1 An IQGAP-related protein controls actin ring assembly and septation in Ashbya gossypii. Jorgen Wendland. Friedrich Schiller-University of Jena, Dept. of Microbiology.
The organization of the actin cytoskeleton is highly ordered in hyphae of filamentous fungi during polarized growth. Cortical actin is clustered in hyphal tips and actin cables serve as tip-directed tracks to promote vesicle delivery. Additionally, at sites of septation actin-ring structures occur. Actin-based contraction processes have been shown to be critical during cytokinesis of yeast-like and mammalian cells. In order to analyze these events in a filamentous fungus the A. gossypii homolog of an IQGAP-related protein encoded by the Saccharomyces cerevisiae CYK1/IQG1 gene was isolated. The AgCyk1p encodes a protein of 1553 aa which is 53 aa longer than its yeast homolog. Both proteins are about 30% identical and share CH-domains (involved in actin filament binding), eight IQ-repeats (presumed to interact with calmodulin) and GAP-domains (GTPase activation). In addition, AgCyk1p possesses a PE-repeat domain which is absent in the yeast homolog. Deletion of AgCYK1 produced viable mutant strains that grew with wt-like rates whereas in S. cerevisiae CYK1 deletion results in lethality. Agcyk1 mutants, however, showed an increased rate of lysed hyphae as well as a sporulation defect. Septation was compared between wild-type and Agcyk1 mutants by fluorescence microscopy. The absence of actin-ring structures in these mutants was found to be accompanied by a lack of chitin accumulation at presumptive sites of septation. Our results indicate that Agcyk1 controls actin-ring assembly and is also required to direct chitin deposition at sites of septation. In contrast to yeast-like fungi in filamentous ascomycetes polarization of the actin cytoskeleton in the growing hyphal tips and at sites of septum formation can occur at the same time and lack of septation does not interfere with polarized hyphal growth.
2 Searching for additional components in the mating-type associated vegetative incompatibility response. Megan D. Hiltz1,Patrick K.-T. Shiu2,N. Louise Glass1.. 1UC Berkeley, PMB, Berkeley CA USA. 2 Stanford University, Biology, Palo Alto CA
Vegetative incompatibility is a common and ubiquitous phenomenon in
many filamentous fungi, from Ascomycetes to Basidiomycetes. In our lab, we use
Neurospora crassa to characterize the components and mechanisms behind
vegetative incompatibility to improve the understanding its function and its role as a non-self
recognition system. In the vegetative incompatibility response, there is a prevention of vigorous
heterokaryons, which is mediated when there is genetic dissimilarity between the fusing
individuals at a heterokaryon incompatibility (het) locus. Incompatibility is noted
in heterokaryons that exhibit growth inhibition, hyphal compartmentation and death.
A and a represent the two mating-types (at the mat
locus) in N. crassa required to fuse in the initiation of the sexual cycle. During
vegetative phase, the mat locus also functions as one of the 11 het
loci. The tol mutation causes a recessive suppression of mating-type vegetative
incompatibility. While the molecular function of TOL is not apparent, it contains putative
protein-protein interaction domains.
To determine the role of TOL in mating-type vegetative incompatibility and to identify other proteins involved in the process, TOL was used as bait in a yeast 2-hybrid system. Of the identified positive cDNA clones, one was characterized to deduce its role in mating-type associated vegetative incompatibility. Ncvip1 has homology with vip1,which was identified in a p53 expression library screen of Schizosaccharomyces pombe. RIP (repeat-induced point) mutational analyses of the Ncvip1 was attempted and the progeny from the cross examined as to their effects on mating-type associated vegetative incompatibility.
3 Molecular genetic basis of resistance to the DMI fungicide in Tapesia yallundae. Henry M. Wood, Paul S Dyer, Matt J Dickinson, John A Lucas*. University of Nottingham, Life & Environmental Sci. Nottingham, , UK. * IACR Long Ashton, Bristol UK
Resistance to DMI fungicides in fungal plant pathogens has previously been correlated with point mutations in the CYP51 gene encoding the target enzyme eburicol 14alpha-demethylase. The CYP51 gene was isolated from a genomic library of the cereal eyespot pathogen Tapesia yallundae using a PCR-based strategy with degenerate primers. The gene has a coding region of 1708 bp, containing two putative introns, and a predicted 526 amino acid polypeptide product which exhibits 55 - 68 % homology to CYP51s from other filamentous fungi. The CYP51 gene was fully sequenced from four further field isolates of T. yallundae either sensitive or resistant to the DMI fungicide prochloraz. CYP51 was also partially sequenced from two additional field isolates and eight progeny from a cross between prochloraz sensitive and resistant parents. Two allelic forms of the gene were detected termed CYP51-1 and CYP51-2. These differed by 0.7 % over the coding region and exhibited differences in sequence upstream of the putative start codon. No correlation was found between change in sequence and fungicide sensitivity, with CYP51-1 and CYP51-2 alleles found in both sensitive and resistant isolates. Thus, contrary to frequent reports for resistance to other DMI fungicides, resistance to prochloraz in the isolates of T. yallundae examined in this study involved some other mechanism than mutation in the gene encoding the target site enzyme. There was also no evidence of increased copy number of CYP51 as a possible basis of prochloraz resistance in T. yallundae
4 Symmetry of branching in Neurospora crassa. Michael K. Watters. Valparaiso University, Valparaiso Indiana.
In a previous study of branching in Neurospora, it was determined that branching at the tip is not a tip autonomous process, but that branching is controlled at least in part by factors at or near the previous branch point. This was determined by the demonstration of a statistical correlation between lengths of branch intervals (the distance between two tandem branch points) having a common origin. That study was unable to determine the nature of that common factor. Namely, the correlation could have been due to the physical bisection of some structure or resource (such as the Spitzenkörper) at the time the common branch event occurred. It could have also been caused by the division, at the common branch point, of the flow of some factor (such as tip growth vesicles) toward the growing tips. This study seeks to distinguish between these two alternatives by extending the examination of branch interval correlation and comparing branch intervals which share a common origin, one step removed. The observation of branch length correlation at this level demonstrates that the previously observed correlation results from the division of some factor flowing toward the tip and not the singular division of some structure at the time of branch formation.
5 The extracellular soluble polysaccharide (ESP) from Aspergillus kawachii improves the stability of extracellular enzymes and is involved in their localization. Kazuhiro Iwashita, Nao Harada, Hitoshi Shimoi, Kiyosi Ito. National Research Institute of Brewing, 3-7-1, Kagamiyama, Higashihiroshima, Hiroshima, 739-0046, Japan
Aspergillus kawachii produces two extracellular beta-glucosidases (EX-1 and EX-2) and one cell wall-bound (CB-1) beta-glucosidase, which are derived from the same bglA gene. Extracellular beta-glucosidases (EX-1 and EX- 2) are quite stable in crude solution, but they become unstable in purified form under moderate condition. Purified extracellular beta-glucosidases bind to a cell wall fraction from mycelia, even though these enzymes are released into the medium under solid culture conditions. A. kawachii produces an extracellular soluble polysaccharide (ESP) under solid culture conditions. Addition of purified ESP remarkably stabilized the beta-glucosidases. ESP directly interacted with the purified extracellular beta-glucosidases but it did not affect the Km value of these enzymes. Moreover, ESP inhibited the adsorption of purified extracellular beta-glucosidases to the cell wall fraction and extracted them from it. To examine the effect of ESP to other extracellular enzyme, we purified extracellular alpha-glucosidase and alpha- galactosidase of A. kawachii. As expected, ESP stabilized these two enzymes and inhibited their adsorption to the cell wall fraction. These results indicate that ESP plays an important role in the stability and localization of extracellular enzymes. ESP from A. kawachii directly binds to the enzymes and releases them to the medium from the cell wall layer and then stabilizes them.
6 Cell cycle progression and cell polarity require sphingolipid biosynthesis in fungi. Jijun Cheng, Anthony Fischl and Xiang S. Ye. Infectious Diseases Research, Lilly Research Laboratory, Eli Lilly and Company, Indianapolis, IN 46285
Sphingolipids are major components of the plasma membrane of eukaryotic cells but their physiological functions are not well understood. Here we show that sphingolipid biosynthesis is required for cell cycle progression in G1 and cell polarity in Aspergillus nidulans. Genetic or pharmacological inactivation of aurA encoding inositol phosphorylceramide (IPC) synthase causes cell cycle arrest in G1 and also prevents polarized hyphal growth of germinating spores. Inactivation of IPC synthase eliminates IPC synthesis but also leads to the accumulation of its upstream intermediate, ceramide. Serine palmitoyl CoA transferase (SPT) is the first committed step of the sphingolipid biosynthesis pathway. Inactivation of SPT also prevents polarized growth but not nuclear division of germinating spores, indicating that IPC is required for cell polarity and that accumulation of ceramide causes G1 arrest. Interestingly, inhibition of sphingolipid biosynthesis in germlings promotes rapid multiple branching at the hyphal tip, which normally never occurs. Hyphal tip branching is closely associated with a dramatic rearrangement of the actin cytoskeleton and is dependent on the actin function. Sphingolipids have no effect on the microtubule. The results indicate that sphingolipids regulate fungal cell polarity via the function of actin cytoskeleton.
7 Visualization of nuclei in Aspergillus oryzae by expressing EGFP. Jun-ichi Maruyama, Harushi Nakajima, and Katsuhiko Kitamoto. Department of Biotechnology, The University of Tokyo, Tokyo, Japan.
Aspergillus oryzae is an important fungus in the fermentative industry and has been used in sake, soy sauce and miso manufacture as well as production of commercial enzymes. In spite of its fermentative and industrial use little is known about the cellular and developmental processes. Nuclear migration is one of the most important processes because A. oryzae forms multinucleate cells in conidia as well as hyphae. We focused on nuclear migration in A. oryzae. In order to stain nuclei in A. oryzae, we fused A. nidulans histone H2B with EGFP. The fusion protein, H2B::EGFP, successfully visualized nuclei in hyphae and conidia. Time-lapse observation exhibited that apical nuclei migrated toward tips of growing hyphae. In a conidium varied numbers of nuclei, ranging from one to three or four, were observed. FACS analysis also supported this result. In filamentous fungi cytoplasmic dynein and dynactin complex are required for nuclear migration. We have cloned arpA and dhcA genes, encoding Arp1 in dynactin complex and cytoplasmic dynein heavy chain, respectively, and analyzed phenotypes of these disruptants. Time-lapse observation by expressing H2B::EGFP revealed that both arpA and dhcA disruptants showed a defect in nuclear migration.
8 Determinants of fungal morphogenesis: isolation and analysis of Neurospora crassa mutants defective in the maintenance and regulation of cellular polarity. Stephan Seiler and Michael Plamann. UMKC, 5100 Rockhill Road, Kansas City, MO 64110
Elongating at rates up to a micrometer per second, filamentous fungi are among the fastest growing cells known and are a good system to study cellular polarity. By filtration enrichment and a colony overlay method, we have isolated a large collection of temperature sensitive mutants in Neurospora crassa defective in the maintenance and regulation of growth polarity as a basis to study cellular developement in filamentous fungi. Phenotypical analysis and complementation tests of ca 800 mutants have allowed us to identify 20 morphological classes that define more than 80 genes involved in polar or directed growth. The observed phenotypes of polarity mutants range from a total loss of polarity over the whole hypha, generating chains of spherical cells, to more specific defects localized to hyphal tips or to subapical regions. These mutant phenotypes suggest that fungal morphogenesis is the result of the well balanced action of proteins at the tip as well as subapically to produce and maintain the characteristic tube-like structure of the hypha. Additional mutants, defective in the regulation of polarity and growth directionality, exhibit abnormal side branches and highly irregular growing hyphae, implying defects in the establishment of mature tips and Spitzenkörper positioning, respectively. Currently we are isolating and sequencing genes defined by representative mutants of each phenotypic class to understand tip growth and polarity in filamentous fungi
9 Unraveling G-protein mediated signaling in Phytophtora infestans. Maita Latijnhouwers1, Ana Laxalt2, Teun Munnik2, and Francine Govers1. 1Wageningen University, Phytopathology, Wageningen, Netherlands. 2University of Amsterdam, Swammerdam institute, Amsterdam, Netherlands
Many fungal plant pathogens develop highly specialized infection structures in response to signals detected upon contact with the plant. In addition, proliferation within host tissue requires both metabolic and morphological adaptation to the plant environment. Work on several plant-pathogenic fungi revealed a role for G-protein- and cAMP-mediated signal transduction in these processes. The general finding is that mutants with defects in components of the cAMP-signaling pathway have attenuated virulence. Our aim is to study the role of G-protein signaling in pathogenesis of the oomycete Phytophthora infestans, the causal agent of potato late blight. We isolated and characterized P. infestans genes coding for Galpha (Pigpa1) and Gbeta (Pigpb1) protein subunits, with up to 42% and 62% identity with known G-protein subunits in the database, respectively. Expression studies showed that both genes are differentially expressed in various stages of the life cycle. In an EST database, clones encoding putative catalytic and regulatory subunits of cAMP-dependent protein kinase (PKA) were selected. Through homology dependent gene silencing PIGPA1 and PIGPB1 deficient transformants will be obtained and this will enable us to determine the function of Pigpa1 and Pigpb1 in P.infestans. Phospholipid signaling was investigated by treating sporangiospores with the G-protein activator mastoparan. This treatment resulted in increased levels of the phospholipids PA and DGPP, as was detected using TLC analysis. This shows that mastoparan activates PLD in P. infestans and strongly suggests that in P. infestans, phospholipid and G-protein signaling are linked. PLC activation was not detected.
10 Microtubules in the fungal pathogen Ustilago maydis are highly dynamic and determine cell polarity. Irene Schulz1, Roland Wedlich-Söldner1, Marianne Brill2, Gero Steinberg1. 1MPI Terrestrial Microbiology, Marburg, Germany. 2LMU Institut für Med. Mikrobi, Munich, Germany
Many fungal pathogens undergo a yeast-hyphal transition during their pathogenic development that requires the rearrangement of the cytoskeleton, followed by directed membrane traffic towards the growth region. The role of microtubules and their dynamic behavior during this process is not well understood. Here we set out to elucidate the organization, cellular role and in vivo dynamics of microtubules in the dimorphic phytopathogen Ustilago maydis. Hyphae and unbudded yeast-like cells of U. maydis contain bundles of spindle pole body-independent microtubules. At the onset of bud formation two spherical tubulin structures focus microtubules towards the growth region, suggesting that they support polar growth in G2, while spindle pole body-nucleated astral microtubules participate in nuclear migration in M and early G1. Conditional mutants of an essential a-tubulin gene from U. maydis, tub1, confirmed a role of interphase microtubules in determination of cell polarity and growth. Observation of GFP-Tub1 fusion protein revealed that spindle pole body-independent and astral microtubules are dynamic with elongation and shrinking rates comparable to those found in vertebrate systems. In addition, very fast depolymerization was measured within microtubule bundles. Unexpectedly, interphase microtubules underwent bending and rapid translocations within the cell suggesting that unknown motor activities participate in microtubule organization in U. maydis.
11 Isolation and characterization of novel Ran-binding proteins in the yeast Saccharomyces cerevisiae. Andreas Braunwarth1, Thomas Gerstberger1, Micheline Fromont-Racine2, Pierre Legrain2, Ed Hurt1 and Markus Kunzler1. 1Biochemie-Zentrum Heidelberg (BZH), Ruprecht-Karls-Universitat, Im Neuenheimer Feld 328, 4. OG, D-69120 Heidelberg, Germany; 2Institut Pasteur, Genetique des Interactions Macro-moleculaires, Departement des Biotechnologies, 25-28 rue du Docteur Roux, F-75724 Paris cedex 15, France
The Ran GTPase switch plays a key role in nucleocytoplasmic transport and, as shown more recently, in the formation of microtubule asters and the nuclear envelope. Despite the apparent functional diversity of this GTPase only a limited number of Ran-targets is known mostly implicated in nucleocytoplasmic transport. We applied a 2-hybrid approach in order to identify novel Ran-binding proteins in the yeast Saccharomyces cerevisiae. As baits we used both wildtype Ran, Gsp1, as well as two mutant forms, Gsp1(G21V) and Gsp1(T26N), which are locked in the GTP- or GDP-bound form, respectively. We will present the results of these screens by which we identified known Ran-binding proteins, such as Ran-binding protein 1 (Yrb1), NTF2, Mog1 and various members of the beta-karyopherin family, as well as a number of uncharacterized ORFs which are candidates for novel Ran- binding proteins. We will present the characterization of one of these ORFs, termed Yrb30, which was identified as the only prey besides Yrb1 in the Gsp1(G21V) screen. Consistent with its way of isolation, we could demonstrate that Yrb30 binds, like Yrb1, specifically to the GTP-bound form of Gsp1. This was shown both by in vitro pull-down experiments using recombinant proteins as well as by copurification of the proteins from yeast. Interestingly, Yrb30 localizes, like Yrb1, to the cytoplasm. In contrast to Yrb1, however, which acts as a coactivator of RanGAP, Yrb30 inhibits RanGAP-mediated GTP-hydrolysis by Ran. We are currently trying to understand the cellular function of this novel RanGTP- binding protein.
