386 Differential gene expression in germinating basidiospores. Lori G. Baker and Sarah F. Covert. Department of Genetics and School of Forest Resources University of Georgia, Athens, Georgia USA
Cronartium quercuum f.sp. fusiforme is the causative agent of the southern pine disease known as fusiform rust. This disease is characterized by woody, spindle-shaped galls, which weaken pine trees and reduce their value. In a pathogenic fungus such as C. q. fusiforme, spore germination directly impacts dissemination and the spread of disease. Depending upon their environment, C. q. fusiforme basidiospores can germinate either directly or indirectly. When C. q. fusiforme basidiospores are cast upon certain surfaces, such as pine needles or plastic, they germinate directly by sending out a long, thin germ tube. In contrast on other surfaces, such as oak leaves or glass, C. q. fusiforme basidiospores germinate indirectly by sending out a very short, wide germ tube that leads to the formation of a secondary basidiospore. This study utilizes subtractive hybridization techniques to compare gene expression patterns between these two germination fates. By identifying genes that are differentially expressed in these distinct germination events, we will expand our understanding of C. q. fusiforme's germination, sporulation, and infection processes and perhaps contribute to our general understanding of the same events in other rust/pathogenic fungi.
387 Interactions between mating-type proteins from the homothallic ascomycete Sordaria macrospora. Jacobsen S, and Pöggeler, S. Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Uni-Bochum D-44780 Bochum, Germany
Mating type genes control sexual development in ascomycetes. Few is know about their function in homothallic species, which are self-fertile and do not need a mating-partner for sexual reproduction. The mating-type proteins are thought to act as transcription regulator proteins and via protein-protein interaction. To elucidate the function of the mating type genes in the homothallic fungus Sordaria macrospora, we have tested them in the yeast TWO HYBRID system. In this system the mating-type proteins SMTA-1 and SMTa-1 have domains capable of activating transcription of yeast reporter genes. The TWO HYBRID analysis for mutual heterodimerization as well as for homodimerization revealed the ability of SMTA-1 to interact with SMTa-1, two proteins which are encoded by different mating types in the related heterothallic species Neurospora crassa. Moreover, we gained evidence for homodimerization of SMTA-1. The interaction between SMTA-1 and SMTa-1 was specified by interaction experiments with truncated derivatives of SMTA-1. Furthermore we raised antibodies against SMTA-1 and SMTa-1, respectively and are currently underway to confirm the interaction between SMTA-1 and SMTa-1 by means of co-immunoprecipitation. In the light of these findings possible functions of mating-type proteins in the homothallic S. macrospora will be discussed.
388 Pheromone genes of Sordaria macrospora. Pöggeler, S. Lehrstuhl fur Allgemeine und Molekulare Botanik, Ruhr-Uni-Bochum D-44780 Bochum, Germany
In heterothallic fungi an essential step in the mating process is the recognition of mating-partners via mating pheromones. Sordaria macrospora is a homothallic ascomycete and as such a homokaryotic ascospore gives rise to hyphae that can complete the sexual life-cycle. Thus in contrast to heterothallic ascomycetes, a mating-partner with an opposite mating-type is dispensable during the sexual development. In order to analyze the involvement of pheromones in cell recognition and mating in a homothallic fungus, two putative pheromone precursor genes, named ppg1 and ppg2, were isolated from S. macrospora. The ppg1 gene is predicted to encode a precursor pheromone that is processed by a Kex2p-like protease to yield a peptide 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. Both genes are expressed during the life-cycle of S. macrospora. This is the first description of pheromone precursor genes encoded by a homothallic fungus.
Pöggeler, S. (2000) Two pheromone precursor genes are transcriptionally expressed in the homothallic ascomycete Sordaria macrospora. Curr Genet 37: 403-411
389 Signal transduction of light through ndk-1 to the morphogenesis of perithecia in Neurospora crassa. Kohji Hasunuma, Yasunobu Ogura, Yusuke Yoshida, Naoto Yabe. Yokohama City Univ. Kihara Inst. Biol. Res. Yokohama Japan
By use of a crude membrane fraction of mycelial extract of Neurospora crassa an in vitro system was developed. The irradiation of the extract by blue light increased the phosphorylation of 15 kDa protein, which was purified and identified to be nucleoside diphosphate kinase and designated to be NDK-1. NDK-1 showed i) gamma-phosphate transferring activity, ii) autophosphorylation activity and iii) protein kinase activity phosphorylating myelin basic protein. A mutant psp (phosphorylation of s mall protein) with no phosphorylation of NDK-1 was isolated, and the cDNA included ndk-1Pro72His mutation. His tagged NDK-1 and His tagged NDK-1Pro72His showed equivalent activity of i) gamma-phosphate transferring activity. However, the mutant protein showed very reduced activity of ii) autophosphorylation and iii) protein kinase activities. The psp mutant lacked ability to stimulate light induced polarity of perithecia. However, the transformation of ndk-1Pro72Hsi by cDNA and genomic DNA for NDK-1 restored the ability to stimulate the light induced porality of perithecia. Thus psp was identified to be ndk-1 Pro72His.
390 Transcription of the Achlya ambisexualis hsp90 gene is regulated by the steroid hormone antheridiol and the encoded Hsp90 protein is a component of specialized heteromeric protein complexes associated (in vertebrates) with steroid receptors. Shelley A. Brunt and Julie C. Silver. Department of Molecular and Medical Genetics and Division of Life Sciences, University of Toronto at Scarborough, Toronto, Ontario, Canada.
In the oomycete Achlya ambisexualis , hyphae of the male strain undergo sexual differentiation upon exposure to the steroid hormone antheridiol, which is produced and secreted by the female strain. Northern hybridization established that the level of hsp90 transcripts, as well as the level of transcripts for several other chaperones (hsp70, hsp60), increased in hormone-treated cells. Nuclear run-on studies established that these increases resulted from the increased transcription of these genes in the antheridiol-treated mycelia. Ongoing studies are addressing the identification of the regulatory proteins and sequences involved in the expression of the hsp90 during steroid mediated development. Earlier studies demonstrated that antheridiol binds with high affinity to a 9S multiprotein complex from A. ambisexualis cytosols. In vertebrates, steroid hormone receptors are found in 9S heteromeric complexes (aporeceptors) that exhibit high affinity ligand (steroid hormone) binding. The vertebrate aporeceptors complexes contain several distinct proteins, including Hsp90, Hsp70, p60 (Sti 1), immunophilins (e.g. FKBP51) and others. Hsp90-containing heteromeric complexes isolated from Achlya, where shown to contain Hsp90, Hsp70, 61 kDa and 64 kDa proteins, a 56 kDa protein related to the immunophilin FKBP51, as well as several other proteins similar to those observed in vertebrate steroid receptor complexes. Although the antheridiol receptor has not been isolated, the ability of antheridiol to regulate gene transcription and the presence of aporeceptor-like complexes in the mycelium suggest that the antheridiol receptor has functional ( and perhaps structural) similarity to members of the steroid hormone receptor superfamily. (Supported by grants to J.C.S. from NSERC Canada)
391 A member of the p450 superfamily is induced during steroid mediated sexual development in the oomycete Achlya ambisexualis. Shelley A. Brunt, Thilaka Sritharan, Evangalia Tomai, Paul Milman, Chai Chen and Julie. C. Silver. Department of Medical Genetics and Microbiology and Division of Life Sciences, University of Toronto at Scarborough. Toronto, Ontario, Canada, M1C1A4.
Sexual differentiation in male and female strains of Achlya ambisexualis is controlled by the steroid hormones antheridiol and oogoniol. The Achlya steroid hormone antheridiol induces antheridial branching in male strains. These specialized branches subsequently differentiate into antheridia (male gametangia). Coincident with antheridium development male strains synthesize and secrete the steroid oogoniol which mediates the development of oogonia (female gametangia) in female strains. Differential hybridization screens using cDNAs from antheridiol-treated and vegetative hyphae of the male strain E87 identified a hormone-inducible cDNA that was not expressed in vegetative mycelia. This cDNA was found to encode a member of the p450 superfamily. The rapid induction of p450 transcripts requires no new protein synthesis and is therefore a primary response to antheridiol. The deduced amino acid sequence of the mature protein encoded by this cDNA has a predicted molecular weight of 59446 and a pI of 8.52. Analyses of the deduced amino acid sequence suggest that the protein encodes a soluble mitochondrial protein, i.e., is located in the mitochondrial matrix. To our knowledge this is the first p450 superfamily member isolated from an oomycete. A genomic clone containing the promoter has been isolated and is presently being studied to address the regulation of the p450 gene. (Supported by grants to J. C. S. from NSERC Canada)
392 Cru1, a fizzy-related protein from Ustilago maydis is required to mating. Tatiana García-Muse, Sonia Castillo, and José Pérez-Martín. Department of Microbial Biotechnology, Centro Nacional de Biotecnología (CSIC), Campus de cantoblanco-UAM, 28049 Madrid SPAIN
We have cloned a gene from U. maydis which encodes a protein which belongs to the Frizzy-related family of cell-cycle regulators. Deletion of this gene affects vegetative growth in Ustilago maydis and ectopic expression induces cell-cycle arrest. This cell-cycle regulator is required to arrest cell-cycle before mating takes place in Ustilago maydis. A signal emanating from the pheromone receptor which requires the participation of a MAP kinase cascade is involved in the activation of the Cru1 protein. Potential roles in mating regulation of this protein will be discussed.
393 Crk1, a CDK-like kinase is required for hyphal development in Ustilago maydis. Elia Garrido and José Pérez-Martín. Department of Microbial Biotechnology, Centro Nacional de Biotecnología (CSIC), Campus de Cantoblanco-UAM, 28049 Madrid, SPAIN
Using an oligonucleotide probe-based screen we have cloned several CDK-like proteins from Ustilago maydis. One of these genes, crk1, encodes a protein with sequence similarity with the Cdc2 protein from the same organism. Deletion of this gene in U. maydis led to a defective hyphal growth in nitrogen-limited medium. Ectopic expression induces hyphal growth in rich medium. Our preliminary data suggest that Crk1 is negatively regulated by the cAMP/PKA pathway, which represses filamentous growth in Ustilago maydis. Potential relations between this pathway and filamentation will be discussed.
394 The Cladosporium hydrophobin HCf-1 is required for efficient water-mediated spore dispersal. P D Spanu and J Whiteford. ICSTM, London, UK
The tomato pathogen Cladosporium fulvum has at least six hydrophobins (HCf-1 to 6). Four of these are Class I and two are Class II hydrophobins. The expression of these genes is differentially regulated and we believe they have different functions. In order to investigate hydrophobin function we are creating hydrophobin gene deletion mutants. So far we have obtained deltaHCf-1, deltaHCf-2 and deltaHCf-6 single knockouts and a deltaHCf-1/2 double knockout. We can also down-regulate hydrophobin expression by homology-dependent gene silencing. The degree of silencing varies in different transformants and we have selected a highly silenced strain C11. In C11 all Class I hydrophobins are down-regulated. We believe this is due to expression of antisense HCf-1 RNA; HCf-5 is unaffected and HCf-6 expression is higher than in wild type. Growth and sporulation of the knockout mutants analysed so far does not differ from the wild type isolates. On the other hand sporulation of C11 is both delayed and reduced and germination rate of C11 spores is lower. The deletion of the HCf-1 hydrophobin decreases the hydrophobicity of mycelium and spores in comparison to the other strains. Of particular note is the reduced ability of the spores lacking HCf-1 to be transported on the water/air interface of water droplets. We therefore propose that one of the main roles of HCf- 1 is to aid water-mediated spore dispersal.
395 The LIS1 homolog NUDF of Aspergillus nidulans specifically interacts with subunits of dynein, dynactin and microtubles. Bernd Hoffmann, N. Ronald Morris. Univertity of Medicine and Dentistry of New Jersey, Pharmacology, Piscataway, New Jersey, USA
The human disease lissencephalie or Miller-Dieker syndrome is caused by haploinsufficiency of the Lis1 gene. This disorder results in drastic changes during brain development finally ending in a smooth brain surface with a strongly reduced amount of neuronal cells. This defect seems to be reasoned by a reduced neuronal motility to the brain surface during embryogenesis because of an affected dynein activity in lis1 mutant cells. We used the filamentous fungus Aspergillus nidulans as model organism to study the putative interaction between the Lis1 homolog protein NUDF and its binding protein NUDE with all known dynein, dynactin and microtuble subunits of filamentous fungi. Both proteins interact specifically with subunits of these multiprotein complexes in two-hybrid and coimmuno-praecipitation experiments. While NUDF is able to interact with the intermediate chain of dynein and alpha- or gamma-tubulin, NUDE forms complexes with dynein light and intermediate chain, the actin related protein NUDK of dynactin and, as NUDF, with alpha- and gamma-tubulin. Point mutations in the dimerization domain of NUDF destroy the ability to interact with the intermediate chain and alpha- but not gamma-tubulin. For all observed interactions of NUDE the N-terminus is sufficient. Posttranslational modifications putatively in this area lead to two NUDE subfractions of which the unmodified form has a high affinity to alpha-tubulin while the modification leads to a drastic change in affinity from alpha- to gamma-tubulin. These data argue for a direct protein-protein interaction between the LIS1 homolog protein NUDF of A. nidulans and subunits of dynein/dynactin and for a functional involvement of the protein complex NUDE/NUDF in mitosis via gamma-tubulin binding.
