complementation. A new gene in primary hyphal knot formation appears to encode a protein related to hetE ofPodospora, the product of a gene in secondary hyphal knot formation has cyclopropane fatty acid synthase function.
147. The dst1 gene responsible for a photomorphogenetic mutation in Coprinus cinereus encodes a protein with high similarity to WC-1. Katsuyuki Yuki1, Masashi Akiyama1, Hajime Muraguchi2 and Takashi Kamada1. 1Department of Biology, Faculty of Science, Okayama University, Okayama, Japan. 2Department of Biotechnology, Faculty of Bioresource Sciences, Akita Prefectural University, Akita, Japan.
Light is one of the environmental cues that regulate mushroom (fruit body) development in the basidiomycete Coprinus cinereus. When the fungus is grown under a light-dark cycle, normal, fertile fruit bodies develop. However, when the fungus is grown in continuous darkness, ethiolated, slender fruit body primordia, called "dark stipe" form, in which the cap (pileus) remains rudimentary and never develops to produce the fertile fruit body. We isolated and genetically analyzed several blind mutants, which produce "dark stipe" even if proper light conditions are given. From the genetic analysis, we identified two genes, named dst1 and dst2, responsible for the blind phenotype. We then cloned one of the genes, dst1, as a DNA fragment that rescues the "dark stipe" phenotype by screening a cosmid library of C. cinereus. Sequencing analysis revealed that thedst1 gene encodes a protein with high similarity to WC-1 of Neurospora crassa, which has been proposed to be the blue light photoreceptor.
148. The eln3 gene responsible for cellular morphogenesis during mushroom development in Coprinus cinereus encodes a membrane protein with a general glycosyltransferase domain. Toshihide Arima, Yoshinori Morimoto, and Takashi Kamada. Department of Biology, Faculty of Science, Okayama University, Okayama, Japan.
The hypha grows at the tip during the vegetative phase. However, during mushroom (fruit body) development in homobasidiomycetes, the component hyphal cells of the fruit-body exhibit diffuse extension growth as well as tip growth to produce a proper shape of the fruit body. In Coprinus cinereus, the fruit-body primordium exhibits remarkable expansion during the last phase of development, resulting a slender fruit body with a long stipe (stalk). We isolated a developmental mutant (eln3-1) of C. cinereus, which produces an aberrant fruit body with a very short stipe, after REMI mutagenesis. Microscopic analysis revealed that the eln3-1 mutant phenotype is due to the defect in diffuse extension growth of the component cells of the stipe. After plasmid rescue from the genomic DNA of the eln3-1mutant strain, we identified the eln3 gene as a DNA fragment that rescues the eln3-1 mutation. The eln3 gene encodes a novel membrane protein of 927 amino acids with seven transmembrane helices and a general glycosyltransferase domain. The level of the eln3 transcription was much higher in the stipe than in other tissues of the fruit body and in the vegetative hyphae. Also, the level of the eln3 transcription in the stipe changed in parallel with the elongation rate of the stipe in the course of development.
149. Isolation and characterization of nsdD suppressor mutants in Aspergillus nidulans. Jee Hyun Kim, Dong Beom Lee, Jung Youl Min, Kap-Hoon Han1, Kwang-Yeop Jahng2, Dong-Min Han. Dept. Biol. Wonkwang University, Iksan, Korea, 1Dept.Food Microbiol.and Toxicol., University of Wisconsin-Madison, Madison,U.S.A, 2Div. Biol. Science, Chonbuk Univ. Chonju, Korea
The nsdD gene encodes a GATA type transcription factor, carrying a type IVb zinc finger DNA binding domain, which functions in activating sexual development of A. nidulans. A number of suppressor mutants of nsdD were isolated and characterized. According to the phenotypes of growth rate and developmental pattern, nine mutants were selected and recombinants carrying suppressor mutation but free of nsdD mutation were isolated through genetic crosses with wild type. Most of suppressor mutants developed plenty of cleistothecia even under the conditions, such as high osmolarity(e.g.1 M KCl), visible light or acetate as a sole C source, where sexual development of wild type is completely inhibited, implying that the genes might be involved in the regulation of sexual development in response to the presence of those environmental factors. All mutant alleles were recessive to that of wild type. They were grouped into five complementation groups and the respective genes were designated as sndA, sndB, sndC, sndD and sndE. ThesndA and sndC were linked to linkage group II, sndB and sndD to linkage group V and sndE to linkage group VII. Some of those genes includingsndB were cloned and sequence-analyzed.