12 Molecular mechanism of resistance to terbinafine: cloning of a fragment that turns Aspergillus nidulans resistant to this antifungal agent. Graminha, M.A.S.; Rocha, E.M.F. and Martinez-Rossi, N.M.. Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brazil.
The allylamine antimicotic agent terbinafine is employed both orally and topically for the treatment of fungal infections of the skin, nails and hair. Its mechanism of action is related to a specific inhibition of squalene epoxidase, an enzymatic step essential in the synthesis of ergosterol, resulting in deficiency and accumulation of intracellular squalene that leads to cell death. Little is known about the molecular mechanisms involved in the resistance to this antifungal agent. Thus, in order to understand the mechanisms involved in resistance to terbinafine we studied resistant mutants isolated in our laboratory by UV light irradiation. Genetic analysis revealed that a co-dominant gene located on chromosome IV is responsible for resistance. In an attempt to clone this gene we constructed a mutant genomic library and isolated a clone carrying a 6.5 kbp fragment that complements a sensitive GR5 strain by sib selection. After subcloning, a 3.5 kbp fragment still had the ability to make GR5 resistant. The clone was sequenced and Blast analysis revealed the presence of an entire ORF (accession number AF316427) homologous to the Pseudomonas putida salicylate 1- monooxygenase gene and part of an ORF homologous to the Candida albicans fatty acid synthase beta subunit. We are currently attempting to define which of these two genes is involved in terbinafine resistance. Financial support: FAPESP, CNPq and CAPES
13 The peroxisome biogenesis gene PEX6 ofColletotrichum lagenarium is required for appressorium-mediated plant infection. Akiko Kimura, Yoshitaka Takano, Iwao Furusawa, and Tetsuro Okuno. Kyoto University, Graduate School of Agriculture, Kyoto, Japan.
Restriction enzyme-mediated integration (REMI) mutagenesis of Colletotrichum lagenarium, the causal agent of cucumber anthracnose, led to the identification of a pathogenicity gene ClaPEX6. The ClaPEX6 gene encodes a protein showing high homology to Pex6 proteins involved in peroxisome biogenesis. clapex6 mutants, generated by gene replacement, lacked pathogenicity to the host plant whereas they showed normal growth and conidiation on nutrient-rich medium. Conidia of clapex6 mutants germinated and formed appressoria which were smaller than those of the wild type and which showed severe reduction of melanization. The appressoria of the mutants failed to form penetration hyphae into the host plant.The clapex6 mutants caused lesion formation when inoculated through wound sites. These results indicate that the pathogenicity defect of clapex6 mutants is due to formation of appressoria defective in penetration. To investigate whether clapex6 mutants show impaired peroxisome biogenesis, the green fluorescent protein (GFP) containing a peroxisome targeting signal type 1 (PTS1), designated GFP-PTS1, was expressed in both wild type and clapex6 mutant. In the clapex6 mutant, the GFP-PTS1 proteins showed no specific localization whereas they localized in peroxisomes in the wild type. clapex6 mutants failed to grow on oleic acid as the sole carbon source, which indicates a defect in fatty acid beta-oxidation in peroxisomes. Our results show that metabolic functions of peroxisomes are necessary for appressorium-mediated infection by C. lagenarium.
14 Branching of Achlya ambisexualis hyphae results in concomitant alterations in the levels of heat-shock protein chaperones. Julie C. Silver, Lianna Kyriakopoulou, Chai Chen and Shelley A. Brunt. Department of Medical Genetics and Microbiology and Division of Life Sciences, University of Toronto at Scarborough, Toronto, Ontario, Canada M1C 1A4.
Heat shock protein chaperones, assist in protein folding, oligomerization and translocation in the cytoplasm, nucleus, mitochondria and endoplasmic reticulum in all eukaryotic cells. In addition, the chaperones Hsp90 and Hsp70 along with specific other chaperones and co chaperones, are essential for maintaining high level ligand binding ability by steroid receptors and for the delivery of these receptors to the nucleus. Basal levels of the Achlya heat shock protein chaperones Hsp90, Hsp70-1, cytoplasmic Hsp70, mitochondrial Hsp70 (Ssc1), endoplasmic reticulum Hsp70 (Bip/Kar2/Grp78) and mitochondrial Hsp60 (GroEL), are present in tip-growing vegetative hyphae of A.ambisexualis. However, with the exception of one of the two tunicamycin and calcium ionophore- inducible Hsp70 proteins (Hsp70-1) which decreased, the levels of each of the above chaperones and of their respective mRNAs, increased significantly when the normally smooth and unbranched hyphae, develop numerous lateral branches as a result of treatment with the Achlya steroid hormone antheridiol. Very similar changes in the chaperone transcript levels tested were observed when branching was induced by casein hydrolysate, even though the casein-induced branches have a different morphology and function than antheridiol-induced branches. Nuclear run-on assays demonstrated that the transcription of genes encoding at least three of the above chaperones, was increased in antheridiol- treated cells. Supported by grants to J.C.S from NSERC Canada.
15 Characterization of the cAMP signalling pathway in Aspergillus nidulans and its role in conidiospore germination. S. Fillinger, M.-K. Chaveroche, and C. d'Enfert. Unite de Physiologie Cellulaire, Institut Pasteur/Paris, France
In the yeast Saccharomyces cerevisiae cAMP signalling is relatively well understood; its implication in such diverse cellular processes like differentiation, mating, stress response or nutrient sensing has been proven. In filamentous fungi cAMP signalling has been involved in developmental processes, therefore contributing to the virulence of pathogenic fungi. However a holistic view of cAMP signalling in filamentous fungi is still missing. We have choosen the model fungus A. nidulans to rapidly characterize the elements involved in cAMP signalling and to establish a first interaction network. We have cloned and inactivated the major elements of this signalling cascade: the adenylate cyclase gene, cya, the gene encoding the regulatory subunit of protein kinase A, pkaR, and the gene encoding another protein kinase of type A, schA. Using genetic and phenotypic analyses we have investigated the role of CyaA, SchA, PkaC (K. Shimizu and N. Keller/Texas A&M), and Ras during spore germination and colony establishment. In particular, we have monitored in single and double mutants the kinetics of trehalose breakdown which is a landmark of early stages of spore germination and the kinetics of germtube formation. Our results show that 1/ PkaC and SchA act synergistically to control trehalose breakdown and spore germination; 2/ cAMP synthesis is required for these two processes as well as proper colony development but that PkaC activity is also regulated by other signals; 3/ the small GTP-binding protein Ras appears to be involved in the control of spore germination independently of cAMP synthesis. A first model for cAMP signalling in A. nidulans will be presented.
16 The Aspergillus nidulans septin AspB localizes to areas of new growth pre- and post- mitotically. Patrick J. Westfall and Michelle Momany. University of Georgia, Department of Botany, Athens, GA.
The septin family of proteins acts as an organizational scaffolding in areas of cell division and new growth. In the filamentous fungus, Aspergillus nidulans, the septin encoding gene, aspB, produces protein that localizes to areas of cellular division and new growth both pre and post-mitotically. AspB localizes at the septum post-mitotically with an underlying polarity evident as cytokinesis progresses. This localization at the septum is dependent on actin, and occurs before the crosswall starts to form. AspB also localizes to areas of new growth including secondary germ tubes and branches. AspB localizes pre- mitotically as a ring at sites of secondary germ tube emergence and branching and is the only known branch site marker. Localization to the secondary germ tube and new branch points is transient, and disappears as the hypha extends. In addition, AspB is found at several stages during the development of the asexual reproductive structure, the conidiophore. It localizes transiently to the vesicle/metulae and metulae/phialide interface, and persistently to the phialide/conidiospore interface.
17 Cloning of the Aspergillus nidulans lipA gene, where mutation confers resistance to undecanoic acid. Ana G. Brito1, Ana L. Fachin2, Monica S. Ferreira-Nozawa2, Nilce M. Martinez-Rossi2 and Antonio Rossi1. 1Departamento de Bioquímica e Imunologia, 2Departamento de Genética. Faculdade de Medicina de Ribeirao Preto, Universidade de São Paulo, Brazil
Undecanoic acid may affect respiration, pigment formation or fatty acid synthesis in some fatty acid-sensitive fungi. Trichophyton rubrum, a fungus causing superficial mycoses in skin, hair and nails, depends on the secretion of some enzymes (lipases, keratinases, etc.) to parasitize its host, since these enzymes have not been observed in non-pathogenic strains (udar). Furthermore, these strains, in contrast to the pathogenic ones (udas), are resistant to undecanoic acid. We selected mutants resistant to undecanoic acid from both A. nidulans and T. rubrum, and two resistant A. nidulans mutants (designated lipA1 and lipA2) were mapped on chromosome VIII and showed a dominant character. A genomic library of lipA1 has been constructed in plasmid pUC18 (BamHI/BAP) and used to transform sensitive A. nidulans strains. The clone containing the gene for resistance to undecanoic acid was recovered from E. coli transformants and sequenced (accession number AF315651) and its aligment with sequences of GenBank revealed homology to triacylglicerol lipases, indicating that this enzyme could be a determinant of fungal pathogenicity. Financial Support: FAPESP, CAPES, CNPq and FAEPA.
18 The role of Aspergillus nidulans scaA gene on S and G2 mitotic checkpoints induced by DNA damage. Renata Castiglioni Pascon, Marcia Regina von Zeska Kress Fagundes, Marcelo A. Vallim, Maria Helena S. Goldman1 and Gustavo H. Goldman. Faculdade de Ciencias Farmaceuticas de Ribeirao Preto and 1Faculdade de Filosofia, Ciencias e Letras de Ribeirao Preto, Universidade de Sao Paulo, Av. do Cafe S/N, 14040-903 Ribeirao Preto, SP, Brazil
The DNA damage response in A. nidulans is mediated through cdc2 tyrosine 15 phosphorylation. Several other genes have been shown to play a role in DNA damage induced checkpoints, as uvsB (ATM kinase homologue) and uvsD (Rad 26 homologue). Mutation in these genes cause premature mitosis in the presence of damaged and unrepaired DNA. The Aspergillus nidulans scaA gene was initially isolated as a genetic determinant for camptothecin resistance, an anti-tumor drug known to stabilize the DNA/Topoisomerase I complex. The cloning and sequencing of this gene showed that the protein has moderate similarity with the human nibrin gene. This protein is the target of the ATM Kinase and forms a protein complex with Rad50 and Mre11 in higher eukaryotes. In humans, when this gene is mutated, it causes the Nijmegen Breakage Syndrome. Here, we show that A. nidulans scaA gene is necessary not only for camptothecin resistance,but also to respond to a large range of DNA damage agents, such as 4-nitroquinoline oxide, UV-light, gamma-rays, berberine, the DNA cross-linking agent mitomycin C, and the DNA replication blocking agent, hydroxyurea. The scaA mutant strain does not undergo mitotic delay in the presence of DNA damage as the wild type. Actually, it acts similarly to a cdc2AF strain (FRY 20), suggesting that this protein has a role in G2/M checkpoint control. Surprisingly, a S-phase delay that is caused by 6 mM of hydroxyurea activates a S/G2 checkpoint, impairing development of germ tubes and aberrant nuclear morphology and decreased viability in the disrupted scaA strain. This suggests that scaA operates a very important function in S-phase, however is not part of a checkpoint in this stage of the cell cycle. Financial Support: FAPESP and CNPq, Brazil.
19 Genetic analysis of cytokinesis mutants in Ustilago maydis. Leonora Leveleki and Michael Bölker. University of Marburg, Dept. of Biology, Karl-von-Frisch-Strasse, D-35032 Marburg
The main interest of our group is the study of morphological and
cytokinesis mutants in the dimorphic basidiomycete Ustilago maydis. The cells of
the don mutants (don1 and don3), which have been investigated in
our laboratory, are not able to form a secondary septum, which is required for cell separation.
Thus, these cells stay together and form tree-like structures.
Don1 and Don3 play a role in a GTPase cycle. Don1 acts as a guanine nucleotide exchange factor specific for GTPases of the Rho/Rac family and activates Cdc42. Active Cdc42 is supposed to activate the Don3 protein, a Ste20-like protein kinase, which presumably effects a protein kinase cascade. We are currently investigating the role of this signalling cascade for cell separation.
To identify additional genes, that play a role in the same pathway upstream or downstream of don1 and don3, we performed a genetic screen to isolate additional mutants with the same, tree-like phenotype. By UV irradiation, 51 don-like mutants could be isolated. Complementation analysis of these mutants demonstrated that 40 of them carry the don1 mutation, none of them the don3 mutation and 11 of them carry mutations in novel genes.
We have complemented some of these mutants by transformation with a U. maydis cosmid library. Restriction mapping and subcloning of the cosmids were carried out to analyze the newly identified genes in more detail.
The cytokinesis defect in the mutant cells was further characterized by localizing the septin homolog Cdc10 as a GFP fusion protein. In addition, the mutants were analyzed by immunostaining and confocal laser scanning microscopy (CLSM) for the structures of the septum and the microtubule cytoskeleton, and the localization of endosomal vesicles which are supposed to be involved in cell separation.
20 The snxA suppressor of the Aspergillus nidulans nimX2 mutation affects S-phase checkpoint control. Sarah Lea McGuire, Ann Long, Allison McElvaine. Milsaps College Biology Department, Jackson MS
We have recently isolated extragenic suppressors of the Aspergillus nidulans nimX2cdc2 mutation, designated snxA-snxD, for suppressors of nimX (see McGuire et al., Genetics 156, December 2000). The snxA1 mutation is allele-specific, causes cold-sensititivity and leads to aberrant nuclei when conidia are germinated at 20C. Reciprocal shift experiments suggest that snxA1 leads to arrest of nuclear division during G1 or early S at the restrictive temperature. The snxA1 mutation is pleiotropic in that in addition to causing nuclear division defects at 20C, the mutation allows cells to divide in the absence of a normally functional NIMXCDC2 (e.g., it was isolated as a suppressor of nimX2). In order to better understand the interactions of SNXA with NIMXCDC2, we are analyzing the effects of two drugs which affect checkpoint control, hydroxyurea (HU) and methylmethanesulfonate (MMS), on snxA1/nimX2 double mutants at 42C (the restrictive temperature for nimX2). In the presence of 10mM HU at 42C, 58% of snxA1/nimX2 cells enter a lethal premature mitosis, compared to 0% of either single mutant or wild type cells. In addition, in the presence of 0.02% MMS at 42C, 62% of snxA1/nimX2 cells enter premature lethal mitosis, compared to 0% or 3% of wild type cells or single mutants, respectively. We are in the process of analyzing NIMXCDC2 activity and tyrosine-15 phosphorylation of NIMXCDC2 in single and double mutants under various conditions to determine the effects of the snxA1 mutation on these. These data suggest that snxA1 affects S-phase checkpoint control of NIMXCDC2. Supported by NIH grant R15GM55885.
21 Circadian rhythms in Neurospora crassa: A new way to study rhythms in frq-less strains. Tabitha Granshaw and Stuart Brody. Biology Dept. UCSD, La Jolla, CA.
The frq gene is an important component of the Neurospora clock. A strain carrying frq10, a deletion of this gene, expressed a conidiation rhythm (banding) only after 5 to 7 days of growth. Although it had a period of 24 to 27 hours at 20C, the banding was not very robust or synchronous, making it difficult to study the frq-less rhythm. To circumvent these problems, a set of culture conditions was developed to produce synchronous, robust and reproducible banding in Petri dishes. A key aspect of the improved culture conditions is the addition of trans-farnesol or geraniol, both of which are related to intermediates in the steroid pathway. The addition of trans-farnesol, at a final concentration of 5x10-5 M, produced a period between 23 and 26 hours, whereas geraniol, at a final concentration of 40x10-5 M, lowered the period to between 17 and 21 hours. The growth rate was reduced 30% by trans-farnesol and 20% by geraniol. Other properties of the circadian rhythm missing in frq10, i.e. light sensitivity and temperature compensation, were not restored when frq10 was grown on geraniol or trans-farnesol. Neither chemical altered the period (21 hours) of the control strain, bdcsp, but the growth rate was inhibited 60% by trans- farnesol and 40% by geraniol. The existence of some part of the circadian system remaining in frq10 has lead to the idea of a two-oscillator system, composed of the frq/wc oscillator and the frq-less oscillator. The first part of the system is presumably disabled by the loss of the frq gene. Since frq10 bands robustly and synchronously on both trans-farnesol and geraniol, the use of these culture conditions could potentially allow the study of the "frq-less" oscillator.
