46. Msb3p and Msb4p, a pair of Rab GAPs, link actin organization to secretion in S. cerevisiae. Erfei Bi, Dept. of Cell & Dev. Biol., Univ. of Pennsylvania, Philadelphia, PA 19104-6058
Budding in S. cerevisiae occurs by polarized secretion to the daughter cell, which is directed by the polarized actin cytoskeleton. Cdc42p, a small Rho-type GTPase, plays an essential role in the polarization of the actin cytoskeleton, whereas Sec4p, a Rab GTPase, plays an essential role in the transport and tethering of the post-Golgi vesicles to the daughter cell. Previously, we have shown that Msb3p and Msb4p could function as dosage-dependent suppressors of cdc42 mutants and displayed GTPase-activating protein (GAP) activities for a number of Rab GTPases in vitro, including Sec4p. Now, we provide evidence to indicate that Msb3p and Msb4p function as "arginine-finger" GAPs for Sec4p in vivo and the GAP activity of Msb3p and Msb4p is essential for their functions in secretion and actin organization. In addition, we have shown that Msb3p and Msb4p interact directly with Cdc42p and are in the same complex with Bni1p, Spa2p, and Bud6p, all of which are involved in the formation and/or attachment of actin cables to the presumptive bud site. Thus, Msb3p and Msb4p may coordinate actin organization to secretion by interacting with both the Cdc42p and the Sec4p GTPase modules.
47. The accumulation of cytoplasmic dynein and dynactin at microtubule plus-ends in Aspergillus nidulans is kinesin dependent. Jun Zhang*, Shihe Li*, Reinhard Fischer** and Xin Xiang* *Department of Biochemistry, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, U. S. A **Max-Planck-Institute for Terrestrial Microbiology, Karl-von-Frisch-Str. D-35043 Marburg, Germany
The mechanism(s) by which microtubule plus-end tracking proteins are targeted is unknown. In the filamentous fungus Aspergillus nidulans, both cytoplasmic dynein and NUDF, the homolog of the LIS1 protein, localize to microtubule plus ends as comet-like structures. Here we show that NUDM, the p150 subunit of dynactin, also forms dynamic comet-like structures at microtubule plus ends. By examining GFP-fusion proteins in different loss-of-function mutants, we demonstrate that dynactin and cytoplasmic dynein require each other for microtubule plus-end accumulation, and the presence of cytoplasmic dynein is also important for NUDF's plus-end accumulation. Interestingly, deletion of NUDF increases the overall accumulation of dynein and dynactin at plus ends, suggesting that NUDF may facilitate minus-end directed dynein movement. Finally, we demonstrate that a conventional kinesin, KINA, is required for the microtubule plus end accumulation of cytoplasmic dynein and dynactin, but not of NUDF.
48. The mitogen-activated protein kinase Osc1 confers the response to high osmotic stress and fungicide sensitivity in Colletotrichum lagenarium. Kaihei Kojima, Yoshitaka Takano, Taisei Kikuchi, and Tetsuro Okuno. Department of Agriculture, Kyoto University, Kyoto, Japan
Colletotrichum lagenarium, the causal agent of cucumber anthracnose disease, invades the host plant using specialized infection structures called appressoria. We have shown two mitogen-activated protein kinase (MAPK) genes, CMK1 and MAF1, are required for fungal infection process including appressorium formation in this fungus. Here, we identifiedOSC1 encoding a MAPK of C. lagenarium that belongs to the S. cerevisiae Hog1 MAPK subfamily. OSC1 encodes a 357 amino acid protein with 96% identity to the Magnaporthe grisea Osm1 MAPK and 80% identity to the S. cerevisiae Hog1 MAPK. Target disruption of OSC1 had no detectable effect on mycelial growth, conidiation, and pathogenicity to cucumber plants. However, the osc1mutants exhibited reduced growth on high osmotic media compared with the wild-type strain, indicating that Osc1 is involved in response to high osmolarity like yeast Hog1. We also found that the osc1 mutants were resistant to a phenylpyrrole fungicide fludioxonil, indicating that the Osc1 MAPK pathway confers sensitivity of the fungicide. When conidia of the wild-type strain were inoculated on cucumber leaves in the presence of fludioxonil, they failed to form lesions efficiently. In contrast, the osc1 mutants exhibited normal pathogenicity even in the presence of fludioxonil, indicating that inhibition of fungal infection by fludioxonil depends on OSC1. These suggest that proper regulation of the Osc1 MAPK is required for fungal pathogenicity of C. lagenarium.
49. Altered ionic homeostasis and phenotypic suppression of the Neurospora crassa COT1 kinase mutant by environmental stresses. Oded Yarden and Rena Gorovits. Department of Plant Pathology and Microbiology and The Otto Warburg Center for Agricultural Biotechnology, Faculty of Agricultural, Food and Environmental Quality Sciences. The Hebrew University of Jerusalem, Rehovot 76100, Israel
Neurospora crassa grows by forming spreading colonies. cot-1 belongs to a class of N. crassa colonial temperature-sensitive (cot) mutants, and encodes a Ser/Thr protein kinase that is structurally related to the human myotonic dystrophy kinase, which when impaired confers a disease that involves changes in cytoarchitecture and ion homeostasis. When grown at restrictive conditions, cot-1 cultures exhibited enhanced medium acidification rates, increased relative abundance of sodium and higher intracellular glycerol content, indicating an ionic homeostasis defect in the hyperbranching mutant. The presence of ion transport blockers, increased medium osmoticum (NaCl or sorbitol), H2O2 or ethanol, suppressed the cot-1 phenotype to various degrees, suggesting that COT1 is linked with stress signaling. Environmental suppression of the cot-1 phenotype was accompanied by a reduction in PKA activity. Direct inhibition of PKA with KT-5720 partially suppressed the cot-1 phenotype, but in contrast to that observed with extragenic ropy suppressors of cot-1, did not change COT1 polypeptide expression patterns in the mutant. We suggest that COT1 function is linked to the PKA pathway, which is altered in a cot-1 background and that reducing PKA activity bypasses the requirement for a fully functional COT1. This work was supported by the Israel Science Foundation.
50. Protein Kinase C interacts with and regulates the protein levels of the Neurospora photoreceptor WC-1. Franchi L., Fulci V., Macino G. Department of Biology, Universita' La Sapienza, Rome, Italy
The Neurospora photoreceptor WC-1 is transiently phosphorylated in response to light and this correlates with the transient increase of the light-regulated mRNAs levels, and with a progressive, light induced degradation of WC-1. Here we have investigated the function of PKC activity on the Neurospora photoreceptor WC-1, and on the regulation of light responses. We used PKC activators and inhibitors to test the effects on WC-1 phosphorylation and stability. We show that PKC activators keep WC-1 hyperphosphorylated even after several hours of constant light, inducing a degradation of the WC-1 protein. We show that PKC interacts with WC-1 in vivo and that endogenous PKC phosphorylates WC-1 on the zinc-finger region in vitro. To understand the physiological meaning of these observations, we studied the function of PKC using dominant negative and constitutively active mutants of the kinase. We found that a constitutively active PKC induces a dramatic decrease in WC-1 protein levels, and a decrease in the light induced transcription of the al-2 mRNA. We see clear opposite effects in the presence of the dominant negative PKC. Furthermore, we have seen that PKC kinase activity is transiently light induced and it has an important function in the light regulated inhibition of mycelial growth. Our data indicate that the protein levels and the activity of the Neurospora photoreceptor WC-1, and the light regulated mycelial growth are regulated by PKC, which is, therefore, a novel component of the light signal transduction pathway directly interacting with and phosphorylating WC-1.
51. Aspergillus nidulans MATE mobile elements: evidence of RIPping? John Clutterbuck. Institute of Biomedical and Life Sciences, Anderson College, University of Glasgow, Glasgow Scotland,
MATE elements were identified as a result of investigations into the AMA1 sequence which allows autonomous replication of plasmids in A. nidulans (Aleksenko & Clutterbuck 1996 Mol. Microbiol. 19: 565-574). Five MATE elements can be identified in the Glasgow strains of A. nidulans, while wild isolates contain variable numbers, inserted at different sites. MATE sequences obtained from the Cereon Microbial Sequence Database show elements of 6097 bp, flanked by 9 bp target site repeats. The most notable sequence features are numerous "Spe" motifs, [(RWCTAGWYNNN)2-4], scattered throughout, and potential stem-loop structures at each end of the elements. No ORFs with significant homology have been identified, and the mechanism of transposition and relationship to plasmid replication are both unknown. MATE1a and MATE1b are inserted back-to-back on chromosome IV, separated by 374 bp of unique sequence. Partial sequencing indicates that these two copies are identical, but solitary elements on other chromosomes differ from MATE1 by numerous transition mutations. Comparisons between these solo copies indicate greater divergence, suggesting independent origins from MATE1 or its precursor. The mutated copies show both T-C and G-A substitutions in patterns suggesting independent mutation of each strand. The doublet preference for mutation is CpG > CpA, with other doublets rarely mutated. It is suggested that mutation may have occurred by a RIP-like mechanism, from which MATE1 elements are protected by their putatively telomeric chromosomal location.
52. Characterization of the Aspergillus nidulans morphogenetic mutants hypA1and hypA6. Sha Yu and Susan Kaminskyj Department of Biology, University of Saskatchewan, Saskatoon, Canada.
Aspergillus nidulans has tubular hyphae with highly polarized tip cell growth, and quiescent basal cells separated from the tip by a septum. This morphogenetic pattern requires genes including hypA to promote growth of tip cells and suppress growth of basal cells. Fungal homologues of hypA include Saccharomyces cerevisiae TRS120, which is essential and is required for Golgi transit, a stage in secretion required for polarized growth.A. nidulans hypA is not essential, but a hypA knockout strain grows very poorly hypA has two temperature sensitive, nonlethal, recessive alleles, hypA1 andhypA6, which produce similar restrictive phenotypes: wide slow growing cells with thick walls. The hypA1 lesion is G329R; sequencing shows the hypA6 lesion is K885F plus E932K, which is being confirmed by complementation. The effect of hypA1 and hypA6 on secretion polarity being studied using the in vivo dye, FM4-64, that tags endomembranes. As expected, wildtype hyphae and the permissive phenotype of hypA1 and hypA6 have well organized endomembrane systems culminating in a Spitzenkörper. In contrast, hypA1 and hypA6 restrictive phenotypes have diffuse endomembrane arrays that are somewhat more concentrated at their growing tips, consistent with their poorly polarized wall deposition pattern.
53. Regulation of septation in Aspergillus nidulans. Bo Liu, and Y.-R. Julie Lee. Plant Biology, UC-Davis, Davis, CA.
Septation initiation network (SIN) involving a MAP kinase-like kinase cascade plays a pivotal role in temporal regulation of septation in fungi. But it is unclear how the SIN network initiates septation. In the filamentous fungus Aspergillus nidulans, after conidial germination, the first septum is assembled at or near the basal end of the hypha after three rounds of mitosis. Among SIN molecules, the novel evolutionarily conserved MOB1 protein associates with the last kinase in this kinase cascade. We have cloned cDNA of the AnMOB1 gene in A. nidulans. By using a functional GFP-AnMOB1 fusion, the AnMOB1 protein was detected at the spindle pole body in interphase. In M phase, however, GFP-AnMOB1 appeared not only at the spindle pole body, but also along the central spindle and later at the septation site. Down-regulation of AnMOB1 expression or deletion of AnMOB1 allowed hyphal growth, but abolished septation and conidiation. We are now exploring whether over-expression of AnMOB1 or other SIN molecules allows coupling of septation and mitosis after conidial germination in this filamentous fungus. Interestingly, AnMOB1 localization to the spindle pole body and the septation site was dependent on intact microtubules. Previously, we have shown that the cytoplasmic dynein is required for correct positioning of the septum, implying that this microtubule motor may play a role in the localization of SIN molecules. Therefore, we are testing whether the localization of AnMOB1 and other SIN proteins is dependent on microtubule-based motor proteins including dynein.