150. veA-dependent expressions of indB and indD encoding proteins that interact with NSDD, a GATA-type transcription factor required for sexual development inAspergillus nidulans. Nak-Jung Kwon, Dong-Min Han1, Suhn-Kee Chae. Research Center for Biomedicinal Resources and Division of Life Science, Paichai University, Daejeon 302-735, Korea and 1Division of Life Science, Wonkwang University, Iksan 570-749, Korea
In nsdD mutants of Aspergillus nidulans, no sexual organs including cleistothecia, Hull cells, and primodia were ever found. Molecular cloning of the nsdDgene revealed that nsdD encoded a putative GATA type transcription factor carrying a type IVb zinc finger motif at the C-terminal end. To clarify the NSDD function, proteins interacting with NSDD have been screened using the yeast two-hybrid system. Two IND (Interactor of NSDD) proteins, INDB and INDD were isolated and characterized further. Determination of indB and indD cDNA sequences revealed open reading frames of 648 bp and 642 bp, encoding polypeptides of 215 and 213 amino acids, respectively. INDB and INDD showed 42% amino acid sequence identity and also shared similarities to an ORF in Neurospora crassa. NSDD-INDB and NSDD-INDD interactions were confirmed in vitro using a GST-pull down assay. Both proteins interacted with the Zn-finger domain of NSDD. The N-terminus of INDB and the C-terminus of INDD were responsible for the NSDD interaction. INDB and INDD was able to interact each other. Self association of INDB but not for INDD was observed, too. In Northern analysis, 1.2 kb transcripts were detected for both genes. The indB transcripts expressed to similar levels during asexual development, but increased at 20-30 hour after induction of sexual development, then decreased. In contrast, the indD transcript was the most abundant at the initial asexual developmental stage. Furthermore, both kinds of transcripts were highly produced in veAmutant strains, but repressed in veA+ wild type background, indicatingveA-dependent expressions. veA mutations conferred defectiveness in sexual differentiation. Our results suggest that increased expressions of INDB and INDD in veAbackground might affect NSDD function negatively by binding to the Zn-finger region of NSDD to block DNA binding. [Supported by grants from KOSEF]
151. NpgAp encoding the homolog of 4'-phosphopantetheinyl transferase is required for cellular growth and conidiation in Aspergillus nidulans. Jung-Mi Kim, Dong Min Han1, Keon-Sang Chae, Hwan-kyu Kim and Kwang-Yeop Jahng. Division of Biological Sciences, Chonbuk National University, Chonju, Korea; 1Division of Life Science, Wonkwang University, Iksan, Korea
In Aspergillus nidulans, the npgA gene has been known that it plays an important role in pigmentation. We previously isolated and sequenced the DNA fragment that complemented npgA1 mutation from genomic cosmid library of A. nidulans. ThenpgA gene encoded a putative protein of 344 amino acids that has 42% of similarity with phosphopantetheinyl transferase encoded by the LYS5 gene of Saccharomyces cerevisae, which transfers the 4'-phosphopantetheinyl (P-pant) moiety of coenzyme A. The cDNA of npgA of A. nidulans recover the growth defect of the lys5strain of S. cerevisae, indicating that npgA could functionally complementlys5 deletion of S. cerevisae. The deletion mutant of npgA neither grew nor produced secondary metabolites such as the sterigmatocystin and the siderophore. The defect of growth and conidiation in npgA knock-out mutant was cured when provided with the culture filtrate of wild type. These results suggested that the NpgAp might be involved in the posttranslational modification of enzymes required for primary growth or the synthesis of secondary metabolites such as pigment and antibiotics in A. nidulans.
152. Dissecting the blue light response in Trichoderma atroviride. Casas-Flores, S.1., Rosales-Saavedra, M., Rios-Momberg, M., Bibbins, M., Ponce-Noyola, P., and Herrera-Estrella, A2. Departament of Plant Genetic Engineering, CINVESTAV Unidad Irapuato. Apartado postal 629. 36500., Irapuato, Guanajuato, México.