22 Rho GTPases in Aspergillus nidulans. Gretel M. Guest and Michelle Momany. Department of Botany, University of Georgia Athens, GA
Rho GTPases are enzymes that link extracellular growth signals or intracellular stimuli to the assembly and organization of the actin cytoskeleton. They function as binary switches that cycle between the active GTP-bound form and inactive GDP-bound form. Rho GTPases have been studied in a variety of systems including Saccharomyces, Fucus, Dictyostelium, and human neutrophils. In S. cerevisiae the Rho GTPases establish and maintain polarity by affecting downstream targets which influence actin organization. Little is know about genes controlling polar growth of filamentous fungi. Actin undergoes constant reorganization especially at the tip of a growing hypha. The signals triggering this are unknown, but given the Rho GTPases function in other organisms, it seems likely that they may play a part in the actin reorganization process in filamentous fungi. In an effort to identify the possible role of Rho GTPases in A. nidulans, a 400 bp region of RHO 1 and RHO 3 from S. cerevisiae was used to identify the putative homologs from an A. nidulans cosmid library. rho1 and rho3 have been identified as being on chromosomes 5 and 6, respectively. Currently sequencing, targeted gene replacement, and cytological studies are underway to understand the function of these genes.
23 A domain in Tom40 required for assembly and stability of the TOM complex in Neurospora crassa. Rebecca D. Taylor and Frank E. Nargang. University of Alberta, Biological Sciences, Edmonton, Alberta, Canada.
Mitochondria are nearly ubiquitous in eukaryotes and are the site of many important biological functions. Most proteins required by mitochondria are encoded by nuclear genes and are synthesized on free cytosolic ribosomes before being imported into mitochondria. The TOM complex (translocase of the outer membrane) is responsible for initial recognition and translocation of mitochondria targeted precursor proteins across the outer mitochondrial membrane. Recognition of preproteins is mediated by the receptors of the TOM complex, Tom20, Tom22 and Tom70. Tom40, the major component of the TOM complex, is thought to form a pore through which preproteins traverse the outer membrane. Alignment of Tom40 from various species reveals a conserved domain in the amino terminus of the protein that is thought to be found in the intermembrane space of mitochondria. We have made specific mutations in this region via site directed in vitro mutagenesis. Our results suggest that this conserved amino terminal domain is required for assembly of Tom40 into the TOM complex, or required to maintain the stability of Tom40 within the complex, or both.
Acknowledgements: This work was supported by studentships from the Alberta Heritage Foundation for Medical Research and the Natural Sciences and Engineering Research Council of Canada to RDT, and a grant from the Medical Research Council of Canada to FEN.
24 Fungal cell biology profiling technology for high throughput functional genomics analysis in plant pathogenic fungi. Sanjoy K. Mahanty, Matthew Tanzer, and Jeffrey R. Shuster. Department of Microbial Phenomics , Paradigm Genetics Inc., 108 Alexander Drive, RTP, NC 27709
At Paradigm Genetics Incorporated, the Microbial Research Group is obtaining information about the interplay between a microorganism and its internal and external enviornment through gene function analysis (using TAG-KOTM technology) for antifungal / fungicide discovery. Towards this end, we have developed a sophisticated cell biology profiling (CBP) platform. Our initial investigation is initiated by generating signatures through cell biology profiling technology in the budding yeast Saccharomyces cerevisiae. Cell Biology Profiling through microscopy plays a key role in defining specific targets affecting cell cytoskeleton, cell physiology and cell cycle. Microscopic images of various cell biological structures (actin, microtubule cytoskeleton structure, vacuolar, nuclear, mitochondrial and cell wall structure), data visualization, and quantitation provide the best understanding of the cellular processes and have greater impact on target discovery. CBP is the only technology that provides information on multiple interacting or independent components within or between living cells. The information enables more qualified new chemical entities to come out of the early drug discovery pipeline. Here, the microscopic images of various cell biological structures of plant pathogenic fungi, Magnaporthe grisea, Mycosphearella graminicola and Botrytis cinerea will be presented. Results with cell biology profiling (CBP) in discovery of novel and known cell biological targets, as well as the power of cell biology profiling in defining gene modulation studies with filamentous fungi will be presented.
25 Stress-induced calcium responses in Aspergillus nidulans. Diana C. Bartelt, Vilma Greene, and Adrienne Dolberry. Dept. of Biol. Sci., St. John's University, Jamaica, NY11439.
We have examined the response of the filamentous fungus Aspergillus nidulans to conditions of environmental stress using strains expressing apoaequorin in the cytoplasm and mitochondria to monitor intracellular [Ca2+] in vivo. Exposure to acute hypertonic, cold, and oxidative stresses elicit transient increases in cytoplasmic [Ca2+]. Hyperosmotic conditions brought about by the addition of osmotica such sorbitol, glycerol, glucose, KCl and NaCl at a concentration of 0.2 M cause a rapid transient in cytoplasmic [Ca2+] but have little effect on the mitochondrial Ca2+ pool. The response requires the presence of Ca2+ in the external medium and is unchanged with increasing osmotic strength. Exposure to hypotonic conditons has no effect on cytoplasmic [Ca2+]. Cold shock causes a prolonged elevation of cytoplasmic [Ca2+] which is not dependent upon the presence of Ca2+ in the external medium but is dependent upon prior exposure to calcium. All of the above responses can be inhibited by the presence of 5F-BAPTA in the external medium suggesting that, at least in part, the source of Ca2+ is extracellular. Oxidative stress induced by exposure to 10mM H2O2 causes an increase in cytoplasmic [Ca2+] without prior exposure of cells to calcium and an even greater increase in mitochondrial [Ca2+], neither of which is affected by the presence of 5F-BAPTA. Resting levels of [Ca2+] have been determined to be below the level of detection, (50 nM). Exposure to Ca2+ causes a dose-dependent transient increase in cytoplasmic [Ca2+]. The duration, but not the onset of this response is temperature-dependent. Supported by N.I.G.M.S. R15GM52630 and an I.M.S.D. grant to St. John's University.
26 Isolation of mutants of Aspergillus nidulans showing hypersensitivity to calcofluor white. Terry W. Hill1, Darlene M. Loprete1, Midu Bagrodia1, Amit Mirchandani1, Jennifer A. Livesay1, and Michelle Momany2. 1Department of Biology, Rhodes College, Memphis, TN. 2Department of Botany, University of Georgia, Athens, GA.
The hyphal wall is the cell structure most responsible for mediating the varied interactions between the fungal cell and its environment. It is a dynamic organelle, assembled in situ from exported precursors and subject to developmentally regulated modulation during such processes as branching and sporulation. The architectural relationships between the numerous polysaccharides and glycoproteins of the wall are little known, as are the steps by which the complex fabric of the wall is assembled and modified. In an effort to isolate mutants deficient in any of a wide range of cell wall assembly steps, we have identified 52 strains from an Aspergillus nidulans mutant collection, which show hypersensitivity to the chitin synthase inhibitor Calcofluor White (Blankophor BBH) at concentrations having little effect on wildtype strains. The phenotype appears as reduced hyphal growth rate, blocked or delayed conidium germination, or abnormalities of hyphal form. The screening strategy is based upon the premise that mutants with already-weakened walls will be less able to endure an additional disturbance from a sublethal concentration of the inhibitor. To date, ten single-gene mutations have been identified, and one (designated calA) has been shown to be closely linked to the argB locus (chromosome III). Assignment of the remaining nine mutations to linkage groups is underway, and all are being assigned to complementation groups.
27 A PAK-like protein kinase is required for maturation of young hyphae and septation in the filamentous ascomycete Ashbya gossypii. Yasmina Ayad-Durieux, Philipp Knechtle, Fred Dietrich and Peter Philippsen. Biozentrum, University of Basel, Applied Microbiology, Basel, Switzerland
Filamentous fungi grow by hyphal extension, which is an extreme example of polarized growth. In contrast to yeast species, where polarized growth of the tip of an emerging bud is temporally limited, filamentous fungi exhibit constitutive polarized growth of the hyphal tip. In many fungi including Ashbya gossypiipolarized growth is reinforced by a process called hyphal maturation. Hyphal maturation refers to the developmental switch from slow- growing hyphae of young mycelium to fast-growing hyphae of mature mycelium. This process is essential for efficient expansion of mycelium. We report for the first time on the identification and characterization of a fungal gene important for hyphal maturation. This novel A. gossypiigene encodes a presumptive PAK-like (p21-activated kinase) kinase. Its closest homolog is the S.cerevisiaeCla4 protein kinase, the A. gossypiiprotein is therefore called AgCla4p. Agcla4deletion strains are no longer able to perform the developmental switch from young to mature hyphae, and GFP (green fluorescent protein)-tagged AgCla4p localizes with much higher frequency in mature hyphal tips than in young hyphal tips. Both results support the importance of AgCla4p in hyphal maturation. AgCla4p is also required for septation, indicated by the inability of Agcla4deletion strains to properly form actin rings and chitin rings. Despite the requirement of AgCla4p for the development of fast-growing hyphae, AgCla4p is not necessary for actin polarization per se, because tips enriched in cortical patches and hyphae with a fully developped network of actin cables can be seen in Agcla4deletion strains. The possibility that AgCla4p may be involved in regulatory mechanisms that control the dynamics of the actin patches and/or actin cables is discussed.
28 Molecular analysis of the Aspergillus nidulans morphogenesis locus, hypA. Susan Kaminskyj1, Xianzong Shi1, John Hamer2. 1Biology Dept. U Saskatchewan, SK Canada, and 1Paradigm Genetics, Research Triangle Park NC, USA.
Aspergillus nidulans hypA1 strains are wildtype at 28 C but at 42 C grow slowly with wide hyphae, frequent branches and septa, and delayed sporulation. hypA was cloned by complementation, encoding a predicted 1418 amino acid peptide with a molecular weight of 155.9 kDa and a pI of 5.77. Motif searching suggests that the hypA product is cytoplasmic and may be regulated by phosphorylation via cAMP- and/or Ca-dependent kinases. A. nidulans hypA has closely related sequences in Neurospora crassa and Schizosaccharomyces pombe whose function is unknown, and Saccharomyces cerevisiae TRS120, which is a putative regulatory subunit in the TRAPP complex that mediates ER to cis-Golgi transport. The N. crassa and S. pombe genes repaired the hypA1 phenotype by homeologous recombination but did so in different ways; repair of hypA1 by TRS120 was unsuccessful. Repair by the S. pombe sequence created a new hypA allele. We deduced six conserved blocks amongst these four sequences and showed that hypA1 is due to a point mutation causing a non-conservative amino acid change in block A. Unlike TRS120, which is essential in S. cerevisiae, hypA knockout strains grow slowly, but only on 1M sucrose agar at 28 C, and produce sparse but viable spores after three weeks. Shifting hypA knockout strains to 42 C was lethal. hypA has roles in hyphal growth and morphogenesis that may be mediated through regulated secretion.
29 Differential expression of two novel Aspergillus fumigatus putative drug efflux genes in mutant strains with high level resistance to itraconazole. Adriana M. Nascimento1, Steven Park, S.A.E. Marras, R. Kashiwazaki, Gustavo H. Goldman1, and David. S. Perlin. Public Health Research Institute, USA and 1FCFRP - Universidade de Sao Paulo, Brazil
Aspergillus fumigatus is an opportunistic pathogen that can cause life-threatening invasive disease in severely immunocompromised patients. For such patients, the treatment options are limited to therapy with amphotericin B and/or itraconazole. As a preliminary step to investigate the underlying resistance mechanisms in this clinically important pathogen, A. fumigatus mutants resistant to itraconazole (RIT) were selected in vitro following UV irradiation. A total of fifty-five strains were isolated that grew in presence of up to 100 mg/ml of itraconazole. To evaluate whether ATP Binding Cassete (ABC) multiple drug resistance (MDR) pumps contributed to the observed drug resistance phenotype of the RIT mutants, PCR amplification using degenerate primers to a conserved region within the ABC superfamily was used to identify genes encoding potential MDR efflux pumps. The PCR fragments obtained were cloned and sequenced, and six of these clones corresponded to potential MDR type genes. Two of these novel A. fumigatus putative drug efflux genes, designated mdr3 and mdr4, showed differential, drug-induced, gene expression and were evaluated in more detail. Real-time assays with molecular beacon probes were used to quantitatively measure mRNA levels in the 23 mutant strains grown in the presence and absence of itraconazole (10 or 100 ug/ml). Two RIT strains, RIT11 and RIT13, were identified that showed up to 200-fold increased levels of mdr3 and mdr4 mRNA after treatment with itraconazole. Our results suggest that these novel drug efflux genes may contribute mechanistically to the itraconazole resistance phenotypes displayed for the A. fumigatus RIT mutant strains, and may have clinical significance.
Financial support: CAPES, PHRI and FAPESP.
30 Molecular responses in Aspergillus nidulans to treatment with Streptomyces-produced antibiotics. Petter Melin1, Johan Schnurer1 & Gerhart Wagner2. 1Department of Microbiology, Swedish University of Agricultural Sciences 2Institute of Cell and Molecular Biology, Uppsala University
In the search for fungal-antagonistic bacteria we previously identified a strain of Streptomyces halstedii that produces the macrolide-antibiotics bafilomycin B1 and C1. Bafilomycins are known to inhibit vacuolar ATPases. Filamentous fungi exposed to these antibiotics characteristically display reduced growth rate and increased branching frequency. We previously developed an experimental model to study the molecular response in fungi exposed to bafilomycin B1. By using mRNA differential display, we have identified five genes in Aspergillus nidulans whose expression changes upon bafilomycin treatment. We are now proceeding to test for the biological function of the detected genes, starting with binB, whose expression had been shown to be up- regulated approximately 30 times. We have also initiated a proteome analysis to monitor expression changes at the protein level. We expect that the complementary results from RNA (differential display) and proteome analysis will be useful to identify more putative targets of the antibiotics, and also will provide an estimate of the sensitivity of the two screening methods. In the proteomics study we used the antibiotic concanamycin A, which induces phenotypic changes in the fungus that are virtually identical to those observed with Bafilomycin. Among the 130 most prominent proteins spots analyzed by 2D gel electrophoresis, 23 proteins show a change in abundance of at least two-fold. The identity of two proteins with decreased abundance was identified as CPCB (involved in regulation of sexual development) and GPD (a central protein in glycolysis), respectively. We cannot, at this point, provide a biological explanation for the reduced abundance of these protein as a function of antibiotics treatment. The remaining proteins are still under investigation.
31 The MAP kinase cascade and cAMP signaling modulate pH-induced yeast to mycelium change in Ustilago maydis. Alfredo D. Martinez-Espinoza 1,2, Jos Ruiz-Herrera2, Claudia G. Leon-Ramirez and Scott E. Gold1. 1Plant Pathology Dept. University of Georgia, Athens GA, 30602-7274, USA. 2Departamento de Ingenieria Genetica, Centro de Investigacion y de Estudios Avanzados del Instituto Politecnico Nacional. Unidade Irapuato, Apartado Postal 629, 36500 Irapuato, Gto. Mexico.
Ustilago maydisis a worldwide smut pathogen of maize. In its saprophytic phase, the fungus is haploid and grows as a budding yeast. A pathogenic dikaryotic mycelium is produced after mating of compatible sporidia. Alternatively to mating, acidic pH of the media can induce the dimorphic yeast- mycelium switch of this fungus. A mutational analysis of two important signal transduction pathways showed how the pH mycelial induction might be operating. Mycelial formation in vitrowas inhibited by the addition of exogenous cAMP in all growth conditions. cAMP levels drop dramatically and were maintained at low levels when the fungus was grown at pH 3.0. Mutants affected in the regulatory subunit of PKA still showed a mycelial phenotype at low pH, indicating an alternative effector of the cAMP signal. With respect to the MAP kinase pathway, it was observed that mutations in any of the members: MAPK, MAPKK, MAPKKK or a putative adaptor protein, lost their capacity to form mycelium in vitro. Interestingly, a mutation on the transcriptional regulator prf1, was still capable of forming mycelium under acidic conditions. These results suggest that the normal condition of growth of U. maydisis budding, which is maintained at least in part, by a mechanism of repression exerted through PKA signaling. When levels of cAMP descend, the fungus exhibits a filamentous morphology using a mechanism that requires the MAPK pathway. This pathway can be stimulated under natural conditions in the dikaryon by a b-dependent mechanism or in vitro under low pH conditions.
32 Expression of green fluorescent protein in Neurospora crassa. Michael Freitag, Lynda Ciuffetti* and Eric U. Selker. Institute of Molecular Biology, University of Oregon, Eugene, and *Department of Botany and Plant Pathology, Oregon State University, Corvallis
Since the first report on heterologous expression of GFP in Escherichia coli and Caenorhabditis elegans, GFP has become an amazingly versatile and useful protein marker in many experimental systems. GFP and GFP fusion proteins have been successfully expressed under the control of both endogenous and heterologous promoters in some fungi, but not previously in N. crassa. After transformation of N. crassa with pCT74, a plasmid that carries a modified form of the GFP gene under the control of the Pyrenophora tritici-repentis ToxA promoter and the E. coli hph gene as a selectable marker, we detected high GFP levels in the cytoplasm and nuclei of both hyphae and macroconidia. GFP appeared excluded from vacuoles and mitochondria. Transformants exhibited variable GFP expression levels and patterns, presumably because of differences in their copy number and site of integration. We therefore targeted a single copy of GFP to the his-3 locus. The resulting transformants showed uniform and high-level expression of GFP. To assess the sexual stability of these transformants and expression of GFP in sexual tissues, we crossed single copy GFP transformants with wildtype strains; GFP was easily detected in developing ascospores, proving GFP to be a useful ascospore color marker. One explanation for previous difficulties in creating successfully expressing Neurospora-GFP fusion proteins may be the relative strength of the available promoters. We are presently testing this and are performing a promoter trap screen to identify useful Neurospora promoters. Low levels of GFP expression may also be explained by gene silencing, for example by DNA methylation. This possibility is also being examined.