396 Meiosis and ascospore development in nonlinear asci of Neurospora pannonica. Namboori B. Raju, Stanford University.
Neurospora pannonica is homothallic, with eight-spored asci. Immature asci are usually swollen and noncylindrical while the mature asci are narrow and cylindrical. The two meiotic divisions resemble those of other Neurospora species. However, the orientation of third-division mitotic spindles and the distribution of nuclei in the swollen asci are irregular. Ascospores are arranged irregularly at first, but as the ascospores enlarge and mature the asci gradually become cylindrical, with the ascospores aligned in single file. The asci cannot be considered ordered tetrads, because ascospore order does not reflect the assortment of chromosomes at the first and second meiotic divisions. Contrary to the original species description, ascospores require heat shock for germination and hyphae are sent out at both ends of germinating ascospores. Supported by MCB-9728675 from the National Science Foundation.
397 The genetic basis of abnormal ascospore morphology in Neurospora. Namboori B. Raju1 and Anna Geng Burk2. 1Stanford University. 2Cornell Univeristy.
A recessive ascospore mutant, named bud, was isolated from a wild- collected, multi-component, heterokaryotic strain of Neurospora tetrasperma. When bud is homozygous, meiosis is apparently normal but postmeiotic events are abnormal. Orientation of spindles at the postmeiotic mitosis is often abnormal and this results in failed pairwise association of nuclei and their irregular distribution along the length of the ascus prior to spore delimitation. Consequently, many asci cut out more than four ascospores; some contain no nuclei while others contain more than two nuclei. The most dramatic effect of bud is on ascospore delimitation itself. Many ascospores are irregularly shaped and are often interconnected, because of incomplete spore delimitation. Ascospores also show one or two lobes or bud-like extensions of varying sizes. Over 90% of ascospores from bud X bud remain white or tan and are inviable. The interaction of bud with a dominant Eight spore mutant (E) was examined in both heterozygous and homozygous crosses. When both are heterozygous, bud has no effect on ascospore delimitation or on the phenotype of E because bud is recessive, and many asci produce 5 to 8 ascospores just as in E X E. And when bud is homozygous and E is heterozygous, ascospore delimitation is less affected than when E is absent. Moreover, when both bud and E are homozygous, the effect on ascospore development is less extreme than when either mutant is homozygous singly. Supported by MCB-9728675 from the National Science Foundation.
398 Characterization of ras mutant alleles from Coprinus cinereus homokaryon AmutBmut. Alan P. F. Bottoli, Robert P. Boulianne, Markus Aebi and Ursula Kües. ETH Zurich, Institute for Microbiology, Schmelzbergstr. 7, CH-8092 Zurich, Switzerland
Using the ras gene of C. cinereus homokaryon AmutBmut, we produced mutant alleles for a dominant activated Ras protein (Ras-val19) and a dominant inhibitory Ras protein (Ras-asn24). Both ras alleles impaired mycelial growth of transformants of homokaryon AmutBmut on selective minimal medium. This failure to grow was due to loss of the co-transformed selection marker, together with the mutant ras gene copies. In the two monokaryons PG78 and 218, a specific growth phenotype was identified only for Ras-val19. Ras- val19 transformants of monokaryon PG78 have a reduced growth rate and show enhanced invasive growth, whereas Ras-val19 transformants of monokaryon 218 exhibit a disoriented pattern of hyphal growth with a general increase in branching. The aerial mycelium has a fleecy appearance with regularly distributed regions of highly proliferating hyphae. These areas are different from the small, more compact primary hyphal knots, structures of local intense branching that give rise to sclerotia (multicellular resting structures). In fact, formation of primary hyphal knots, the initiating process of fruiting body development as well as sclerotia development, was found to be repressed in Ras-val19 transformants on complete medium.
399 An essential gene for fruiting body initiation in the basidiomycete Coprinus cinereus is homologous to bacterial cyclopropane fatty acid synthase genes. Yi Liu, Sabine Loos, Markus Aebi and Ursula Kues. Institute of Microbiology, ETH Zurich, Schmelzbergstrasse 7, CH-8902 Zurich, Switzerland
Homokaryon AmutBmut is a specific strain of the basidiomycete Coprinus cinereus that, due to mutations in the mating-type loci, produces fruiting bodies without prior mating to another strain. The homokaryon has therefore been used for creating mutants in fruiting body development. Early stages of fruiting body development include the dark-dependent formation of primary hyphal knots and the light-induced transition from primary hyphal knots to the more compact secondary hyphal knots (fruiting body initials). UV mutant 6-031 forms primary hyphal knots but development arrests at the transition stage. Genetic analysis indicated that this phenotype is caused by a single recessive defect (skn1). Using a SIB-selection transformation procedure, a cosmid was isolated from a genomic DNA library that complemented the defect. The responsible gene (referred as cfs1) on this cosmid encodes a protein product highly similar to cyclopropane fatty acid synthases, a class of enzymes so far characterized only in prokaryotes. The cfs1 allele of mutant 6- 031 carries a T to G transversion, leading to an amino acid substitution (Y441D) in a domain suggested to be involved in the catalytic function. The mutant allele of cfs1 was unable to complement the fruiting deficiency in strain 6-031, indicating that this gene is essential for fruiting body initiation in C. cinereus.
400 The MAP kinase gene MAF1 is essential for infection structure formation of Colletotrichum lagenarium. Kaihei Kojima, Taisei Kikuchi, Yoshitaka Takano, and Tetsuro Okuno. Kyoto University, Graduate School of Agriculture, Kyoto, Japan.
Colletotrichum lagenarium, the causal agent of cucumber anthracnose disease, invades the host plant using a specialized infection structure called appressorium. It has been demonstrated that the MAP kinase (MAPK) gene CMK1 , showing homology to the FUS3/KSS1 MAPK genes of Saccharomyces cerevisiae, is required for diverse aspects of fungal pathogenesis including appressorium formation in C. lagenarium. Here we report that the SLT2 type MAPK gene MAF1 is essential for appressorium differentiation. MAF1 encodes a 418 amino acid protein with 91% identity to Magnaporthe grisea Mps1 MAPK and 80% identity to S. cerevisiae Slt2 MAPK. maf1 null mutants were created by targeted gene replacement. maf1 mutants showed normal growth and reduction in conidiation. Pathogenicity of maf1 mutants was dramatically reduced. On glass and host plant surfaces, conidia of maf1 mutants germinated but failed to form appressoria. Germ tubes of the mutants continued to elongate without any swollen structures. When conidia of the wild type are incubated on nutrient agar, they elongate germ tubes without appressoria, which is similar to the phenotype of maf1 mutants on glass. This suggests a possibility that the MAF1 MAPK pathway is inactive in conidia of the wild type incubated on nutrient agar. The phenotype of maf1 mutants is distinct from that of cmk1 mutants that produce swollen structures from germ tubes but fail to complete appressorium formation. These results indicate that the two MAPK genes, MAF1 and CMK1, regulate the distinct steps of appressorium formation independently.
401 Genetic and molecular analysis of mutations affecting the propagation of two non-conventional genetic elements in Nectria haematococca. Stéphane Graziani, Philippe Silar and Marie-Josée Daboussi. Université Paris Sud, IGM, Orsay FRANCE
One strain of the ascomycete Nectria haematococca can display two different morphological modifications caused by two specific cytoplasmic determinants, the 'Anneau' and the 'Secteur'. Once initiated, these modifications spread through anastomoses leading to a growth alteration along its path with a speed (up to 4mm/h) that dictates their form. Recently, it was shown that similar phenomena in fungi are controlled by prion-like proteins. In the light of this discovery, infectious modifications in Nectria are now being reconsidered. Both 'Secteur' and 'Anneau' are under the control of nuclear genes. Two kinds of mutations have been detected. Some do prevent expression of both modifications and these map to at least four loci (called nas). They are characterized by dense white aerial mycelia and female sterility. The other mutations prevent specifically the formation of either one of the modifications. They are located at a unique locus, locus S for the 'Secteur' and locus A for the 'Anneau'. These loci are supposed to be direcly involved in the generation of the determinants and thus have to be cloned. The SIB selection failed, but insertional mutagenesis as an alternative cloning strategy allowed the recovery of 10 mutants impaired in the ability to express the 'Secteur'. Sexual crosses indicated that the S gene and several nas genes are tagged. Presently, a cosmid that restores the ability to differentiate sectors when introduced in a strain mutated at the S locus is under characterization. Results on the molecular analysis of nas mutants will also be presented.
402 Characterization of differential gene expression in a haploid filamentous mutant of the anther smut, Microbotryum violaceum. Carolyn F Hughes, Malia A Ray, and Michael H Perlin. University of Louisville, Louisville, KY, USA
M. violaceum is dimorphic in that the yeast-like haploid sporidia can mate with other sporidia of opposite mating-type to produce infectious dikaryotic hyphae. This switch is an absolute requirement for infectivity and the remaining stages of development are obligately parasitic. Normally the switch requires a signal from the host plant and this signal does not elicit hyphal production from non-mated sporidia. Thus, the appearance of sporidia that grow filamentously raises interesting questions about differences in gene expression among the corresponding cell types: i.e., wildtype sporidia, filamentous mutants, and dikaryotic hyphae. Strain NINE is a haploid sporidial isolate from the wildflower Silene latifolia. Prolonged growth of this strain at room temperature allowed the identification of a mutant, NINE-fil, which produces long filaments. Initially, NINE-fil cells appear elongated, but after more than two weeks at room temperature, filamentous growth is obvious on rich media. On low-nitrogen media, the phenotype is more pronounced, with filaments produced after 1-2 days. There is no evidence that NINE-fil is infectious alone, but it is capable of mating. This allowed the demonstration that the filamentous phenotype segregates after passage through the plant. A subtractive cDNA library was constructed using cDNA from the wildtype strain as driver, so as to identify genes up-regulated in the NINE-fil mutant. The results of characterization of these genes as well as comparison with genes known to be up-regulated in dikaryotic hyphae of this organism will be presented.
403 Surprise, surprise: The smut fungi, Ustilago maydis and Microbotryum violaceum contain STE20 homologues. Wei Hong1, David Smith1, Scott Gold2, and Michael H Perlin1. 1University of Louisville, Louisville, KY, USA, 2University of Georgia, Athens, GA, USA
The mitogen-activated protein kinase (MAPK) pathways are conserved from fungi to humans and have been shown to play important roles in mating and filamentous growth for both yeast and dimorphic fungi, and in infectivity for pathogenic fungi. Upstream of the typical 3-kinase module, STE20, encodes a protein kinase of the PAK family that regulates more than one of these cascades in yeasts. We hypothesized that a STE20 homologue would play a similar role in the dimorphic plant pathogens U. maydis and M. violaceum. Using degenerate PCR primers, portions of the genes for STE20 homologues were amplified from genomic DNA of both fungi and the resulting fragments were sequenced to confirm their identities. The full-length copy of the U. maydis gene was obtained from a genomic library and was found to contain a 1782 bp coding region, yielding a predicted protein of 594 amino acids. Two regions of the predicted protein were particularly conserved compared to other STE20 proteins: one in the N-terminal portion, expected to be the binding region for a regulatory factor and the other in the C-terminal portion, corresponding to the kinase catalytic region in the S. cerevisiae protein. No intron was found for this gene. The sequence was also obtained for 1299 bp of the coding region for the STE20 homologue from M. violaceum, predicted to encode 433 amino acids which also showed homology to the STE20 proteins in GenBank. A knock-out construct was made for the U. maydis homologue- to decrease the likelihood of non-homologous recombination, it was used as a PCR product containing only the STE20 gene and a selectable marker inserted within the gene to disrupt its function.
404 Methylammonium permeases in Ustilago maydis and Microbotryum violaceum - connection to the cAMP-pathway? David Smith, Michael H. Perlin. University of Louisville, Louisville, Kentucky, USA
We are investigating the effects of nutrient limitation and its role in regulating fungal dimorphism in plant pathogens. Nutrient stress, such as low ammonium, may be one of many initial signaling events that stimulates mating and hyphal growth. We isolated full-length cDNA clones for ammonium transporters in the corn smut, U. maydis, and in the anther smut, M. violaceum: Ump1 and MEPa, respectively. These proteins are predicted to have 10-12 transmembrane spanning regions and are believed to have functional similarity to other known ammonium transporters. Their function was tested in a heterologous host. S. cerevisiae mutants with no functional ammonium transporters have a severe growth defect on low ammonium media. When transcribed from a yeast expression vector, both Ump1 and MEPa could complement this growth defect. The possible roles of these genes in regulating dimorphism was studied further by creating knock-outs of the Ump1 gene in both mating types of U. maydis. These strains are unimpaired in their ability to mate and show typical signs of disease. However, after prolonged culture some knock-out strains showed a consistent filamentous phenotype similar to that seen in strains lacking the ability to produce cAMP. The addition of exogenous cAMP to the media restores growth to a budding phenotype. This suggests that Ump1 regulates the switch to filamentous growth in a cAMP-dependent manner.