The biocontrol agent Trichoderma atroviride responds to blue light by developing conidia in the colony perimeter where the light pulse was received. Additionally, an increase of the transcripción of phr1 (photolyase gene) and tpka1 (protein kinase gene) is normaly detected. In Neurospora crassa, white collar-1 (wc-1 ) andwhite collar-2 (wc-2 ) genes are required for blue light response and circadiam rhythms. The corresponding homologous genes to wc-1 and wc-2 were cloned inT. atroviride (twc1 and twc2 respectively). Expression analysis of bothtwc1 and twc2, showed no significative changes when Trichodermacolonies were shifted from dark to light conditions. twc1 and twc2 null mutants were generated by gene replacement. Both mutants were unable to produce conidia in response to blue light. Conidiation driven by any other conditions was not affected. Light-induced transcription of phr1 was completely dependent on functional Twc1 and Twc2. On the other hand, tpk1 induction was not affected in twc1- andtwc2- strains. Interstingly, both mutants seem to have a differencial behaviour when growing under light or dark conditions These results suggest the presence of at least two different photoreceptors for blue light in Trichoderma. Addittionally we are employing an strategie based on a massive molecular analysis of four subtractive cDNA libraries that were obtained during blue light exposure.
153. GanBp, a homolog of G protein alpha subunit, negatively regulates the asexual sporulation in Aspergillus nidulans. Mi-Hee Chang1, Dong-Min Han2, Keon-Sang Chae1 and Kwang-Yeop Jahng1. 1Division of Biological Sciences, Chonbuk National University, Chonju; 2Division of Life Sciences, Wonkwang University, Iksan, Korea.
We isolated ganA and ganB encoding G alpha protein homologs fromAspergillus nidulans. To investigate the cellular function of these G proteins, we have constructed various mutants of ganA and ganB by gene targeting. The viscosity of culture broth of ganA dominant-activating mutant in which GanAp is constitutively activated was lower than that of wild type. The protoplasts were generated more rapidly in this mutant. However deletion, over-expression, or dominant interfering mutants of ganAhave shown no clear phenotype in growth and development. Meanwhile, ganB deletion or dominant-interfering mutants showed hyperactive sporulation phenotype and derepressedbrlA expression in submerged culture. Constitutive activation of GanBp caused reduction of hyphal growth and asexual sporulation. Cleistothecium formation was accelerated inganB over-expression mutants but reduced in ganB deletion and interfering mutants. Consequently, we suggest GanBp probably negative regulator of asexual sporulation resulting in progressing sexual development. In addition, ganB deletion or dominant-interfering mutation delayed conidial germination rate and dominant-activating mutation caused precocious germination even without carbon source, implying that GanBp might be involved in carbon source sensing required for conidial germination in A. nidulans.
154. Conidiation genes of the plant pathogenic fungus Fusarium oxysporum. Toshiaki Ohara, and Takashi Tsuge. Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
Fusarium oxysporum is a soil-borne facultative parasite that causes economically important losses on a wide variety of crops. F. oxysporum produces three kinds of asexual spores, microconidia, macroconidia, and chlamydospores. Ellipsoidal microconidia and falcate macroconidia are formed from phialides; globose chlamydospores with thick walls are formed acrogenously from hyphae or by the modification of hyphal cells. We identified the F. oxysporum homologs of medA and stuA, which have been identified to encode the developmental regulators for asexual and sexual sporulation in Aspergillus nidulans. The medA homolog, named REN1, was identified by restriction enzyme-mediated integration mutagenesis. The stuA homolog, named FoSTUA, was isolated by a PCR-based cloning. Although the REN1 mutants exhibit normal growth and form chlamydospores, they lack microconidia and macroconidia and form rod-shaped, conidium-like cells. Thus, REN1 is required specifically for development of microconidia and macroconida. The FoSTUA mutants produce microconidia, macroconidia, and chlamydospores. Mutation in FoSTUA, however, quantitatively affects the development of macroconidia and chlamydospores: numbers of macroconidia and chlamydospores were significantly reduced and increased, respectively, by the mutation. We propose that REN1 is comprised in the core pathway for development of microconidia and macroconidia in F. oxysporum.