54. Is a 1,3 - beta - glucan synthase homolog an essential gene in Coccidioides posadasii? Ellen M. Kellner1, Kris I Orsborn1, Erin M Siegel1, Marc J Orbach2 and John N Galgiani1. 1The Valley Fever Center for Excellence, Southern Arizona VA Health Care System, Tucson, AZ. 2Department of Plant Pathology, University of Arizona, Tucson, AZ.
Beta glucans are major components of the cell walls of many fungi including Coccidioides spp. Because of this, they are targets for antifungal drugs and the 1,3 beta - glucan synthase inhibitor, caspofungin, has shown potential therapeutic benefit for treatment of coccidioidomycosis. In Paracoccidioides brasiliensis, Aspergillus nidulans, Aspergillus fumigatus and Cryptococcus neoformans, the FKS subunit of 1,3 beta - glucan synthase which is the target of caspofungin, appears to exist as an essential single copy gene. We identified, cloned and sequenced the gene, GS1, from the Silveira strain of Coccidioides posadasii which showed a high degree of similarity to FKS genes in other species. In order to validate GS1 as a target for antifungal therapy in Coccidioides spp., we sought to create a null allele through homologous gene replacement. We replaced the GS1 coding sequences of C. posadasii with a dominant selectable marker for hygromycin resistance using the Agrobacterium tumifaciens T-DNA transfer technique. PCR and Southern blot analyses showed all 24 hygromycin resistant transformants harbored a wild-type allele of GS1. However, 6 (25%) also contained a homologous gene replacement and therefore were likely heterokaryons. Heterokaryotic transformants that included homologous GS1 replacements were defective in arthroconidiation as compared to strains with ectopic construct integrations or wild-type strains. Homologous and ectopic transformants were purified through streaking for single colonies and subsequent purification of arthroconidia from isolated colonies. All purified strains maintained a wild-type allele of GS1. These findings support an essential role for GS1 in Coccidioides spp. an attractive target for further antifungal drug discovery.
55. The phenotype of the Neurospora crassa cot-5 mutant, defective in a mannosyltransferase, can be suppressed by increased medium osmoticum. Zipora Resheat-Eini, Rena Gorovits, Oded Yarden. Department of Plant Pathology and Microbiology, Faculty of Agricultural, Food and Environmental Quality Science, The Hebrew University of Jerusalem, Rehovot 76100, Israel
Neurospora crassa colonial temperature sensitive mutants, (cot-1, cot-2, cot-3, cot-4 and cot-5) form compact, highly branched colonies. As the defects in these mutants also involve apparent alterations in cell wall morphology, we examined the effect of changes in environmental osmoticum on the mutants. When grown in the presence of 0.75-1M sorbitol the hyper branching phenotypes of cot-1, cot-4 and cot-5 were suppressed to various degrees. cot-5 exhibits an altered COT1 polypeptide expression pattern, which suggests that COT5 and COT1 may be functionally linked. We have cloned cot-5, by complementation, and have determined it encodes a mannosyltransferase which is highly similar to the Saccharomyces cerevisiae dolichol pathway ALG2 protein. The mutation in cot-5 consists of a single base substitution leading to the formation of an amber termination codon at amino acid 37. Treatment with Tunicamycin (a specific inhibitor of N-glycosylation) inhibits hyphal elongation and induces hyperbranching in a manner that mimics the cot-5 phenotype. Even though COT1 has several potential N-glycosylation sites, we have not found evidence for altered COT1 glycosylation in a cot-5 background.
56. Isolation and characterization of genes encoding dynein heavy chain, dhc1 anddhc2, and Ras GTPase-activating protein, gap1, from Schizophyllum commune. D. Schubert and E. Kothe, FSU, Jena, Germany.
In the homobasidiomycete Schizophyllum commune mating is in part controlled by a pheromone receptor system. Signal transduction starting from an activated pheromone receptor results in fast nuclear migration and is a prerequisite for establishment of the fertile dikaryon. We are studying the roles of cytoplasmic dynein and Ras GTPase-activating protein in these processes. The heavy chain of cytoplasmic dynein is encoded by two separate genes in S. commune, dhc1 and dhc2. Unlike dyn1 of Ustilago maydis, dhc1 comprises only the N-terminal dimerization domain of the protein. ATP-binding sites and microtubule-binding domain are encoded by the second gene, dhc2. Deletion of dhc2 led to viable monokaryotic strains. They showed slow growth and a tendency of hyphal knot formation resulting in colonies having a complex morphology. In matings with wildtype strains deltadhc2 mutants were able to accept nuclei, but dikaryotization was slower than in the wildtype. The Ras GTPase-activating protein gene gap1 showed the highest identity togap1 from Schizosaccharomyces pombe. gap1 deletion mutants were able to mate both with wildtype and delta gap1 strains. Dikaryons heterozygous for delta gap1developed normally. However, homozygous delta gap1 dikaryons showed abnormal clamp cell formation which affected the backward bending of clamp cells. In addition, development of lateral branches from clamp cells could be observed.
57. A homologue of Ste6p, the a-factor transporter in Saccharomyces cerevisiae, functions bilaterally in Cryptococcus neoformans. Yen-Ping Hsueh and Wei-Chiang Shen. Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, Taiwan
Fungal pheromones have been demonstrated to function in the initial recognition of the fungal mating process. One type of peptide pheromones, identified in the Ascomycetes and Basidiomycetes, terminate in a conserved CAAX motif which triggers sequential post-translational modifications of the pheromone precursors. Among this type of peptide pheromones, a well-studied one is the a-factor of Saccharomyces cerevisiae. The mature a-factor is exported from the cell via an alternative mechanism involving the ATP-binding cassette transporter Ste6p, which is distinct from the typical secretory pathway utilized by most peptides. Unlike the Ascomycetes, the basidiomyceteous fungi produce only CAAX motif containing lipopeptide pheromones. Cryptococcus neoformans, a human pathogenic basidiomycetous yeast, causes the life-threatening meningoencephalitis mainly in individuals with compromised immune functions. Virulence studies of the congenic pair of C. neoformans strains have shown that MATalpha cells are more virulent thanMATa cells. Characterization of mating pheromone genes in the MATalpha strains have suggested an autocrine signaling loop may function and contribute to the virulence of the MATalpha cells. To further address the role of pheromone in the signaling loop, we have identified STE6 homologue in the C. neoformans genome project at SGTC and begun to dissect its function. By disrupting theSTE6, we found that ste6 mutants in either MATalpha orMATa background showed partially impaired mating function, although slight differences were noticed. However, when ste6 MATalpha andMATa mutants cross with each other, the mating process was nearly completely abolished. Our data indicates that the STE6 functions bilaterally and is required but not essential for mating in C. neoformans.
58. Expression profiling of the ectomycorrhizal interaction between birch and different compatible strains of Paxillus involutus. Andres Schützendübel1, Antoine Le Quere1, Thomas Johannson1, Susanne Erland1,Anders Tunlid1 1Department of Microbiol Ecology , University of Lund, Sweden
A lot of investigations led to the assumption that a continuum between parasitic, saprophytic and mutualistic living basidiomyctes exists and the Ectomycorrhizal-(ECM) fungi have evolved from saprophytic precursors, but there are also multiple reversals to a free living lifestyle (Hibett et al., 2000).Differences in the form of interaction exist not only between different species and groups also between different strains of the same species mutualistic as well as saprophytic lifestyles and intraspecific strong host specificity has been observed (Brundett, 2002). The aim of this study is to investigate the different forms and changes of interactions between roots and ECM-fungi. Birch and Paxillus involutus were chosen as a model system. We screened several strains of P. involutus on its ability to develop a functioning mycorrhizal interaction with birch. Two strains, isolated from different hosts showed strong differences in establishing mycorrhizal structures. In the non-mycorrhizal strain no Hartig-net was formed and no inhibition of lateral root growth during the early phase of the interaction (one day – seven days) was observed. Additional differences in physiological interaction were detected. By microarray analysis we are now investigating differences in gene expression between the two strains during mycorrhiza development.
Literature cited: Hibett et al., (2000) Nature 407: 506-508 Brundett, (2002)New Phytol 154: 275-304
59. Checkpoint control genes in Neurospora crassa. Chizu Ishii and Hirokazu Inoue, Laboratory of Genetics, Saitama University, Saitama, Japan
We have searched for genes related to cell cycle functions on the Neurospora genome database. Among 28 ORFs that showed homology in blast search, five candidates were located close to known genes. By transformation using genome fragments, we confirmed thatun-14, mus-9 and mus-21 were homologs respectively of CDC25,MEC1, and TEL1 of Saccharomyces serevisiae. As a tel1 mutant in yeast has no apparent phenotype as a DNA repair mutant, we characterized the mus-21mutant in particular. The mus-21 mutant was highly sensitive to methyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, tert-butyl hydroperoxide and slightly sensitive to UV, camptothecin, hydroxy urea and hydrogen peroxide. The mus-21 mutant was sterile when crossed homozygously. Cytological investigation revealed that the defect(s) was at meiosis I. The mus-21 mutation induced synthetic lethality in combination with the mus-9 mutation. Epistatic grouping of the mus-21 mutation based on mutagen sensitivity of double mutants showed that this mutation was involved in the uvs-6 (RAD50 homolog) group, but synergistic to one member of this group, the mei-3 (RAD51 homolog) mutation. These results indicate that Neurospora uses two redundant but essential signaling pathways for checkpoint control and that, as in yeast, recombination repair has two independent pathways with the Neurospora Tel1 homolog plays a role in the Rad51 independent pathway.
60. A class V myosin involved in chitin synthase delivery towards growth sites ofUstilago maydis. Isabella Weber, Gagan Gupta and Gero Steinberg. Max-Planck-Institute for terrestrial Microbiology, Karl-von-Frisch Strasse D-35043 Marburg, Germany.
Class V myosin motors utilize F-actin to support intracellular traffic in eukaryotic cells. Our knowledge about their existence and function in fungi is restricted to the yeasts S. cerevisiae and S. pombe. In the former, a class V myosin is thought to support polar growth by delivering chitin synthase towards the growth region. We identified a class V myosin in U. maydis (Myo5) that is involved in polar growth and pathogenicity (see poster by Gupta et al.). We checked for a role of Myo5 in polar depositioning of chitin and localization of chitin synthase by wheat-germ agglutinin staining and a cross reactive antibody against Chs2p from S. cerevisiae (Sietsma et al. 1996). Both chitin and chitin synthase were found to form a gradient towards the growing bud of wild-type cells. This localization was insensitive to microtubule disruption by Benomyl, but both the actin inhibitor Latrunculin and the myosin drug BDM severely affected chitin and chitin synthase distribution. This suggests that the actomyosin system supports directed transport of chitin synthases towards the growth region. A temperature sensitive Myo5 mutant was normal at 20̊C but showed defects in morphogenesis at 28̊C, a temperature that did not affect the actin cytoskeleton. Interestingly, polar distribution of chitin and chitin synthase was disrupted in Myo5ts cells upon temperature shift, but chitin and chitin synthase became repolarized after 3h. This redistribution of chitin and chitin synthase in Myo5ts cells was also sensitive to Latrunculin and BDM, indicating that after 2-3 hours an actin-based transport mechanism compensates for the inactivation of the temperature sensitive allele of myo5 . This suggests that, in contrast to the yeast S. cerevisiae, other myosins participate in polar growth and chitin synthase distribution in Ustilago maydis. Recent progress in elucidating the mechanisms underlying this phenomenon will be presented.
61. Cloning and characterization of a two-component histidine kinase gene of Cochliobolus heterostrophus involved in osmotic adaptation and dicarboximide resistance. Akira Yoshimi, Chihiro Tanaka and Mitsuya Tsuda. Laboratory of Environmental Mycoscience, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan.