33 Characterization and cloning of the Neurospora crassa DNA repair gene, upr-1. Wataru Sakai, Chizu Ishii and Hirokazu Inoue. Saitama University, Urawa, Japan
Two mutagen-sensitive mutants, upr-1 and mus-26, share the same phenotype; (1) sensitive to UV light and 4-NQO and only slightly sensitive to MMS and gamma rays, (2) low reversion frequency and (3) partial defective in photoreactivation. Genetic analysis showed that they belong to the same repair pathway. However, there is no other mutant that shows epistasis to upr-1 and mus-26. Therefore the function of these products is unknown. The upr-1 gene locates at the left arm of linkage group I, near mating type ( mt). In order to isolate the upr-1 gene, we did chromosome walking from the mt locus to the right direction and contiguous clones covered about 300-400 kb genomic region. Some clones complemented the temperature- sensitive mutant un-16, which is mapped between the mt locus and the upr-1 locus. Based on the obtained un-16 gene sequence, the MIPS Neurospora crassa database was screened for contiguous sequence. We found that the homolog of budding yeast REV3 is next to the un-16 gene. Phenotype of the yeast rev3 mutant is similar to that of the upr-1 mutant. We are now testing whether the upr-1 gene is identical to the REV3 homolog, and characterizing RIP mutants of the N. crassa REV3 homolog.
34 Characterization of mutations in the two-component histidine kinase gene of dicarboximide-resistant field strains of Botrytis cinerea. Michiyo Oshima1, Makoto Fujimura1, Noriyuki Ochiai1, Akihiko Ichiishi1, Takayuki Motoyama2, and Isamu Yamaguchi2. 1University of Toyo, Dept of Life Science, Itakura, Gunma, Japan. 2Microbial Toxicology Lab., RIKEN Institute, Wako, Saitama, Japan
The mutations in putative osomosensor histidine kinase gene (os-1) in Neurospora crassa confer not only osmotic sensitivity but also resistance both to dicarboximides and phenylpyrroles. In a plant pathogenic Botrytis cinerea, the dicarboximide-resistant strains isolated from practical fields are still sensitive to phenylpyrroles without osmotic sensitive phenotype, although most dicarboximide-resistant mutants isolated under laboratory condition exhibit both osmotic sensitivity and resistance to phenylpyrroles. To investigate the possibility that mutations of histidine kinase gene (BsOS1) confer dicarboximide-resistance in field strains of B. cinerea, we cloned BsOS1 genes from both dicarboximide-sensitive and resistant field strains by PCR amplification and compared their sequences. The overall domain organization was conserved between the os-1p and BcOS1p including the N-terminal domain that contains tandem repeats of 90 amino acids. All dicarboximide-resistant field strains of B. cinerea were found to contain a single base pair mutation in their osmosensor histidine kinase gene that resulted in an amino acid substitution in second unit of 90 amino-acids tandem repeats of BcOS1p. In four resistant isolates, coden-76 of second amino-acid repeat, which encodes isoleucine in sensitive strains, was converted to a coden for serine. The coden-70 of second repeat for leucine was converted to a coden for proline in one resistant strain. These results strongly suggests that the amino acid substitutions within second repeat of BcOS1p are responsible for phenotypes of field resistant isolates (resistant to dicarboximides, sensitive to phenylpyrroles, and insensitive to osmotic stress) in B. cinerea.
35 Characterization of he kex2 endopeptidase genes from Aspergillus oryzae and Aspergillus nidulans. Y.Yamgata1, M. Yamamoto1, A. Nojima1, Y. Katsuno1, K. Abe1, T. Nakajima1, T. Akao2, O. Akita2, M. Sano3, M. Machida3. 1Tohoku University Sendai, Japan. 2National Resarch Institute of Brewing, Saijo, Japan. 3National Institute of Bioscience and Human-Technology, Tskuba, Japan
The details of fungal protein expression system have been not clear, and it has been thought as same as the yeast secretion system. As the first step to clarify the protein expression system of Aspergillus, we cloned and sequenced genes for processing protease of Aspergillus nidulans and A. oryzae. As a result of BLAST search against EST database of A. nidulans, we found 600-bp sequence homologous to yeast kex2 gene, and the sequence was amplified with PCR. Complementary DNA sequence was obtained by screening a lambda-ZAP cDNA library of A. nidulans A69 strain with the amplified fragment. Obtained gene contained ORF of 2460 bp, and it encoded 819 amino acid residues. It was 40% identical with yeast kex2 protease. Then as we searched for homologous sequences with the A. nidulans kex2 homologue in A. oryzae EST database, the sequence of 70 % identical with kex2 homologue was found. The sequence contained ORF of 2511 bp, and it encoded 836 amino acid residues. These two enzymes and A. niger kex2 protein were 70 % identical with each other, and 40 % identical with yeast kex2 protease. The amino acid residues of catalytic triad with serine protease, the amino acid sequences of transmenbrane domain and p-domain were well conserved. The gene from A. oryzae was introduced into E. coli with pET expression vector, the hydrolytic activities against synthetic peptides were observed in E. coli lysate. The facts above indicated that these two genes encoded kex2 maturating protease of A. nidulans and A. oryzae.
36 Molecular cloning and characterization of a novel two-component signaling histidine kinase gene tcsB (NHK1) of Aspergillus nidulans. Yasuaki Katsuno, Kentaro Furukawa, Keietsu Abe*, and Tasuku Nakajima. Department of Molecular and Cell Biology, Graduate School of Agricultural Science, Tohoku University, Sendai, JAPAN
We cloned and characterized a novel Aspergillus nidulans histidine kinase gene, tcsB (NHK1) encoding a two-component signaling protein homologous to the yeast osmosensor synthetic lethal of N-end rule 1 (SLN1) which transmits signals through the high-osmolarity glycerol response 1 (HOG1) mitogen- activated knase (MAPK) cascade in yeast responding to environmental osmotic stimuli. In the A. nidulans EST database (http://www.genome.ou.edu/ fungal.html), we found three EST sequences homologous to histidine kinase conserved regions, H-boxes, N-boxes, and regulator domains respectively observed in yeast SLN1 or Neurospora crassa NIK-1. Thus, DNA fragments containing either of the three EST sequences were amplified by PCR and used for screening of an A. nidulans cDNA library as probes. From the screening, we isolated a positive clone containing a 3,210 bp long open reading frame that gave a putative protein consisted of 1,070 amino acids, and designated the gene tcsB . The predicted tcsB protein has two putative transmembrane regions in the N-terminal half and has structural similarity to the yeast Sln1p. Overexpression of the tcsB cDNA suppressed the lethality of the temperature-sensitive osmosensing-defective yeast mutant sln1-ts. By contrast, tcsB cDNAs in which conserved histidine or aspartate residues had been substituted (H552Q, D989N) failed to complement the sln1-ts mutation, indicating that the tcsB protein functions as a histidine kinase.
37 Nuclear control of mtDNA maintenance: identification and characterization of two orphan genes in Podospora anserina. M. Dequard-Chablat, V. Contamine and M. Picard. Institut de Genetique et Microbiologie, CNRS-UMR 8621 Universit Paris-Sud, Bat 400 91405-ORSAY cedex FRANCE
In P. anserina, a degenerative process (premature death) is linked to the accumulation of mtDNA molecules (delta1) carrying a large and specific deletion. It occurs only in the presence of some mutations of the AS1 gene encoding a cytosolic ribosomal protein. Our working hypothesis is that these mutations alter translation, notably for mRNAs encoding proteins required for the maintenance/distribution of the mitochondrial genome. Search for mutations which either delay or suppress the accumulation of delta1 should help to identify the relevant genes. Unexpectidly, the first two genes identified by this procedure are probably not the targets of the AS1 effect: mutations in these genes might indirectly counteract this effect through modifications in the process for protein import into mitochondria. In contrast, the SMP1 gene is a good candidate for an AS1 target. It was identified as a gene which, in two copies, delays premature death. This is an orphan gene which probably encodes a HMG protein located into mitochondria. The rmp1 gene is also an orphan gene. It was discovered as a gene exhibiting natural allelic forms which belong to two classes: class 1 alleles lead to the premature death syndrome per se while class 2 alleles delay accumulation of delta1. Sequencing of six class 1 and two class 2 alleles has shown that the second forms encode a truncated protein. Experiments are in progress to understand the role(s) of these two genes in mtDNA maintenance and distribution.
38 Peroxisomal ABC transporters, inter-organelle cross-talk and development in Podospora anserina. Stéphanie Boisnard, Gwenaël Ruprich-Robert, Véronique Berteaux-Lecellier, Denise Zickler and Marguerite Picard. Institut de Génétique et Microbiologie, UMR 8621, Universit Paris-Sud, Bat. 400, 91405 Orsay Cedex, France
Although recent advances have greatly expanded our knowledge of peroxisome biogenesis, the connection between peroxisomal deficiencies and developmental defects remains largely unknown. We have previously shown that pex2 mutants of the filamentous fungus Podospora anserina, impaired in peroxisome assembly, are also defective in sexual differentiation: dikaryotic mutant cells remain in a proliferative stage and are unable to differentiate into meiocyte and enter meiosis. To confirm the involvement of peroxisomes in sexual differentiation, we took advantage of the unexpected link found in mammals between PEX2 (general loss of peroxisomal function; Zellweger syndrome) and peroxysomal ABC transporters: overexpression of either ALDP (Adrenoleukodystrophy) or PMP70 (whose role(s) remain unclear) suppresses defects in peroxisomal assembly in cells that are deficient for PEX2. We show that the human PMP70 protein can restore sexual differentiation and peroxisome biogenesis in a Podospora peroxisome-deficient pex2 mutant. These observations confirm that the developmental defect observed in the mutant is a consequence of a peroxisomal dysfunctionment and rule out a role of PEX2 per se. Moreover, we show that expression of the human PMP70 cDNA in a Podospora wild-type strain causes developmental defects. To precise the peroxisomal requirements during sexual development, we also performed a search for extragenic suppressors of pex2 mutants. This led to the characterization of six complementation groups among which the mitochondrial citrate synthase gene (cit1) has been identified. cit1 mutations act as partial suppressors of pex2 mutants (via a process which probably involves an inter-organelle cross-talk) and disclose a meiotic checkpoint at the diffuse stage.
39 Apical growth and mitosis do not compete for the same microtubule resources in Aspergillus nidulans. M. Riquelme1, R. Fischer2 and S. Bartnicki-Garcia1. 1Department of Plant Pathology. University of California, Riverside. Riverside, CA 92521. 2 Max- Planck-Institut f r Terrestrische Mikrobiologie. Marburg, Germany.
We conducted a simultaneous analysis of nuclear division and apical growth in living fungal hyphae by fluorescence and phase-contrast microscopy, respectively, to determine if the key parameters of apical growth (elongation rate and Spitzenkörper behavior) are affected during mitosis. We used Aspergillus nidulans strain SRS27, in which nuclei are stained with GFP (Suelmann et al., 1997). During mitosis, nuclei became invisible as the GFP was released from the nuclei and the entire hypha became diffusely fluorescent. Interphase nuclei and their disappearance during mitosis could also be observed by phase-contrast microscopy but with considerable difficulty. Contrary to earlier predictions, our analysis showed that there is no disruption of apical growth during mitosis. There was no decrease in the rate of hyphal elongation or any alteration in Spitzenkörper behavior before, during or after mitosis. The distribution of cytoplasmic microtubules (MTs) in young hyphae of A. nidulans was examined by immunofluorescence to get a better understanding of the status of the microtubular cytoskeleton during mitosis. In addition to the typical mitotic spindles, cytoplasmic MTs were clearly evident in cells arrested at metaphase. Our findings disprove the common belief that apical growth and mitosis compete for the same pool of MTs. Presumably, the population of cytoplasmic MTs involved in apical growth operates independently of that involved in mitosis.
40 Comparative morphometric analysis of lateral and apical branching in hyphae of Neurospora crassa. M. Riquelme1, G. Gierz2, and S. Bartnicki-Garcia1. Departments of 1Plant Pathology and 2 Mathematics. University of California, Riverside. Riverside, CA 92521.
We have used high-resolution video-enhanced light microscopy (VELM) to measure hyphal growth kinetics, with high accuracy, and to examine, in fine detail, the intracellular events that lead to lateral and apical branching in hyphae of Neurospora crassa. We found remarkable differences in the events preceding lateral vs. apical branching. Lateral branches emerged without affecting the growth of the main hypha. Lateral branch formation did not interfere with either the elongation rate of the primary hypha or the behavior of its Spitzenk÷÷rper (Spk). During emergence of a lateral branch, a new Spk was formed (at 15-30 m behind the apex) without affecting the behavior of the primary Spk. In sharp contrast, apical branching was preceded by a series of intracellular events at the parental hyphal apex. The sequence involved: 1) Cytoplasmic contraction, followed by 2) retraction/dislocation, and disappearance of the Spk. During this period, hyphal elongation decreased sharply accompanied by a transient phase of non-polar isotropic growth. About 2 min after the Spk retraction, active growth resumed with the formation of two or more apical branches, each one with a Spk formed de novo by gradual condensation of phase-dark material (vesicles) around an invisible nucleation site.
41 Regulation of cytokinesis in Ustilago maydis. Helge Hudel, Britta Abel, Marisa Piscator, Michael Bölker. University of Marburg, Department of Biology, Marburg, Germany
The phytopathogenic fungus Ustilago maydis exhibits a dimorphic life style. Haploid sporidia grow yeast-like by budding and are non-pathogenic. By UV mutagenesis we have identified two genes that are involved in the regulation of morphogenesis and dimorphism. don1 and don3 mutant cells fail to separate after nuclear division and septum formation. The don1 gene encodes a guanine exchange factor (GEF) specific for members of the Rho/Rac/Cdc42 family, the don3 gene codes for a member of the STE20 like kinases that are known to be activated by Rho/Rac proteins. The Ras-like GTPase Cdc42 is supposed to be involved in this regulatory cascade. By using an in vitro kinase assay we could demonstrate that Cdc42 is able to activate the Don3 protein kinase. To characterize the role of Cdc42 in more detail we are currently testing specific mutants of Cdc42 for their effect on cytokinesis and cell separation. We propose that Don1 and Don3 control via Cdc42 the growth of a secondary septum which is required to form a fragmentation zone.
42 Pheromone discrimination of chimeric Balpha receptors in Schizophyllum commune. Susanne Gola, and Erika Kothe. Dept. Microbiology, Friedrich-Schiller-University, Jena, Germany.
The Balpha mating type locus of the basidiomycete Schizophyllum commune contains a pheromone receptor system which is capable of ligand discrimination to confer mating specificity to a haploid strain. Each pheromone receptor induces B-dependent development upon activation by pheromones of eight non-self specificities, while self-pheromones are not bound. In order to define specificity domains of receptors, chimeric receptors were constructed using the receptor genes bar1 of Balpha1 specificity and bar2 of Balpha2 specificity. Chimeras were transformed into a Bnull strain lacking endogenous pheromone and receptor genes. Specificities of the chimeric receptors were analysed by mating to tester strains of all nine Balpha specificities. Matings revealed four phenotypes of the chimeras: Two of the receptors carried Balpha1 specificity. One showed an altered, highly discriminative exclusion profile. The remaining three receptors were promiscuous, one of them was also constitutive. The group of promiscuous receptors showed activation by pheromones of all nine Balpha specificities, i.e. no discrimination between self and non-self mates. To distinguish activation profiles of chimeric receptors single pheromone genes were expressed under control of the strong tef1 promotor in the Bnull recipient strain and then mated to transformants expressing chimeric receptors. Distinct pheromone-receptor pairs detected differential responses of the promiscuous receptors to specific pheromones. Thus, the receptors are not accepting any pheromone as expected for truly promiscuous phenotypes. They still show differential activation profiles, albeit with a new assortment of pheromones. In addition, the profiles of activation vs. rejection differed between the promiscuous receptors analyzed.
43 A linear plasmid from Blumeria graminis f. sp. hordei. Henriette Giese1, Bjarne Stummann2 and Solveig Christiansen3. 1Royal Agricultural and Veterinary Universitya, Ecology, Copenhagen, Denmark. 2Vet. Agri. University , Ecology, Copenhagen Denmark. 3Risoe National Laboratory, Plant Biology, Roskilde, Denmark.