406 Molecular cloning of the laeA gene regulating aflR expression in Aspergillus nidulans. Jin Woo Bok and Nancy P. Keller. Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas 77843-2132
AflR is a C6 zinc cluster protein positively regulating the expression of biosynthetic genes required for sterigmatocystin (ST) production in A. nidulans and aflatoxin (AF) in A. flavus and A. parasiticus. Loss of expression of aflR leads to loss of ST/AF production. Here we describe progress in characterizing an A. nidulans loss of aflR expression (lae) mutant. LaeA1 was complemented by transformation with a trpC library. Subcloning of the cosmid rescued from the laeA wild type transformant showed that the complementary DNA fragment contained an ORF of ca. 3.0 kb. This ORF has no homology to any gene in the database. mRNA analysis reveals a 2.3 kb transcript that is developmentally regulated and appears prior to aflR expression. Two putative AflR binding sites are found in the promoter of laeA.
407 Morphological mutants of Neurospora from nature. Alka Pandit and A. J. Griffiths. Department of Botany, University of British Columbia, 6270 University Boulevard, Vancouver, B. C., Canada, V6T 1Z4.
In order to understand growth and morphogenesis of fungi Neurospora has been extensively used for genetic investigations. Most of the attention has been focussed on mutants derived in the laboratory. Strains from nature have been rarely investigated for their morphological abnormalities. We have screened a local population of Neurospora from India for morphological defects. Surprisingly many of these natural strains show distinct morphological defects. Ujjain ûû1 is one such N. crassa strain which shows morphological abnormalities like erratic growth rate and defect in branching. Upon crossing this strain with ORS-6a an interesting mutant has been recovered in the progeny (V2-17). This strain (V2-17) has a peculiar growth pattern which is unlike any other known mutant. The main hypha bends down at the tip and the fungus grow forward by the extension of the lateral branch, which again bends at tip after some growth and the pattern is repeated. We are trying to characterise these mutants to determine the number of loci involved and their possible functions. The existence of these morphological mutants in nature raise many interesting questions, like their origin and possibility of being carried in heterokaryons. Although it may appear far fetched the idea that these mutations may be adaptive can not be ruled out. The study of these mutants may help in answering these questions about the natural populations of Neurospora.
407 Neurospora crassa cold-sensitive mutants potentially significant for elucidation of hyphal morphogenesis. Aleksandra Virag, and Anthony J.F. Griffiths. University of British Columbia, Vancouver, Canada.
The goal of this study is to identify a class of cold-sensitive mutants that will be useful for investigating hyphal morphogenesis. Sites of tip growth and branching in N. crassa are locations at which complex processes occur. They result in a characteristic dynamic change in the distribution of hyphal components. Among these components are microfilaments organized into a cortical network. When shifted to low temperatures, wild type strains respond with a cold-shock reaction that transiently changes the branching frequency and pattern (Watters et al., 2000). Low temperatures also affect polymerization of F-actin filaments. Because of its presence at all active sites of growth, actin in the form of microfilaments could, directly or indirectly, contribute to tip growth and branching processes both in standard (25 degrees C) and low temperature conditions (7 degrees C). Cold-sensitive mutants may point to the presence of modifications in actin or actin related proteins. However, a wide range of molecular and cellular events not related to actin and microfilaments can also result in sensitivity to cooling. Selection for mutations causing cold sensitivity as a result of microfilament modification was achieved by selecting for mutant strains that in addition to cold sensitivity are resistant to cytochalasin A, or show a different branching behaviour. Three main categories of mutant strains were obtained by UV mutagenesis, and identified. Beside cold-sensitivity, at standard temperature conditions these strains showed higher branching frequency than wild type, lower branching frequency than wild type, or cytochalasin A-resistance coupled with a transient loss of hyphal orientation upon shift to low temperatures. Characteristics of these strains are described. This work was supported by NSERC.
409 The role of G-proteins in the regulation of dimorphic switching in Penicillium marneffei. Sophie Zuber, Michael J. Hynes and Alex Andrianopoulos Department of Genetics, University of Melbourne, 3010 Victoria, Australia
The opportunistic human pathogen P. marneffei is a dimorphic ascomycete able to switch between two growth forms in a temperature dependent manner. At 25 C, P. marneffei exhibits filamentous growth by producing unconstricted branching hyphae, whereas at 37 C it grows as unicellular yeast dividing by fission. We are interested in understanding the mechanisms controlling the dimorphic switch and in particular the temperature sensing mechanism and the signal tranduction pathway it triggers. Heterotrimeric G- proteins control developmental programs in fungi and other organisms acting as signal transducers that couple cell surface receptors to cytoplasmic effector proteins and have been implicated in dimorphic switching in other fungal species such as Saccharomyces cerevisiae and Ustilago maydis. Therefore, we are interested in determining if heterotrimeric G-proteins are involved in the temperature-dependent dimorphism exhibited by P. marneffei. We have cloned by homology genes encoding three G-protein alpha-subunits from P. marneffei, designated gasA, gasB and gasC, representing one of each major subgroup within the family of fungal G-protein alpha-subunits. GasA and GasC are members of subgroup I and III, respectively, which are related to the mammalian Galphai and Galphas proteins. GasB falls into subgroup II which has no corresponding mammalian counterpart. GasA is highly homologous to the Aspergillus nidulans FadA, a key component in the regulation between growth and development. The gasB gene shows strongest homology to the A. nidulans ganA gene. GasC is closely related to the A. nidulans GanB, Magnaporthe grisea MagA and the S. cerevisiae Gpa2 proteins, the latter playing a crucial role in signalling leading to pseudohyphal growth. Using mutational analysis we have examined the function of these genes by (I) construction of gene-replacement knock-out mutants in P. marneffei and (II) generation of dominant activating and dominant interfering alleles producing constitutively active or inactive proteins, respectively. We have introduced these into P. marneffei to characterise the role of these signalling components in dimorphic switching, asexual development and growth.
410 Fruiting body size regulation via a factor which modulates cAMP signal transduction. Richard H. Gomer, Lei Tang, Robin Ammann, Celine Roisin, and Wany Jang. Howard Hughes Medical Institute and Department of Biochemistry and Cell Biology, MS-140, Rice University, 6100 S. Main Street, Houston, TX 77005-1892
When starved, cells of the primitive fungus Dictyostelium discoideum aggregate in dendritic streams which break up into groups of ~2 x 104 cells. A secreted 450 kDa protein complex, counting factor (CF), regulates the size of these groups. High levels of CF cause the streams to break into small groups, while cells lacking CF form huge groups, with the resulting fruiting bodies toppling over. The aggregation is mediated by relayed pulses of extracellular cAMP. cAMP activates a receptor which activates G proteins, which activate PI3 kinase. This then modifies plasma membrane lipids, allowing a protein called CRAC to bind to the membrane and activate adenylyl cyclase. The G proteins also cause a transient activation of guanylyl cyclase. We find that CF slowly down-regulates the guanylyl cyclase activity. In contrast, a one-minute exposure of cells to CF increases the cAMP-induced cAMP pulse. This is accomplished by potentiating adenylyl cyclase via a pathway that is downstream of the cAMP receptors/ G protein/ PI3K/ CRAC pathway. The internal pulses of cGMP and cAMP regulate cell motility and the expression of cell-cell adhesion molecules. We find that via this pathway CF modulates adhesion and motility. Low levels of CF decrease motility and increase adhesion, resulting a stream staying intact. High levels of CF increase motility and decrease adhesion, causing the stream to break up. Computer simulations indicate that as a general principle, a secreted signal regulating motility and adhesion can regulate group size. Similar pathways may thus regulate tissue size in other systems.
411 Cat-1, an endurable large catalase of Neurospora crassa conidia. Díaz, A., Lledías, F., Michán, S., Rangel, P., Montes de Oca, Y. and Hansberg, W. Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ap. postal 70-242, México 04510 D.F.
Cat-1 constituted 0.6% of total protein in conidia, where it was localized mainly at the cell wall. Cat-1 activity decreased during germination and was substituted by Cat-3 during growth. Activity increased with conidiation, heat shock, light and singlet oxygen. Cat-1 is a homotetramer of 88 kDa subunits containing each a 679 Da chlorin instead of protoheme IX. The tetramer pI was 5.25. The protein had 30 hexoses per subunit. Cat-1 crystal structure showed a 'flavodoxin-like' domain at the C-terminal and the N-terminal arm of a monomer slipping through of the 'wrapping domain' of the Q-related subunit and vice versa. The enzyme was stable in organic solvents, high salt or SDS concentrations. Urea 9 M or 3.5 M guanidine-HCl partially inhibited the enzyme, but activity was recovered upon dialysis. A Km of 22 mM was obtained with less than 100 mM H2O2 and of 233 mM at molar H2O2 concentrations. It exhibited no pH optimum and was inactivated by 3-amino-triazole, azide and hydroxylamine. KCN was a competitive inhibitor with a Ki of 0.76 mM. Cat-1 is modified by singlet oxygen giving rise to more acidic active conformers. Modification site is at the chlorine. Compared to the non-oxidized conformer, the fully oxidized enzyme had a lower Km at molar concentration of H2O2, was more sensitive to KCN, and less stable at an acidic pH and in the presence of guanidine-HCl. The gene has one intron, codifies for a 737 amino acid polypeptide. It is 64% similar to Aspergillus nidulans CatA, 66% to A. fumigatus CatA and 53% to N. crassa Cat-3. Accumulation of the mRNA was observed at the stationary growth phase and late during conidiation.
412 Cat-3, a light-inducible catalase in Neurospora crassa mycelia. Michán, S., Díaz, A., Lledías, F., Beltrán, M., Montes de Oca, Y., Martínez, C. and Hansberg, W. Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ap. postal 70-242, México 04510 D.F.
Cat-3 activity increased during the growing and pre-stationary phase, adhesion of hyphae and growth of aerial hyphae. The enzyme was localized at the cell wall but also in mitochondria and the cytosol. Cat-3 activity increased with depletion of the carbon source, paraquat, light and singlet oxygen. Cat-3 is a homotetramer of 79 kDa subunits, probably containing a chlorin instead of protoheme IX. The tetramer pI is 4.80. The protein was slightly glycosylated. The enzyme was stable in organic solvents, high salt or SDS concentrations. Cat-3 was resistant to molar concentrations of H2O2. A Km of 74 mM was obtained with less than 100 mM H2O2 and of 209 mM at molar H2O2 concentrations. It exhibited no pH optimum and was inactivated by 3-amino-triazole, azide and hydroxylamine. KCN was a competitive inhibitor with I50 of 60 microM. Cat-3 is also modified by singlet oxygen giving rise to more acidic active conformers. The gene has two introns, codifies for a 719 amino acid polypeptide. It is 67% similar to Claviceps Cat1, 65% to Aspergillus nidulans CatB and 64% to A. fumigatus CatB and 53% to N. crassa Cat-1. Accumulation of the mRNA was observed at the pre-stationary growth phase. Accumulation of the mRNA by light was dependent on both 'white collar' transcription factors. Initiation sites and the polyadenilation site were determined. Paraquat regulated cat-3 expression at the transcriptional level.
413 Neurospora crassa catalase-2 is a developmentally regulated catalase- preoxidase. Peraza, L., Montes de Oca, Y., Michán, S., and Hansberg, W. Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ap. postal 70-242, México 04510 D.F.
Catalase-2 (Cat-2) activity was detected in mycelia at the stationary growing phase or when deprived of the carbon source. Activity increased in aerial hyphae and in conidia. High levels of Cat-2 activity were found in heat shocked or H2O2- treated mycelia or when conidiation takes place in a medium with ethanol, methanol, uric acid, acetate or fatty acids as sole carbon source. Immunodetection and cell fractionation experiments indicated that the enzyme is located partially in the cell wall. Cat-2 was purified to homogeneity. The enzyme is a homodimer of 89 kDa subunits. Absorption spectrum and activity inhibition with cyanide indicates that it is a heme protein. Cat-2 has both catalase and peroxidase activities. O-dianisidine, diaminobenzidine, guaiacol and ascorbate are accepted as electron donors but not NADH, NADPH, glutathione or cytochrome c. Cat-2 has a Km of 9.02 mM and a high catalytic efficiency. Optimum pH for catalase and peroxidase activity is 6.3 and 4.7 respectively. The kinetic constants, pH optimum, and a low inhibition with 3-amino-triazole are features similar to other catalase-peroxidases. The sequence of an internal polypeptide confirmed that Cat-2 is a catalase-peroxidase. The sequence obtained coincided with part of the predicted peptide from the cDNA sequence NM6H12 (GenBank Acc: AA901970 & AA901741) which has high similarity with bacterial catalase-peroxidases. Cat-2 mRNA accumulation was observed in the aerial hyphae 18 h after induction of the conidiation process.