A two-component histidine kinase gene was cloned and sequenced from a plant pathogenic fungus Cochliobolus heterostrophus. The predicted protein possessed the conserved histidine kinase domain, the response regulator domain and the six-tandem repeats of 92 amino acids in the N-terminus. The deficiency of C. heterostrophus Dic1 mutant was complemented with introduction of the wild type histidine kinase gene, suggesting the Dic1 gene encoded the putative histidine kinase. All the Dic1 mutant alleles exhibit osmotic sensitivity and resistance to dicarboximide and phenylpyrrole fungicides, and they can be classified into three types on the basis of their phenotypes. To elucidate molecular basis of their allelic nature, we sequenced the genes from ten Dic1 mutants. The null mutants of Dic1p and the mutants with deletion or point mutation in the N-terminal repeat region were highly sensitive to osmotic stress and highly resistant to both fungicides. The single amino acid change within the kinase domain or the regulator domain conferred various sensitivity to osmotic stress and moderate resistance to the fungicides. These results suggest that this predicted protein, especially the amino acid repeat region, has an important function in the osmotic adaptation and the fungicide resistance. Further functional analysis of the histidine kinase based on site-directed mutagenesis is in progress.
62. Characterization of the mechanism of action of PAF, an antifungal protein secreted byPenicillium chrysogenum. Florentine Marx1, Christoph Oberparleiter1, Wolfgang Burgstaller2, Renate Weiler-Görz1 and Lydia Kaiserer1. 1Department of Molecular Biology, Innsbruck, Austria.2Intstitute of Microbiology, Innsbruck, Austria
The filamentous fungus Penicillium chrysogenum secretes the antifungal protein PAF. So far, no information is available on the exact mode of action of this small, highly basic and cysteine rich protein. We characterized the protein function in more detail and show that secreted PAF retains its activity over a broad pH range and resists to high temperature and protease digestion. The protein is effective against the growth of a variety of phytopathogenic and opportunistic human pathogenic molds. PAF reduces spore germination and hyphal extension in a dose dependent manner and induces stress responses, including morphological changes, plasma membrane leakage, generation of radicals and metabolic inactivity. These detrimental effects cannot be antagonized by cations and can be neutralized only to some extent by hypertonic growth conditions, suggesting that the cell wall or membrane are not the primary target sites of PAF. This finding is supported by the intracellular localization of PAF in sensitive fungi. Our results demonstrate that the mechanism of action of PAF differs from that of other known antifungal proteins. Thus PAF represents a new antifungal agent potent to inhibit the growth of harmful molds.
63. The WASP-homolog Wal1p is required for morphogenesis, hyphal growth and full virulence of the human fungal pathogen Candida albicans. Andrea Walther, Nir Osherov and Jürgen Wendland Dept. of Microbiology, Friedrich-Schiller Univ. and Hans-Knoell-Institute for Natural Products Research, Jena, Germany;Dept. of Human Microbiology; Sackler School of Medicine; Tel-Aviv Univ., Tel Aviv, Israel.
The dynamic reorganization of the actin cytoskeleton underlies processes of cell polarization, morphogenesis and cytokinesis. Cell proliferation and morphogenesis requires two distinct steps (i) polarity establishment to define new areas of growth eg for bud formation and (ii) maintenance of polarized growth eg to form hyphae. Previously we characterized the essential role of Rho-type GTPases in these processes in the filamentous fungus Ashyba gossypii. Here we report the identification and characterization of a gene required for the maintenance of polarized growth in Candida albicans. This gene, encoded by CaWAL1, belongs to the family of Wiskott-Aldrich syndrome-Like proteins that are known for their role in actin filament nucleation via activation of the Arp2/3 complex. Homozygous C. albicans wal1/wal1 cells diplay several mutant phenotypes that are all based on defects in cortical actin cytosekeleton assembly. (i) Mutant cells display a random budding phenotype instead of the wildtype bipolar budding pattern. (ii) Mutant cells exhibit a cytokinesis defect. (iii) Yeast cells appear round in shape instead of ellipsoidal as wildtype (or heterozygous WAL1/wal1 cells) indicating loss of poalrized secretion. (iv) hyphal induction of wal1/wal1 cells using either spider medium or serum both in liquid medium or on plates only results in the formation of pseudohyphae with a drastic decrease in filament formation. (v) And, finally, mutant cells display a decreased virulence (about 1% of wildtype level) in a mouse model. Our results demonstrate that cortical actin cytoskeletal organization and polarized secretion are crucial for morphogenesis, hyphal formation and virulence in C. albicans.
64. Maintenance of polarized hyphal growth requires a WASP family member protein in the filamentous fungus Ashbya gossypii. Andrea Walther and Jürgen Wendland. Dept. of Microbiology, Friedrich-Schiller University and Hans-Knoell-Institute for Natural Products Research, Jena, Germany
Mycelium formation in filamentous fungi is based on a two step process requiring the actin cytoskeleton for both stages. First, the polarity establishment machinery is involved in chosing a site at which a new hyphal tip is formed. Second, maintenance of polarized cell growth keeps secretion of cell wall compounds polarized to the hyphal tip and results -in contrast to yeast cells- in hyphal elongation. Previously, we identified two genes required for the first step, i.e. polarity establishment, in Ashbya gossypii, encoded by AgCDC42 and AgCDC24. Here we report the identification and characterization of a gene required for the maintenance of hyphal growth. This gene, AgWAL1, encodes a protein with similarity to the Wiscott-Aldrich Syndrome protein family. WASP family members regulate the organization of the cortical actin cytoskeleton via interaction with the Arp2/3 complex and activation of actin filament nucleation. Agwal1 mutant spores germinate unilaterally and do not display the bipolar branching pattern of wildtype spores. Growing hyphal tips exhibit severe defects in hyphal elongation after tip formation. This results in swollen hyphae that reveal extended periods of isotropic growth as determined by time lapse microscopy. Our results indicate that an AgWal1p-independent mechanism ensures the reorganization of the actin cytoskeleton during hyphal tip formation whereas polarized organization of actin cortical patches and the maintenance of hyphal growth is dependent on AgWal1p.
65. Characterization of a secretion-related small GTPase encoding gene in Aspergillus niger. A.F.J. Ram1,2, X.O. Weenink1, P.J. Punt2 and C.A.M.J.J. van den Hondel1,2. 1)Leiden University, IMP, Wassenaarseweg 64, 2333 AL, Leiden, The Netherlands, 2) TNO-Nutrition, AMGT, 3700 AJ Zeist, The Netherlands.
Filamentous fungi, such as Aspergillus niger, have the capacity to secrete large amounts of enzymes into their environment. Transport of these proteins through the secretion pathway is mediated by vesicles and controlled by highly conserved secretion related small GTPases of the Rab/Ypt family. Based on this conservation, we have cloned seven secretion related small GTPases (srg genes) from A. niger (Punt, et al., 2001). For the A. niger srgC gene, a putative homologue of the S. cerevisiae ypt6 gene, a disruption mutant was generated. The srgC disruption mutant is viable but grows slower and more compact at 30̊C compared to the wild-type strain. At higher temperatures this phenotype becomes more severe and results in a conidiation defect. In order to monitor protein secretion and protein transport to the vacuole, we have made GFP-based secretion reporter proteins. In both the wild type strain and in the srgC deletion strain, the CPY-GFP fusion protein is efficiently targeted to the vacuole. However, the disruption of the srgC gene resulted in a fragmentation of vacuoles, indicating a function of SrgC at a later step than the Golgi to vacuole transport step, e.g. a function in vacuolar fusion. Microscopical analysis of the srgCdisruption mutant using the Glucoamylase-GFP fusion protein showed partial intracellular accumulation of the GFP fusion protein indicating that the disruption of srgC also affects protein secretion.
66. Molecular characterization of the ramosa-1 mutant in Aspergillus niger. A.F.J. Ram1,2, M. Arentshorst1, C. G. Reynaga-Pena3, S. Bartnicki-Garcia4, C.A.M.J.J. van den Hondel1,2. 1)Leiden University, IMP, Leiden, The Netherlands,2)TNO-Nutrition AMGT, Zeist, The Netherlands, 3Universidad de Guanajuato, Mexico, 4Centro de Investigacion Cientifica y de Educacion Superior de Ensenada, Mexico.
Polarized growth of a leading hyphe of many filamentous fungi is characterized by the presence of a Spitzenkörper or Vesicle Supplying Centre (VSC). Molecular mechanisms underlying the establishment and maintenance of the Spk are unknown. To identify proteins involved in this process we have characterized the previously isolated apical branching mutant, ramosa-1 (1). We have cloned the ramosa-1 gene by complementation of the temperature sensitive phenotype. Homology searches indicate that the protein belongs to an evolutionary conserved family of proteins. For the S. pombe homolog, it has been shown that the protein interacts with a stress activated MAP kinase. Overexpression of the ramosa-1 cDNA could rescue the S. cerevisiae gene deletion mutant, indicating a functional conservation between the two homologs. 1) C.G. Reynaga-Pena and S. Bartnicki-Garcia. 1997. Apical branching in a temperature sensitive mutant of Aspergillus niger. Fungal Genetics and Biology 22, 153-167.
67. Identification and role of peroxisomal ABC transporters in the sexual development of the filamentous fungus Podospora anserina. S. Boisnard, D. Zickler, M. Picard and V. Berteaux-Lecellier. Institut de Génétique et Microbiologie, UMR 8621 Université Paris-Sud Bat. 400, 91405 Orsay cedex France.
Peroxisomes play essential roles in cellular metabolism, their deficiencies are responsible for severe (and often lethal) human disorders. In the filamentous fungus Podospora anserina, we have previously observed that the peroxisome assembly pex2 mutants (Zellweger Syndrome in humans) are impaired in sexual differentiation (Berteaux-Lecellier et al., 1995). Moreover, we have shown that expression of the human ABC transporter PMP70 in a P. anserina pex2 mutant strain partially restores both peroxisome biogenesis and sexual differentiation (Boisnard et al., submitted). This result ascertains a role for peroxisomes in sexual development in this fungus. Interestingly, our study has also disclosed an unexpected detrimental defect of h PMP70cDNA expression in a wild-type background (pex2+): it leads to an abnormal distribution of both nuclei and peroxisomes during sexual differentiation. This last discovery prompted us to clone and sequence the two "expected" genes encoding peroxisomal ABC transporters in P. anserina. The experiments performed with the first peroxisomal ABC transporter pABC1, which display 35% identity with PMP70, confirm the results obtained with the human transporter. Further studies are in progress with the second gene pABC2 and should help to understand the involvement of peroxisomal ABC transporters in differentiation as well as their physiological role(s).
68. The CBEL glycoprotein of Phytophthora parasitica var. nicotianae is involved in cell wall deposition and cellulose sensing. E. Gaulin1,2, A. Jauneau1, F. Villalba3, Mt. Esquerre-Tugaye1, M. Rickauer1 and A. Bottin1. 1 Pôle de Biotechnologie Végétale,31326 Castanet-Tolosan, FRANCE. 2 Dept Plant Pathology, OARDC, 1680 Madison Avenue, Wooster, OH 44691-44096, USA. 3Bayer Cropscience,69263, Lyon, FRANCE
Phytophthora parasitica var. nicotianae, an oomycete pathogen of tobacco, produces a Cellulose-Binding Elicitor Lectin (CBEL) which is cell wall-localized and induces defense in plants. The biological role of CBEL in Phytophthora was investigated by examining the phenotype of strains where CBEL expression had been silenced by transformation. When grown on cellophane membranes, the silenced strains were strongly reduced in substrate adhesion, whereas the CBEL-expressing strains adhered strongly to the membrane, in contact with which they formed lobed hyphal structures. Electron microscopy observations showed abnormal paramural appositions in hyphae of the silenced strains ; furthermore these strains did not branch in contact to flax cellulose fibers, in contrast to CBEL-expressing strains. The data indicate that CBEL is involved in cell wall apposition in Phytophthora, in adhesion to cellulose and in differenciation of specialized structures in response to this polysaccharide (1). 1- Gaulin, E. et al. (2002). J Cell Science, 115, 4565-4575
69. Identification of photoreceptors that sense light and inhibit differentiation ofCryptococcus neoformans. Alexander Idnurm and Joseph Heitman. Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710.
Cryptococcus neoformans is a heterothallic basidiomycete yeast that infects animals, including humans. The infective particle of the fungus is hypothesized to be the basidiospore or spore resulting from haploid (monokaryotic) fruiting. Both mating and haploid fruiting are inhibited by light. This work aims at identifying the photoreceptor(s) involved in inhibition. Three genes (encoding a putative opsin, white collar 1 and phytochrome-like protein) have been identified as single copy genes in a serotype D MAT alpha strain of C. neoformans. The three genes have been mutated by targeted disruption, and mutants in theMATa mating type generated by crossing. Strains with two or three of the genes mutated are being constructed. The ability of these strains to mate and haploid fruit in the light compared to the dark, and the wavelength of inhibition, will be examined. Transcription of genes in the mating type locus and others identified through microarray analysis will also be examined in the wild-type and mutant strains grown in the light and dark.