A 8.0 kb linear plasmid from the obligate biotrophic fungus Blumeria graminis f.sp. hordei (synonym Erysiphe graminis) (Bgh), causing the disease powdery mildew on barley, was characterised by sequence analysis. The plasmid contains two identical terminal inverted repeats (TIR) of 610 bp. Two open reading frames on opposite strands and with start codons 570 bp from the plasmid ends were identified, one encoding a DNA polymerase with 1030 aa and the other an RNA polymerase with 961 aa. These genes are transcribed throughout fungal development. A putative 11 bp ARS consensus sequence ATTTATATTTA was identified within the TIR elements. Sexual transmission of the plasmid was studied in a cross between two Bgh isolates characterised by presence and absence of the plasmid and with different mitochondrial genotypes. The plasmid was transferred to 75% of the 87 progeny isolates and 66% of the isolates had the mitochondrial genotype of the isolate without the plasmid. The mitochondrial genotype originally harbouring the plasmid may have a dependence on the plasmid as only 4% of the isolates had this genotype without the plasmid. No transfer of the plasmid was observed between two Bgh isolates co-cultivated for over a year on a common host variety.
44 Isolation and characterization of sexual sporulation mutants of Aspergillus nidulans. K. Swart, D. van Heemst, M. Slakhorst, F. Debets and C. Heyting. Laboratory of Genetics, Department of Plant Sciences, Wageningen University, 6703 HA Wageningen, The Netherlands.
For the genetic dissection of sexual sporulation in Aspergillus nidulans, we started a collection of ascosporeless mutants. After mutagenization of conidiospores with high doses of UV, we isolated 20 mutants with defects in ascospore formation. We crossed these mutants in two successive rounds with the wildtype strain. Eighteen of the 20 isolated mutants produced progeny with the original mutant phenotype in these crosses, and these mutants were further analyzed. All 18 analyzed mutations were recessive to wildtype. We assigned them to 15 complementation groups, based on crosses between mutants. The mutants could be classified as follows according to their cytological phenotype: (1) no croziers; (2) arrest at pre-karyogamy; (3) arrest in early meiotic prophase; (4) arrest in late meiotic prophase; (5) arrest in meiotic metaphase I; (6) defective post meitotic mitosis and/or deliniation of ascospores; and (7) slow progression through the post meiotic stages of ascospore formation. A large proportion of the mutants, namely 11 out of 18, arrested in meiotic prophase or metaphase I. We discuss a possible approach for isolating the wildtype aleles of the genes that carry the sexual sporulation mutations.
45 Fungal cell death. Charlotte Thrane and Stefan Olsson. Ecology, Royal and Veterinary University Coppenhagen, Denmark
In plants, controlled cell death with similarities to animal apoptosis is thought to occur and biochemical signs like caspase 3-like activities and PARP- cleavage have been observed during patogen infection or after toxin treatment. In fungi, it is still speculative but parts of the protein machinery (Bcl-2 and Bax) were identified by chemical treatment of Mucor racemosus. Some form of controlled cell death has also been suggested in cord formation, heterokaryon incompatibility in Neurospora crassa, and in the development of primordia in Agaricus bisporus. We have studied fungal cell death in two different systems; toxin (or antimicrobial) induced and developmental. Our data indicate that proteins central in animal apoptosis are present in fungi. However, there are several large differences in the basic cellular organisation between fungi and animal cells that might signify differences in a programmed cell death pathway. The fungal cell wall is one such difference which is significant in toxin or antimicrobial induced stress: The fungal cell wall can function to protect the fungus from extracellular stress by sealing of the boundary to the environment, and the wall is, further, a site for accumulation of antimicrobial compunds. This suggests that the signs of fungal cell death as a response to stress differ from wall-less animals and more resemble stress responses in plants. On the other hand, developmental cell death in fungi and animals might be more similar.
46 Stress induced responses in Fusarium culmorum. Henriette Giese, Stefan Olsson, Jakob Skov, Carsten Tobiasen, Morten Grell, and Bjarne Stummann. Ecology, Royal and Veterinary University Coppenhagen, Denmark
Fusarium culmorum is the most frequent Fusarium species in Danish soils and is believed to be the major cause for Fusarium head blight of barley in Denmark. Fusarium species are also used as production organisms by the biotechnological industry and it is important to gain insight into the processes that lead to mycotoxin production. The aim of our research is to characterize the processes leading to toxin production in Fusarium culmorum when the fungus is subjected to different stress conditions. Defined culture systems for F. culmorum have been developed. Stress applications such as nutrient and oxygen starvation, heat, pH variation and chemical compounds are tested. The entire mycelium is stressed in a liquid culture system to get a definite starting point to facilitate molecular analyses of fungal stress reactions as a series of events triggered by a specific external factor. The fungal stress responses are characterised by microscopy to monitor cytological changes that can be used to verify reproducible stress treatments. 2-D electrophoresis of proteins extracted from fungal cultures subjected to different types of stress is carried out to detect the resulting change and develop a databank of protein patterns derived from fungal cultures of different age and stress treatments. A long term perspective is to analyse individual protein spots by mass spectrometry to identify the proteins that are characteristic for specific culture conditions. A transformation system for the fungus is under development and initially a vector containing a constitutive promoter in front of a Ca2+ responsive reporter gene will be used. This will permit direct assessment of stress induction in Fusarium culmorum. Promoters from genes that are induced by specific treatments will at later stages be used in transformation experiments to study gene expression in stressed fungal cultures.
47 Characterization of nop-1 expression during development in Neurospora crassa. Jennifer A. Bieszke, Donald O. Natvig, and Katherine A. Borkovich. University of New Mexico, and the University of Texas-Houston Medical School.
Opsins are seven-transmembrane helical apoproteins that form light absorbing pigments upon binding retinal. Genes encoding opsins were found exclusively in animals and the archaea until the discovery of nop-1 in the filamentous fungus Neurospora crassa. Previously, we have shown that heterologously expressed NOP-1 in P. pastoris membranes could bind all-trans retinal (lambdamax = 534nm), and undergo a photochemical reaction cycle similar to archaeal rhodopsins. Also, we found that nop-1 is expressed to high levels under conditions that favor conidiation. Further analysis of nop-1 mRNA levels during conidial development demonstrates that the nop-1 transcript first appears early in conidiation (4 hrs) and persists throughout conidiophore development and conidial maturation (16 hrs). Since several genes specific to conidiation are dependent on blue-light, nop- 1 expression was evaluated in the wc-1 and wc-2 strains. Preliminary evidence suggests that nop-1 expression is independent of blue-light control. Finally, a NOP-1 antibody has been generated and the expression and localization of NOP-1 throughout N. crassa development is currently being investigated.
48 Cloning of the Neurospora uvs-3 gene. Schroeder, Alice L1,Kazama, Yusuke2, Ishii, Chizu 2 and Inoue, Hirokazu 2. 1School of Molecular BioSciences, Washington State Univ.,USA, 2 Lab. of Genetics, Saitama Univ., JAPAN
A genomic DNA fragment which complemented the methyl methanesulfonate (MMS) sensitivity of the DNA repair mutant, uvs-3, was cloned. The 4.5 Kb XhoI-BglII fragment of the cosmid X:18E9 contains two long ORFs divided by a putative intron. The deduced protein is 883 a.a. long with no detectable termination codon, and shows about 35 % similarity and 25% identity to the Aspergillus nidulans uvsD gene product. The uvsD mutant of Aspergillus is thought to be involved in checkpoint control and shares many characteristics with the uvs-3 mutant, including a broad mutagen specificity and retention of photoreactivation ability for at least 4 hours after UV irradiation. The introduction of uvsD cDNA partially restored MMS and UV resistance to the uvs-3 mutant. This suggests that uvs-3 plays a role in checkpoint control in Neurospora. Putative null mutants created by ripping shared a similar phenotype with the original uvs-3 mutant. Full resistance to MMS was restored in the ripped mutant by introduction of the whole cosmid fragment, while the original mutant gained only partial resistance with the cosmid. This result indicates that original mutation in the uvs-3 mutant may be a semi- dominant. Northern blot analysis of the uvs-3 gene indicated that the uvs-3 gene was constitutively expressed at a low level. The expression was reduced after UV irradiation but recovered within 60 min. By 90 to 120 min after irradiation it had increased to a level several times higher than the constitutive level. This work was supported in part by a NSF US - Japan Cooperative Research Grant.
49 Protein kinase a control in glucose and nitrogen sensing in Saccharomyces cerevisiae. Inge Holsbeeks, Monica C.V. Donaton, Ole Lagatie, Marion Crauwels, Joris Winderickx, Johan M. Thevelin. Laboratory of Molecular Cell Biology, K.U. Leuven, Kardinaal Mercierlaan 92, B-3001 Leuven, Flanders, Belgium.
Protein kinase A (PKA) and the PKA-related pathways play a major role in nutrient induced signal transduction in Saccharomyces cerevisiae. These PKA-pathways are important for control of a variety of growth related metabolic properties such as regulation of trehalose and glycogen content, expression of STRE-controlled genes and ribosomal gene expression (1). Addition of glucose to cells deprived for glucose triggers a cAMP-increase which is dependent on the Gpr1-Gpa2 G-protein coupled receptor system. This cAMP-signal triggers a PKA-mediated protein phosphorylation cascade that affects the different targets of the cAMP-PKA-pathway. When cells starved for an essential nutrient in presence of glucose are replenished with the essential nutrient, a similar PKA-mediated protein phosphorylation cascade occurs, but without cAMP-increase. Because of requirement of glucose and complete growth medium this pathway is called "fermentable growth medium induced" (FGM) pathway (2). This pathway has been studied in nitrogen starved cells on a glucose medium to which a nitrogen source was added again. Activation of the FGM-pathway is largely dependent on the general amino acid permease Gap1. In a gap1 strain some amino acids can still be transported into the cell but they can't activate the PKA-targets. Furthermore some C-terminal truncations of Gap1, which have no effect on the transport capacity of Gap1, resulted in reduced trehalase activation after addition of nitrogen. On the other hand these truncated alleles of Gap1 showed a constitutive activation of all other PKA-targets measured irrespective of the presence of the nitrogen source. These results provide strong evidence that Gap1 acts as a sensor for amino acid activation of the FGM-pathway.
1. Thevelein, J.M. et al. (2000). Nutrient-induced signal transduction through the protein kinase A pathway and its role in the control of metabolism, stress resistance, and growth in yeast. Enzyme and microbial technology, 26: 819-825. 2. Thevelein, J.M. (1994). Signal transduction in yeast. Yeast, 10: 1753-1790.
50 het-c vegetative incompatibility causes well-timed mycelial growth arrest and hyphal compartmentation and death in Neurospora crassa. Qijun Xiang, Magdalen Barton, Gopal Iyer, and N. Louise Glass. University of California, Plant and Microbial Biology, Berkeley, CA,
In filamentous fungi, the formation of stable heterokaryons between different strains is genetically controlled by het loci. The het-c locus is one of eleven het loci in Neurospora crassa. Vegetative incompatibility mediated by differences at het-c has at least three phenotypic aspects: mycelial growth arrest, suppression of conidiation, and hyphal compartmentation and death (HCD). Hyphal compartmentation death rates are relatively stable, not cumulative over time, and are not related to hyphal fusion events in an established colony. By contrast, mycelial growth arrest is well timed and is not related to an increase in hyphal compartmentation and death. Using a temperature sensitive het-c mutant, we determined that HCD could be triggered, in mycelial heterokaryons, after 2 hours incubation at permissive temperatures. Conidia and ascospores, however, are not competent to undergo HCD until 12 to 14 hours after germination. The temperature sensitive het-c allele has been cloned and sequenced and is currently being analyzed. Transcriptional analyses of het-c revealed a temporal expression in a compatible reaction. Competitive RNA analyses in a reation to Beta-tubulin revealed a peak accumulation of het-c transcript at 16 h. These data suggest that vegetative incompatibility mediated by het-c is a well-timed and programmed process and is associated with the transition from germination to mycelial growth.
51 No evidence for endocytosis in Neurospora by electron microscopy. Torralba S., Heath I.B.. York University, Biology, Ontario, CANADA
During tip growth of filamentous fungi, hyphal extension is confined to the hyphal apex and involves highly polarized exocytosis. It is well documented that secretory vesicles deliver membrane, cell wall precursors and wall- building enzymes to the hyphal tip. However, the presence or absence of endocytosis in these organisms is not yet clear. Although endocytosis is well known in animal cells and there is evidence for an endocytotic pathway in yeast and plant cells, reports of the existence of endocytosis in filamentous fungi have been conflicting. Uptake of fluorescent markers such as FM4-64 by living hyphae of various species, including Neurospora, has been taken as positive evidence for endocytosis. However, other endocytosis markers were not internalized by hyphae of Pisolithus and neither clathrin-coated vesicles nor any other definitive ultrastructural indicator of endocytosis have been found in fungal hyphae. In this work, we have investigated endocytosis in Neurospora by electron microscopy, using lanthanum as an opaque endocytosis marker. Neurospora hyphae grew at normal rate in the presence of lanthanum. Lanthanum was able to penetrate the cell wall and treated hyphae presented electron dense deposits on the plasma membrane. Some deposits were associated with vesicle-like structures, but such structures were shown to form part of invaginations of the plasma membrane, when followed by serial sections. We failed to observe any internalization of lanthanum into the Neurospora hypha and no deposits were present in the cytoplasm or associated with any organelles, questioning the occurrence of endocytosis, as opposed to molecular internalization of FM4- 64, in this fungus.
52 Towards an understanding of the mechanisms which generate variability in Colletotrichum lindemuthianum. Raul Rodríguez-Guerra, Maria-Teresa Ramírez-Rueda, June Simpson. CINVESTAV, Unidad Irapuato, Apdo. Postal 629, Irapuato, Gto. Mexico.
Characterization of pathotypes and use of molecular markers has indicated a high level of variability between isolates of Colletotrichum lindemuthianum. This variability could be due to spontaneous mutation or sexual or parasexual recombination. To investigate these posibilities, 19 C. lindemuthianum isolates from different regions of Mexico were analyzed. Initially all isolates were confronted with themselves and with all other isolates in order to determine their capacity to produce perithecia and/or undergo anastomosis. A small number of isolates produced perithecia when confronted with other isolates however these structures were found to be infertile. A larger number produced spherical structures assumed to be protoperithecia. All isolates anastomosed with themselves but differed in their capacity to anastomose with other isolates. A group of 6 isolates with different capacities for anastomosis was chosen to determine whether new genotypes due to mitotic recombination are formed following confrontation of two distinct isolates. Following confrontation, around 40 spores were isolated individually and subjected to AFLP anaylsis. In confrontations where isolates were capable of anastamosis, new genetic fingerprints different to those of the original isolates were observed. In combinations of isolates unable to anastomose, genotypes corresponded mainly to one of the original isolates. Analysis of 40 individual spores from individual isolates showed identical genotypes, strongly suggesting that the variability observed in genotype patterns is due to mitotic recombination and not to spontaneous mutation. We are grateful to CONACyT (K0195B) and SIHGO
53 Apoptosis occurs in the Basidia of Mutants of Coprinus cinereus. Benjamin C. Lu1, Natasha Gallo1, and U. Kues2. 1University of Guelph, Guelph, Canada. 2ETH Zurich, Switzerland.
Apoptosis occurs in the basidia of Coprinus cinereus when mutations occur that cause defects in (1) meiotic progression, or (2)basidiospore formatino. Mutants that are defective in meiotic progression, such as the assembly of synaptonemal complex, homologous chromosome pairing, or DNA repairs, trigger apoptosis as soon as the meiotic nuclei attempt to enter metaphase I. These mutants will produce some spores varying from extremely low number of tetrad to almost gray cap; the number varies from fruiting body to fruiting body even when they are on the same culture dish. Mutants that are defective in spore formation trigger apoptosis in the tetrad stage after completion of meiosis. Apoptosis follows a set pattern regardless of the type of mutations. It starts with chromatin condensation and DNA fragmentation, followed by plasmolysis, cytoplasmic fragmentation and cell shrinkage of basidia, first in a few cells then gradually spread to nearly all basidia. The end stage is represented by extreme shrinkage and total degradation of DNA of meiotic nuclei. Apoptosis is cell type specific; it occurs only in the basidia and not the surrounding somatic tissues. Apoptosis can be inhibited when meiosis progression is arrested by a checkpoint control that prevents entry to the division program.
54 Characterization and phylogenetic analysis of the septin gene family from Aspergillus nidulans and other fungi. Michelle Momany, Jiong Zhao, Rebecca Lindsey, Patrick J. Westfall. Department of Botany, University of Georgia, Athens, Georgia 30602
Members of the septin gene family are involved in cytokinesis and the organization of new growth in organisms as diverse as yeast, fruit fly, worm, mouse, and man. We have cloned and sequenced five septins from the model filamentous fungus A. nidulans. As expected the A. nidulans septins contain the highly conserved GTP binding and coiled-coil domains seen in other septins. Hybridization of clones to a chromosome specific library and correlation with an A. nidulans physical map showed that the septins are not clustered, but are scattered throughout the genome. Phylogenic analysis showed that most fungal septins could be grouped with one of the prototypical S. cerevisiae septins, Cdc3, Cdc10, Cdc11, and Cdc12. Intron-exon structure was conserved within septin classes. Examination of asp message levels during asexual spore formation showed that A. nidulans septin gene family members are differentially expressed. Our results suggest that most fungal septins belong to one of four orthologous classes.