414 het-s: an heterokaryon incompatibility gene encoding a fungal prion. Coustou V, Maddelein ML, Dos Reis S, Schaeffer J, Saupe SJ and Begueret J. Laboratoire de Genetique Moleculaire des Champignons, IBGC UMR, 5095 CNRS Universit de Bordeaux 2, 1 rue Camille St Sains,33077 Bordeaux cedex, France
Prions are proteinaceous infectious particles that cause spongiform encephalopathies in mammals (1). Proteins that behave as prions (infectious proteins) have been described in yeast and in the filamentous fungus Podospora anserina. They correspond to the Ure2p, Sup35 (2) and HET-s proteins (3, 4). They have been termed prion proteins because they exist as two alternate states: a soluble form and a transmissible (infectious) and aggregated form. The het-s gene is one of the heterokaryon incompatibility genes of P. anserina. Strains of the het-s genotype can exist as two alternate incompatibility phenotypes : the neutral [Het-s*] and the active [Het-s] phenotype. A [Het-s*] strain is converted to the [Het-s] phenotype upon contact with a [Het-s] strain. Using GFP fusion proteins, we show that the transition from the [Het-s*] to the [Het-s] state (prion state) leads to in vivo aggregation of the protein. Purified his-tagged recombinant HET-s protein which is initially soluble and monomeric, forms aggregates after several days at 4 C. These aggregates trigger the aggregation of fresh soluble protein. They bind congo red and show birefringence under polarised light, a property of amyloids. Characterization of these aggregates by electron microscopy indicated that they are composed of ordered unbranched fibrils of about 25 nm in width and up to several micrometers in length. (1) Prusiner (1998) Proc. Natl. Acad. Sci. USA 95: 13363 (2) Wickner et al. (1999) Microbiol. Mol. Biol. Rev. 63: 844 (3) Coustou et al (1997) Proc. Natl. Acad. Sci. USA 94: 9773 (4) Coustou et al (1999) Genetics 153: 1629
415 Regulation of spore germination in Colletotrichum graminicola by environmental signals. L. Vaillancourt 1, J. Chaky 2, and E. Nuckles1. 1Department of Plant Pathology, University of Kentucky, Lexington KY 2 Department of Plant Pathology, University of Nebraska, Lincoln NE
Spore germination is not only an interesting problem in developmental and cell biology; it represents a potentially important target for novel antifungal therapies. However, the molecular regulation of spore germination in filamentous fungi is poorly understood, even in the better-studied model systems like Neurospora and Aspergillus. In our laboratory, we have initiated a study to elucidate the molecular mechanisms that control germination in the plant-pathogenic fungus Colletotrichum graminicola. Spore germination in C. graminicola is environmentally responsive. Breaking of spore dormancy occurs only when specific environmental cues are received. Subsequent germ tube development is also controlled by environmental cues, particularly by physical aspects of the substrate. We have begun dissecting the response of the spore and the germling to its environment, using a four-fold approach. First, we are characterizing and quantifying the spore germination process as it occurs in various artificial environments. Second, we are using pharmacological inhibitors to indicate which molecular signaling pathways might be involved. Third, we are creating random insertional mutants and screening for those that are deficient in spore germination. And fourth, we are cloning C. graminicola homologues of genes that are known or suspected to play a role in spore germination and development in other fungi. The roles of these genes in C. graminicola will be determined by knockout experiments. Our progress in using these approaches to elucidate molecular mechanisms of spore germination and subsequent development will be presented.
416 Demonstration of circadian rhythms in Aspergillus Andrew Greene*1,2, Nancy Keller1, 3, and Deborah Bell- Pedersen1, 2. 1Program For the Biology of Filamentous Fungi, 2Department of Biology, 3Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77843
Endogenous circadian oscillators have been described in a variety of organisms ranging in complexity from cyanobacteria to humans. The filamentous fungi Neurosporaand Aspergillusare ideal organisms in which to study the molecular basis of the circadian clock because of their well-developed genetics and ease of manipulation in the laboratory. Furthermore, in Neurospora, the circadian clock controls asexual spore production, providing an easy means to assay rhythmicity. Most studies of circadian rhythms in fungi have been carried out in Neurospora; no rhythms in Aspergillus have been reported. Unlike Neurospora, Aspergillus is of extreme agricultural importance because of its production of the potent carcinogen aflatoxin, which causes billions of dollars of crop losses each year. Previous studies have demonstrated that development and toxin production are closely associated. Therefore, our goals are to develop Aspergillus as a model system for studies of circadian rhythms and to investigate the link between the circadian clock, development and toxin production. As first steps towards these goals, we have established the presence of a circadian rhythm in sclerotial production in Aspergillus flavus. Additionally, we have demonstrated that the A. nidulans gpdA gene, which encodes glyceraldehyde-3-phosphate dehydrogenase, shows rhythmic expression in constant darkness with a period of approximately 28 hours. Several approaches are currently underway to investigate the circadian clock in Aspergillus and these will be presented.
417 A STE12 homolog from the asexual, dimorphic fungus Penicillium marneffei complements the defect in sexual development of an Aspergillus nidulans steA mutant. Anthony R. Borneman, Michael J. Hynes and Alex Andrianopoulos. Department of Genetics, University of Melbourne. Victoria, Australia. 3010.
Penicillium marneffei is an opportunistic fungal pathogen of humans and the only dimorphic species identified in its genus. At 25 C P. marneffei exhibits true filamentous growth while at 37 C, P. marneffei undergoes a dimorphic transition to produce uninucleate yeast cells which divide by fission. Members of the STE12 family of regulators are involved in controlling mating and yeast-hyphal transitions in a number of fungi. We have cloned stlA, a homolog of the S. cerevisiae STE12 gene from P. marneffei. The stlA gene, along with the A. nidulans steA and Cryptococcus neoformans STE12alpha genes, form a distinct subclass of STE12 homologs which have a C2H2 zinc finger motif in addition to the homeobox domain that defines STE12 genes. To examine the function of stlA in P. marneffei, targeted deletion of the stlA gene was used to create a stlA mutant strain. Loss of StlA function had no detectable effect on vegetative growth, asexual development or dimorphic switching in P. marneffei. Despite the lack of an apparent function, the P. marneffei stlA gene complemented the sexual defect of an A. nidulans steA mutant and in addition, substitution rate estimates indicate that there is a significant bias against nonsynonymous substitutions between stlA and steA. These data suggest that P. marneffei may have a previously unidentified cryptic sexual cycle in which stlA plays a role therefore accounting for the high degree of conservation observed.
418 Cloning of chitin deacetylase gene from
Phycomyces blakesleeanus. Imamura,
Kengo* , Kondo, Hisae*, Yoshida, Shuji* ,
Murayama, Tadako*, Miyazaki Atsushi**, Otaki,
Tamotsu** (*Coll. of Engn., Kanto-gakuin Univ. ,
**IGE, Tohoku Univ.)
The cell wall of the Zygomycetes fungus Phycomyces blakesleeanus is mainly composed of chitin and chitosan. Chitin is converted to chitosan by chitin deacetylase. It has been reported that the biosynthesis of chitosan in these fungi proceeds by the coordinated action of chitin synthase and chitin deacetylase. In the pilD mutant which was induced by 4-nitroquinoline-1- oxide and forms swollen and short sporangiophores similar to those of Pilobolus, the content of chitin was lower than in the wild type and the content of chitosan was much higher than in the wild type. It suggests that chitin deacetylase activity is higher in the pilD mutant than in the wild type. We cloned the genomic DNA fragment (218bp) encoding the homologous amino acid sequence to the consensus sequences in chitin deacetylase of Mucor rouxii (MrCDA), nodB protein of Rhizobium leguminosarum, and polysaccharide deacetylase of Bacillus stearothermophilus by PCR. Southern analysis with this DNA fragment suggested there is only one copy of the chitin deacetylase homologue in this fungus. A positive cDNA clone was isolated from the cDNA libraries made from exponentially growing mycelia of pilD. The clone is composed of 1,613bp and encodes a predicted polypeptide (PbCDA) of 459 amino acids. Amino acid sequence of PbCDA is 45% homologous to that of Mr.CDA in the central part of the molecule. The sequence analysis of genomic DNAs of the wild type and the pilD mutant showed that there is no intron in the coding region of PbCDA gene and there is no difference of the nucleotide sequence in the ORFs between the wild type and pilD. These results suggest that pilD mutation is located outside the coding region.
419 The Role of small GTPases in the growth and development of the dimorphic fungus Penicillium marneffei. K.J. Boyce, M.J. Hynes and A. Andrianopoulos. Department of Genetics, University of Melbourne, Royal Parade, Parkville 3010, Australia.
The opportunistic human pathogen Penicillium marneffei exhibits a temperature dependent dimorphic switch. At 25 C, P. marneffei grows vegetatively as multinucleate filamentous hyphae, which can differentiate to produce asexual spores (conidia). At 37 C, P. marneffei grows as uninucleate yeast cells which divide by fission. We are interested in elucidating the pathways regulating dimorphic switching. Small GTPases have been shown to regulate morphological transitions in numerous organisms and we are therefore investigating the role played by small GTPases in P. marneffei. Using heterologous probes and degenerate PCR, we have cloned homologues of the Ras and Rho GTPase family from P. marneffei. Sequence comparisons with other cloned small GTPase genes has shown that we have isolated a highly conserved CDC42 (cflA = cdc fortytwo like gene A), Rac (cflB = cdc fortytwo like gene B) and RAS2 (rasA) homologue. cflA was cloned by low stringency hybridisation using the Aspergillus nidulans modA gene and encodes a highly conserved CDC42 homologue. The cflA gene expresses two transcripts during vegetative growth, in asexually developing cultures at 25 C and during yeast like growth at 37 C. We have generated dominant negative and activated cflA alleles, based on Saccharomyces cerevisiae CDC42 mutations, and introduced these into P. marneffei. Dominant negative transformants have a delay in germ tube emergence, whereas, the dominant activated cflA transformants showed an increase in the rate of germ tube emergence. Both dominant negative and activated transformants possessed abnormally swollen and misshapen hyphae. At 37 C, the yeast cells produced by the dominant negative and activated transformants were swollen and misshapen and possessed cell division defects. Therefore, at 25 C, CflA is involved in the control of germ tube emergence and in the maintenance of polarised hyphal growth and at 37 C, CflA controls the morphology of yeast cells. CflA does not affect dimorphic switching in P. marneffei, as both transformants were capable of undergoing the morphological transition. Interestingly, the cflA dominant negative and activated mutations do not detectably affect conidiation in P. marneffei, which is in contrast to A. nidulans. Conversely, when either of the mutant P. marneffei cflA alleles were transformed into A. nidulans, the result was a complete lack of conidiation.
420 Role of pheromone precursor genes of Magnaporthe grisea in mating. Piotr Bobrowicz, Wei-Chiang Shen, and Daniel J. Ebbole. Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77843, USA.
Sexual reproduction in heterothallic ascomycetes requires the interaction between two mating partners of the opposite mating type. As was shown in model yeast species the recognition between cells during mating involves a pheromone signaling system. To better understand the role of pheromones and the pheromone response pathway in filamentous ascomycetes we cloned two pheromone precursor genes of an important plant pathogen, Magnaporthe grisea. The genes are present in both mating types but they are expressed in a mating type specific manner. Mat1-1 strains of the fungus express the MF1-1 gene that is predicted to encode a short polypeptide with a terminal CAAX motif found in lipopeptide pheromones. The MF2-1 gene, expressed in Mat1-2 strains, is related in gene organization to the alpha-factor genes of Saccharomyces cerevisiae and is probably processed by a Kex2-like protease. Analysis of the null mutants in sexual crosses indicates that pheromones are important, however not essential, for male fertility and that they are dispensable for the female fertility.
421 TupA, the Penicillium marneffei TUP1 homologue, represses asexual development and yeast-like growth to allow vegetative filamentous growth. Richard B. Todd, Michael J. Hynes and Alex Andrianopoulos. The University of Melbourne, Parkville 3010, AUSTRALIA.