155. Roles of a ras homologue in apical growth of Neurospora. Tadako Murayama,Tomomi Edo, and Yasuhiro Ishibashi. College of Engineering, Kanto-Gakuin University, Yokohama 236-8501 Japan
A morphological mutant smco7 is a null mutant of one of the ras homologues in Neurospora, NC-ras2. The extension growth of the smco7 mutant was considerably lower than that of the wild type. The hyphae of the mutant were thinner and more crowded than those of the wild type. The apical cells of smco7 were shorter, thinner, and more fragile than those of the wild type. The smco7 mutation seemed to cause the defects in cell wall synthesis. The cell wall precursors in the apical vesicles have been reported to be transported to the apices, and secreted there through the common mechanisms to those in the transport and exocytosis of vesicles containing extracellular enzymes. The extracellular invertase and trehalase activities were much lower in smco7 than in the wild type. An actin inhibitor, Cytochalasin A (CA), considerably inhibited the hyphal growth, made hyphae thinner and more crowded, and lowered the level of extracellular invertase after the mycelia of the wild type were shifted to the medium containing CA. The smco7 mutant was much more sensitive to CA than the wild type. These results suggest that the actin plays important roles in the apical growth of the hyphae and the secretion of extracellular enzymes and the NC-ras2 protein plays some roles in the regulation of function of actin in Neurospora. The region where the Nc-ras2 protein functions and roles of the Nc-ras2 protein will be discussed
156. Morphological mutants of Neurospora crassa. I. From sequence to phenotype in Neurospora morphogenesis. David D. Perkins, Stanford University.
This is the first of three posters that give different views of mutants with altered morphology (or pigmentation), as seen (1) in living cultures, (2) scanning EM photographs, and (3) conventional micrographs of altered hyphal growth. Living cultures of ~50 representative morphological mutants will be displayed, with primary gene products specified when they are known. Prior to the Neurospora genome project, nearly one-tenth of the predicted 10,000 genes had already been identified and mapped using mutations and classical genetic methods. Many of these mutations were recognized by their effects on morphology. Thirty years ago, E. L. Tatum and others attempted to identify the defects of various morphological mutants and to use them in studies of morphogenesis, but they were frustrated for want of appropriate molecular tools. Molecular identification of primary products of some of the morphologicals was eventually accomplished by other workers, using cloned genes at loci that had previously been defined using mutant phenotypes and classical genetic methods. For example, the wild type allele ofcrisp, one of the first mapped Neurospora mutants, was cloned, sequenced, and shown to specify adenylate cyclase. Now, ESTs and information from the genome project enable us to go in the reverse direction, from DNA sequence to phenotype. Inactivation of genes that were originally defined by molecular sequence reveals that many of them affect morphology. Predicting mutant morphology solely from a priori knowledge of sequence will usually be difficult or impossible. However, it may be possible to rationalize the phenotypic effect once the mutant morphology is known. Unravelling the web of morphogenetic events that lead from primary gene product to visible phenotype remains a daunting challenge.
157. Morphological mutants of Neurospora crassa. II. Scanning EM photographs. Matthew L Springer, Stanford University.
This is the second of three posters giving different views of mutants with altered morphology. Examination of morphological mutants by scanning electron microscopy provides information to supplement what is known from gross morphology. The photographs of morphological mutants that are shown here can be accessed via the FGSC web site at http://www.fgsc.net/neurosimages/neuimage.htm or via the American Society for Microbiology web site at http://www.microbelibrary.org/Visual/page1.htm. Methodology used in preparing material for photography is described in Springer and Yanofsky 1989, Genes and Development 3:559-571. Information on the individual mutants, and references to published sources, are given in Perkins, Radford, and Sachs, 2001. The Neurospora Compendium: Chromosomal Loci (Academic Press), available on line at http://www.fgsc.net/. Living cultures of all the mutants pictured here can be seen in the preceding poster.