55 A novel putative transmembrane protein is involved in vegetative incompatibility and hyphal fusion in Neurospora crassa. Qijun Xiang and Louise Glass. Department of Plant & Microbial Biology, Berkeley, CA 94720-3102, USA
Vegetative incompatibility triggered by allelic differences at the het-c locus in Neurospora crassa is characterized by growth inhibition and arrest, suppression of conidiation and hyphal compartmentation and death. To genetically dissect the pathway of het-c vegetative incompatibility, we allowed incompatible transformants to escape from growth inhibition and suppression of conidiation. Three mutants were identified that contained a recessive suppressor of vegetative incompatibility, all of which were allelic to the vib-1 locus, which is located between lys-2 and ilv-2 on chromosome V. A common phenotypic hallmark of Vib mutants is that all of them can block het- c vegetative incompatibility. Another common characteristic is that all conidiate (nearly) constitutively. vib-1(2) and vib-1(3) mutants have no other obvious defects, while vib-1(1) mutants have additional morphological and developmental defects. The vib-1(1) mutant shows aerial hyphae defects and dramatically reduces hyphal fusion events. Crosses between the vib-1(1) mutant and wild-type strain show dominant ascospore lethal affects, which may be caused by meiotic transvection. vib-1 was cloned by complementation of conidiation defect in vib-1(1) mutant. It encodes a novel 1085 aa putative protein. Primary analyses indicate that VIB-1 has three putative transmembrane domains. It does not display a high level of identity with proteins of known function in other organisms, except a ~150 aa region in the C-terminal that is similar to a number of hyphothetical proteins in eukaryotic organisms. This conserved region is required for VIB-1 function.
56 Co-localization of alternative HET-C alleles in hyphae undergoing vegetative incompatibility by confocal microscopy. Sovan Sarkar and N. Louise Glass. Plant and Microbial Biology Department, University of California, Berkeley CA 94720-3102
In N. crassa, vegetative incompatibility is mediated by genetic differences at het loci. The het-c locus has been previously characterized and initial data indicated that non-self recognition is mediated by the formation of a HET-C heterocomplex. Formation of the HET-C heterocomplex leads to hyphal compartmentation and death (HCD). We demonstrated the co- localization of alternative HET-C proteins in hyphae undergoing vegetative incompatibility by using fluorophore conjugated affinity purified antibodies (cy3 and cy5) to tagged het-c alleles, het-c OR ::GFP and het-c PA::HA using confocal microscopy. Co-localization was observed in the plasma membrane suggesting that formation of the HET-C heterocomplex triggers HCD. Permeabilization studies of cells with 0.1% triton destroyed co-localization indicating that the C-terminus of HET-C is located on the outside of the cell. We have been unable to detect HET-C by itself or in homocomplex. The reason for this is unclear but it maybe due to the instability of HET-C in the absence of heterocomplex formation. HET-C is predicted to have a signal peptide. However, a het-c construct missing the signal peptide triggers vegetative incompatibility, providing one HET-C protein retains a signal peptide. Co-transformation of alternative het-c alleles, both of which lack the signal peptide, resulted in only compatible transformants. Studies to examine the localization of HET-C in signal peptide deleted strains are currently under investigation.
57 Rapamycin and Tor in the pathogenic fungi Cryptococcus neoformans and Candida albicans. Jill Blankenship, M. Cristina Cruz, Maria E. Cardenas and Joseph Heitman. Duke University Genetics Dept. Durham, NC
Cryptococcus neoformans and Candida albicans are opportunistic human pathogens that effect primarily immunocompromised patients. Several drug treatments have been developed to combat the serious fungal infections caused by these pathogens, but due to a significant rate of drug resistance and toxicity, novel drug treatments need to be established. The macrolide drug rapamycin is a good candidate for such therapy because it has potent antifungal effects, and non-immunosuppressive rapamycin analogs that retain antifungal activity have been identified. In complex with the protein FKBP12, rapamycin binds to and inhibits the TOR proteins which are conserved from yeast to mammals. Unlike S. cerevisiae and S. pombe, C. albicans and C. neoformans have only a single Tor kinase homolog. Thus, these systems provide an opportunity to dissect the functions of Tor that are important for the shared and unique roles of the two Tor homologs in budding and fission yeasts and to examine the role of Tor in pathogenicity and antifungal drug action. We have analyzed the effects of rapamycin on C. neoformans in vitro and are determining whether the TOR1 gene is essential using diploid strains we have recently identified. We have also identified a gene encoding a novel TOR-like kinase Tlk1, which shares significant sequence identity to the Tor kinase domain but lacks the conserved FKBP-rapamycin binding domain. A tlk1 mutant strain generated by homologous recombination via biolistic transformation is viable and as yet has no discernable phenotype. Finally, we have constructed a rapamycin resistant Tor1-1 mutation in Candida albicans, and are disrupting the TOR1 gene in this organism to compare and contrast the functions of the Tor kinases in pathogenic and model yeasts.
58 Tubular vacuoles in the hyphal tip region of oomycetes. William G. Allaway and Osu Lilje. School of Biological Sciences, The University of Sydney, NSW 2006, Australia
The tip region of oomycete hyphae contains a network of fine, slow-moving tubular structures which have been identified as tubular vacuoles by their ability to accumulate and retain fluorescent probes. We describe our investigations of the connections of this tubular network in the tip region with larger vacuoles further back from the tip, using fluorescence, DIC and confocal microscopy.
60 Identification of G1/S regulators which interact physically and genetically with the Aspergillus nimODbf4protein. Steve James1, Carey Connelly1, Nitin Malhotra1, Holly Massimilla1, Karen Messner2, & Peter Mirabito3 1Department of Biology, Gettysburg College, Gettysburg, PA USA. 2Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, NC USA. 3T.H. Morgan School of Biological Sciences, University of Kentucky, Lexington, KY USA.
nimODbf4 of Aspergillus nidulans encodes the regulatory subunit of a conserved eukaryotic enzyme known as Dbf4-dependent kinase (DDK). In budding yeast, Dbf4p initiates DNA synthesis by activating a catalytic subunit, Cdc7p, and escorting it to origins of replication. DDK then triggers origin firing through phosphorylation of DNA helicase subunits. We have identified three Aspergillus genes whose products associate directly with nimOp or indirectly influence its function: (1) the Cdc7Asp homolog was isolated. Overexpression of alcA::Cdc7Asp rescued nimO18 ts-lethality, but did not rescue G1/S mutations in other genes. Two-hybrid analysis revealed that N- and C-terminal fragments of nimOp interact specifically with Cdc7pAsp. Furthermore, single amino acid replacements in the C- terminal nimO zinc finger abolished the interaction, indicating that this novel motif is essential for nimOp-Cdc7p association; (2, 3) we generated partial nimO18 suppressors in two loci, snoA and snoB (suppressor- of-nimO). The suppressors were tested in a background where the only copy of nimO is under the control of the alcA promoter. Such strains are normally ethanol-dependent and glucose-inviable. However, recessive snoA suppressors appeared to bypass the requirement for nimO by rescuing growth on glucose. Semi-dominant snoB alleles did not bypass the need for nimO, suggesting that snoBp and nimOp may associate directly or act in the same complex. Efforts are underway to isolate snoA and snoB, and to explore the functions of nimOp and its suppressors by using an epitope- tagged allele of nimO. (Supported by NSF-RUI: MCB 95-07485 to SWJ)
61 Integration profiles of transforming vector in the null mutants of Rad51 homologue in filamentous fungi. Ichioka Daisuke, Toyoaki Natsume, and Yasuo Itoh Faculty of Science, Shinshu University, Matsumoto, Japan
Some species of filamentous fungi, including the model organism Aspergillus nidulans, are useful for studying mechanisms that catalyze the exogenous DNA integration during genetic transformation. Both homologous and ectopic integrations are detected with comparable frequency, in contrast to the bias for either type of integration observed in Saccharomyces cerevisiae and animals. To control these types of DNA integration by regulating the catalyzing machinery, we were interested in Rad51 of S. cerevisiae. Rad51 has been extensively studied on its involvement in homology search between DNA strands during meiosis and recombinational repair. Site specific disruptants were obtained for A. nidulans and Penicillium paxilli by gene replacement and integration profiles of transforming vector were analyzed. No homologous integration was detected with a 1.7-kb fragment that targeted for argB2 locus in the uvsC null mutant of A. nidulans. Furthermore number of transformants that had ectopic / illegitimate integration at multiple genomic sites was significantly increased by inactivating Rad51 homolog in both species when selection was done with nutrient markers. Geneticin resistance was also used for P. paxilli transformation and in this case, evident effect of Rad51 inactivation was decreased occurrence of direct repeat formation. These results were consistent with the established function of Rad51. Increased number of integration events by RAD51 disruption could be explained by the inefficiency of double-strand break repair apparatus that does not require
62 Genetic analysis of the calcineurin signal transduction pathway in Cryptococcus neoformans. Deborah S. Fox and Joseph Heitman. Departments of Genetics, Pharmacology and Cancer Biology, Microbiology, and Medicine, and the Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC
Cryptococcus neoformans is an opportunistic fungal pathogen that causes life-threatening meningoencephalitis in immunocompromised patients. Calcineurin is a Ca2+-calmodulin-activated protein phosphatase and a target of the immunosuppressive drug FK506, which inhibits calcineurin activity. In other organisms, calcineurin regulates cell polarity, mating, and cytokinesis. Previously, we have shown that disruption of the genes encoding the catalytic or regulatory subunits of calcineurin in C. neoformans abolishes growth at elevated temperature, virulence, hyphal elongation, and haploid filamentation. In this study we examined the hypothesis that calcineurin-dependent effectors are present in C. neoformans, and that their over-expression will suppress the temperature-sensitive growth defect conferred by a calcineurin mutation. To identify components of the calcineurin signaling pathway in C. neoformans, we isolated multicopy suppressors of the temperature-sensitive defect of a calcineurin-deficient strain. A multicopy genomic library was introduced into a calcineurin mutant strain. Plasmid-dependent transformants were isolated which restored growth at high temperature, revealing a novel gene, named CTS1 for calcineurin temperature suppressor. CTS1 encodes a C2 domain- containing protein, and may function as an effector of calcineurin. Over- expression of Cts1 conferred resistance to FK506 in wild-type organisms and suppressed the temperature-sensitive phenotype of calcineurin mutant strains. Disruption of the CTS1 gene by homologous recombination resulted in a cytokinesis defect, synthetic lethality with calcineurin mutations, enhanced sensitivity to FK506, and a reduction in mating and haploid filamentation. Ongoing studies aim to elucidate the functions of Cts1 in the calcineurin signal transduction pathway and may further our understanding of the roles of calcineurin in virulence and differentiation.
63 Isolation and characterization of the npgA gene involved in pigment formation in Aspergillus nidulans. Jung-Mi Kim, Dong Min Han1, Keon-Sang Chae, Suhn-Kee Chae2, and Kwang-Yeop Jahng. Division of Biological Sciences, Chonbuk National University, Chonju, South Korea; 1Department of Molecular Biology, Wonkwang University, Iksan, South Korea; 2Division of Life Science, Paichai University, Taejon, South Korea.
The npgA gene has been known that it plays an important role in constructing cell wall structure or depositing pigment in Aspergillus nidulans. To study the biological function of npgA gene, we isolated and sequenced the nucleotide of the DNA fragment that complemented npgA1 mutation from the genomic cosmid library of Aspergillus nidulans. Sequence data of the full-length npgA showed that there was an open reading frame (ORF) possibly encoding a polypeptide of 344 amino acid. The amino acid sequence showed 30-40% similarity to the amphipatic protein surfactin of Bacillus subtilis. The sequence analysis revealed that the npgA is linked to the gene aatA encoding the homolog of acyl transferase on the chromosome 1. The 269th codon (TTA) for leucine of wild type ORF was replaced by termination codon (TGA) in the npgA1 mutation. The transcript of npgA gene appeared throughout the whole life cycle. The deletion mutant of npgA neither grew well nor showed normal pigmentation throughout the whole life cycle suggesting that the full length of NpgAp might be required for the celllular growth as well as metabolite formation. However over-expression mutant of npgA showed no distinguishable phenotype in growth and pigmentation except the conidiopores formation was earlier. The protoplasts were generated more rapidly and in larger quantity from npgA deletion mutant. The viscosity of culture filtrate of npgA deletion mutant was similar to npgA1 mutant. These phenotypes of deletion mutant and the amino acid homology to the surfactin imply that the protein NpgAp might be involved in the synthesis of secondary metabolites such as pigment in A. nidulans.
64 Isolation and characterization of two genes encoding mitogen-activated protein kinase homologs, mpkB and mpkC, from Aspergillus nidulans. Sei-Jin Lee, Hyun-Joo Park, Dong Min Han1, Keon-Sang Chae, Suhn-Kee Chae2, and Kwang-Yeop Jahng. Division of Biological Sciences, Chonbuk National University, Chonju, South Korea; 1Department of Molecular Biology, Wonkwang University, Iksan, South Korea; 2Division of Life Science, Paichai University, Taejon, South Korea.
Mitogen-activated protein kinases (MAPKs) play an important role in regulation of diverse cellular functions such as growth, differentiation, osmoregulation and cell wall biosynthesis in fungi. Two genes encoding mitogen- activated protein kinase homologs, mpkB and mpkC, were isolated from Aspergillus nidulans by PCR with degenerated primers and colony hybridization. Each nucleotide sequence of mpkB and mpkC consisted of presumptive ORF containing 457 and 347 amino acids and 3 and 6 introns, respectively. The amino acid sequence of MpkBp showed 84% of identities to the gene pmk1 which is involved in differentiation and pathogenicity in Magnaporthe grisea suggesting that the gene mpkB might play some roles in development in A. nidulans. However the amino acid sequence of MpkCp showed 50% of identities to the gene HOG1, which was known to regulate the osmostress in yeast, implying that the gene mpkC might be a member of stress-activated protein kinases (SAPK). To identify and characterize the function of the gene mpkB, it was disrupted and over-expressed. The phenotype of disruptant of mpkB showed not forming of cleistothecium when induced sexual development. However over- expression mutant did not show any phenotypic changes. From these results, we suggest that the MAPK gene of A. nidulans, mpkB should be required for the formation of sexual organ, cleistothecium. In contrast, the deletion mutant and over-expression mutant of mpkC showed no significant phenotypes in the various conditions of environmental stress. From our results, we learned that MAP kinases are possibly involved in sexual development in A. nidulans.
65 Cellular function of a gene encoding G protein alpha subunit homolog in the development of Aspergillus nidulans. Mi-Hee Chang, Dong Min Han1, Keon-Sang Chae, Suhn-Kee Chae2 and Kwang-Yeop Jahng. Division of Biological Sciences, Chonbuk National University, Chonju, South Korea; 1Department of Molecular Biology, Wonkwang University, Iksan, South Korea; 2Division of Life Science, Paichai University, Taejon, South Korea.
Signal transduction via G protein coupled receptor plays important roles in various cellular responses in eukaryotic organism. Recently many fungal G proteins have been identified in various species and most of them are involved in mating, development, growth and morphogenesis of fungi. In Aspergillus nidulans, a G alpha gene, fadA has been known to have a major role in determining the balance between growth and sporulation. We have previously isolated the gene ganA that presumably encodes G protein alpha subunit homolog from A. nidulans. However its biological function is not clear yet. Here we present that another G protein alpha subunit homolog, ganB, is involved in growth and development in A. nidulans. Putative GanBp is consisted of 356 amino acids and closely related to Gs alpha subfamily. It has consensus myristoylation site at amino terminal. Transcription of ganB was likely to be dependent on the developmental stage. The transcript of ganB increased as sexual development was proceeded. To investigate cellular function of ganB we constructed various mutants of ganB by site-specific targeting. Constitutive activating mutation of ganB significantly reduced asexual sporulation and led abnormal mycelium growth.
66 Characterization of secretion related small GTPase encoding genes in Aspergillus niger. X.O. Weenink1, A.F.J. Ram1,2, B. Seiboth3, B. Luken2, P.J. Punt2 and C.A.M.J.J. van den Hondel1,2. 1)Leiden University, Inst. Mol. Plant Sciences/Centre for Phyto-techn., Wassenaarseweg 64, 2333 AL, Leiden, The Netherlands, 2) Dep. Appl. Microbiol. Gene Techn., TNO-Nutrition, 3700 AJ Zeist, The Netherlands, 3)TU Wien, Inst. for Biochem. Microbiol., Wien, Austria.
