Poster Abstracts

1
Characterization of efflux of basic amino acids from the vacuole of Neurospora crassa.
Kelly A. Keenan, Ryan Cinalli and Chris Sondey. Richard Stockton College, Pomona, NJ.

The vacuole stores large amounts of the basic amino acids--arginine, lysine and ornithine--and it has been observed that conditions of nitrogen starvation cause an efflux or movement of these amino acids out of the vacuole. The amino acids presumably serve as a reserve nitrogen source. The components involved in efflux--the proteins as well as the signals-have never been characterized. The cupric ion permeabilization method has recently been developed to measure efflux and several strains with an increased level of efflux have been identified. RSC-44 has an increased arginine efflux compared to wild type while both arginine and ornithine efflux are increased in RSC-63. These strains offer a way to characterize this efflux process. The strains were characterized by backcrossing with wild type and mapping the location of the mutation. The number of genes involved in efflux can be identified this way as well as the chromosomal location. The response of the strains to nitrogen starvation was also characterized since this is known to produce increased efflux. Unlike wild type, there was no increase in activity of the arginase during nitrogen starvation in the strains and an altered response in the vacuolar amino acids.

2
Photoactivation of con-10 gene expression in Neurospora crassa.
Luis M. Corrochano, Maria Olmedo, and Laura Navarro-Sampedro. Departamento de Genética, Universidad de Sevilla, Spain

The gene con-10 of Neurospora is expressed during conidiation and following illumination of mycelia with light. The photoactivation of con-10 disappears after two hours of illumination (light adaptation). To investigate the molecular nature of light adaptation in Neurospora, we have designed a protocol to isolate mutants altered in the adaptation of con-10 photoactivation. We are using a strain of Neurospora with a fusion of the con-10 promoter to the gene conferring resistance to hygromycin. This strain is sensitive to the drug when the promoter is inactive, i.e. during vegetative growth either in the dark or under continuous light. We have isolated three mutants that grow in the presence of hygromycin under continuous light but not in the dark. Presumably this is due to a defect in the mechanism controlling light adaptation. The promoter of con-10 is composed of DNA segments involved in repression in the dark and during mycelial growth, and DNA segments required for the activation during conidial development. We are using a series of fusions between specific segments of the con-10 promoter and the lacZ gene to investigate in detail the DNA sequences involved in regulating the expression of con-10.

3
RIP of pacC-1 affected both the glycosylation of secreted alkaline phosphatase and conidiation in Neurospora crassa.
S.R. Nozawa¹, M.S. Ferreira-Nozawa², M. Duarte³, N.M. Martinez-Rossi², A. Videira³ and A. Rossi⁴. ¹Departamento de Química, FFCLRP-USP, ²Departamento de Genética, FMRP-USP and ³Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal and ⁴Departamento de Bioquímica e Imunologia, FMRP-USP, Ribeirão Preto, Brazil.

 A conserved pacC-dependent pH response pathway has been identified in many fungi like N. crassa and A. nidulans. Gene pacC codes for a Zn-finger transcription factor that undergoes proteolysis at alkaline pH, yielding a functional protein responsible for the induction of genes expressing products with optimal activity at alkaline pH (e.g. alkaline phosphatases) and repression of those with optimal activity at acidic pH (e.g. acid phosphatases). Transcription of pacC is itself induced under alkaline growth conditions, and the pal genes promote the proteolytic activation of PacC, because expression of C-terminal truncated PacC derivatives suppresses pal mutant effects. Thus, if pacC gene governs positively the expression of alkaline genes, pac C mutants inactivated by RIP should not secrete alkaline phosphatase and the properties of the secreted acid phosphatase should be identical to that secreted by wild-type strains. Indeed, pacC-null mutants (RIP) were affected in conidiation at pH 4.0, and secreted alkaline phosphatase at pH 7.8 with altered thermal inactivation, electrophoretic and isoelectric focusing patterns, and reduced sugar content, as compared to the properties of the wild-type enzyme, indicating additional roles for pacC gene.
Financial support: FAPESP, CNPq, CAPES and FAEPA.