70. The apsA and apsB genes of Aspergillus nidulans and cytoplasmic dynein. Vladimir Efimov. Department of Pharmacology, UMDNJ-RWJMS. Piscataway, NJ.
The apsA and apsB (anucleate primarysterigmata) mutants of A. nidulans have nuclear distribution defects that
resemble those in the cytoplasmic dynein mutants, but are less severe. The S. cerevisiae homolog of apsA, NUM1,
acts in the dynein pathway. The apsA gene interacts genetically with the nudF (PAC1, LIS1) gene of the dynein
pathway (V.P. Efimov, Mol. Biol. Cell, 2003). Here the relation between the apsA andapsB genes and dynein is
investigated by analysis of double mutants. Mutations in the apsA gene had no effect on the growth of dynein
deletion mutants. In contrast, mutations in the apsB gene suppressed dynein deletion mutants and improved nuclear
migration in their germlings. The suppression may stem from a defect in nuclear fixation that increases nuclear traffic
in the apsB mutants (R. Suelmann et al., Mol. Microbiol., 1998). The results indicate that APSB regulates nuclear
distribution independently of APSA and cytoplasmic dynein.
Financial Support: Scientist Development Grant from the American Heart Association.
71. Expression of a GFP-tagged histone H1 gene during ascus development in Neurospora crassa. N.B. Raju1, M. Freitag2 and R.L. Metzenberg1,3.1Stanford Univ, 2Univ Oregon, 3 UCLA.
A GFP-tagged histone H1 gene (hH1-GFP) under the control of the ccg-1 promoter was inserted at the his-3 locus (his-3+::hH1+-eGFP+), hereafter called H1-GFP. We have examined the expression of H1-GFP in developing asci of homozygous and heterozygous crosses. In homozygous crosses (H1-GFP x H1-GFP), nuclei fluoresce brightly from karyogamy until the production of mature, multinucleate ascospores. In heterozygous crosses (H1-GFP x wild type), expression of H1-GFP is completely silenced, at least until after ascospores are delimited. Silencing does not extend into the autonomously developing ascospores. Nuclei in four of the eight spores begin to fluoresce 18 to 24 h after ascospore delimitation. Fluorescence intensity increases for an additional 24 h as the ascospores form striations and begin to pigment. Additional mitoses occur in mature, black ascospores, and more than 60 nuclei can readily be seen in each of the four H1-GFP ascospores. The four ascospores without H1-GFP do not show fluorescence. Sad-1, which suppresses meiotic silencing of ectopically inserted genes (Shiu et al. 2001, Cell 107:905-916), also suppresses the silencing of H1-GFP in heterozygous asci. In H1-GFP x Sad-1 crosses, expression resembles that of a homozygous H1-GFP cross: Nuclei fluoresce until after ascospores are delimited. The residual H1-GFP in non-H1-GFP nuclei gradually fades while nuclei in the four ascospores that contain H1-GFP continue to fluoresce brightly. These observations provide a clear visual demonstration of meiotic silencing by unpaired DNA. (Support: NSF grant 9728675 to D. Perkins, NIH grant GM35690 to E. Selker and NIH grant GM08995 to RLM.)
72. Characterization of Putative Calcium Transport Proteins in the Vacuole of Neurospora crassa. Stephen Abreu, Emilio Margolles-Clark and Barry Bowman. University of California, Santa Cruz CA 95064
The vacuole stores high concentrations of calcium and may play a central role in regulating calcium levels within fungal cells. We are investigating the role of three proteins, CAX, NCA-2 and NCA-3, that are hypothesized to transport calcium across the vacuolar membrane. The genes encoding these proteins were identified by similarity to calcium transport proteins in other organisms. Analysis of the genes indicates that CAX encodes a 12-helix protein that facilitates the exchange of calcium for protons. NCA-2 and NCA-3 both appear to be homologs of the PMC1 gene of S. cerevisiae. These genes appear to encode P-type ATPases that pump calcium into the vacuole. It is not known why N. crassa has two of these genes whileS. cerevisiae has only one. To investigate the function of these genes we used the RIPing procedure to generate mutant strains. Inactivation of the cax gene produces no obvious growth phenotype, but the mutants are deficient in calcium transport into the vacuole when assayed in vitro. Mutant strains that lack the nca-3 gene also grow like the wild type. However, inactivation of thenca-2 gene causes slow growth and abnormal morphology. The cax nca-2 double mutant is even more impaired. Other multiple mutants are being generated. A preliminary interpretation of these results is that the cax and nca-2 gene products have partially redundant functions in the vacuole. The role of the nca-3 gene is unknown.
73. NPKA is a novel putative p34cdc2-related PITSLRE gene from Aspergillus nidulans, associated with the cellular DNA damage response. Gustavo H. Goldman, Marcela Savoldi, Maria Angela Castro Dani, Marcelo Afonso Vallim, Roy E. Larson1, Maria Helena S. Goldman2 FCFRP, 1FMRP, and 2FFCLRP, Universidade de São Paulo, Brazil
The DNA damage response is a protective mechanism that ensures the maintenance of genomic integrity during cellular reproduction. We have been using A. nidulans as a model system to genetically characterize the DNA damage response caused by the anti-topoisomerase I drug, camptothecin. Here, we report the molecular characterization of a novel p34cdc2-related PITSLRE gene, npkA, from A. nidulans. The PITSLRE kinases are a superfamily of protein kinases related to p34cdc2 that have been implicated in apoptosis. ThenpkA gene is transcriptionally induced by campthotecin and other DNA-damage agents, and its induction in the presence of camptothecin is dependent on the uvsBATR gene. Down-regulation of the npkA caused an increased induction of the uvsCRAD51, scaANBS1, and Anmre11 genes when A. nidulans is grown in the presence of camptothecin, suggesting that gene expression induced by DNA damage is dependent on the npkA gene. Furthermore, NPKA levels are apparently controlled by proteolysis when A. nidulans is grown in the presence of MMS. Our results suggest that the npkA gene may play a role in the DNA-damage signal transduction pathway in A. nidulans. Financial support: CNPq and FAPESP, Brazil
74. Different roles of the mre11 complex in the DNA damage response in Aspergillus nidulans. Gustavo H. Goldman, Camile P. Semighini, Márcia R. V.Z. Kress Fagundes, Joseane C. Ferreira, Renata C. Pascon, Maria Helena S. Goldman1FCFRP and 1FFCLRP, Universidade de São Paulo, São Paulo, Brazil
The hMRE11-hRAD50-NBS1 protein complex has emerged as a central player in the cellular DNA damage response. Mutations in scaANBS1, which encodes the apparent homolog of human nibrin in A. nidulans, inhibit growth in the presence of anti-topoisomerase I drug camptothecin. We have used the scaANBS1cDNA as a bait in a yeast two-hybrid screening and report the identification of the A. nidulans Anmre11 homologue. The inactivated Anmre11 strain (TMRE11) was more sensitive to several DNA damaging and oxidative stressing agents. Septation in A. nidulans is dependent not only on the uvsBATR gene but also on the mre11 complex. scaANBS1 and Anmre11 genes are both involved in DNA replication checkpoint while Anmre11 gene is involved in the intra-S-phase checkpoint. SCAANBS1 and ANMRE11 also participate in G2-M checkpoint upon DNA damage caused by 4-NQO. Homologous recombination is affected by the scaA1 mutation since this mutant does not have the ability of integrating exogenous DNA introduced by DNA-mediated transformation in a homologous way. The scaANBS1 gene is important for ascospore viability while Anmre11 gene is essential for the sexual fertility in A. nidulans. In addition, the mre11 complex and uvsCRAD51 genes are highly expressed at mRNA level during the sexual development. Financial support: CNPq and FAPESP, Brazil
75. The new subfamily of CuZn Superoxide Dismutase is involved in Branching and Hyphal Growth in Candida albicans. Mikhail Martchenko, Doreen Harcus, and Malcolm Whiteway. Biotechnology Research Institute (NRC), Montreal, Canada.
Candida albicans is an opportunistic human pathogen that can cause life-threatening systemic infections in immunocompromised. It is possible that in order to protect against damaging superoxide radicals produced in the phagosome C. albicans express different classes of superoxide dismutases (SOD). Superoxide dismutase converts superoxide radicals into less damaging H2O2. C. albicans is known to express Cu/ZnSOD (SOD1) and MnSOD (SOD3) in the cytosol and MnSOD (SOD2) in the mitochondria. Through the use of cDNA Microarray technology and the sequence of C. albicans genome we report the existence of three additional Cu/Zn superoxide dismutases, CaSOD4, CaSOD5 and CaSOD6. The transcription of CaSOD5 is upregulated in yeast to hyphal transition of C. albicans, which is thought to be involved in the pathogenesis of this organism.CaSOD5 is highly expressed when C. albicans cells are challenged with osmotic or with oxidative stresses at 30C but not at 37C. Efg1 and Rim101 transcription factors are involved in CaSOD5 regulation. Deletion of CaSOD5 produced a viable mutant strain, which showed the same resistance to macrophage attack as its parental strain. CaSOD5 mutant strain showed a temperature sensitive lysine auxotrophy, andCaSOD5 was found to be involved in hyphal branching and in hyphal growth. This work defines a new subfamily of CuZn superoxide dismutases, and characterizes a member that is primarily involved in morphogenesis, rather than in defense mechanisms of C. albicans.
76. Calcium mobilization to cell wall in Neurospora crassa. P.Maruthi Mohan and Naveena Lavanya Latha, Department of Biochemistry, Osmania University, Hyderabad – 500 007 (A.P.), India
Calcium was found in significant quantities (25%) on mycelial surface of N. crassa. Removal of surface-bound calcium by EGTA resulted in complete replacement until 75% of the intracellular calcium pool is depleted. Loss of intracellular calcium pool was observed in vacuolar and mitochondrial fractions. A 60% decrease in calcium mobilization was observed with metabolic inhibitor (azide), while channel blockers; verapamil and nifidipine inhibited this almost completely. Concanamycin-A, a specific inhibitor of vacuolar ATPase inhibited Ca mobilization by more than 90%. Neomycin a general inhibitor of IP3 signal transduction pathway also inhibited Ca mobilization completely. Vacuolar mutants of N. crassa (vma-5) with defect in Ca transport failed to mobilize Ca on to surface. Toxic metal ion bound to surface by proportionally displacing Ca and Mg ions. Scanning Electron microscopy showed gross structural changes on hyphal surfaces of EGTA treated mycelia as compared to controls. Transmission EM pictures showed increased number, size and fusion of vacuoles. Periplsamic marker enzymes (invertase and alkaline phosphatase) and small molecular weight cell wall proteins/peptides were released when mycelia were treated with EGTA. The implications of Ca mobilization on to the cell wall and its relation to toxic metal ion interactions at both surface and possible role of Ca in maintenance of cell wall structure will be discussed. Acknowledgments: The work was supported by grants from the Department of Science & Technology(PMM)and UGC-SAP(DRS-I)
77. Regulation of Asynchronous Mitoses within a Multinucleated Cell. Amy S. Gladfelter, Katrin Hungerbuehler, Hans Peter Helfer, and Peter Philippsen. Molecular Microbiology, University of Basel Biozentrum, Basel, Switzerland.
We use the filamentous fungus, Ashbya gossypii, as a model organism to study the regulation of nuclear division in multinucleate cells. In this organism, mitosis occurs asynchronously such that only 1 or 2 nuclei divide in a cell compartment with 6 or 7 nuclei. We have generated GFP labels to visualize nuclei, spindle pole bodies, and septins in live cells to analyze the dynamics and regulation of asynchronous mitotic events. Using these tools, we have determined that: 1.) All nuclei have the capacity to divide, 2.) Neighboring nuclei reside in different cell cycle stages, and 3.) Mitotic frequency is highest at branch points. Furthermore, we have shown that septin proteins are arranged in elongated bar structures rather than rings as observed in other fungi. Interestingly, Swe1p, a wee1 homologue, may function to maintain this asynchronous nuclear division because swe1 mutants display increased nuclear density. We speculate that Swe1p is locally stabilized due to the distinct structure of the septin scaffold in these cells.