Filamentous fungi, including Aspergillus niger, have the capacity to secrete large amount of enzymes into their environment. Transport of these proteins through the secretion pathway is mediated by vesicles. The formation, budding and fusion of vesicles is regulated by highly conserved secretion related GTP-binding proteins. Based on this conservation, we have cloned seven secretion related small GTPases (srg genes) from A. niger. Transcript analysis of the different GTPases revealed unusually long 5ÆÆ and 3ÆÆ untranslated regions for all srg genes. Expression of the several srg genes was independent of different carbon sources used. Multiple transcripts were detected for srgA, srgC, srgE and sarA. The largest transcripts of sarA, srgC and srgE were abundant throughout different stages of growth, whereas their smaller transcripts were only detected during early growth. This latter behaviour was also found for srgD and srgF. In contrast, both srgA transcripts and the single srgB transcript were abundant throughout the whole cultivation period. We have started to make mutants (deletion or conditional) to systematically inactivate the various srg genes to generate a collection of secretion mutants with defined blocks in the secretion pathway. To monitor protein secretion both in wild type and various secretion mutants strains we have deleveloped in a GFP- based secretion reporter system which is used for the analysis of the secretion mutants.
67 The roles of CHS1, CHS8 and BNI4 in cell wall synthesis in C. albicans. Munro, C.A., Winters, K., Rella, M., Rowbottom, L., Bulawa, C.E. and Neil A.R. Gow. Dept of Molecular and Cell Biology, University of Aberdeen, Scotland, UK. Millenium Pharmaceuticals, 75 Sidney Street, Cambridge, MA, US.
Chitin is a critical component of fungal cell walls. Here we present recent advances in the molecular analysis of chitin synthase in the human pathogen Candida albicans. In this fungus there are 4 chitin synthases. We have shown recently that CaChs1p synthesizes the septal chitin and contributes to chitin in the lateral cell wall. A conditional deltachs1 mutant was generated by regulating the expression of CaCHS1. Under repressing conditions yeast cells no longer separated from each other after budding and grew as septum-less chains for several generations before growth ceased. Hyphae grew for several hours with a normal morphology. Eventually hyphae stopped growing, the cell wall ballooned at various positions and cells lysed. CaCHS1 is the first example of an essential chitin synthase in fungi. CaChs2p encodes the major chitin synthase activity in vitro, and deltachs2 null mutants have marginally less chitin in hyphal cells. CaChs3p synthesizes the majority of cell wall chitin and the ring of chitin at the site where a new bud emerges. Recently we identified a fourth gene encoding a chitin synthase in C. albicans we have named this CHS8 (Chs4-7p are apparently regulators of Chs3p). CaChs8p has the highest identity to CaChs2p at the amino acid level. The function of CaChs8p is currently under investigation and latest progress will be presented . Several proteins have been identified as regulators of Chs3p in S. cerevisiae. One regulator Bni4p is found at the mother-bud neck associated with septins and Chs4p, which in turn is linked to Chs3p. A homologue of BNI4 was identified in C. albicans. The deltaCabni4 null mutant has a 30% reduction in cell wall chitin compared to wild type cells. Calcofluor staining revealed the chitin ring was still synthesized at the bud neck and bud scars were evident suggesting that CaBni4p has a redundant function in targeting Chs3p to the mother-bud neck in C. albicans.
68 Quantitative Analysis of the expression of ABC-transporter genes in Aspergillus nidulans by Real-Time RT-PCR Assay. Camile P. Semighini, Marcelo A. Vallim, Mozart Marins, Renata Castiglioni Pascon, Maria Helena de Souza Goldman1, and Gustavo Henrique Goldman. Faculdade de Ciências Farmaceuticas de Ribeirão Preto and 1Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Brazil
The frequency of life-threatening fungal infections is rising world-wide. Drug treatment failures in fungal infections combined with improvements in performance and standardisation of antifungal-susceptibility testing have drawn attention to the problem of antifungal drug resistance. It is now clear that antifungals can create clinical and epidemiological situations that are analogous to those found with antibiotic-resistant bacteria. Typical gene products responsible for multiple drug resistance (mdr) are ABC (ATP-Binding-Cassette) transport proteins which are responsible for the efflux of toxic compounds. Here, we evaluate the potential of real-time RT-PCR to quantify the mRNA expression of four ABC-transporter genes from A. nidulans, named AtrA, AtrB, AtrC and AtrD. The Atr-genes were shown to be induced by several structurally different drugs, a hallmark of the mdr phenotype. These genes displayed a very complex expression pattern in different ima genetical backgrounds. The imaB mutant has a higher basal level of expression of AtrB and -D than the wild-type strain and these two genes have comparable levels of expression when the imaB mutant is grown in the presence and absence of imazalil. This dependence between the presence of imaB and expression of AtrB and -D indicates that the imaB locus regulates AtrB and -D expression, possibly acting as a a regulatory gene. The imaB mutant has a higher basal level of expression of AtrB and -D than the wild-type. These results showed a complex net of interactions among expression levels of the different ABC-transporter genes in the ima mutants background. Quantitative analysis of the mRNA expression of these genes in multidrug-resistant of clinical isolates, will allow us to access their behavior in several pathogenic fungi leading to the improvement of antifungal therapy. Financial support: FAPESP-Brazil
69 Molecular analysis of three kinesins in Aspergillus nidulans. Natalia Requena, Patricia Rischitor, Else Winzenburg, Ralf Liese and Reinhard Fischer. Max-Planck-Institut for terrestrial Microbiology, Karl-von-Frisch-Str., D-35043 Marburg, Germany.
Motor proteins are involved in a variety of different cellular processes such as mitosis, cytokinesis or organelle movement. In A. nidulans and several other fungal species dynein is required for nuclear migration. Recently, it was shown that in S. cerevisiae in addition to dynein, several kinesins are involved in nuclear positioning and nuclear migration. Since the processes of nuclear migration could be rather different in yeast cells and in filamentous fungi, we wanted to investigate the roles of different kinesins in A. nidulans. Taking advantage of the A. nidulans sequencing project at Cereon Genomics (Cambridge, USA) several partial kinesin sequences were obtained. Further sequence analysis revealed that one of them displayed strong homology to conventional kinesin identified before in several fungi. The complete sequence was obtained, the gene (kinA) deleted and the kinesin deficient strain analysed for vesicle, mitochondrial and nuclear movement using the GFP technology. We present evidence that this motor protein affects the stability of microtubules. In addition, homologues of S. cerevisiae Kip2 and Kip3, both of which are involved in nuclear migration, are studied. Deletion strains of the corresponding genes in A. nidulans were constructed as well as double and triple mutants. These strains are phenotypically analysed with respect to nuclear migration and microtubule stability.
70 The effects of ropy-1 mutation on cytoplasmic organization and intracellular motility in mature hyphae of Neurospora crassa. Robert Roberson. Arizona State University Plant Biology
To better understand the role(s) of dynein during hyphal tip growth, we have used light and electron microscopy to document the cytoplasmic effects of the ropy (ro-1) mutation in mature hyphae of Neurospora crassa. Transmission electron microscopy (TEM) showed that the apical cytoplasm of wild-type hyphae contained a typical Spitzenkörper (Spk) composed of a spherical aggregation of secretory vesicles ranging in size from 120 (macrovesicles) to 50 (microvesicles) nm diam. In most Spk, these vesicles surrounded a central core composed primarily of microvesicles embedded in a dense granular/fibrillar matrix. ro-1 hyphae contained Spk of reduced size and unclear pattern of vesicle distribution. Video-enhanced light microscopy was used to characterize intracellular behavior of multivesicular bodies (MVBs) and mitochondria in wild-type and ro-1 hyphae. In wild-type cells, MVBs traveled along paths that were generally parallel to the longitudinal axis of the cell. Anterograde and retrograde motility was observed, with most travel being anterograde. A small population of mitochondria displayed rapid anterograde and retrograde movements, while most maintained a constant position relative to either the advancing cytoplasm. In ro-1 hyphae, the motility and/or positioning of MVBs and mitochondria was significantly altered relative to the wild-type. Immunofluorescence confocal microscopy revealed that the microtubule cytoskeleton was severely disrupted in the ropy mutant. Clearly, dynein deficiency in the mutant causes profound perturbation on cytoskeleton organization and organelle dynamics. These perturbations impact negatively on the organization and stability of the Spk, which, in turn, leads to severe reduction in growth rate and altered hyphal morphology.
71 G gamma subunit homolog identified in Neurospora crassa. S. Krystofova, K.A. Borkovich. University Texas HSC Houston, Dept. Microbiology and Molecular Genetics, Texas, USA.
Heterotrimeric guanine nucleotide-binding proteins (G proteins) consisting of alfa, beta and gamma subunits mediate signalling between cell surface receptors and intracellular effectors in eukaryotic cells. Upon agonist binding to the receptor, the G protein alpha subunit releases GDP, binds GTP, and dissociates from the G protein beta gamma subunit dimer. Depending on the system, either G alfa or G beta gamma go on to activate downstream effectors. In Neurospora crassa it is a G alfa homolog (GNA-1) that positively regulates adenylyl cyclase activity. Three G alfa subunits genes, gna-1, gna-2 and gna-3, and one G beta subunit gene, gnb-1, have been identified in Neurospora crassa. We identified a putative G protein gamma subunit, gng-1, in N.crassa during homology searches of the University of Oklahoma EST database. The full length of gng-1 cDNA (273bp) has been found in the cDNA clone. Our data have shown that the introns are in positions which are conserved in the mammalian G gamma subunits. The putative protein sequence of GNG-1 shows a COOH-terminal CAAX motif of isoprenylation commonly found in G gamma subunits (CVVM). GNG-1 is closely related to Saccharomyces cerevisiae G gamma subunit, STE18 (35% identity) and non-visual mammalian G gamma subunits (30-33% identity).
72 Live cell imaging of hyphal fusions in growing Neurospora colonies Patrick C. Hickey1, David J. Jacobson2, Nick D. Read1 and N. Louise Glass2. 1 Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh, EH9 3JH, UK. 2 Department of Plant and Microbiological Science, University of California at Berkeley, California 94720, USA.
The process of hyphal fusion (anastomosis) in growing colonies of Neurospora has been visualized using confocal microscopy. A variety of fluorescent dyes were used to follow different cell components during anastomosis. These include the plasma membrane, the Spitzenkörper, mitochondria, vacuoles, and nuclei. Time-lapse movies illustrate the dynamics of these cell components in each of the discrete physiological states of the participating hyphae: pre-contact, post-contact, and post-fusion. Fusion-competent hyphae were generally morphologically distinct and often showed forms of remote sensing, resulting in branch initiation and re-direction of growth to facilitate contact. Intense membrane activity was seen where fusion-competent hyphae met (post-contact), possibly due to vesicles delivering compounds to first agglutinate cell walls at the point of contact and subsequently to soften cell walls only at the fusion pore. Three-dimensional reconstruction of confocal images clearly showed the structural changes of hyphae at the fusion pores. De novo septum formation, at a distance from the fusion site, often accompanied anastomosis. Dramatic post-fusion changes in cytoplasmic flow were frequently visible. The role of anastomoses in the dynamics of cytoplasmic flow in the mycelium is being assessed. Taken together, these observations form a mechanistic model of the events accompanying anastomosis. Imaging of mutants with known defects in hyphal fusion will be used to test this model. In addition, post-fusion events in compatible anastomoses provide the framework for future studies of programmed hyphal compartmentation and death caused by the genetically controlled vegetative incompatibility system.
73 Characterization of MKK1, a putative MAP kinase kinase in Pneumocystis carinii. Donald J. Ferguson Jr. and A. George Smulian. VA Medical Center, Cincinnati, OH.
Signal transduction pathways in various fungi have been extensively studied. However, much remains to be elucidated regarding the signal transduction pathways of the opportunistic fungal pathogen Pneumocystis carinii. Recently, the gene encoding a putative mitogen-activated protein kinase kinase (MAPKK or MEK) referred to as mkk1, was cloned and is predicted to encode the MEK in the stress response signal transduction pathway previously defined in P. carinii. Sequence alignments displayed homology between the P. carinii MKK1 and the known MEK homologs of Saccharomyces cerevisiae. Sequence homology predicts that MKK1 would complement a mkk1deletion/mkk2deletion defect in the cell integrity pathway of S. cerevisiae. The wild type MKK1-GST was unable to complement the pbs2deletion defect in the high osmolarity growth pathway or the ste7deletion defect in the pheromone response pathway of either mating type a or type alpha S. cerevisiae. Site directed mutagenesis was used in an attempt to generate constitutively active and inactive mutants of MKK1. The wild type MKK1 as well as the mutants were cloned into a modified pYX 213 yeast expression vector containing a TPI promoter and a glutathione-S- transferase (GST) tag. Western blotting using anti-GST antibodies revealed products of the appropriate size to indicate expression of the GST-fusion Mkk1 proteins. The MAP kinases MKP1 and MKP2 of P. carinii have been cloned and expressed in our laboratory and will be examined for their ability to serve as substrates for the P. carinii MKK1.
74 Isolation of sepA enhancer mutations in Aspergillus nidulans. Claire L. Pearson and Steven D. Harris. Dept. of Microbiology, University of Connecticut Health Center, Farmington, CT.
In Aspergillus nidulans, the sepA gene is required for the formation of septa and polarized morphogenesis. SEPA is a member of the formin family, which is characterized by the presence of multiple functional domains that mediate interactions with other proteins involved in organization of the actin cytoskeleton. The goal of this project is to identify proteins that interact with SEPA or, function in parallel pathways. To identify these proteins, we have initiated an enhancer screen using a strain in which the only copy of sepA is expressed under the control of the inducible alcA promoter. To date, we have identified ten mes (morphological defective enhancer of SEPA) mutants that display severe morphological defects on repressing media. One of the mes mutations (mesA1) causes pronounced defects in hyphal morphogenesis in a wildtype background. We have demonstrated that this mutation also enhances the phenotypes caused by the Ts sepA1 allele, which suggests that mesA and sepA may function in parallel genetic pathways to control tip growth in A. nidulans. The mesA1 mutant displays defects in the maintenance of hyphal polarity and in the pattern of cell wall deposition. We have mapped mesA1 to chromosome II, and have identified a genomic clone that appears to complement the morphological defects. Preliminary sequence analysis of this clone reveals homology between mesA and an uncharacterized gene in Schizosaccharomyces pombe. Further characterization of this clone, as well as the phenotypes caused by the mesA1 mutation, will be presented.
75 Characterization and localization of the Aspergillus nidulans Formin SEPA. Kathryn E. Sharpless and Steven D. Harris. Dept. of Microbiology, University of Connecticut Health Center, Farmington, CT 06030-3205
SEPA is a member of the formin family of proteins, which are thought to act as molecular scaffolds that organize signaling and structural proteins involved in actin-dependent processes. In Aspergillus nidulans, SEPA is required for actin ring formation at septation sites, and is also involved in polarized tip growth. SEPA is thought to organize actin filaments in septal rings and at hyphal tips by interacting with other proteins through multiple distinct domains, including the conserved FH1, FH2, and FH3 domains. To identify functionally important domains in SEPA, we determined the sequence of the temperature sensitive sepA1 and sepA3 alleles. The sepA1 mutation maps to a conserved residue in the FH2 domain, whereas the sepA3 mutation lies in a region of SEPA that potentially binds Rho family GTPases.
To further characterize SEPA, a functional SEPA::GFP fusion protein has been constructed and localized to both septation sites and hyphal tips. Using live imaging, we have found that SEPA forms a ring that constricts and disappears at septation sites. Simultaneously, at hyphal tips, SEPA is found as a dynamic dot or crescent-shape patch. We have also determined that SEPA co-localizes with actin at both septation sites and hyphal tips. Furthermore, we have determined that the amino terminus of sepA is sufficient for the localization of SEPA to both sites, and that the sepH gene product is required for SEPA localization at septation sites but not for localization at hyphal tips.
76 Isolation of a white collar-1 homolog gene from Trichoderma harzianum. Rios-Momberg, Mauricio; Bibbins, Martha and Herrera-Estrella, Alfredo. Departamento de Ingenieria Genetica, CINVESTAV, Irapuato, Mexico
The biological control agent Trichoderma harzianum responds to blue light forming a ring of green conidia at what had been the colony perimeter at the time of the light pulse. The action spectrum of this phenomenon is similar to the absorption spectrum of flavins and to that of many responses to blue/UV-A light in several species of fungi, to that of photomorphogenesis of lower plants and to that of higher plants phototropism. The photoreceptor for phototropism is a LOV domain-flavin based protein, as the white collar-1 protein, the putative blue light photoreceptor of Neurospora crassa. In order to determine its possible role in the Trichoderma photoresponses, we describe in this work the isolation and sequence analysis of a white collar-1 homolog gene from Trichoderma harzianum. The putative protein is 150 aa shorter and 60% identical to WC1 over the 800 aa carboxi-terminal region spanning the LOV, PAS and zinc-finger domains.