The opportunistic human fungal pathogen Penicillium marneffei is dimorphic, showing growth as two different morphological forms. At 25 C the free-living saprophyte grows vegetatively as multinucleate filamentous hyphae and may undergo asexual development (conidiation) by the production of conidia-bearing conidiophores. At 37 C growth occurs as uninucleate yeast cells that are pathogenic. Little is known about the molecular events involved in the establishment and maintenance of the developmental states in P. marneffei. We have cloned tupA, the homologue of the Saccharomyces cerevisiae pleiotropic repressor TUP1, from P. marneffei. The TupA protein is highly conserved with homologues in other filamentous and yeast- like fungi. Deletion of the tupA gene results in pleiotropic effects. The tupA deletion mutant shows partial derepression of a subset of carbon catabolite (glucose) repressible metabolic activities, at 37 C formation of irregularly shaped yeast-like cells, and at 25 C inappropriate developmental switching. At 25 C prolific asexual development and production of uninucleate yeast-like cells occurs, indicating that tupA plays a role in vegetative filamentous growth by repression of both asexual and yeast developmental programs. These data contrast with both the lack of asexual development and the constitutive filamentation observed in the equivalent mutants in the mono-morphic fungus Neurospora crassa and in the dimorphic fungal pathogen Candida albicans, respectively. Although TUP1 homologues in fungi are structurally conserved and regulate analogous developmental and metabolic processes they appear to differ in their specific roles within these processes.
422 Use of GFP variants to study conidial germination in Colletotrichum gloeosporioides. Sima Barhoom, Iris Nesher, and Amir Sharon. Department of Plant Sciences, Tel Aviv University, Tel Aviv 69978, Israel
Colletotrichum gloeosporioides f. sp. aeschynomene (C.g.a.) is pathogenic on the weed Aeschynomene virginica. A biocontrol product named Collego that is used to control A. virginica is produced from C.g.a. conidia. The mature conidia are dormant but submerging of conidia in pea extract induces instant germination. Upon induction the conidia divide and then a single germ tube is formed. Staining of cell walls and nuclei revealed that the pea extract promotes fast cytological changes in the conidia. After 1 hour the nucleus divides and then a septum is formed. About 2.5 h after the induction one of the two nuclei is no longer detected by propidium iodide staining while the other nucleus divides, and a germ tube is formed.In the current work we used C.g.a.-transgenic strains that express various forms of GFP to further characterize the germination process. Work with C.g.a.isolates that express YFP in the cytoplasm and GFP in the nucleus revealed new details of the cellular events. Less than 30 minutes after induction the nucleus migrates to one side of the conidium, it then divides and the two new nuclei move each to an opposite pole. A septum is formed and the nuclei return to the center of the cell. Finally, a germ tube and a new nucleus are formed, the new nucleus migrates to the tip of the germ while the nuclei in the original conidial cells start to degrade. The current data suggest that the germination process in C.g.a. may involve a cell cycle controlled germination process in one cell and a programmed cell death in the other.
423 Isolation and genetic characterization of Sordaria macrospora mutants, defective in fruiting body development. Sandra Masloff, Stefanie Pöggeler, Severine Mayrhofer, Ulrich Kück. Lehrstuhl für Allgemeine und Molekulare Botanik, Fakultät für Biologie, Ruhr-Universität Bochum, D - 44780 Bochum, Germany
The homothallic ascomycete Sordaria macrospora shows an apandrous fruiting body formation during sexual development. In order to understand the genetic control of this developmental process, we have started a program to generate mutant strains, which are defective in different steps of fruiting body development. This includes the formation of fruiting body initials, the transition of protoperithecia into perithecia and the maturation of ascospores. Using UV- and EMS-mutagenesis, we have generated a set of 110 mutants, showing different developmental blocks during fruiting body formation. All developmental mutants have in common that they do not show any disposal of mature ascospores through the ostiole (pre-formed opening) of the perithecia. From characterization using scanning and light microscopy, these mutants can be divided into different classes according to their morphological phenotypes: 1. asc mutants are characterized by the lack of any ascogon-like structures, which can be considered as the primary initials of fruiting body formation. 2. pro mutants form only protoperithecia with a size of about 20-50 µµm. Thus, this type of mutants is unable to form any perithecia or ascospores. 3. per mutants are able to generate mature perithecia (size about 500 µµm), however, ascospore formation is completely prevented. These mutants have a defect in meiotic or post-meiotic nuclear division. A detailed analysis providing genetic and morphological data from all mutants will be presented. Allelic tests, for example, showed that beyond this large set of mutants several strains carry allelic mutations, indicating that our attempts are close to a saturated mutagenesis.
424 Molecular analysis of developmental genes in Sordaria macrospora: Identification and characterization of putative components of signal transduction pathways. Sandra Masloff, Stefanie Pöggeler, Sabine Jacobsen, Henriette Bruggemann, Ulrich Kück. Lehrstuhl für Allgemeine und Molekulare Botanik, Fakultàt für Biologie, Ruhr- Universitàt Bochum, D- 44780 Bochum, Germany
Fruiting body development in the homothallic ascomycete Sordaria macrospora is controlled by multiple developmental genes. In order to understand these morphogenic processes on the molecular level, we provide the detailed analyses of genes, which are involved in the transition of protoperithecia into mature fruiting bodies (perithecia). One example is the pro1 gene, which encodes a putative transcription factor. PRO1 carries a DNA-binding domain, which shows homology to the Zn(II)2Cys6-cluster of other fungal C6 zinc finger proteins. Using a PCR-based cloning strategy, we succeeded in isolating homologues of the pro1 gene from two related heterothallic species, namely Neurospora crassa and Sordaria brevicollis. On the DNA level, the genes from the two heterothallic species show a 91 % and a 90 % similarity, respectively, to the pro1 gene from Sordaria macrospora. Amino acid sequence comparisons of the predicted PRO1 polypeptides from the three ascomycetes showed typical DNA-binding domains. We demonstrate by DNA-mediated transformations that the N. crassa homologue is able to restore fertility in the sterile S. macrospora pro1 mutant. Using in vitro mutagenesis, we are able to demonstrate that single nucleotide base pair substitutions in the coding region of the DNA binding domain of the wild type pro1 gene result in mutant allels, which are unable to restore the sterile phenotype. Fusions of parts of the PRO1-polypeptide to the GAL4 DNA binding domain led to the identification of a domain, which is able to transactivate gene expression in yeast. The molecular analysis of other developmental mutants with a defective fruiting body formation will be presented.
425 Meiotic deletion at the Magnaporthe grisea BUF1 locus is controlled by interaction with the homologous chromosome, Mark L. Farman. University of Kentucky, Plant Pathology, Lexington, KY, USA
The Magnaporthe grisea BUF1 gene suffers high frequency mutation in certain genetic crosses, resulting in buff-colored progeny. Analysis of 16 buf1- mutants arising from a cross with a mutation frequency of 25% revealed that, in every case, the BUF1 gene was deleted. The deletions occurred in only one of the parental chromosomes and were due to intrachromosomal recombination. Tetrad analysis revealed that deletions occurred in 44% of meioses and usually affected both chromatids of the mutable chromosome. This suggests they happen before the pre-meiotic round of DNA synthesis. However, they were also almost entirely restricted to crosses in which the homologous chromosome carried the non-mutable allele. This, together with the discovery of extensive structural polymorphism between the stable and unstable BUF1 loci, suggests that the deletion process is governed by pairing interactions between homologous chromosomes. Given that karyogamy is not supposed to occur until after pre-meiotic DNA replication in Pyrenomycetous fungi like M.grisea, this latter observation would place the time of deletion during, or after, DNA synthesis. These conflicting results suggest that karyogamy might actually precede replication in M.grisea, or that parts of the genome remain unreplicated until after karyogamy and subsequent chromosome pairing have taken place. Results of experiments to determine the timing of BUF1 deletion will be presented.
427 The ubc2 gene of Ustilago maydis encodes a novel adaptor protein required for filamentous growth, pheromone response and virulence. Scott E. Gold, Alfredo D. Martinez-Espinoza and Maria E. Mayorga*. Department of Plant Pathology, University of Georgia, Athens, GA 30602-7274 *Current address: Microbia Inc., 840 Memorial Drive, Cambridge, MA 02139
The Basidiomycete fungus Ustilago maydis, causes corn smut disease and alternates between a budding haploid saprophyte and a filamentous dikaryotic pathogen. Previous work demonstrated that haploid adenylate cyclase (uac1) mutants display a constitutively filamentous phenotype. Suppressor mutants of a uac1 disruption strain, named ubc for Ustilago bypass of cyclase, no longer require cAMP for the budding morphology. The ubc2 gene was isolated through complementation by virtue of its requirement for filamentous growth. The deduced amino acid sequence encoded by ubc2 shows localized homology to Sterile Alpha Motif (SAM), Ras Association (RA) and src homology 3 (SH3) protein-protein interaction domains. A K78E missense mutation within the SAM domain, revealed a genetic interaction between ubc2 and ubc4, a pheromone responsive MAP kinase kinase kinase. This indicates involvement of ubc2 in the pheromone responsive MAP kinase cascade and ubc2 is required for pheromone responsive morphogenesis. The ubc2 gene is a critical virulence factor. Thus ubc2 encodes a novel adaptor protein acting directly upstream of the pheromone responsive MAP kinase cascade in U. maydis.
428 Differential gene expression in dimorphism and pathogenesis in Ustilago maydis. María D. García-Pedrajas, David L. Andrews, Karen E. Snyder and Scott E. Gold. Department of Plant Pathology, University of Georgia, Athens, GA 30602-7274
Ustilago maydis displays dimorphic growth alternating between a budding haploid and a filamentous dikaryon formed by the fusion of two haploid cells. This morphological switch plays a critical role in pathogenicity since only the filamentous dikaryon can infect corn plants. 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 several genes upregulated during filamentous or budding growth and have confirmed differential expression by northern blot analysis. We are now in the process of fully characterizing some of these genes. We plan the production of disruption mutants to determine the biological roles of select genes 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. In order to analyze a large number of clones after the construction of the subtractive cDNA libraries, we are using robotic devices to automatically pick and array colonies on nylon membranes. In this way we can efficiently analyze several thousand colonies simultaneously.
429 Suppressors of the meiotic defects of spo11-1 in Coprinus cinereus. Martina Celerin, Jana Stone, Rachel Shiffrin, Kelly K. Meeks, Sandra T. Merino, Miriam E. Zolan. Department of Biology, Indiana University, Bloomington, Indiana, 47401
In the mushroom caps of wild-type Coprinus cinereus, the basidial nuclei undergo meiosis, and each of the four products migrates into a basidiospore. These basidiospores give the cap its black appearance. In contrast, the C. cinereus spo11-1 mutant is defective in meiosis and produces a virtually sporeless cap, which is white. Spo11, a type II topoisomerase, likely is required universally for the initiation of meiotic recombination, and C. cinereus spo11-1 fruits white. We screened for genetic suppressors of the spore formation defect by mutagenizing oidia from spo11-1 and looking for surviving strains that were able to produce basidiospores. Of the 1385 isolates that produced fruiting bodies, eight produced grey caps. All eight produced more spores than the spo11-1 mutant, and the percent viability of these spores was higher than that of the starting strain. These eight mutants represent four complementation groups, termed sse1-sse4, for suppressors of spo eleven. Previous studies have shown that suppression of spo11-1 by artificially created DNA breaks (caused by ionizing irradiation) or by the absence of a sister chromatid induce formation of synaptonemal complex (SC). Surprisingly, none of the suppressors isolated in this study induces substantial amounts of SC formation in a spo11-1 background. Thus, they presumably suppress the sporulation defects in C. cinereus spo11-1 by completely different mechanisms.
430 Factors affecting meiotic stability of the BUF1 gene in Magnaporthe grisea. Yun-Sik Kim and Mark L. Farman. University of Kentucky, Plant Pathology, Lexington, KY, USA
Meiotic stability of the BUF1 gene in Magnaporthe grisea strain Guy11 is controlled by interaction with the homologous chromosome and appears to be correlated with the homologous BUF1 loci having different physical organizations (see abstract by M. Farman). In this abstract, we describe the testing of two hypotheses related to BUF1 instability: Hypothesis 1: BUF1 instability is caused by mis-pairing between the homologous BUF1 loci. Physical mapping was used to identify strains with different physical organizations at their BUF1 loci. Genetic crosses were then used to incorporate these loci into both the MAT1-1 and MAT1-2 mating type backgrounds. Compatible strains were then mated so that the loci were paired in all possible combinations. Regardless of origin, the BUF1 gene was stable in most crosses involving BUF1 loci with identical organizations. In the one exceptional cross it was lost at a low frequency. Loci that were originally from strains 2539 and Arcadia were perfectly stable in all crosses. However, the loci from ML33 and Guy11, which both suffered deletion in crosses with strain 2539, were stable in crosses with Arcadia. Arcadia has a BUF1 locus structure that is quite different from that of ML33 or Guy11, indicating that mis-pairing per se is insufficient to induce instability at the BUF1 locus. Instead, we propose that specific structural differences promotes the loss of BUF1. Hypothesis 2: The frequency of deletion is determined by the physical organization of the BUF1 locus. This was tested by crossing strains with different BUF1 locus structures to the same tester strain and measuring deletion frequency. Surprisingly, the frequency varied significantly between different crosses of the same strains. Furthermore, if the BUF1 gene survived deletion through crosses, it became progressively more stable in each subsequent generation. This suggests that BUF1 deletion frequency is determined primarily by genetic background and/or environmental factors.