4
Characterization of pacC-1, a wide domain regulatory gene responsive to ambient pH in Neurospora crassa.
N.M. Martinez-Rossi¹, S.R. Nozawa², G.S. May³, M.S. Ferreira-Nozawa¹ and A. Rossi⁴. Departamentos de ¹Genética and ⁴Bioquímica e Imunologia, FMRP-USP, Brazil, ²Departamento de Química, FFCLRP-USP, Brazil and ³University of Texas, M.D. Anderson Cancer Center, Div. Pathol. L.M., Houston, Texas, USA.

An essential step in the conversion of PacC, a conserved wide domain Zn-finger transcription factor, into its fully active form in response to ambient alkaline pH is the proteolytic removal of a C-terminal inhibitory region of the protein as mimicked in the pacC14 mutant of A. nidulans, which was characterized as a gain-of-function mutation. However, pacC14 behaved as a loss-of-function mutation because we have shown that this mutation alters posttranslational glycosylation of both acid and alkaline phosphatases secreted at pH 5.0, indicating that proteolytic cleavage of PacC may not be necessary for its activation. With the aim of characterizing further this adaptive response to ambient pH we cloned gene pacC-1 from N. crassa by screening a genomic library with fragments of genomic DNA generated by PCR amplification. One of the recovered sub-clones of 6.5 Kb revealed a full-length open reading frame of 1960 bp having 100% identity to the open reading frame generated by the N. crassa genomic project. This clone complemented the pacC14 mutation of A. nidulans, including remediation of the glycosylation of both the acid and alkaline Pi-repressible phosphatases secreted at pH 5.0, which confirms the involvement of gene pacC in the glycosylation of both secreted enzymes.
Financial support: FAPESP, CNPq, CAPES and FAEPA.

5
Characterization of the Neurospora "pathogenicity" signaling pathway.
Daniel Ebbole, Piotr Bobrowicz and Dan Li. Department of Plant Pathology and Microbiology, Texas A & M University, College Station Tx.

 A major pathogenicity MAP kinase has been characterized from many plant pathogenic fungi. Although it has been relatively simple to generate MAP kinase mutants in a number of these pathogens it has been less simple to characterize the downstream targets of this pathway that contribute to disease initiation and progression. N. crassa contains all of the pathogenesis signalling components that have been characterized in fungal pathogens to date. Several targets of MAP kinase regulation in N. crassa have been identified in our laboratory and a number of these resemble virulence factors found in pathogens. Based on our interpretation of this data, we argue that it is straightforward for fungi to evolve the major requirements for pathogenesis through minor modifications of the basic pathways found in saprophytic fungi, such as Neurospora.

6
Cloning of ccr100, a Neurospora crassa cytochalasin A resistant mutant.
A. Virag, A.J.F. Griffiths. University of British Columbia, Vancouver BC, Canada.

Although the mechanisms involved in branching regulation are still elusive, branching is clearly affected by a multitude of gene products in filamentous fungi. Among these products actin, in the form of microfilaments, is consistently present at sites of hyphal tip growth and sites of branch emergence. We used this observation as a starting point in a genetic approach to identify genes involved in branching in Neurospora crassa. In a screen for cytochalasin A resistant mutants we isolated a strain that beside cytochalasin A resistance has a different branching pattern than the wild type strain, and is cold sensitive. The mutated locus was mapped to linkage group V. Sib-selection showed that the fragment that complements the ccr100 phenotype contains the gene encoding actin. A collection of mutant strains of the ccr100 locus was obtained by RIP. The analysis and comparison of various characteristics of the obtained mutants is presented.