77 Suppression of ATM kinase defects by mutation of a RecQ helicase in Aspergillus nidulans. Amy F. Hofmann and Steven D. Harris. Genetics, Molecular Biology and Biochemistry, Department of Microbiology
The Aspergillus nidulans uvsB gene encodes a member of the conserved family of ATM-related PI-3 kinases. Members of this family of proteins function as central regulators of the DNA damage response. Mutation of the human ATM gene results in the cancer prone syndrome ataxia telangiectasia. In Aspergillus nidulans, UVSB is required for multiple aspects of the DNA damage response, including, 1.) arrest of nuclear division, 2.) inhibition of septation, 3.) damage induced transcription, and 4.) induction of mutagenesis. The Aspergillus nidulans musN227 and musP234 mutations were originally isolated in a screen for mutants sensitive to methyl methane sulfonate (MMS) (Kafer and Mayor 1986). Both musN227 and musP234 are capable of partially suppressing the poor growth and DNA damage sensitivity of uvsB110 mutants (Kafer and Chae 1994). We have shown that the musN227 mutation partially restores several of the uvsB110 defects, suggesting that MUSN may play a role in recovery from the DNA damage response. musN227 is also capable of partially suppressing the damage sensitivity of a uvsB null mutant, indicating that the suppression is not dependent on any UVSB function. Preliminary results suggest that overexpression of MUSN increases the sensitivity of wildtype to genotoxic agents. This is consistent with a role of MUSN in recovery, as overexpressing MUSN may cause premature recovery before cells have adequately repaired or replicated their DNA. Molecular characterization of the musN gene indicates that it encodes a member of the RecQ family of helicases. These helicases appear to play an important role in maintaining genomic stability in eukaryotic organisms. This is exemplified by the fact that mutations in two related human genes, BLM and WRN, cause a cancer prone syndrome and an aging syndrome respectively. Members of the RecQ helicase family of proteins are reported to be involved in the resolution of Holliday junctions via branch migration. The role of MUSN in recovery from the DNA damage response may be associated with this function. The identity of MUSP remains unknown; however, it appears that expression of musN is capable of partially rescuing the damage sensitivity of the musP234 mutant.
78 Structural analysis of Hsp30, the small heat shock protein of Neurospora crassa. Nora Plesofsky and Robert Brambl. Department of Plant Biology, University of Minnesota, St. Paul, MN 55108
The alpha-crystallin-related heat shock proteins (hsps) comprise an important group of chaperones that are synthesized by all eukaryotic and prokaryotic organisms. These small hsps have been found to strongly enhance resistance to biological and chemical stresses, although they are not essential for survival at normal temperature. We earlier found by targeted mutagenesis that Hsp30 was required for Neurospora crassasurvival at high temperature under conditions of glucose limitation. Furthermore, the mutant cells were strikingly reduced in their glucose phosphorylating activity and in their ability to import proteins into mitochondria. Most small hsps self-assemble into multimeric particles, and this ability to oligomerize correlates with their chaperone activity. We approached the question of how small hsp monomers might be organized in these structures by testing and measuring the binding interactions between pairs of small Hsp30 peptides, using the yeast two-hybrid system. We found that sequences within the most conserved region of these small hsps, in the C-terminal "alpha-crystallin domain," interact with two different domains of Hsp30. There is strong interaction of this conserved region with the complete N-terminal half of Hsp30 and with the complete C-terminal half. Further refinement of these peptide interactions shows that non-identical portions of the conserved domain are required for the N-terminal and the C-terminal interactions. Both conserved and nonconserved sequences within the N-terminal half of Hsp30 are required for interaction with the conserved C-terminal domain. We believe these peptide interactions, detected by two-hybrid assay, may provide the bases for dimerization and oligomerization that characterize small hsps. The involvement of nonconserved sequence in these interactions might provide a basis for the specificity of multimerization for a particular monomer.
79 A heterocomplex formed by alternative HET-C proteins triggers vegetative incompatibility in Neurospora crassa. Gopal Iyer1, Jennifer Wu2 and Louise Glass1. 1111, Koshland Hall, Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720. 2Clinical Research Division, Fred Hutchinson Cancer Center, Seattle, WA 98109.
The rejection of the nuclei in a heterokaryotic fusion cell is controlled by the multiallelic het-clocus. Non-self recognition is triggered by allelic differences at het-clocus, which results in growth inhibition. The introduction of alternative het-callele epitope-tagged constructs by co- transformation into het-cnull strain conferred an incompatible phenotype. Co-immunoprecipitation experiments linked the formation of a heterocomplex of alternative HET-C polypeptides with hyphal compartmentation and death response. Further evidence using organelle fractionation localized the heterocomplex to the plasma membrane. This observation was consistent to the in silico analysis predicted for the HET-C polypetide, which revealed the presence of two transmembrane domains. These findings support a possible model of a signal transduced by HET-C, that initialed the formation of a heterocomplex triggers early biochemical reactions that are essential for hyphal compartmentation and death and activation of unidentified effector machinery.
80 Analysis of the Cochliobolus heterostophus homologs of yeast VAC8 and FAB1. Natalie L. Catlett, Olen C. Yoder, and B. Gillian Turgeon. Novartis Agricultural Discovery Institute, San Diego, California
The fungal vacuole is important for protein degradation, ion and small molecule storage, and osmoregulation. To investigate the role of the vacuole in pathogenesis and developmental processes in filamentous fungi, we disrupted homologs of Saccharomyces cerevisiae VAC8 and FAB1 in the corn pathogen Cochliobolus heterostrophus. In yeast, the armadillo-repeat protein ScVac8p is required for several nonessential vacuolar membrane processes including vacuole-vacuole fusion, cytoplasm to vacuole protein targeting, and transport of the vacuole to the bud. ScFab1p is responsible for production of phosphatidyl inositol 3,5-bisphosphate (PI3,5P2), which is required for normal vacuole morphology and function in yeast. Both the C. heterostrophus vac8 and fab1 deletions are viable and produce viable conidia. Analysis of Chvac8 and Chfab1 deletion strains with the vital dye FM4-64 failed to reveal the gross morphological abnormalities of the vacuole seen in the corresponding yeast mutants. Effects of these deletions on plant pathogenesis and mating are under investigation.
81 SEPB of Aspergillus nidulans is a conserved protein which functions to maintain genome stability. Scott E. Gygax, Amy Hofmann, and Steven D. Harris. Dept. of Microbiology, University of Connecticut Health Center, Farmington, CT. 06030-3205.
A temperature sensitive mutation in the Aspergillus nidulans sepB gene dramatically perturbs chromosomal DNA metabolism. At restrictive temperature, this mutation causes; i) elevated levels of mitotic recombination, ii) enhanced levels of chromosomal loss, iii) progressive delays in nuclear division, and iv) the formation of morphologically aberrant interphase nuclei. Molecular characterization of the sepB gene demonstrates that it encodes a 837 amino acid predicted protein possessing five N-terminal consensus WD-40 repeats, a C-terminal Helix-Loop-Helix motif, and a motif shared with DNA polymerase a. Upon sequencing the sepB3 mutant allele, we identified a single missense mutation (P618S) in a conserved proline residue preceding the C- terminal HLH motif, suggesting that the structural stability of this motif might be essential to its function. Protein sequence homology suggests that SEPB might be a member of a conserved family of proteins (S. cerevisiae Ctf4p, X. laevis AND-1, and H. sapiens hAND-1) required for the maintenance of genome stability. ctf4 mutants have a similar genome instability phenotype, and it has been shown that Ctf4p physically binds to DNA polymerase a suggesting a replication function. We are currently assessing the level of functional conservation within the SEPB family of proteins by testing the ability of hAND-1 and Ctf4p to complement the sepB3 mutant.
Phenotypic characterization of sepB3 double mutants in backgrounds defective in the DNA damage checkpoint (uvsB110, uvsD153, nimXcdc2AF) or DNA recombination (nuv2, nuv4, nuv8, and musN227) revealed several synthetic interactions. The sepB3 mutation also causes an elevated level of both spontaneous and induced mutagenesis at the permissive temperature. Phenotypic characterization of sepB3 double mutants with a hyper-recombinatory mutation such as musN227 (RecQ helicase) demonstrated an increase in mutagenesis compaired to sepB3 alone suggesting that the increased mutagenesis is dependent upon recombination. At semi-permissive temperature, the sepB3 mutation causes enhanced sensitivity to MMS and bleomycin. These data suggest that the SEPB protein family might have a role in DNA replication during S-phase as well the repair of double-strand breaks (DSBs).
82 The vacuolar ATPase in Neurospora crassa: Structure of the enzyme and phenotype of strains that lack specific vacuolar ATPase subunits. Christopher Chavez, Karen Tenney, Emma Jean Bowman, and Barry Bowman. Department of Molecular Cell and Developmental Biology, University of California, Santa Cruz, CA 95064
The vacuolar ATPase is a large multisubunit enzyme that generates an electrochemical gradient for protons across several types of cell membranes. We have identified the genes that encode all 13 of the known subunits of this enzyme. The function of these subunits and a model for their position in the enzyme will be presented. We have also used the RIP procedure to generate strains in which genes for five of the subunits (vma-1, 3, 5, 13, and vph-1) have been inactivated. Inactivation of these genes causes severe morphological changes and also alters the structure of vacuoles within the cell. A characteristic phenotype of strains that lack vacuolar ATPase is the inability to grow in alkaline medium. We have found that mutations in genes that do not appear to encode components of the ATPase can suppress this pH-conditional growth phenotype.
83 Evidence for activation of MAP kinases during vegetative incompatibility in Neurospora crassa. Amita Pandey and Louise Glass. Plant and Microbial Biology Department, University of California, Berkeley, CA 94720-3102
Filamentous fungi exist as a network of hyphal filaments that can undergo frequent fusions within or between individual colonies. After hyphal fusion between genetically dissimilar individuals, heterokaryon formation is restricted by genetic differences at het loci. Hyphal fusion cells are quickly compartmentalized and undergo hyphal compartmentation and death. Phenotypic aspects associated with HCD are similar in various het interactions, which suggests activation of common signaling pathways. MAP kinases have been implicated in cell proliferation, cell cycle arrest and cell death in various organisms. The main objective of the study is to find out the role of MAP kinases in vegetative incompatibility in Neurospora. From the western blot data we show that both ERK1 and ERK2 homologs are phosphorylated when two isolates differing at either het or mat locus form an incompatible heterokaryons. A mutant containing a deletion in the putative erk-2 homolog, mak-2 (kindly provided by P. Bobrovich and D. Ebbole) was assessed for its role in vegetative incompatibility. The mak-2 mutant shows a hyphal fusion defect, is female sterile and has non-repressible conidiation. The mak-2 mutant is missing the erk-2 homolog as observed in the western blots using mammalian ERK1/ERK2 antibodies. We have also used a modified heterokaryon test and have priliminary data that suggests the involvement of MAK-2 in vegetative incompatibility mediated by differences at the mating-type locus. Transformations and protoplast fusion experiments are underway to confirm the role of MAK-2 in mating type and het-c vegetative incompatibility.
84 Characterization of vesicle subsets in CHV1 infected C. parasitica. Massimo Turina, Patricia McCabe, Antonio Prodi, and Neal Van Alfen. UC Davis, Plant Pathology
The filamentous ascomycete Cryphonectria parasitica, the causal agent of chestnut blight, is stopped in its development by Cryphonectria parasitica hypovirus 1 (CHV1) infection, resulting in hypovirulent strains of the fungus. Previous studies on the cytopathological effects of virus infection on its host cells showed a consistent proliferation of host vesicles so far uncharacterized, where virus replication and dsRNA accumulation occur. Isopicnic D2O-Ficoll gradients were used to separate subsets of vesicles from the microsomal fraction of virus infected and uninfected C. parasitica. Vesicle proliferation of viral infected strains was maintained over time (up to six days post inoculation) whereas in healthy mycelia the amount of vesicles is very low and decreases during the same time period. A subset of vesicles shown to contain viral dsRNA and proteins reacting to CHV1 helicase and polymerase antibodies appeared to be coated when negatively stained and observed at the electron microscope. Moreover the vesicle fraction of CHV1 infected C. parasitica contain a many fold enriched protein band reacting with anti bovine clathrin heavy chain antibodies in western blot analysis; the same vesicle preparation did not show enrichment in proteins reacting with beta COP antibodies. This finding that clathrin accumulates in hypovirulent strains of C. parasitica prompted us to clone the C. parasitica clathrin heavy chain gene (CHC) and the middle component of its adapter complex involved in trans Golgi network protein secretion (mu1-adaptin) and to investigate their role in C. parasitica.
85 Conserved SNAREs in Neurospora crassa may regulate exocytosis and intracellular fusions. Gagan D. Gupta and I Brent Heath. York University Biology Toronto, Canada
SNAREs are highly conserved proteins known to be essential for vesicle trafficking and fusion in eukaryotes, but little is known about their presence or role in filamentous fungi. We have identified six SNARE genes in the filamentous fungus Neurospora crassa, either via library screening or analysis of current Neuropsora sequencing databases. Three of these genes, syn, nsyn and nsec9 show significant homology to SNAREs involved in the last step of exocytosis at the plasma membrane in Saccharomyces. Hence syn, nsyn and nsec9 may be important for the establishment of an exocytotic gradient that is required for the generation of hyphal tips. Two other identified genes, ntlg1 and ntlg2, bear strong homology to yeast endosomal SNAREs, suggesting the conservation of a Golgi-endosomal trafficking pathway in Neurospora. Another Neurospora gene, nsyn8, appears to have no counterpart in in the fully sequenced Saccharomyces, but bears similarity to an uncharacterized SNARE in Schizosaccharomyces pombe, which may indicate the presence of an as yet unidentified route in the secretory pathway in fungi.
86 Control of cell morphogenesis in fission yeast. David Wiley, Paola Catanuto and Fulvia Verde. University of Miami, Biochemistry, Miami, FL.
Fission yeast Schizosaccharomyces pombe is an excellent model system for studies of cell morphogenesis because it grows in a polarized fashion with a well-defined cylindrical shape. Moreover, polarized cell growth is tightly regulated during the cell cycle. We have previously identified 19 fission yeast genes important for various aspects of cell morphogenesis, and classified them according to their functions during the cell cycle (Verde et al., 1995). One of these, orb6, is required for maintenance of cell polarity, for polarized localization of the actin cytoskeleton, and for its reorganization during the cell cycle (Verde et al, 1998). Orb6 encodes a protein kinase related to human, C.elegans, and Drosophila Ndr kinases, Saccharomyces cerevisiae CBK1, Ustilago maydis Ukc1 and Neurospora crassa Cot1. These kinases are related to mammalian Rho-kinase but lack the consensus Rho-binding motifs. Like Orb6, Cot1, CBK1, Ukc1 and Ndr have been shown to be required for the regulation of cell morphology. In fission yeast, disruption of the microtubule cytoskeleton induces cell distortion and cell branching, suggesting an important role for microtubules in cell polarity. Another gene, called tea1, encodes a protein which localizes to the cell tips in a microtubule-dependent fashion and is thought to function as a molecular marker for the correct placement of the growth sites (Verde et al. 1995; Mata and Nurse, 1997). In order to investigate the role of Orb6 in the control of cell morphology and to explore the mechanism of Tea1-dependent polarity control, we have conducted genetic screens to identify proteins that interact with Orb6 and Tea1. The results of the characterization of these molecules will be presented.
87 Control of polarity in the filamentous fungus Ashbya gossypii. Philipp Knechtle, Jürgen Wendland and Peter Philippsen. Applied Microbiology, Biozentrum, University of Basel, Switzerland
We used Alexa-Phalloidin staining to localize the actin cytoskeleton in the filamentous fungus Ashbya gossypii. Actin could be observed as patches, cables and as actin rings. Patches localize over the whole cell cortex and polarize at the tips. Patches seem to be interconnected by actin cables. Actin rings localize within the hypha most likely to or at sites of septum formation. Polarized cortical patches could also be observed as a double ring structure close to the neck between germ bubble and germ tube or within hyphae. The gene product of AgBOI seems to be important in the polarization of cortical actin patches because deletion of AgBOI leads to an occasional loss of polarized growth indicated by spherically enlarged tips. The cortical actin in these enlarged tips is delocalized. Polarized growth can be regained in the same axis which is accompanied by a repolarization of cortical actin. To investigate dynamics of polarized growth we identified a polarity marker by sequence similarity to Saccharomyces cerevisiae Spa2p. In Saccharomyces cerevisiae ScSpa2p localizes to sites of polarized growth. AgSpa2p shows significant homology to ScSpa2p in its N- and C-terminal part. The internal domain in AgSpa2p is about 2200aa whereas in ScSpa2p it is only 700 aa in length. Both internal domains carry repetitive sequence towards their end but no homology could be observed within the whole internal domains. The genomic copy of AgSPA2 was labelled with GFP at its C-terminus. AgSpa2p-GFP localized to the tips of hyphae. Occasionally an accumulation of AgSpa2p-GFP could be observed as a double ring structure within hyphae. Using video time lapse microscopy we could show that during the development of a young mycelium AgSpa2p-GFP permanently localized to the growing tips. Upon lateral branch formation first an accumulation of AgSpa2p-GFP at the cortex and then continued localization at the tip of the emerging branch can be observed. A similar localization pattern is seen using a genomic GFP fusion to the F-actin binding protein AgCAP1.
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