78. Requirements for formation of meiotic DNA double-strand breaks in Coprinus cinereus. Alexander M. Many, Sonia Acharya, and Miriam Zolan. Biology, Indiana University, Bloomington, IN
We are using the basidiomycete Coprinus cinereus to investigate genes that play a role in meiosis and DNA double strand break (DSB) repair. Coprinus features a naturally synchronous progression through the early stages of meiosis. We have identified a series of genes, mutant strains of which are sensitive to ionizing radiation and fail to complete meiosis. When cloned, two of these genes were found to encode members of the Mre11 complex, Mre11 and Rad50. This complex has been shown to be important for numerous activities in which an exposed DSB is present. This includes repair of DSBs after ionizing radiation, and the formation and processing of the meiotic DSB. Meiotic recombination requires a programmed DSB that has been found in Saccharomyces cerevisiae to be created by Spo11. We have cloned the Coprinus spo11 gene, and isolated a mutant, spo11-1, which encodes a truncatedspo11 gene product lacking four of the five conserved domains. A spo11-1 strain fails to complete meiosis, but progression through meiosis can be rescued by ionizing radiation-induced DSBs. The rad50-4 strain Coprinus of undergoes an arrest in the diffuse diplotene stage of meiosis. The spo11-1 strain of Coprinus exhibits a programmed cell death phenotype. A double mutant was made using these two strains, and it exhibits a spo11-1 like phenotype. Upon irradiation in early prophase the double mutant reverts to a rad50-4 like phenotype. This indicates that the formation of a meiotic DSB by Spo11 does not require wild -type Rad50 protein. To complement these studies anmre11-1;spo11-1 double mutant is being constructed. A mre11-1 strain of Coprinus undergoes meiotic arrest at a condensed metaphase I-like state, enabling an analysis similar to that performed for the rad50;spo11 double mutant. Co-immunoprecipitation of the Mre11 complex and immunohistological studies of Mre11, Rad50, and Spo11 will also be carried out to gain further insight into the early meiotic roles of these genes.
79. The snxA1 gene of Aspergillus nidulans affects the DNA damage checkpoint. Sarah Lea McGuire, Jim Goode, Allison McElvaine, and David Norris. Millsaps College, Jackson, MS, USA.
ThesnxA1 mutation of A. nidulans was identified as an extragenic suppressor of the nimX2cdc2 mutation. In addition to this suppression, snxA1leads to a late G1/early S arrest at 20C. Protein kinase assays and western blots suggest thatsnxA1 does not suppress the nimX2 mutation by increasing the activity or protein level of NIMX. Double mutant analysis has shown that snxA1 causes a synthetic lethal phenotype in combination with a deleted ankAwee1 and partially suppresses the nimT23cdc25 mutation. To determine if snxA1 is involved in the DNA damage checkpoint response, we germinated snxA1 mutant cells and then exposed them to 0.02% MMS at 32C and 44C to induce DNA damage. Under these conditions,snxA1 mutants had a significantly elevated mitotic index, similar to checkpoint-deficientbimE7 cells: at 32C, 28% of snxA1 cells and 29% of bimE7 cells were in mitosis, and at 44C 47% of snxA1 cells and 55% of bimE7 cells were in mitosis. In both cases, 0% of wild-type cells were in mitosis. These data suggest that snxA1 leads to a DNA damage checkpoint defect. Western blots indicate that the level of Tyr15 phosphorylation of NIMX is decreased in snxA1 cells compared to wild type cells exposed to MMS, suggesting that the checkpoint defect caused by snxA1 ultimately functions through Tyr15 phosphorylation of NIMX. Supported by NIH R15GM55885.
80. Functional analysis of the Aspergillus nidulans HbrB protein. Gatherar, I. M.1, Pollerman, S.1, Dunn-Coleman, N.2 and Turner, G.1 1Department of Molecular Biology and Biotechnology, University of Sheffield, UK. 2Genencor International, Palo Alto, CA. US.
An orphan gene specific to the filamentous fungi, hbrB, has been characterised. The gene was isolated by complementation of a temperature sensitive allele, hbrB3, which results in a hyperbranching phenotype. Though absent from yeasts, HbrB has apparent homologues in N. crassa and A. fumigatus (49% and 67% identity respectively). In order to study the gene function, a promoter exchange with the conditionally expressed gene,alcA, was undertaken. The alcA promoter is induced by ethanol, ketones and threonine and is repressed by glucose. Approximately 1 Kb of the 5' end of hbrB was cloned into the alcA expression vector, pAL3, which was used to transform wild type A. nidulans (1). A Southern blot to confirm correct integration was carried out on the strains with a regulatable phenotype on glucose and threonine. One strain appeared to have the correct integration pattern. Downregulation of this promoter replacement strain gives a loss of polarity phenotype, which is different from that of the temperature sensitive phenotype. A sexual cross between the promoter replacement strain (alcA::hbrB) and the temperature sensitive mutant (hbrB3) showed that there was no recombination between hbrB and hbrB3 indicating that both alleles are at the same locus. To determine cellular localisation of the hbrB product, the gene will be fused with the GFP (Green Fluorescent Protein) gene under control of the alcA promoter. This vector will be used to transform the hbrB3 temperature sensitive strain in order to visualise the location of HbrB, and to confirm function of the GFP-HbrB fusion product through growth at restrictive and permissive temperatures. References
1. Waring, R. et al. (1989). Characterisation of an inducible expression system in Aspergillus nidulans using alcA tubulin-encoding genes. Gene, 79, 119-130.
81. Cell wall remodeling in response to cell wall stress in Aspergilus niger. R. A. Damveld1, M. Arentshorst1, F.M. Klis3, C.A.M.J.J. van den Hondel1,2 and A.F.J. Ram1,2.1)Leiden University, IMP, Leiden, The Netherlands, 2)TNO-Nutrition, AMGT, Zeist, The Netherlands, 3) Fungal Research Group, University of Amsterdam, Amsterdam, The Netherlands.
The fungal cell wall is a highly dynamic structure: both its composition and architecture respond to internal and external stimuli to ensure the integrity of the cell wall. Using Aspergilus niger as a model fungus we have studied how the fungus responds to the presence of an antifungal compound, Calcofluor White, which interferes with normal cell wall assembly. The addition of Calcofluor White (CFW) to just germinated spores resulted in a retardation of growth and swelling of hyphal tips. After 2 to 4 hours, depending on the concentration CFW used, growth resumed. Using PCR, we have isolated several A. nigergenes that encode proteins involved in cell wall biosynthesis. Northern analysis indicated that the mRNA level of agsA, encoding an alpha-1,3-glucan synthase, and gfaA, encoding the glutamine:fructose-6-phosphate aminotransferase enzyme involved in de biosynthesis of UDP-N-acetyl-glucosamine, were induced 20-fold an 4-fold respectively. This finding suggests important roles for the cell wall polymers alpha-1,3-glucan and chitin in response to cell wall stress to ensure cell wall integrity.
82. Characterization of genes involved in the histidine kinase TcsB-HogA MAPK cascade inAspergillus nidulans. Kentaro Furukawa, Keietsu Abe, Youhei Yamagata, and Tasuku Nakajima. Department of Molecular and Cell Biology, Graduate School of Agricultural Sciences, Tohoku University.
We have cloned and characterized a novel Aspergillus nidulans histidine kinase gene, tcsB, encoding a membrane-type two-component signaling protein homologous to the yeast osmosensor Sln1p, which transmits signals through the Hog1p-MAPK cascade in yeast in response to osmotic stimuli. Overexpression of the tcsB cDNA suppressed the lethality of the temperature-sensitive osmosensing-defective yeast mutant sln1-ts. However, tcsB cDNAs in which the conserved phosphorylation site His552 or phosphorelay site Asp989 had been substituted failed to complement the sln1-ts mutant. Introduction of the tcsB cDNA into the yeast double mutantsln1-delta sho1-delta, which lacks two osmosensors, suppressed lethality in high-salinity media and activated the HOG1 MAPK. These results imply that TcsB functions as an osmosensor histidine kinase. We constructed an A. nidulans strain lacking the tcsB gene (tcsB-delta) and examined its phenotype. However, unexpectedly, the tcsB-delta strain did not exhibit a detectable phenotype in either hyphal development or morphology on standard or stress media. The result suggests that A. nidulans has more complex and robust osmoregulatory systems than the yeast SLN1-HOG1 MAPK cascade. Functional analysis of other histidine kinase genes homologous to yeast YPD1 and SSK1 will be also discussed.
83. Characterization of Aspergillus nidulans alpha-subunits of heterotrimeric G-proteins and their role in the control of conidial germination and cAMP signaling. Anne Lafon(1), Mi-Hee Chang(2),Patrick van Dijck(3), Johan Thevelein(3), Kwang-Yeop Jahng(2), Christophe d'Enfert(1). (1)Institut Pasteur, France, (2) Chonbuk National University, South Korea, (3)K.U. Leuven, Belgium
In an effort to identify the cellular components involved in the activation of adenylate cyclase at the onset of A. nidulans condial germination, we have focused our interest on the role of 3 Galpha proteins, FadA, GanA and GanB. Phenotypic analyses have been performed with fadA, ganA and ganB null mutants and show that only the ganB mutant is defective for trehalose breakdown and for the kinetics of germ tube formation, suggesting a direct role of GanB in the activation of adenylate cyclase. Attempts to complement through expression of ganA, ganB or fadA the defects that are observed in a S. cerevisiae strain with a null mutation in the GPA2 gene which encodes the Galpha subunit responsible for activation of adenylate cyclase in response to glucose were unsuccesful despite, in particular, the close relationship of GanB and Gpa2. We suggest that this inability of GanB to fulfill the functions of Gpa2 results from a defect in the interaction with either adenylate cyclase or the S. cerevisiae G-protein coupled receptor Gpr1 which acts upstream of Gpa2. Experiments aimed at testing this hypothesis will be presented. Physical interactions between GanB and adenylate cyclase are currently analyzed using two-hybrid experiments involving GanB, adenylate cyclase and a putative cyclase associated protein.
84. Cloning and characterization of two nucleotide excision repair genes, ncRAD10 and ncRAD14, in Neurospora crassa. Akihiko Ichiishi, Masahito Sato, Takaharu Niki, and Makoto Fujimura. Dept of Life Sciences Toyo University, Izumino, Japan.
In Neurospora crassa, nucleotide excision repair (NER) mechanism has not been well characterized in molecular level. To study NER mechanism in N. crassa, we tried to clone Neurospora genes involved in NER. In searching the genome database of N. crassa, several genes which homologues to Saccharomyces cerevisiae and Schizosaccharomyces pombe NER genes were found. Two genes encoding NER genes homolog, ncRAD10 and ncRAD14, were isolated by PCR. The amino acid sequence of ncRad10p showed 48% of identities to the S. pombe Swi10 protein which was homologues to S. cerevisiae Rad10p and human ERCC1 protein. The predicted amino acid sequence of ncRad14p showed 33% homology to S. cerevisiae Rad14p with a well conserved zinc finger motif. To characterize the function of ncRAD10 and ncRAD14, we disrupt these two genes by the RIP. Both RIP mutants showed sensitivity to UV light and 4-NQO but not to MMS on a spot test. This phenotype is similar to that ofmus-38 mutant which is defective in NER in N. crassa.