431 Two G-alpha proteins, GNA-1 and GNA-3, regulate adenylyl cyclase in Neurospora crassa. Ann M. Kays, F. Douglas Ivey, and Katherine A. Borkovich. University of Texas Health Science Center - San Antonio and University of Texas - Houston Medical School.
Cellular responses to external stimuli, such as odorants and pheromones, are mediated by heterotrimeric G protein coupled receptors in eukaryotic systems. Our laboratory has identified and characterized three G alpha subunit genes, gna-1, gna-2, and gna-3, from the saprophytic fungus Neurospora crassa. N. crassa G alpha proteins display remarkable conservation of sequence and function with G alpha proteins from the pathogenic fungi Magnaporthe grisea and Ustilago maydis. All three G alpha subunits have been shown to regulate different aspects of sexual development. GNA-1 is a positive regulator of apical extension and GNA-3 is a negative regulator of asexual sporulation. Deletion of gna-1 in an adenylyl cyclase null mutant confers increased thermotolerance and elevated expresssion of stress proteins. Biochemical analysis indicates that GNA-1 and GNA-3 differentially regulate adenylyl cyclawe activity and levels, respectively. A delta gna-1 delta gna-2 delta gna-3 and a delta gna- 1 delta gna-3 mutant have been constructed by a sexual cross to explore the role of each G alpha protein in signaling. Studies exploring the roles of these mutants in Neurospora crassa development will be presented.
432 Analysis of two genes that are highly expressed in starved and sexual tissues of Neurospora crassa. Hyojeong Kim and Mary Anne Nelson. The University of New Mexico, NM, USA
Two novel and highly expressed genes were identified by the Neurospora Genome Project at UNM (Nelson et al., Fungal Genetics and Biology 21, 348-363, 1997). These genes are tentatively named poi-1 and poi-2 (for plenty of it), since they are the most highly expressed genes in the starved mycelial and/or perithecial tissues of N. crassa. The most abundantly expressed of the two genes, poi-1, is expressed as an approximately 1.2 kb transcript and contains an ORF of 24 aa near the 5' end. The mostly non-coding poi-1 mRNA is predicted to form an unusual secondary structure. Analysis of the poi-1 mutants generated by repeat-induced point mutation (RIP) shows that not only the ORF but also the rest of the transcript is required for normal vegetative and sexual development. The second most abundantly expressed gene, poi-2, contains an ORF with good codon bias. It has been shown by in vitro transcription and translation to encode a 27 kDa protein, and the putative protein is predicted to contain a transmembrane helix and a signal peptide. The putative poi-2 protein also contains a novel 16 tandem repeat of 13-14 amino acid residues, the partial loss of which results in defective vegetative development.
433 Etiology of senescence associated with retroplasmid-containing strains of Neurospora. A. Nicole Fox, Jonamani Saud and John C. Kennell. Department of Biological Sciences, SMU, Dallas, TX
Senescence of filamentous fungal cultures is invariably associated with mitochondrial dysfunction. Unlike Podospora, in which senescence appears to be part of the life cycle, growth decline in Neurospora is uncommon and generally limited to strains that harbor certain mitochondrial plasmids. We have been studying the etiology of senescence in strains of Neurospora crassa containing the Mauriceville mitochondrial retroplasmid. Previous studies have shown senescent cultures contain variant forms of the retroplasmid that contain cDNA copies of mt tRNAs. Here, we evaluate the rate at which variant plasmids arise in subcultures of the Mauriceville strain and their association with the senescent phenotype. Although variant plasmid formation always preceded senescence, subcultures were found to tolerate the variant plasmids for variable lengths of time and no correlation could be made between the specific sequence inserted in the plasmids and the rate or frequency of senescence. We also report the isolation of a strain in which senescence occurs in the absence of variant plasmid formation or detectable alterations in mtDNA. Spectral analysis of mitochondria isolated from pre-senescent cultures show greatly reduced levels of cytochrome c and genetic studies indicate the senescent phenotype is associated with a single nuclear mutation. Interestingly, the mutation appears to function in a dominant negative fashion. Together with other studies, our findings indicate that there are at least three separate pathways which lead to senescence in Neurospora.
434 A. nidulans SteA a Homeodomain/Zn+2 finger protein binds DNA by its homeodomain. Marcelo A. Vallim1,2 and Bruce L. Miller2. Universidade de Sao Paulo, Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Ribeirao Preto-SP, 14040-903. Brazil1. University of Idaho, Microbiology, Molecular Biology and Biochemistry Depart., Moscow, ID 83843,USA2
The homeodomain (HD) protein Ste12p plays a key role in determining cell identity, mating response and dimorphic transitions in S. cerevisiae by coupling MAPKinase signal transduction pathways to gene-specific transcriptional activation. Ste12p homologues have been identified in other dimorphic yeast, and its has been reported to be required for invasive, filamentous growth and pathogenesis. steA, a ste12 homologue, was isolated from the homothallic, filamentous fungus A. nidulans (Vallim et al., 2000, Mol. Microbiol. 36:290-301,) SteAp has an N-terminus HD similar to Ste12p, and in add tion, it has a C-terminus C2/H2-Zn+2 finger domain. Inactivation of steA resulted in sterility and inability to differentiate ascogenous tissue and cleistothecia, but did not interfere with either filamentous growth or polymorphic transitions associated with conidiation. Here we report the results of an oligonudeotide library screening, which shows that SteAp HD actually binds specifically to a DNA sequence with 7 bases, in vitro. A strong transcription activation domain was characterized at the N-terminus portion of SteAp by a yeast two-hybrid assay. The transcription of medA and stuA which are absent in the fungus hyphal stage, are de-repressed in the disrupted steA backgrond. However, gel shift assays using medA and stuA promoter sequences showed that they carry no target for the homeodomain. In vivo assays showed that over expression of SteAp inhibited the expression of a reporter gene placed downstream the promoter sequence of stuA during develpoment suggesting that SteAp might be involved in activating the transcription of a factor engaged in repressing the transcription of this gene.
435 Components and function of the Fus3/Kss1-related MAP kinase pathway in Neurospora crassa. Piotr Bobrowicz and Daniel J. Ebbole. Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77843, USA.
In yeast Saccharomyces cerevisiae two closely related MAP kinases Fus3p and Kss1p regulate pheromone response and filamentous growth. Recently, close homologues of those kinases were isolated in several pathogenic filamentous ascomycetes and shown to be important pathogenicity factors. Compared to the situation in yeast our knowledge about signaling pathway(s) regulated by those proteins and their target genes is still very limited. We decided to use Neurospora crassa, a well-characterized heterothallic ascomycete, to isolate more components of the Fus3/Kss1-related MAP kinase pathway and try to identify genes regulated by it. The MAP kinase similar to yeast Fus3p and Kss1p is encoded in N. crassa by the mak-2 gene. The mak-2 gene replacement mutants show significant reduction in growth rate and have poorly developed aerial hyphae. Mutants are fertile as a male but fail to produce protoperithecia and therefore are completely sterile as a female. Ascospores with deleted mak-2 gene do not germinate. Initial results from Western blot experiments indicate that mak-2 gene might be the only FUS3/KSS1 homologue in N. crassa. Using subtractive cloning we identified 5 novel genes that are not express or show reduced expression in mak-2 mutant as well as 2 genes that are overexpressed in mutant. The second gene from the pathway that we currently analyze is the STE12 homologue. The sequence of the predicted protein is very similar to the SteAp from Aspergillus nidulans with homeodomein in N-terminal part and C-terminal C2/H2 zinc finger motif. The characterization of this gene will determine if there is an epistatic relationship with mak-2.
436 Roles of NC-ras2 in apical growth of Neurospora. Tadako Murayama, Junko Yoshie and Junichi Kawakami (Kanto-gakuin Univ. Yokohama, 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 relationship between NC-ras2 and mcb which has been suggested to regulate growth polarity through organizing actin patches at the cell cortex will be discussed.
437 A homolog of the transcriptional repressor Ssn6p antagonizes cAMP signalling in Ustilago maydis. Michael Feldbrüggee1, Gabriel Loubradou2, Andreas Brachmann2, and Regine Kahmann1. 1Max-Planck-Institute for Terrestrial Microbiology, Karl-von-Frisch-Str., D-35043 Marburg, 2Institute of Genetics and Microbiology, Ludwig Maximilians University, Maria-Ward-Str. 1a, D-80638 München
In Ustilago maydis cAMP signalling is crucial for successful infection of maize plants. Strains are nonpathogenic if mutated in all of the so far identified components of the cAMP signalling pathway. Deletion of signalling components resulting in low cAMP levels trigger filamentous growth and high cAMP conditions cause the formation of glossy colonies such as conferred by the allele gpa3Q206L encoding a constitutively active form of the G alpha subunit. By screening a multicopy plasmid library in gpa3Q206L mutant cells we identified sql1 as suppressor of the glossy colony phenotype. Interestingly, only alleles encoding a C terminal truncated version of Sql1 were able to complement the mutant phenotype. Sql1 is a functional homologue of the yeast transcriptional repressor Ssn6p and contains 10 TPRs of which the first six are important for suppressor function. Wild type strains expressing various C terminal-truncated versions that are able to complement glossy colonies of gpa3Q206L show filamentous growth in liquid culture. Filament formation is reversed in the presence of cAMP. According to our model Sql1 is part of an evolutionary conserved Sql1/Tup1 transcriptional repressor complex that antagonizes cAMP signalling by repressing cAMP-regulated genes.
438 A pcl-like cyclin of Aspergillus nidulans is transcriptionally activated by developmental regulators and is involved in sporulation. Niklas Schier, Ralf Liese and Reinhard Fischer. Philipps-Universitat Marburg and Max-Planck-Institut fur terrestrische Mikrobiologie, Marburg, Germany.
The filamentous fungus Aspergillus nidulans reproduces asexually through the formation of spores on a multicellular aerial structure, called conidiophore. A key regulator of asexual development is the TFIIIA-type zinc finger containing transcriptional activator Bristle (BRLA). Besides BRLA, the transcription factor ABAA, which is located downstream of BRLA in the developmental regulation cascade is necessary to direct later gene expression during sporulation. We isolated a new developmental mutant and identified a leaky brlA mutation and the mutated Saccharomyces cerevisiae cyclin homologue pclA, both contributing to the developmental phenotype of the mutant. pclA was found to be 10-fold transcriptionally upregulated during conidiation and a pclA deletion strain was 3-5-fold reduced in conidia production. Expression of pclA was strongly induced by ectopic expression of brlA or abaA under conidiation suppressing conditions indicating a direct role for brlA and abaA in pclA regulation. PCLA is homologous to yeast Pcl cyclins, which interact with the Pho85 cyclin-dependent kinase. However, PCLA function during sporulation was independent of the A. nidulans Pho85 homologue PHOA. Besides the developmental regulation, pclA expression was cell cycle-dependent with peak transcript levels in S-phase. Our findings suggest a role for PCLA in mediating cell cycle events during late stages of sporulation. To identify interacting proteins of PCLA, especially the cyclin-dependent kinase, a Two-Hybrid screening and biochemical approaches are underway.
439 Mating-type genes control fertilization, internuclear recognition, developmental arrest and recovery in Podospora anserina. Sylvie Arnaise, Evelyne Coppin, Robert Debuchy, Denise Zickler and Marguerite Picard. Institut de Génétique et Microbiologie, UMR 8621, bâtiment 400, Université Paris-Sud, F-91405 Orsay cedex, France
A common feature of the sexual cycle of fungi is the formation of a large number of different progeny from one mating event. This feature is achieved in Euascomycetes after fertilization, by a series of mitotic divisions of parental nuclei within a syncytium. The development proceeds then to a transition from the syncytial stage to a cellular stage, which requires that a male and a female nucleus recognize each other (internuclear recognition, IR) and migrate from the plurinucleate cell into a specialized hypha where they divide synchronously. Caryogamy, meiosis and ascospore formation take place within these specialized hyphae. The heterothallic euascomycete Podospora anserina has two idiomorphs, mat+ and mat-, which control the two recognition steps of the sexual cycle: fertilization and IR. Fertilization is regulated by FMR1 (mat- specific gene) and FPR1 (mat+ specific gene). Mutations in FMR1 indicate that it operates as an activator of mat- fertilization functions and repressor of mat+ fertilization functions. FPR1 has opposite functions in mat+ strains. IR is under the control of three regulatory genes: FMR1 and SMR2 (mat- specific genes), and FPR1 (mat+ specific gene). Mutations in these genes suggest that FMR1 and SMR2 operate as activators of the mat- IR system and repressors of the mat+ IR system in mat- nuclei, whereas FPR1 have reversed effects in mat+ nuclei. Theses results are in agreement with a model of IR based on a nucleus-limited expression of these genes. Vegetative overexpression of the IR regulatory genes suggests that they also induce a developmental arrest which is overcome by the expression of the third mat- gene, SMR1.