7
An on-line comprehensive gene list of Neurospora crassa.
Alan Radford, School of Biology, The University of Leeds, Leeds, UK

While compiling "The Neurospora Compendium", it became apparent that ambiguities had arisen, making our work more difficult where two totally different genes had been given the same symbol and where the same gene had been given more than one. Table 1 in the Compendium lists all the symbols that have been replaced and that are now obsolete synonyms, covering up to mid-2000. Use of these symbols should be avoided. Although the normal rules of precedence apply, there was no comprehensive list of gene symbols and names to use for verification that a symbol or name was available, or in a series of genes with the same root symbol to discover what was the next available number in the series. The FGSC lists only those genes for which it has strains. I have compiled an updatable list in XML format, with sufficient information for identification, which currently requires a recent version of Internet Explorer. The list gives gene name and symbol, plus map location, phenotype, gene product and sequence code if available. It is being updated as information on new loci and relevant new data on existing loci become available. Updating would be greatly assisted if Neurospora workers routinely sent me details of new genes (a.radford@leeds.ac.uk). A form for convenient data submission is being developed.

8
Locating genes of the cellulase complex of Neurospora crassa.
A Radford, School of Biology, The University of Leeds, Leeds, UK.

 Yazdi et al (1991) Enzyme Microb Technol 12:120-123 demonstrated the cellulolytic potential of Neurospora crassa, and partially characterised members of the complex. Taleb and Radford (1995) Gene 161:137-138 subsequently cloned and sequenced one gene of the complex, the cellobiohydrolase cbh-1. Neurospora being a close relative of Trichoderma reesei, the cellulase enzyme sequences of the latter were used to probe version 2 of the Neurospora genome database for homologues, using the integral BLAST facility. Five homologues for the EG1/CBH1 family were identified, on contigs 2.521, 2.343, 2.341, 2.681 and 2.236, with the first being the the best match for CBH1 and the second the best for EG1. The other three homologues lacked a cellulose-binding domain, a feature also absent in some members of the complex in other species. Contigs 2.585, 2.732 and 2.237 were homologues of CBH2 with the first of the three being the closest match. Contig 2.20 was the only homologue for EG2. A number of additional homologues were found for the cellulose-binding domain itself.

9
Microsampling and mass spectroscopy of cytoplasm from single fungal hyphae.
Richard J.A. Goodwin¹, C. Logan Mackay², Patrick R.R. Langridge-Smith², Frank Moffatt³, Dave Bartlett³, and Nick D. Read¹. ¹Fungal Cell Biology Group, Institute of Cell and Molecular Biology, University of Edinburgh, Rutherford Building, Edinburgh EH9 3JH, UK²The Experimental Measurement Science Group, Department of Chemistry, University of Edinburgh, EH9 3JJ, UK³Syngenta, Jeallot's Hill International Research Centre, Bracknell, Berkshire, RG42 6ET, UK

 We have developed techniques to analyse the biochemistry of Neurospora crassa at the single cell level. This radically new approach to fungal biochemistry is fundamentally different from the more traditional "mince-and-measure" analytical techniques. Procedures for microsampling from a single hypha, transferring the microsampled cytoplasm to a capillary electrophoresis column and analysis of the separated sample using mass spectrometry, have been accomplished. Both the Neurospora wild type and hex-1 mutant, which is unable to block its septal pores allowing larger volumes of cytoplasm to be microsampled, have been used in these studies. Typically the volume of cytoplasm analysed has varied between 100 and 1000 pl.

10
Measuring calcium dynamics in living Neurospora hyphae using recombinant aequorin.
Alex Zelter^1,2, Oded Yarden² and Nick D. Read¹. ¹Fungal Cell Biology Group, Institute of Cell and Molecular Biology, University of Edinburgh, Rutherford Building, Edinburgh EH9 3JH, UK.²Department of Plant Pathology and Microbiology, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel.

 Indirect evidence in the literature has suggested the involvement of Ca²⁺ signalling in regulating hyphal branch formation in Neurospora crassa. For example, hyperbranching can be induced by various Ca²⁺ modulators, or by genetical impairment or pharmacological perturbation of calmodulin/calcineurin. To obtain more direct evidence regarding the role of Ca²⁺ signalling in hyphal branching we have developed a technique using the Ca²⁺-sensitive luminescent protein aequorin, which allows cytosolic free Ca²⁺ ([Ca²⁺]c) measurement in living hyphae. Aequorin, originally from the jellyfish Aequorea victoria, responds to free Ca²⁺ by emitting light in a dose-dependent manner. Several strains of N. crassa were transformed with the codon optimised gnaeqS gene encoding aequorin. Based on their in vivo aequorin activity, the successful transformation of wild type, cot-1, frost, spray, and T3 (harboring an inducible cna-1 antisense expression cassette) strains of N. crassa has been shown. These strains are now being used to analyse the role of Ca²⁺ signalling in regulating hyphal branching.