85. Interaction of mitochondria with microtubules in the filamentous fungusNeurospora crassa. Florian Fuchs, Walter Neupert and Benedikt Westermann. Institute for Physiologcal Chemistry, Ludwig-Maximilians-University Munich,
The establishment and maintenance of the three-dimensional structure of eukaryotic cells depends on active transport and positioning of organelles along cytoskeletal elements. The biochemical basis of these processes is only poorly understood. We analysed the interaction of mitochondria with microtubules in the filamentous fungus Neurospora crassa. Mitochondria were fluorescently labelled by expression of matrix-targeted green fluorescent protein (mtGFP). Upon isolation, mitochondria collapsed to round spherical structures that were, however, still able to interact with microtubules in vitro. Binding of mitochondria to microtubules was dependent on peripherally associated proteins on the organellar surface, and was sensitive to adenine nucleotides. MMM1, a mitochondrial outer membrane protein important for maintenance of normal mitochondrial morphology, was not required. This suggests that the interaction of mitochondria with the cytoskeleton is independent of MMM1. Furthermore we detected a novel kinesin-related protein on mitochondria and outer membrane vesicles, which might be a mediator of the interaction. Taken together we conclude that mitochondrial morphology is maintained by a complex interplay of extrinsic and intrinsic factors, including ATP-dependent proteins on the organellar surface.
86. PHOA interacting proteins are involved in regulation of development in Aspergillus nidulans. Dongliang Wu, Shahr B. Hashmi, Xiaowei Dou, and Stephen A. Osmani Department of Molecular Genetics, The Ohio State University, Columbus, OH43210, USA
PHOA was identified as a non-essential cyclin-dependent kinase which controls the developmental program of Aspergillus nidulans in an environment dependent manner (Bussing and Osmani, EMBO J 1998 17, 3990). Four genes interacting with PHOA have been isolated by a yeast two-hybrid system and have been provisionally named pipA ̈C pipD (PHOA interactive protein A ̈C D). In vitro transcription, translation and coimmunoprecipitation results confirmed the physical interactions between PHOA and PIPA, PIPB, PIPD proteins. We used the pyroA marker gene to replace the four structural genes and got the knockout mutant of pipA and pipB which were viable indicating that these genes are not essential. Deletion of pipA (which encodes a potential transcription factor) caused some phenotypes similar to that caused by deletion of phoA, suggesting pipA may be involved in regulating development downstream of phoA. pipB encodes a PCL-like cyclin and its deletion caused increased conidiation but lack of sexual sporulation when compared to wildtype or phoA deleted strains when grown under different growth conditions. The data indicate that this PCL-like cyclin protein may repress asexual development but be necessary for sexual development. Deletion of pipC could not be achieved in normal haploid cells but could be maintained in heterokaryons carrying some nuclei with deleted pipC and others with wild type pipC. Conidia generated from these heterokaryons were unable to germinate under selective conditions indicating that deletion of pipC causes lethality and an inability to germinate. Sequence analysis indicates that pipC encodes a recently isolated helix-loop-helix transcription factor termed AnBH1 (Caruso et al., 2002 J. Mol. Biol. 323, 425). Finally, pipD encodes another cyclin like protein with similarity to yeast Pho80 and this gene has not been successfully deleted yet. As expected, we have been able to identify cyclin-like partners for the PHOA cyclin dependent kinase and also potential transcription factors with which PHOA may interact. Importantly, deletion analysis indicates that some of these genes play roles during development. Further analysis of PHOA and its interactive proteins should shed further light on how the environment can modify development in multicellular eukaryotes.
87. Localization of recombinant gene products in mycelial fungi by means of egfpreporter plasmids. Mayrhofer S, Hoff B, Masloff S, Kück U, Pöggeler S. Department for General and Molecular Botany, Ruhr-University Bochum, 44780 Bochum, Germany
In recent years, the gfp gene encoding the green fluorescent protein (GFP) has become an important reporter gene for in vivo studies. The development of the enhanced gfp gene (egfp), which is adapted to altered codon usage in mammals, make the efficient expression of this reporter gene in ascomycete fungi possible. Here, we describe the construction and application of a series of plasmids, which support the expression of the egfp gene in the two ascomycetes Sordaria macrospora and Acremonium chrysogenum. The vectors assist the study of diverse developmental processes. The plasmids presented here, include a promoterless egfp vector for monitoring the expression of cloned promoters/enhancers in fungal cells, and vectors for creating translation fusions to the N-terminus of EGFP. The vectors were further modified by introducing a variant hygromycin B phosphotransferase (hph) gene, lacking the commonly found NcoI site. Instead, the single NcoI site, containsing an ATG start codon, can be used for cloning translational fusions in front of the egfp gene. The applicability of these vectors is demonstrated by analysing transcription regulation as well as protein localization and secretion in the two ascomycetes Acremonium chrysogenum and Sordaria macrospora. In the latter, the heterologous egfp gene is stably inherited during meiotic divisions as can easily be seen from fluorescent ascospores.
88. The role of myosin I during hyphae formation in C. albicans. U. Oberholzer, T. Iouk and M. Whiteway. Biotechnology Research Institute, Montreal, Canada.
Myosin I is required for hyphal formation in the pathogenic yeast Candida albicans, due to its role in organizing the cortical actin patches and hence its role during endocytosis. We have investigated the role of myosin I tail domains and found that the putative membrane binding domain TH1 and the calmodulin interacting IQ motifs are required for optimal vegetative growth, for hyphae formation and for fluid phase endocytosis. Deletion of these domains allows the formation of pseudohyphae but not of hyphae. Moreover, deletion of either domains leads to depolarized cortical actin patches in these pseudohyphal structures and the corresponding myosin-GFP fusion proteins are mislocalized, i.e. they are not found in patches at the pseudohyphal tip. In contrast, we found that the SH3, acidic A- or ATP independent actin binding domain TH2 are not required for the formation of hyphae. Consistent with this observation, the actin cytoskeleton of these hyphae is not affected by the deletion of the SH3 or A- domains, and the localization patterns of the corresponding myosin-GFP fusion proteins appear normal. Surprisingly, cortical actin patches are depolarized in the hyphae where the TH2 domain of myosin I is deleted. Moreover, fluid phase endocytosis is defective in this mutant suggesting that defective and depolarized cortical actin patches are not essential for hyphae formation. Finally, in all of these mutants except in those where the SH3 or A domains have been deleted, Arp3-GFP patches are depolarized, i.e. are not localized in the hyphal tips. We propose a model for how hyphae formation in C. albicans is dependent on myosin I.
89. A role for the Ubiquitiin Conjugating Enzyme, Ubc4, in G2 and Mitosis in Aspergillus nidulans. Peter Mirabito, Kuttalaprakash Chudayalandi, and Alberto Ribes-Zamora. Dept of Biology, University of Kentucky, Lexington, KY.
The long-term goal of this research is to determine the mechanisms regulating mitosis in Aspergillus nidulans. Previous work demonstrated that Aspergillus Anaphase-Promoting Complex (aka Cyclosome or APC/C) has a novel role in G2 in addition to its well-conserved function in mitosis and G1. As part of our investigation of APC/C regulation in G2, we have identified a recessive lethal mutation in nadH, the A. nidulans UBC4 gene, which phenocopies APC/C mutations in G2 and mitosis. nadHUBC4 is required for chromosome segregation but not chromosome decondensation, whereas APC/C is required for both. This suggests that NadHUbc4 acts with APC/C in G2 and in chromosome segregation, whereas another ubiquitin-conjugating enzyme acts with APC/C in chromosome decondensation. These results suggest ubiquitin-conjugating enzymes may play a role in the specificy of APC/C function.
90. Depletion of a polo-like kinase in Candida albicans activates hyphal-like growth and transcription. Catherine Bachewich (1), David Thomas (2), Malcolm Whiteway (3). (1) Health Sector, Biotechnology Research Institute (BRI), National Research Council of Canada (NRC), Montreal, Canada; (2) Department of Biochemistry, McGill University, Montreal, Canada; (3) Health Sector, BRI/NRC, Montreal, Canada
Morphogenesis in Candida albicans is an important virulence-determining factor, since the environmentally-stimulated switch between yeast and hyphal growth can increase pathogenesis. We identified CaCDC5, a cell cycle regulatory polo-like kinase inCandida albicans, and demonstrate that shutting off its expression induced cell cycle defects and dramatic changes in morphology. Cells lacking CaCdc5p were blocked with short spindles and unseparated chromatin. GFP-tagged CaCdc5p localized to unseparated spindle pole bodies, the spindle, and chromatin, consistent with a role in spindle formation. The cell cycle defects were accompanied by the formation of hyphal-like filaments under yeast growth conditions. Similar spindle defects and a corresponding induction of filaments occurred when yeast cells were exposed to hydroxyurea, suggesting a link between spindle function and filament formation. The filaments resembled serum-induced hyphae with respect to many cytological features, and microarray analyses demonstrated that the filaments expressed several factors normally modulated in serum-induced hyphae. Filament formation required CaCdc35p, but not Efg1p or Cph1p, of the hyphal-signaling pathways. Thus, an internal cell cycle-related cue can activate hyphal regulatory networks in Candida.
91. Cdc42 regulates cytokinesis and morphology in Ustilago maydis. Michael Mahlert, Leonora Leveleki, Björn Sandrock and Michael Bölker, Dept. of Biology, University of Marburg, Karl-von-Frisch-Strasse 8, D-35032 Marburg, Germany,
Small ras-like GTPases are known to act as molecular switches regulating diverse processes such as cytoskeleton organization, cell cycle and vesicular traffic. In U. maydis, the GTP binding protein Cdc42 plays a central role in regulating cytokinesis and cell polarity. A signalling module triggers cell separation and consists of a Rho/Rac-GEF (Don1), which activates Cdc42, and a Ste20 like kinase (Don3), which is likely to be a target for Cdc42. We could show that expression of the dominant active version Cdc42-Q61L rescues the cytokinesis defect of don1 mutants but not that of don3 confirming the role of Cdc42 in this signalling pathway. Expression of dominant negative Cdc42-T17N in wild type cells leads to deformation of cells and lateral budding. This demonstrates that Cdc42 plays an additional role in polarized growth of U. maydis. We could identify another Ste20 like kinase, Cla4, which regulates in a parallel Cdc42 containing pathway polarized growth and septum formation. We were able to generate a deletion mutant for cdc42, which surprisingly is viable. Depending on the genetic background, the mutants exhibit either only a cell separation defect or additional morphological aberrations. Since the phenotype of a conditional cdc42 mutant strain is less pronounced, even a low level of Cdc42 expression may be sufficient to maintain a proper cell shape.
92. Cell wall synthesis in Aspergillus nidulans. Gerald Hofmann1, Mhairi McIntyre1, and Jens Nielsen1. 1Center for Process Biotechnology, BioCentrum-DTU, Denmark.
Since the beginning of fungal research the cell wall of fungi has been subject of many studies. Despite these efforts, our knowledge about the synthesis and the regulation of its composition is still incomplete. One important step in the synthesis of cell wall compounds is the formation of the precursor UDP-glucose, since this metabolite is used in the anabolism of glucans, and as glucosyl donor for the glycosylation of proteins incorporated in the cell wall. Furthermore this step is involved in the catabolism of galactose, and the formation of trehalose. Therefore the gene ugpA, encoding the enzyme UDP-glucose pyrophosphorylase (EC 184.108.40.206), was cloned from Aspergillus nidulans and characterised. The derived protein sequence comprises 514 amino acids and shows a very high degree of conservation to other eukaryotic UDP-glucose pyrophosphorylases. In silico promotor analysis revealed several putative regulatory elements, including binding sites for CreA, PacC, and one site that might be recognised by a homologue of the Gal4 protein from S. cerevisiae. Northern blott analysis was carried out to further investigate possible regulation through these sites. These results and further characterisation of the gene and its role in the cell wall metabolism, which are currently underway, will be presented.