440 Genetic interactions between a mating-type gene and a gene involved in nuclear distribution in Podospora anserina. Khaled Bouhouche, Sylvie Arnaise, Denise Zickler and Robert Debuchy. Institut de Génétique et Microbiologie, UMR 8621, bâtiment 400, Université Paris-Sud, F-91405 Orsay cedex, France.
In Podospora anserina, sexual reproduction is under the control of one locus with two alternate alleles mat- and mat+. Fertilization leads to the formation of plurinucleate cells with intermingled parental mat+ and mat- nuclei. Further development requires an internuclear recognition (IR) between mat+ and mat- nuclei so that mat+/mat- pairs migrate into specialized hyphae, that develop in croziers, in which caryogamy, meiosis and ascospore formation take place. FPR1 (mat+ specific gene), FMR1 and SMR2 (mat- specific genes) control IR step, which is followed by a developmental arrest. SMR1 (mat- specific gene) is required for recovery from the block in development. The ami1 gene is the functional homologue of the Aspergillus nidulans apsA gene (Gra a et al, 2000, Genetics, 155, 633-646). ami1 is involved in nuclear distribution events throughout the life cycle. A loss of function of ami1 causes abnormal distribution of nuclei in microconidia and mycelial filaments. A loss of function of ami1 in both parents does not preclude the production of a progeny but frequently causes abnormal distribution of nuclei in crozier cells and asci. Genetic interactions between ami1 and the mat genes have been analyzed in double mutant strains. Preliminary results indicate that a fmr1-r mutation, leading to self-recognition of mat- nuclei at the time of IR, is epistatic on the ami1-2 mutation (partial deletion of ami1). Surprizingly, association of the ami1-2 and smr1-r mutations leads to complete arrest of the development of the perithecia which look like unfertilized prothoperithecia, whereas the smr1-r mutant is able to start fruiting body development and displays an arrest of development before the formation of ascogenous hyphae. This suggests that SMR1 and ami1 participate to the same process during early development of the perithecium.
442 Forcible discharge of ascospores. F. Trail, H. Xu, I.Gaffoor, and C. Andries. Department of Botany and Plant Pathology, Michigan State University, E. Lansing MI
In many Pyrenomycetes and Loculoascomycetes, ascospores are forcibly discharged from perithecia. The mechanism of forcible discharge of ascospores has not been well studied in any ascomycetous fungus. It is clear that osmotic pressure builds up within the ascus and serves as the force behind discharge. Physiological and genetic methods have been used to elucidate this mechanism. Physiological studies in Gibberella zeae have identified several factors that are likely involved in this increase in pressure. The genetic basis of these factors is being studied. Generation of 5000 insertional mutants yielded one that formed morphologically normal perithecia that had lost their ability to discharge ascospores. Analysis of the discharge minus mutant is in progress.
443 Characterization of the racA gene in Aspergillus niger. A.F.J. Ram1,2, M. Arentshorst1, R.A. Damveld1, P.J. Punt2 and C.A.M.J.J. van den Hondel1,2. 1Leiden University, Inst. Mol. Plant Sciences, Leiden, The Netherlands,2Department of Appl. Microbiol. Gene Techn., TNO- Nutrition, Zeist, The Netherlands.
The members of the Rho-subfamily of Ras related GTP binding proteins (Rho, Rac and Cdc42) are signalling molecules originally identified as regulators of actin cytoskeleton reorganization. More recent evidence has implicated a wider function of the rho-subfamily that also include activation of kinase cascades and regulation of enzymes activities. Using PCR-based cloning approaches we searched for Cdc42-like GTPases in A. niger. Both a Cdc42 (Cdc42A) homologue and a Rac homologue (RacA) were isolated. The presence of a Rac homologue seems to be unique for filamentous fungi, since homologues of this gene are not present in yeasts like S. cerevisiae and S. pombe. Deletion of the racA showed that the gene is not essential. The mutant showed alterations in hyphal morphology and a reduced conidiation. Overexpression of the racA gene from the inducible glucoamylase promoter also resulted in reduced conidiation, indicating a role for RacA in conidiophore initiation or formation. A GFP-RacA fusion protein has been constructed which is currently used to localize RacA in the fungal hyphae.
444 The role of beta-1,3-glucanase in Phytophthora infestans. Adele McLeod, Christine D. Smart, and William E. Fry. Department of Plant Pathology, Cornell University, Ithaca, NY 14853
Phytophthora infestans is an oomycete pathogen that causes late blight of potato and tomato. Oomycetes have a cell wall that is comprised of 80-90% beta-1,3-glucan, therefore beta-1,3-glucanases could play an important role in the biology of these organisms. Suggested functions of beta-1,3-glucanase are the hydrolyses of cell-associated reserve material, hydrolysis of extra-cellular nutrients in the environment, cell wall growth and extension, breakdown of plant cell walls and breakdown of plant defense structures. The aims of our study are to characterize beta-1,3-glucanases from P. infestans and to attempt to investigate their biological function in the pathogen by gene silencing. Two putative beta-1,3-glucanase fragments, one endo-beta-1,3-glucanase and one exo-beta-1,3-glucanase, were amplified from P. infestans genomic DNA. The putative endo-beta-1,3-glucanase fragment had 35% amino acid similarity to a cell wall endo-beta-1,3-glucanase of Saccharomyces cerevisiae, and the putative exo-beta-1,3-glucanase fragment had 45% amino acid similarity to an exo-1,3-beta-glucanase from Agaricus bisporus. Southern analysis has shown that there is only one copy of the endo-beta-1,3-glucanase and possibly two copies of the exo-1,3-beta-glucanase in P. infestans. Homologous copies of both genes were found in P. palmivora and P. sojae. The PCR products of the putative genes were used to isolate clones from a genomic library of P. infestans, and we are currently identifying full-length genomic sequences. In vitro analysis of expression of the putative exo- and endo-glucanase genes has shown that they are more strongly expressed in mycelia than in sporangia, zoospores or germinating cysts. Both genes are expressed in planta, and can be detected at 72 h after inoculation.
445 A Gbeta Protein from Neurospora crassa. Sheven I. Poole, Qi Yang and Katherine A. Borkovich. Univ. Texas-Houston Medical School, Micro. and Molec. Genet., Houston, TX, USA
Heterotrimeric G protein-mediated signal transduction pathways allow cells to respond to environmental stimuli. Accumulating evidence suggests that G protein beta-gamma dimers as well as Galpha subunits play pivotal roles in these pathways. We have identified a Gbeta subunit from Neurospora crassa, gnb-1. The predicted amino acid sequence of GNB-1 is most identical to Cryphonectria parasitica CPGB-1 (91%) and Gbeta proteins from other fungi. RFLP mapping showed that the gnb-1 gene is located on the right arm of chromosome III near the con-7 and trp-1 genes. Northern analysis reveals two gnb-1 specific transcripts of 2.7 and 1.4 kb. gnb-1 mutants possess many defects, including aberrant growth in submerged culture and female sterility. Loss of gnb-1 also impacts levels of Galpha proteins in N. crassa.
446 Mating-regulated gene expression in Phytophthora infestans. Anna-Liisa Fabritius and Howard, S. Judelson. Department of Plant Pathology, University of California, Riverside, CA 92521, USA.
Interactions between the two mating types (A1 and A2) of the heterothallic oomycete, Phytophthora infestans, result in the formation of gametangia and the sexual spores, which are called oospores. Genes differentially expressed during sexual development were identified by suppression subtractive hybridization-polymerase chain reaction. Nine genes were identified that were induced during mating. Of these, sequence analysis indicated that one was very similar to a previously identified extracellular glycoprotein elicitor gene of P. megasperma. Another gene was highly similar to the pumilio gene of Drosophila melanogaster, which encodes an RNA binding protein that regulates early embryogenesis. Time course analysis indicated that three of the mating-regulated genes of P. infestans were induced very early in sexual development, before defined male- or female-specific structures appeared. The other six genes were induced during later stages, which corresponded to oospore development and maturation. The mating-induced genes were also expressed in self-fertile strains of P. infestans. For one gene, multiple transcripts were detected which showed differential regulation during time-course and between normal matings and self-fertiles. Experiments aimed at silencing the genes in transformants are underway to address their function. Preliminary experiments revealed serious defects in mating behavior among the transformants.
447 Are Gibberella zeae sexual spores the critical inoculum for wheat head blight? Daren W. Brown1, Sung-Hwan Yun2, Theresa Lee3, B. Gillian Turgeon4, and Anne E. Desjardins1. 1Mycotoxin Research Unit, NCAUR, USDA/ARS, 1815 N. University St., Peoria, IL 61604. 2Division of Life Sciences, Soonchunhyang University, Asan, Choongnam, 336-745, Korea. 3School of Agricultural Biotechnology, Seoul National University, Suwon 441- 744, Korea 4Department of Plant Pathology, Cornell University, 334 Plant Science Building, Ithaca, NY 14853
Gibberella zeae (anamorph Fusarium graminearum) causes head blight (scab) in wheat and barley, and ear rot in corn. Since 1991, epidemics of Gibberella head blight have had disastrous effects on wheat and barley production in the Midwestern states. In addition to decreasing yields, the fungus also contaminates grain with trichothecene mycotoxins that are harmful to human and animal health. To understand and control head blight, the factors and conditions that lead to epidemics must be identified. Observations in the field suggest that G. zeae sexual spores (ascospores) produced on plant debris in the soil are a primary source of inoculum for head blight epidemics. Sexual reproduction in G. zeae is controlled by a mating-type (MAT) locus that contains four genes located within 6 kb of genomic sequence. To test the role of ascospores in causing blight, we have constructed an ascospore non-producing strain by deleting the MAT locus using a transformation mediated gene replacement strategy. MAT-null strains appear indistinguishable from wild-type strains in morphology and production of asexual spores (macroconidia). In addition, MAT-null strains are similar to wild-type strains in virulence following injection of individual wheat heads with macroconidia. Experiments planned for Spring 2001 will test a MAT-null strain and the wild- type strain from which it was derived for their ability to cause head blight on wheat following application of infested maize stalk pieces to the soil surface. If ascospores are the major inoculum source, then we predict that exposure of wheat heads to a MAT-null strain that cannot produce ascospores should cause less blight than exposure to an ascospore producing strain.
448 Isolation and expression of a gene encoding the plasma membrane H+-ATPase from the oomycete, Phytophthora infestans, the causal agent of potato late blight. Christine D. Smart1, Nobutaka Kato2, William E. Fry1, and Lawrence B. Smart2. 1Department of Plant Pathology, Cornell University, Ithaca, NY 14853 2Faculty of Environmental and Forest Biology, SUNY College of Environmental Science and Forestry, Syracuse, NY 13210
Phytophthora infestans produces asexual sporangia that can germinate either by forming a germ tube directly or by producing motile zoospores. Progression through the various stages of the pathogen life cycle, including differentiation and germination of zoospores, is apparently regulated by ion flux (K+ and/or Ca2+) across the plasma membrane. These fluxes are, in turn, dependent upon the activity of the plasma membrane (PM) H+-ATPase. In order to gain a better understanding of the regulation of ion transport through P. infestans development, we have initiated molecular studies of the PM H+-ATPase. We utilized degenerate oligonucleotide primers derived from conserved regions of plant H+-ATPases for PCR, then used that PCR product as a probe in screening a genomic library of P. infestans isolate US940480 (a member of the US-8 clonal lineage). The PM H+-ATPase in P. infestans appears to be encoded by a single gene, which is most similar to genes from algae and to a lesser degree from plants. This gene apparently lacks introns and does not contain consensus eukaryotic core promoter elements. We are characterizing the expression of the PM H+-ATPase gene under various pH levels and in the presence of different ion concentrations with the hope of gaining further insight into the developmental biology of this oomycete.