158. Morphological mutants of Neurospora crassa. III. Hyphal morphology mutants. Stephan Seiler, University of Munich and Michael Plamann, University of Missouri-Kansas City
This is the third of three posters giving different views of mutants that alter morphology. Cellular polarity is a fundamental property of every cell. To identify the critical components that contribute to polarized growth, we developed a large-scale genetic screen for the isolation of conditional mutants defective in polar and directed growth in the model fungus Neurospora crassa. Phenotypic analysis and complementation tests of ca. 950 mutants showing defects in hyphal growth identified more than 100 complementation groups that define 20 distinct morphological classes. The phenotypes range from polarity defects over the whole hypha or more specific defects localized to hyphal tips and subapical regions to defects in branch formation and growth directionality. To convert this mutant collection into meaningful biological information, we identified the defective genes in 45 mutants covering all phenotypic classes. These genes encode novel proteins as well as proteins which (i) regulate the actin and microtubule cytoskeleton, (ii) are kinases or components of signal transduction pathways, (iii) are part of the secretory pathway, and (iv) have functions in cell wall formation or (v) membrane biosynthesis. These mutants highlight the dynamic nature of a fungal hypha and establish a molecular model for hyphal growth and polarity.
159. The homolog of yeast NDT80 is involved in vegetative growth in Neurospora crassa. Qijun Xiang and Louise Glass. Dept of Plant&Microbial Biology, University of California, Berkeley, CA94720
The budding yeast protein Ndt80 belongs to a newly defined transcription family. It functions at pachytene of yeast gametogenesis to activate transcription of a large group of genes at the end of meiotic prophase. A putative Neurospora protein shares high similarity with Ndt80p. This NDT80 homolog was mutated by RIP (Repeat Induced Point mutation) and a number of mutants have been obtained. The majority of the mutants have shortened aerial hyphae and profuse conidiation pattern, a phenotype similar to vib-1 mutants that we have reported previously. vib-1 is involved in mediating vegetative incompatibility. Interestingly, The NDT80 homolog and VIB-1 also share a conserved region with ~100 amino acids. We are examining whether or not the NDT80 homolog is also involved in vegetative incompatibility and meiosis.
160. Further analysis of the cAMP pathway in the control of dimorphic growth in Ustilago maydis. Scott E. Gold, John D. Egan*, María D. García-Pedrajas Department of Plant Pathology, University of Georgia, Athens, GA 30602-7274 *Current address, Dept of Biology, Salisbury University, Salisbury, MD
The cyclic AMP pathway is important in the determination of morphological phase in a number of dimorphic fungi. In the corn smut pathogen, Ustilago maydis, activation of the cAMP dependent protein kinase (PKA) pathway generates budding growth. Earlier we had identified specific components of the pathway in a forward genetic approach. Here we report results related to reverse genetic approaches toward further analysis of cAMP regulated morphogenesis. The roles of two protein phosphatases (PP2A and PP2B), hypothesized to counter PKA activity, were tested by mutagenesis and/or inhibitor studies. Calcineurin (PP2B) mutants have phenotypes consistent with those predicted for a protein phosphatase with a role in dephosphorylation of PKA substrates. These included generation of cell clusters of buds defective in cell separation. Deletion mutants for a second protein phosphatase, PP2A were not identified amongst numerous transformants, suggesting that the gene may be essential. Consistent with this result was the fact that addition of the PP2A inhibitor okadaic acid at low concentration caused cells to form clusters of buds similar to hyperactivity of PKA while slightly higher concentrations killed the cells. Two other genes, cap1, the cyclase associated protein and cab1, encoding a putative cAMP binding protein have been identified and their roles in cAMP signaling are being analyzed.
161. Identification of an essential fruiting gene from a REMI mutant in the basidiomycete Schizophyllum commune. Stephen Horton and Ben Wormer. Department of Biological Sciences, Union College, Schenectady, NY 12308 USA
In an effort to identify new genes essential to the process of mushroom development in the basidiomycete Schizophyllum commune, we have utilized the REMI mutagenesis procedure. A homokaryotic fruiting strain was used as a REMI recipient: we describe here the initial characterization of one non-fruiting mutant. Flanking genomic sequence disrupted by the insertion of the transforming plasmid was rescued in E. coli. The mutant was determined to be the result of a true REMI event by Southern hybridization. DNA sequencing of 5' and 3' RACE clones allowed for the prediction of the corresponding polypeptide. The gene product may facilitate hyphal aggregation, based upon some limited similarity found to other polypeptides in the protein databases. The abundance of the corresponding 2 kb transcript may be regulated with regards to fruiting body development. Targeted gene disruption experiments are being performed to confirm the essential nature of this gene to the process of fruiting. We are also in the process of overexpressing this gene to test the hypothesis that this might increase hyphal aggregation, which in turn may enhance fruiting in S. commune.