93. The kinesin motor KipA is required for polarized growth in Aspergillus nidulans. Sven Konzack, and Reinhard Fischer, Max-Planck-Institute for terrestrial Microbiology, Karl-von-Frisch-Str., D-35043 Marburg, Germany
Motor proteins are key factors in various dynamic processes in eukaryotic cells. To analyze organelle movement over long distances, we are studying the function of different kinesins inA. nidulans. So far two mitotic kinesins, BimC and KlpA were described. Taking advantage of the genomic DNA sequencing project at Cereon Genomics, gene fragments corresponding to four new kinesins, designated KifA, KipA, KipB and KinA, were identified. The further results obtained for KipA will be presented here. KipA displayed highest homology to KIF3C from rat, which is involved in vesicle transport, and to Kip2p that is involved in nuclear migration and spindle orientation. KipA was predicted to form a dimer, and a KipA-GFP fusion protein was predominantly found in the cytoplasm. After ATP-depletion of the cell (uncoupling with CCCP), a fraction of KipA localized to cytoplasmic microtubules. In order to assign a function to KipA, we deleted the motor domain of KipA from the genome of A. nidulans and studied the dynamics of microtubules, mitochondria and nuclei by GFP technology. We did not detect any differences in those processes in comparison to wt-strains. However, we observed a strong effect on polarized growth. Whereas the radial colony extension rate appeared similar to wild type, the directionality of the hyphae in kipA mutants was dramatically changed. Hyphae displayed a meandering growth phenotype, suggesting an effect of the kinesin deletion on the positioning of the Spitzenkorper. To further analyse the roles of KipA and possible overlapping functions with other motors, we constructed double and multiple mutations of KinA, KipA, KipB and Dynein. The analyses of their phenotypes are on the way.
94. Oxidative stress-induced Ca2+-signaling in Aspergillus nidulans may be required for survival. Diana Bartelt, Mitra Singh, and Vilma Greene; Department of Biological Sciences, St. John's University, Jamaica, NY 11439.
The effects of oxidative stress on levels of calcium ion (Ca2+) in Aspergillus nidulans were measured using strains expressing aequorin in the cytoplasm (Aeqcyt mt) [Greene, V., H. Cao, F. Schanne, and D. Bartelt. Cell. Signal. 14:437-443 (2002)]. When oxidative stress was induced by exposure to 10 mM H2O2, the mitochondrial calcium response (Ca2+mt) was greater than the change in cytoplasmic calcium (Ca2+c)and was dose-dependent,while the increase in [Ca2+c] did not change with increasing [H2O2]. Ruthenium red (RR) blocked the increase in [Ca2+mt]. Eighteen hour cultures of A. nidulans survived 30 min exposure to 100 mM H2O2 and, following exposure, conidiated normally when grown overnight in YG medium. Treatment with RR alone had no effect on growth and development. Pretreatment with RR prior to 100 mM H2O2 exposure, severely inhibited growth and conidiation. Cytoplasmic and mitochondrial fractions were prepared from cells exposed to H2O2,without and with RR pretreatment. ATP levels decreased, and isocitrate dehdrogenase (IDH) activity was induced in extracts of cells exposed to H2O2 alone. Pretreatment with RR decreased the loss of ATP and inhibited the activation of IDH. (Supported by NIGMS R15 GM52630)
95. Functional analysis of pre-1, a pheromone receptor gene of Neurospora crassa. Hyojeong Kim and Katherine Borkovich. Department of Plant Pathology, University of California, Riverside, CA 92321
Two putative pheromone receptor genes for the heterothallic ascomycete N. crassa, pre-1 and pre-2, were identified during BLAST searches against the entire genome database at the Whitehead Institute Center for Genome Research. The encoded proteins belong to the seven-transmembrane class of G-protein-coupled receptors and show significant sequence similarity to the a-factor receptor Ste3p and alpha-factor receptor Ste2p of Saccharomyces cerevisiae, respectively. Northern analysis showed that the genes were preferentially expressed under mating conditions in a mating type-specific manner. Delta pre-1 mutants have been isolated and characterized in both mating types. They displayed normal vegetative growth, including hyphal fusion. During sexual development, they underwent normal protoperithecial formation and were fertile as males. However, mat A pre-1 strains were completely unable to initiate mating as females in crosses. The female sterility of the mat A pre-1 mutant could be complemented in trans by wild type pre-1+, but not by an activated allele of gna-1. The levels of all G protein subunits were unaffected in mat A pre-1 strains. Thus, the pheromone receptor gene, pre-1, is essential for the fertilization of mat A strains, consistent with a role in initiating the pheromone response signaling pathway.
96. Mutations in snoA and snoB(suppressor-of-nimO) act through different mechanisms to rescue defects in the initiation of DNA synthesis. Steven W. James1, Matthew J. O'Connell2, Syef M. Hoque1, and Brian P. McCarthy1. 1Department of Biology, Gettysburg College, Gettysburg, PA USA. 2Peter MacCallum Cancer Institute, East Melbourne, AUS.
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 unwinding through phosphorylation of DNA helicase subunits. Several approaches are underway to investigate nimO andcdc7 function in Aspergillus. For example, cdc7asp was isolated and, by two-hybrid analysis, cdc7pasp was shown to physically associate with nimOp. Additionally, we generated partial nimO18 suppressors in two loci, snoA and snoB (suppressor-of-nimO). snoA/Balleles partially alleviate the temperature sensitivity of nimO18 by restoring growth at 37oC but not 43oC. snoA31 and snoB59 do not interact genetically with ts-lethal mutations in any of 20 cell cycle control genes in the nim (never-in-mitosis) and bim (blocked-in-mitosis) collection. Thus, the suppressors appear to modify only nimO activity or function. The suppressors were tested in an ethanol-dependent, glucose-inviable strain carrying alcA::nimO as the sole nimO gene copy. Recessive snoA alleles rescued growth on glucose, suggesting that snoA mutations may bypass the requirement fornimO. However, by using a parasexual assay we found that instead of bypass, snoA alleles act by stabilizing or enhancing nimOp produced during leakyalcA::nimO expression on glucose. Conversely, semi-dominant snoB alleles could not rescue growth on glucose, suggesting that snoBp and nimOp may associate directly. This hypothesis is supported by the discovery that snoB59 harbors a missense mutation in the cdc7asp gene. Sequencing of cdc7asp from additional snoB alleles is underway to determine if snoB =cdc7asp. (Supported by NSF-RUI# 01-14446 and by a Research and Professional Development Grant from Gettysburg College).
97. A Neurospora mannosyltransferase required for normal morphology and anastomosis. Amy Marie Piwowar, Shaun M. Bowman, Maria Ciocca, Mashel S. Al Dabbous, Gregory O. Kothe and Stephen J. Free. Dept. of Biol. Sciences, SUNY University at Buffalo, Buffalo, NY.
We have isolated and characterized three mutants affected in a cell wall biosynthetic mannosyltransferase. The levels of cell wall mannose and galactose are reduced by approximately 50% in the mutant cells. The mutant cells grow with an altered hyphal morphology which gives rise to a colonial growth pattern. The mutants are unable to produce normal macroconidia. We have found that the mutants are also affected in the ability to undergo anastomosis (hyphal fusion). The results demonstrate that the mannogalactan portion of the Neurospora cell wall is required for normal growth, development and cell fusion events.
98. The nuclear pore complex component SONB is involved in mitotic regulation and a DNA damage response pathway in Aspergillus nidulans. Colin De Souza and Stephen Osmani. Department of Molecular Genetics, Ohio State University, 802 Riffe Building, 496 W 12th Ave Columbus OH 43210.
The NIMA kinase is essential for mitotic entry in Aspergillus nidulans and is required for nuclear localization of mitotic regulators. We have isolated two nuclear pore complex (NPC) proteins as suppressors of the nimA1 temperature sensitive allele which normally arrests with uncondensed DNA, interphase microtubules and cytoplasmic NIMA. These proteins, SONA and SONB, physically interact and are apparent at the nuclear periphery during interphase but remarkably disperse upon mitotic entry before localizing once again at the nuclear periphery during mitotic exit. We also find that NIMA itself localizes to the nuclear periphery during mitotic entry and that expression of dominant negative NIMA constructs enhances this localization. We propose NIMA regulates mitotic entry by promoting SONA and SONB dissociation from the NPC (see also De Souza and Osmani Wed Mar 19). SONB is a member of the human NUP98/NUP96 family of nucleoporins. Interestingly, at 42oC, the sonB1 allele also displays a remarkably high sensitivity to agents which induce double strand DNA breaks, but less sensitivity to DNA damage induced by UV irradiation which does not cause double strand breaks. We provide evidence that this DNA damage sensitivity is not due to loss of checkpoint regulation over mitotic entry. We will also describe an extra copy suppressor screen aimed at identifying genes genetically interacting with SONB in response to DNA damage.
99. Orp-1, an Opsin Related Protein of Neurospora crassa. Marek Nemcovic and Katherine A. Borkovich Department of Plant Pathology, University of California, Riverside, CA 92521
The Neurospora crassa ORP-1 protein is a member of a newly established class of seven transmembrane proteins termed Opsin related proteins or ORPs. These proteins exhibit significant homology to archaeal rhodopsins, but are missing critical residues in the retinal binding pocket. Fungal ORPs appear to function in cellular responses to pH, organic solvents and stress signals. Here, we report a role for ORP-1 in stress responses. Sequence analysis revealed a HSE and an AP-1 response element in the promoter of the orp-1 gene. Western and Northern analysis support regulation of orp-1 expression by heat shock. The presence of the AP-1 element suggests that ORP-1 is under general amino acid control (cross-pathway amino acid starvation control). Treatment of the orp-1 deletion mutant with 3-aminotriazole resulted in reduced growth compared to wild type.
100. A novel G-protein coupled receptor family identified in Neurospora crassa. Krystofova S., Borkovich KA. University of California Riverside, Department of Plant Pathology.
A family consisting of three G-protein coupled receptors (GPCRs) named GPR-1, GPR-2 and GPR-3 has been identified in both the MIPS and the Whitehead Institute genome databases of Neurospora crassa. The encoded proteins are highly conserved in the seven transmembrane helices, and homology searches show similarity to the Family 2 of GPCRs. Phenotypic analysis of a gpr-2 null mutant did not reveal any particular defects, suggesting possible redundancy. This study is currently focused on disruption of gpr-1 and gpr-3 and characterization of multiple mutants in the gpr-1, gpr-2 and gpr-3 genes.
101. Characterization of the Vo Sector of the Neurospora crassa Vacuolar ATPase. Christopher L Chavez, Kimberly H. Haw, Emma Jean Bowman, and Barry Bowman, U. of California , Santa Cruz, CA 95064
The vacuolar ATPase is a large multi-subunit 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 Vo sector of the enzyme is embedded within the membrane and contains the proton pore. At least four different genes (vph-1, vma-3, vma-11, and vma16) encode for proteins which make-up the Vo sector. In an effort to elucidate the role(s) of these gene products in V-ATPase function, knockouts of the genes were sought. vph-1, vma-11, and vma-16 mutant strains were generated via RIP (Repeat-Induced-Point-Mutation) and modified RIP protocols. Due to meager spore germination rates, these mutants were extremely difficult to isolate. The RIP procedure failed to yield any viable strains in which the vma-3 gene was inactivated. A vma-3 mutant was eventually isolated using homologous gene-replacement in a heterokaryotic host. 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. The major phenotype difference between strains with different V-ATPase mutations is the viability of ascospores. The observation that some subunits are essential for ATPase activity and for ascospore viability suggests that some of the gene products may have additional functions.
102. MesA, a novel fungal protein required for localized cell wall deposition at hyphal tips. Claire Pearson and Steven D. Harris, Plant Science Initiative and Department of Plant Pathology, University of Nebraska, Lincoln, NE, 68588-0660.
In Aspergillus nidulans, SepA is required for septum formation and polarized hyphal extension. SepA is a member of the conserved formin family of proteins, which mediate the formation of actin filaments in response to cellular signals. We have previously shown that SepA functions interdependently with actin to form a contractile ring at septation sites. In addition, SepA displays dynamic localization to hyphal tips. However, the observation that sepA mutants can still establish hyphal polarity and recruit actin to hyphal tips suggests that a parallel pathway contributes to these functions. To identify components of this pathway, we conducted a screen for mutations that enhance the characteristic morphological defects of sepA mutants. Our screen yielded four mutants, mesA-D. The most dramatic synthetic phenotypes are observed in mesA1 sepA double mutants, which at higher temperatures, accumulate as large swollen spores filled with nuclei. Even in an otherwise wildtype background, mesA1 mutants display defects in the maintenance of hyphal polarity. Analysis of mesA mutants revealed that the aberrant morphology is most likely caused by an inability to properly localize spatial determinants required for localized cell wall deposition at the hyphal tip. For example, the normal pattern of SepA::GFP localization at hyphal tips is dramatically perturbed in mesA1 mutants. Molecular characterization of MesA shows that it is a potential transmembrane protein, with homologues found only in other fungi. We propose that MesA functions as a morphological landmark that specifies sites of localized cell wall deposition and recruits the fungal analogue of the polarisome
103. Patterns of germ tube emergence and cell wall deposition in germinating Fusarium graminearum macroconidia. Steven D. Harris, Plant Science Initiative and Department of Plant Pathology, University of Nebraska, N234 Beadle Center, Lincoln, NE, 68588-0660.