449 Identification of the STE20a gene of Cryptococcus neoformans reveals that serotype A MATa strains still exist. Klaus Lengeler and Joseph Heitman. Duke University Genetics
The mating-type loci of the opportunistic human fungal pathogen Cryptococcus neoformans are quite unusual in size, spanning ~50 kb, and encode several allelic but divergent genes, including STE20a and STE20alpha. C. neoformans is classified into three varieties: neoformans (serotype D), grubii (serotype A) and gattii (serotype B and C). Interestingly, environmental and clinical isolates are predominantly of MATalpha mating-type, and MATa strains of variety grubii (serotype A) were thought to be extinct. We recently identified the STE20a gene of serotype D and showed that the gene cosegregates with the MATa mating-type in genetic crosses, maps within the mating-type locus on a 1.8 MB chromosome, and is allelic with the MATalpha locus. During the characterization of unusual clinical isolates we identified a novel STE20 allele from the clinical isolate 125.91. The novel STE20 allele is MATa mating-type specific but not identical to the STE20a gene from serotype D. Capsular antigen analysis, Southern blot analysis, and DNA sequence comparisons revealed that strain 125.91 is serotype A. We characterized clinical and environmental isolates of an unusual serotype AD and find that these strains are aneuploid or near diploid. In addition, we find these strains are heterozygous for the mating-type locus, suggesting that serotype AD strains arise from intervariety crosses between serotype A and serotype D. Moreover, several serotype AD strains inherited the MATa mating-type locus from a serotype A parental strain, suggesting that fertile serotype A MATa strains exist in nature. The serotype A MATa strain 125.91 is sterile under all conditions tested thus far. In addition, the dissection and characterization of basidiospores derived from a self-fertile serotype AD strain that inherited the serotype A MATa locus did not result in the isolation of a fertile serotype A MATa strain. Our long term goal is to construct a congenic pair of fertile serotype A MATa and MATalpha strains for use in genetic analysis of C. neoformans variety grubii .
450 A novel gene from Neurospora crassa involved in sexual development is a member of conserved group of protein methyltransferases. Piotr Bobrowicz and Daniel J. Ebbole. Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77843, USA.
Recently a number of genes encoding members of a conserved family of protein methyltransferases was characterized and shown to play important role in cell cycle control and cellular signaling. For example the product of the human JBP1 gene interacts with Jak kinases involved in cytokine signal transduction. The Hsl7p of Saccharomyces cerevisiae was found to be a component of the Swe1p/Cdc28p morphogenesis checkpoint. Additionally, Hsl7p is a negative regulator of filamentous growth MAP kinase pathway and was shown to compete with Cdc42p for binding to the Ste20p. Using the HSL7 sequence to search the sequence generated by the German Neurospora sequencing project we identified a close homologue of this gene. The predicted protein contains a conserved GXGRGP motif specific for protein methyltransferases that is also present in both JBP1 and Hsl7p. The pp-2 (protoperithecia 2) RIP mutant strains have a slight growth defect and do not produce protoperithecia. Based on the yeast model we have initiated experiments to test the hypothesis that pp-2 is involved in the control of MAP kinase signaling pathway identified by us in N. crassa.
451 Cloning of pheromone genes in Podospora anserina. Evelyne Coppin and Robert Debuchy. Institut de Genetique et Microbiologie, UMR 8621, batiment 400, Universite Paris-Sud, F-91405 Orsay cedex, France
The development of the fruiting body and the production of a progeny in heterothallic Euascomycetes rely on two recognition steps: an intercellular recognition between compatible reproductive organs, which leads to fertilization and then an internuclear recognition (IR) between compatible nuclei. This latter event occurs in plurinucleate cells which contains female nuclei and their responsive nuclei which are derived from the fertilizing male nucleus by mitotic divisions. The compatible nucleus couples migrate from the plurinucleate cell into a specialized hypha (the ascogenous hypha) where take place karyogamy, meiosis and ascospore formation. The heterothallic euascomycete Podospora anserina has two idiomorphs which control fertilization and IR. As idiomorphs encode transcriptional factors, the mechanism of IR remains an enigma. It has been proposed that pheromone - receptor systems may be involved in IR for the production of a cue which would trigger the migration of compatible nuclei into the budding ascogenous hypha. In order to test the role of pheromones in IR, we have decided to clone the pheromone genes in P. anserina. Degenerate primers have been designed from the alignment of Neurospora crassa mfa and Magnaporthe grisea MF1-1 pheromone genes. Low stringency PCR with these primers has allowed us to isolate a pheromone gene in P. anserina. Its functions are under investigation. Cloning of a pheromone gene similar to ccg4 of N. crassa is in progress by heterologous hybridization. Aknowledgement : we are greatly indebted to Dan Ebbole for communicating the sequence of mfa prior to publication and for the gift of plasmids containing mfa and ccg4.
452 Inhibition of a serine protease disrupts development in Cryphonectria parasitica. Patricia M McCabe and Neal K Van Alfen, University of California, Department of Plant Pathology, Davis, CA. 95616, USA.
Infection of the chestnut blight fungus, Cryphonectria parasitica by a dsRNA virus (CHV1) results in loss of development, which is manifested as a reduction in pigmentation, conidial formation, sexual reproduction and virulence. However, there is no change in growth rate and the fungus remains in a healthy juvenile state. Genes that have been identified based on differential expression between CHV1 infected and wild type strains are secreted proteins that play a role in development. The best characterized are a laccase, a mating type specific pheromone and the hydrophobin cryparin. All of the proteins have identical signals for post-translational processing and are thought to be secreted via a vesicle mediated secretory pathway. CHV1 infection causes the accumulation of fungal vesicles inside the cell. Virus dsRNA and RNA-dependent RNA polymerase activity co-purify with these vesicles. One CHV1 down regulated host protein, cryparin, is also associated with this vesicle fraction. Using pulse-chase analysis to study cryparin secretion we found that CHV1 infection reduced the rate of cryparin secretion through cells. This decrease in secretion rate can be mimicked by the addition of a specific serine protease inhibitor. The inhibitor not only slows the rate of cryparin secretion to levels similar to that of CHV1 infection, but phenotypically mimics the virus infection symptoms i.e. it prevents C. parasticia pigmentation and conidia formation without affecting growth, and down-regulates cryparin gene transcription. These remarkable phenotypic similarities suggest that CHV1 is causing symptoms by interfering with a serine protease processed protein secretory pathway.
453 WC-1 and WC-2, the trans-acting factors that mediate expression of the circadian clock gene frq, bind at the frq promoter. Allan Froehlich, Jennifer J Loros, and Jay C Dunlap. Dartmouth Med School, Genetics, Hanover, NH, USA
The frequency (frq)locus is a key component of the Neurospora crassa circadian clock. Under constant environmental conditions, frq mRNA and protein oscillate with an approximate 22 hour period; these oscillations are part of an autoregulatory feedback loop in which frq gives rise to two forms of the FRQ protein which then act to repress the levels of the frq transcript (Dunlap, J. Cell 96:271-290, 1999). Evidence gathered to date suggests that resetting of the clock by light occurs through the rapid induction of frq (Crosthwaite et al. Cell 81:1003-1012, 1995). Resection of the frq promoter has uncovered two cis-acting light response elements (LREs), both of which are necessary for light induction of frq to wild type levels. Using a heterologous reporter, both LREs have been shown to be sufficient for light induction. Deletion of the proximal LRE affects phase following a light to dark transfer, but does not have an effect upon resetting following temperature treatments. Deletion of the distal LRE results in arrhythmia under both light and temperature treatments. The phenotypes of both LRE deletions can be observed at the physiological as well as at the molecular level. White-collar loci products (wc-1 wc-2) have been found to act as global regulators for light perception and as positive components of the circadian clock in Neurospora (Crosthwaite et al. Science 276:763-769, 1997). WC-1 and WC-2 proteins both contain Zn-finger domains with distinct similarity to other transcriptional activators within the GATA factor family. Using electrophoretic mobility shift assays (EMSA), WC-1 and WC-2 have been shown to specifically bind to the frq LREs in two distinct complexes; one complex is present in the light and the other complex is present in the dark. FRQ is not present in either complex. Action spectrum and dose-response data have been generated for changes in binding between the dark and light complexes. These in vitro data correlate well with published in vivo data for light resetting of the Neurospora clock.
454 Analysis of the asd-2 gene of Neurospora crassa. Kelly A. Howe, Mary Anne Nelson University of New Mexico, USA
A genetic approach is being used to the investigate the sexual development of Neurospora crassa, which is poorly understood at the molecular level. The asd-2 (ascus development) gene is essential for sexual development; homozygous asd-2 crosses are blocked shortly after karyogamy. Ascospores are never produced and asci are short and few in number. Small perithecia are formed that lack perithecial beaks. Vegetative growth of asd-2 mutant strains also appears to be deficient. The ASD-2 protein displays significant homology with the AGO/eIF2C2/Zwille family of proteins. This family of proteins has been implicated in two major processes: involvement in stem cell differentiation and involvement in posttranscriptional gene silencing. One member of this family in particular, the N. crassa QDE-2 (quelling deficient) protein, has been shown to be involved in quelling, a particular form of posttranscriptional gene silencing. We are investigating the role of the asd-2 gene product in cell signalling and/or posttranscriptional gene silencing and the relationship of this role to sexual development.
455 Initial characterization of genes involved in mushroom development in the basidiomycete Schizophyllum commune,as isolated by a two-hybrid screen. Stephen Horton and Alaap Shah. Department of Biological Sciences, Union College, Schenectady, NY 12308 USA
The molecular mechanisms controlling the development of mushrooms are incompletely understood in basidiomycetous fungi. The gene FRT1was originally identified in Schizophyllumcommuneby its' ability as a transgene to induce fruiting in certain homokaryotic transformation recipients. In an effort to identify new genes involved in fruiting body development, we have utilized FRT1as 'bait' in a two-hybrid screen. Frt1p has been localized primarily to the outside of the cell wall, and is therefore likely to be post-translationally modified. We therefore decided to employ the Cytotrap Ras rescue two-hybrid approach to isolate genes encoding interacting proteins, rather than a traditional nuclear-based system. 1.5 X 107 transformants were obtained in a screen of two newly-constructed S. communecDNA libraries. 533 initial positives were found, of which 121 passed a second round of screening. Library plasmids were first rescued in E. coli,and the cDNA inserts amplified by PCR. These inserts will be sorted into groups by restriction enzyme analysis. Further characterization of representative members of each group will be presented.
456 Expression of nsdD that controls sexual development of Aspergillus nidulans. Kyu-Yong Han, Kap-Hoon Han1,Seon-Hwa Park, Kwang-Yeop Jahng2, Keon-Sang Chae2 and Dong-Min. Han Division of Life Science, Wonkwang University, Iksan 570-749 ; 1Dept. Microbiology & Molecular Genetics, Oklahoma University Stillwater, OK 74078, USA; 2 Faculty of Biological Science, Chunbuk University, Chunju
The nsdD gene predicted to encode a GATA type transcription factor with the type IVb zinc finger DNA binding domain functions in activating sexual development of A. nidulans. When the nsdD gene was over- expressed, cleistothecia were formed in excess amount even in the presence of 0.6 M KCl that inhibited sexual development of wild type. A lot of suppressors for Delta-nsdD showed the common phenotype similar to that of nsdD over-expressed mutant. Northern blot analysis revealed that the expression of nsdD was repressed by 0.6M KCl. These results strongly suggest that the inhibition of sexual development by salts was carried out via nsdD involved regulatory network. In several allelic mutants of nsdD that resulted in non sense mutations and lacked C terminal zinc finger, the transcription level was greatly increased. Also in over-expressed mutants, the transcription under its own promotor was reduced. These results suggest that the expression of nsdD is negatively autoregulated. The nsdD expression was greatly reduced in flbA deletion mutant, indicating that functional FlbA is required for nsdD expression. However, G-alpha, G-Beta or FluG apparently did not have significant relationship with nsdD expression. This result suggests that FlbA is not specifically required for signaling of asexual sporulation but also required for that of sexual development, while FluG is specific to the asexual signaling.
457 The role of hydroperoxidase (laccase II) and alpha-1,3-Glucanase during sexual development of Aspergillus nidulans. Mario Scherer, Huijun Wei and Reinhard Fischer. Max-Planck-Institute for terrestrial Microbiology, Karl-von-Frisch-Str., D-35043 Marburg, Germany.
Sexual development in Aspergillus nidulans is an
interesting morphogenetic process, which is only poorly understood. We set out to study
differentially expressed genes to use them as tools for the isolation of central regulators for
development. We isolated laccase II, which was described to be specifically expressed in early
developmental structures, such as H lle cells and primordia. The corresponding primary protein
sequence revealed that it is a heme-containing hydroperoxidase rather than a laccase. A
hydroperoxidase-GFP fusion protein was localized in sexual tissues. The expression pattern
revealed an early induction during sexual development and upon carbon starvation. In a
non-targeted approach, differentially expressed genes were isolated using the subtractive
suppressive hybridisation method (Clontech). One of the identified genes was
alpha-1,3-Glucancase, which probably plays a central role in energy supply during sexual
differentiation. The gene is specifically induced early during development and can serve to reveal
the regulatory circuits. In a targeted approach, a homologue of a transcription factor, Pro1, from
Sordaria macrospora, which regulates perithecium formation, was isolated from
A. nidulans. Since overexpression of the gene leads to a misregulation of
development, it appears to play a crucial role in A. nidulans sexual development.
Experiments to analyse a function of this transcription factor for the regulation of the
hydroperoxidase and the glucanase are on the way. These studies also include the analysis of the
roles of veA, nsdD and stuA, which also trigger sexual
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