162. Circadian Rhythms in Development and Gene Expression in Aspergillus. Andrew Greene*1,2, Nancy Keller3, Hubertus Haas4, and Deborah Bell-Pedersen1,2. 1Program For the Biology of Filamentous Fungi, 2Department of Biology, Texas A&M University, College Station, TX 77843, 3Department of Plant Pathology, University of Wisconsin, Madison, WI 53706, 4 Department of Molecular Biology, University of Innsbruck, Fritz-Pregl-Str. 3 A-6020, Innsbruck, Austria
The circadian clock controls daily cycles in biochemical, physiological, and behavioral processes and has been observed in organisms ranging from cyanobacteria to humans. Among the fungi, the circadian clock has been most extensively studied in the filamentous ascomyceteNeurospora crassa, and a detailed understanding of the mechanisms involved in generating circadian rhythmicity has emerged. While daily and circadian rhythms have been documented in other fungi, we know relatively little about the degree of conservation of clock components and mechanisms within the fungal kingdom. In order to begin addressing these questions, we have demonstrated circadian rhythms in development and gene expression in the ascomycetes Aspergillus flavus and Aspergillus nidulans, respectively. The A. flavus sclerotial rhythm has an unusually long free-running period of 33 hours and entrains to light:dark cycles in a unique manner. Additionally, the frequency gene, required for circadian rhythmicity in Neurospora under normal growth conditions, is not present in the sequenced genome of A. nidulans nor the closely related A. fumigatis species. These observations suggest that the circadian clock in Aspergillus is organized differently from that of Neurospora.
163. Identification of Neurospora crassa period-4 (prd-4) may link the circadian clock and cell cycle. António M. Pregueiro, Jay C. Dunlap and Jennifer J. Loros. Departments of Genetics and Biochemistry, Dartmouth Medical School, Hanover, NH 03755
Neurospora crassa is a well established model to study the circadian clock. Among other functions, the clock regulates the developmental program leading to formation of asexual macroconidiophores. This leads to a rhythm in conidiation with a periodicity of approximately 22 hours under constant conditions. In the wild-type strain, this rhythm is endogenous and self-sustained, responds to environmental signals including light and temperature, and maintains an approximately constant periodicity within the organism's physiological range of temperature. This is a fundamental property of circadian clocks and is known as temperature compensation. Several mutants defective in clock properties have been found in Neurospora. One of them, period-4 (prd-4), has a markedly short period length of ~18.5 hours at 25C. The mutant also fails to exhibit temperature compensation, displaying an even shorter period length at higher temperatures. Heterokaryon analyses of the mutant previously showed that the prd-4 mutation is semi-dominant to wild-type making identification of the gene by rescue extremely difficult. With the release of the Neurospora genome sequence, we have sequenced the prd-4 region on Linkage Group I and found a single base-pair change that we now demonstrate is responsible for the prd-4 mutant phenotype. The gene's sequence is consistent with a role as a mitotic cell cycle regulator and may provide an entrée to understanding gating of cell cycles and the connection between the circadian clock and cell division.