The fungus Fusarium graminearum (teleomorph Gibberella zeae) is the causative agent of wheat and barley scab. This disease is initiated and spread via the airborne dispersal of ascospores and macroconidia, respectively. Our ultimate goal is to identify cell wall proteins from F. graminearum spores that are dynamically expressed and play a functional role in host colonization. Towards this goal, we have first characterized the patterns of germ tube emergence and cell wall deposition in germinating macroconidia. F.graminearum macroconidia typically contain three to seven septated cells. When germinated on rich, sucrose-based media, the first germ tube emerges from one of the pole cells ( <90% of the time) in a manner that is independent of nuclear division. In contrast, although internal cells undergo isotropic swelling and nuclear division, they typically produce the first germ tube only if the pole cell is dead or the macroconidia is subjected to stress. The use of fluorescent lectins and other cell wall probes revealed a specific pattern of cell wall deposition in germinating macroconidia. This was most obvious when chitin deposition was examined using FITC-conjugated wheat germ agglutinin. Finally, we have used a combination of 2D gel electrophoresis and concanavalin-A binding assays to identify candidate cell wall proteins that display dynamic expression patterns during macroconidial germination. We propose that these proteins are likely to interact with plant surface components, and thereby play important roles in mediating host colonization.
104. Patterns of Actin Distribution in Neurospora crassa Actin Mutants. Aleksandra Virag, Anthony J.F. Griffiths. University of British Columbia, Botany Department, Vancouver, BC, Canada
Actin is necessary for hyphal morphogenesis, and actin filaments are laid down at sites of tip growth and branch emergence. It is not clear what factors command the localization and dynamics of actin at the hyphal tip. These factors are most likely a part of the machinery that regulates hyphal morphogenesis. By disrupting the actin gene in Neurospora crassa its mode of involvement in hyphal growth and branching can be deduced. N. crassa actin mutants were generated by either UV mutagenesis or RIP (repeat induced point mutations), and caused various morphological abnormalities. Actin filaments were visualized in these actin mutants by immuno-fluorescent labeling. A range of different actin patterns was detected at hyphal tips of the actin mutants. Some of these patterns include an actin distribution similar to the wild type with a mainly round apical actin dot and a peripheral network of smaller actin dots, absence of the apical actin dot, and two or multiple apical actin dots. The results presented indicate a significant role of actin in the shaping of hyphae and their branching.
105. Comprehensive expression analysis of the cell wall related genes in Aspergillus oryzae using the disruption mutant of protein processing enzyme gene (kexB). Osamu Mizutani, Tomonori Fujioka, Youhei Yamagata, Keietsu Abe, and Tasuku Nakajima. Molecular and Cell Biology, Tohoku University, Sendai, Japan
KexB is a processing proteolytic enzyme in fungi. To understand the involvement of KexB in protein processing, we cloned and disrupted kexB gene (DkexB) in A. oryzae. The DkexB strain formed small and shrunk colonies with significantly poor conidia generation on the Czapek-Dox (CD) agar plate. It also showed hyper-branched mycelia in liquid culture. When DkexB was cultivated under the various environmental conditions, we noticed that the phenotypes of the DkexB were restored under high osmolarity in both solid and liquid culture conditions. As a result of the observation of the phenotypes, we speculated that KexB might play an important role in processing of the gene products related to branching and conidia formation in solid cultivation. Then, to investigate the role of KexB in the expression of morphogenesis related genes in A. oryzae, we performed comprehensive gene expression analysis between the DkexB and the wild-type by using DNA microarrays containing 2,000 A. oryzae cDNAs. As a result, expression levels of ca.300 genes in the DkexB were significantly higher than the corresponding genes in the wild-type under solid culture conditions. In particular, the expression levels of chsB, chsC and gel2, which encode the cell wall synthesis enzymes, increased in the DkexB. Expression levels of the cell wall biosynthesis related genes in various growth stages will be also compared between the DkexB and the wild-type.
106. Characterization of two PAK kinases of Pneumocystis and their interaction with Rho-type GTPases. Nichole Heubner, Reiko Tanaka, George Smulian. University of Cincinnati College of Medicine, Cincinnati VA Medical Center, Cincinnati, Ohio
P21 associated kinases (PAKs) are important components of many signal transduction pathways involved in transmitted extracellular signals via MAP kinase cascades and involved in cytoskeletal rearrangement. Two PAKs have been identified in the opportunistic fungal pathogen, Pneumocystis carinii. One gene, identified in a cluster of signal transduction genes, was named ste20 due to apparent homology with the Ste20 gene ofSaccharomyces cerevisiae. Pcste20 is predicted to encode a protein containing both a plecstrin homology (PH) domain, predicted to be involved in membrane localization, and a Cdc42/Rac Interactive Binding (CRIB) domain. A secong PAK gene, Pcpak1, is predicted to encode a p21-associated kinase containing a CRIB domain but lacking a PH domain. On review, pak1 has greater homology to Ste20-like proteins while PcSte20 has greater homology to Cla4-like PAKs. In higher eukaryotes, 3 classes of Rho-type GTPases are found; Rho, Rac and Cdc42. Model fungal organisms such as S. cerevisiae andSchizosacharomyces pombe encode only Rho and Cdc42 homologs and among fungi, a Rac protein has only been identified in Yarrowia lipolytica. P. carinii expresses both Cdc42 and Rac1 homologs. Both Rac1 and Cdc42 were shown capable of interacting with the CRIB domains of PcSte20 and PcPak1 in vitro. Full characterization of the interaction of the PAKs and their cognate GTPase will shed light on the function of these proteins in P. carinii.
107. Genetic and cell biological analysis of cell morphogenesis in Ustilago maydis. Flora Banuett and Wei Wei. Department of Biological Sciences, CSULB, and Department of Biochemistry and Biophysics, UCSF.
Ustilago maydis is a Basidiomycete fungus that exhibits two basic vegetative morphologies: a haploid, yeast-like form that divides by budding and is non pathogenic and a filamentous dikaryon that grows at the tip and is pathogenic. A third form, the teliospore is not capable of vegetative growth but undergoes meiosis to produce the yeast-like form. The filamentous form arises by cell fusion of haploid cells of opposite mating type. This form grows in the plant and induces formation of tumors. The teliospore is produced only within the plant and results from execution of a discrete developmental program that takes place within the tumors. This program is characterized by karyogamy, hyphal fragmentation to produce cylindrical cells that undergo cell rounding, and deposition of a specialized cell wall around the spherical cells to produce mature teliospores. These morphological transitions are likely to involve changes in the organization of the cytoskeleton, which in turn may in part be directed by proteins that control cell polarity and cell wall remodelling events. We are interested in understanding the molecular mechanisms that underlie these morphological transitions. In order to identify some of these components, in particular proteins that organize the cytoskeleton, we have undertaken a multipronged approach involving genetic, cell biological, and biochemical techniques. We carried out a genetic screen to identify temperature-sensitive mutants that exhibit altered cell or colony morphology at the restrictive temperature. We identified approximately 80 mutants, 12 of which have been characterized further. Genetic analysis indicates that each mutant carries a single recessive mutation. The mutants exhibit additional phenotypes with respect to cell wall, sensitivity to drugs, and osmoticum. In addition, the mutants show altered organization of the microtubule or actin cytoskeletons. Here we present the results of the initial characterization of these mutants.
108. Genome defense by mutation and DNA methylation in Neurospora. Michael Freitag, Lakshmi Nimmagadda, Rebecca Williams, Gregory O. Kothe and Eric U. Selker, University of Oregon, Eugene.
During sexual development, Neurospora inactivates genes in duplicated DNA segments by a hypermutation process, RIP (repeat-induced point mutation). RIP introduces C:G to T:A transition mutations and creates targets for subsequent DNA methylation in vegetative tissue. The mechanism of RIP and its relationship to DNA methylation are not fully understood. Mutations in dim-2, a gene encoding a DNA methyltransferase responsible for all known cytosine methylation in Neurospora do not prevent RIP. We disrupted a second putative DNA methyltransferase gene and tested mutants for defects in DNA methylation and RIP. No effect on DNA methylation was detected but the mutants showed recessive defects in RIP. Duplications of several marker genes were stable in crosses homozygous for the mutated potential methyltransferase gene, which we call rid (RIP defective). The same duplications were inactivated normally in heterozygous crosses. The rid genes of various Neurospora species are conserved. We produced full-length or truncated recombinant N. crassa RID in E. coli to assay for DNA methyltransferase activity. (Supported by NIH grant # GM35690 to E.U.S.)
109. Biology of Living Fungi. Patrick C. Hickey and Nick D. Read. Fungal Cell Biology Group, Institute of Cell and Molecular Biology, University of Edinburgh, King's Buildings, Edinburgh, EH9 3JH, UK.
A website and CD-ROM have been compiled which illustrate key aspects of the cell biology of living fungi, visualised at high spatial resolution by confocal microscopy. The aims are to provide a valuable resource for mycologists and a powerful educational tool for schools and universities worldwide. Novel techniques for imaging living fungal hyphae and cells have been developed in our laboratory using confocal microscopy. This advanced imaging technology involves the use of a wide range of vital fluorescent dyes and GFP probes, with particular care to minimise cellular perturbation and provides a unique insight into the biology of living fungi. Examples of movies that we will provide include: hyphal tip growth, branching, septum formation, organelle dynamics, mitosis, and 3-D reconstructions of spores and reproductive structures. The movies are accompanied by simple explanatory text. This resource will be accessible through the internet, complemented by a CD-ROM containing extra material and, in the future, a DVD which can be purchased at minimal costs from http://www.fungalcell.org.
110. Possible role of structural componenets of the secretory pathway in the replication cycle ofCryphonectria parasitica. Massimo Turina, Debora Wilk, and Neal Van Alfen. UC-Davis, Department of Plant Pathology, Davis, CA.
CHV1 infection of the filamentous ascomycete Cryphonectria parasitica, the causal agent of chestnut blight, results in hypovirulent phenotype. Previous studies showed a consistent proliferation of host vesicles where virus replication and dsRNA accumulation occur. Heavy water-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 whereas in healthy mycelia the amount of vesicles dramatically decreases after an early accumulation. A subset of vesicles shown to contain viral dsRNA and proteins reacting to CHV1 helicase and polymerase antibodies appeared to be coated. Moreover the vesicle fraction of CHV1 infected C. parasitica contain an enriched protein band reacting with anti-clathrin heavy chain antibodies and anti- middle component of its adapter complex 1 in western blot analysis. The finding that clathrin coat associated protein accumulates in hypovirulent strains of C. parasitica prompted us to clone the C. parasitica clathrin heavy chain gene and the middle component of its adapter complex 1 in order to investigate their role in CHV1- C. parasitica interaction.
111. Intracellular processing and secretion of the fungal hydrophobin cryparin. Massimo Turina, Pam Kazmierczak, and Neal Van Alfen. Department of Plant Pathology, University of California, Davis, California.
Cryparin is an abundant class II fungal hydrophobin found in the cell walls of fruiting bodies of the fungus Cryphonectria parasitica. This protein is necessary for the eruption of the fungal fruiting body through the bark of infected trees. Large amounts of cryparin are secreted in liquid culture allowing its use to study vesicular protein secretion. The preprocryparin is processed by cleavage of the signal peptide and then the propeptide is cleaved by a Kex2-type of endoprotease. The role of the Kex2-type processing on secretion of this protein was studied by site-specific mutagenesis of the Kex2 recognition site. Antibodies were raised against a His-tag cryparin fusion protein and used in Western blot analysis of sub-cellular fractions and culture fluid of C. parasitica. GFP fusion was also used to study the secretion of this protein. Results indicate that Kex2 processing is not necessary for secretion and that coated vesicles are involved in the secretion of cryparin.