164. Isolation of a mutant with uninucleate conidia from Aspergillus oryzae and its use as a host strain. K. Ishi, J. Maruyama, H. Nakajima and K. Kitamoto. Department of Biotechnology, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Aspergillus oryzae forms multinucleate conidia, which are considered to contribute to genetic stability in Japanese traditional food fermentation. However, this characteristic impedes the efficiency to isolate recessive mutants and homokaryotic transformants. We1) previously reported visualization of nuclei by expressing histone H2B-EGFP fusion protein in A. oryzae. We isolated multinucleate conidia deficient mutants (mun-) using FACS from H2B-EGFP expressing strain (niaD-, niaD::h2b-egfp) by UV irradiation. An isolated mutant, uni10 (niaD-, niaD::h2b-egfp, mun-), formed the highest percentage (approximately 80%) of uninucleate conidia. It showed almost the same phenotype in growth, conidiation and secretion of extra-cellular enzymes as the parent strain, suggesting that uni10 may be a useful host strain to isolate mutants and homokaryotic transformants. After curing of h2b-egfp from uni10 by positive selection method, an auxotrophic strain (niaD-, sC-,mun-) was bred and transformed with plasmids carrying each selection marker (niaD or sC) and heterologous genes. Therefore, it is expected that this strain can be used as a host in protein production. The efficiency of homokaryon formation after transformation will be discussed. 1) Maruyama J, et al. (2002). FEMS Microbiol Lett 206, 57-61
165. An ortholog of the Saccharomyces cerevisiae protein Prm1 is involved in mating of both heterothallic and homothallic Cochliobolus species. S.E. Baker*, S.-W. Lu**, O.C. Yoder*, S. Oide**, B.G. Turgeon**. *Torrey Mesa Research Inst., **Cornell Univ.
Mating of heterothallic ascomycetes involves both cell-cell recognition and fusion between cells of opposite mating type and nucleus-nucleus recognition and fusion, once cells have fused. Cell fusion is required initially when the male cell fuses with the female trichogyne and subsequently when cells in the crozier merge. Are all steps a requirement for sexual reproduction of homothallic species? The process of cell fusion is complex, requiring participation of many proteins, including, ultimately, proteins mediating the actual fusion of cell membranes. We have found orthologs of the yeast gene PRM1 encoding a putative fusase protein, Prm1p, in the filamentous heterothallic ascomycete Cochliobolus heterostrophus and in C. luttrellii, a homothallic species closely related to C. heterostrophus. Deletion of the gene encoding this protein in C. heterostrophus appears to have no effect on mating ability or on fertility; numbers of pseudothecia, asci and ascospores formed when a prm1-mutant is crossed to wild type or to a prm1-mutant of opposite mating type are as in wild type crosses. In contrast, deletion of the gene in C.luttrellii and selfing of transformants yields very small pseudothecia with greatly reduced numbers of mature asci (less than 10% of wild type). The asci and ascospores that are made, appear to be wild type. These results provide evidence that Prm1p may be required by homothallic fungi for cell fusion between hyphae and between crozier cells.
166. A basic-region helix-loop-helix protein-encoding gene (hpa3) involved in the development of Aspergillus nidulans. André Tüncher, Hans Reinke, Goran Martic, Maria Louise Caruso and Axel A. Brakhage Institut für Mikrobiologie, Universität Hannover, Schneiderberg 50, D-30167 Hannover, Germany
By colony hybridization of an Aspergillus nidulans cosmid genomic gene library using a probe which encoded the highly conserved basic-region helix-loop-helix (bHLH) domain of AnBH1, a novel gene designated hpa3 was isolated. HPA3 contains a region with high similarity to the bHLH-domain of different bHLH proteins. The highest similarity was found to an unknown ORF of A. fumigatus and some similarity to ESC1 of Schizosaccharomyces pombe. ESC1 is involved in the induction of sexual differentiation by nitrogen starvation. The analysis of HPA3 using the green fluorescent protein showed that HPA3 is located in the nucleus of A. nidulans. To study the physiological meaning of hpa3 a knock-out mutant was produced which was viable. However, it did not produce conidia on minimal agar plates. Scanning electron microscopic inspection showed that although the conidiophore including the vesicle was formed, hardly any metulae were produced. The addition of KCl (0.6 M) or sucrose (1.1 M) to the medium suppressed the knock-out phenotype. Induction of an alcAp-hpa3 gene fusion in a Dhpa3 mutant led to the wild-type phenotype of the mutant strain, whereas on repressing media (glucose) the mutant strain exhibited the Dhpa3 phenotype. Northern analysis revealed that hpa3 mRNA steady state levels were about the same in sporulating and vegetatively growing mycelia. Moreover, under all conditions tested self-crossing of the Dhpa3 mutant strain was never observed.