11
Meiotic Silencing by Unpaired DNA (MSUD).
Patrick K.T. Shiu¹, Namboori B. Raju¹, Denise Zickler², and Robert L. Metzenberg¹.¹Biological Sciences, Stanford University, CA, USA. ²Institut de Genetique et Microbiologie, Universite Paris-Sud, Orsay, France.

To defend against the invasion of virus, eukaryotes have evolved a number of mechanisms to deal with DNA segments that are present in an inappropriate number. An example is Post-Transcriptonal Gene Silencing, in which a dsRNA species (produced by a RNA-directed RNA polymerase) is cut into fragments of 21-23 nucleotides (siRNA). These fragments act as guide RNAs for the cleavage of homologous cytoplasmic mRNA. We have discovered a related system of targeted gene silencing that operates after karyogamy, Meiotic Silencing by Unpaired DNA (MSUD; Cell 107:905-916). In the MSUD system, DNA unpaired with a homologous sequence during meiotic prophase generates a sequence-specific signal, presumably an RNA. This signal destroys existing transcripts originating both from unpaired and paired DNA sequences homologous to it. We have isolated a mutant that fails to perform MSUD. Sad-1 suppresses several classical ascus-dominant mutants, suggesting that these, too, owe their ascus dominance to the MSUD mechanism. In addition, Sad-1 suppresses the near-absolute barren phenotype of crosses between wild type and a variety of strains in which a segment of DNA is duplicated. Finally, interspecies crosses within the genus Neurospora that are normally almost completely infertile become much fertile if the N. crassa parent is a Sad-1 mutant, suggesting that MSUD due to numerous small mispairings play a role in reproductive isolation of these species. sad-1+ encodes a RNA-directed RNA polymerase.

12
A quick and easy way to determine gene function in meiosis using MSUD.
Patrick K.T. Shiu¹, Namboori B. Raju¹, Denise Zickler², and Robert L. Metzenberg¹. ¹Biological Sciences, Stanford University, CA, USA. ²Institut de Genetique et Microbiologie, Universite Paris-Sud, Orsay, France.

DNA unpaired in meiosis causes silencing of all DNA homologous to it, by a process called Meiotic Silencing by unpaired DNA (MSUD). MSUD can be triggered in a cross containing three copies of a gene, i.e., between a wild-type strain and a strain containing two copies of a gene. Silencing by an ectopic transgene using the MSUD system is potentially a quick and easy tool to determine the role of genes in meiosis and ascospore development. The advantages can be summarized as follows. 1) Isolating a strain containing a duplicated gene segment is considerable faster and easier than isolating a RIP or gene-replacement mutant. 2) MSUD can be performed on genes for which a knockout construct would be lethal in the vegetative phase. 3) The effect of meiotic silencing will not begin until after karyogamy, allowing the investigators to determine the true role of a gene during sexual development. Clearly, this method can used to idendity gene functions in Neurospora that govern meiosis, post-meiotic mitoses and ascospore maturation. The stage at which development is arrested has been determined for several genes using the MSUD system. They include asm-1 (white aborted ascospores), mei-3, which encodes the RecA/RAD51 protein (blocked in meiosis I), and genes that encode beta-tubulin (arrest before metaphase I), actin (lollipop asci and giant spores), histones H3/H4 (ascospores do not mature), and plasma membrane ATPase (bubble asci that do not mature).

13
Cloning and characterization of scon-3⁺: A new member of the Neurospora crassa sulfur regulatory system.

Steven T. Sizemore and John V. Paietta. Biochemistry and Molecular Biology, Wright State University, Dayton, OH, USA.

In Neurospora crassa, a group of sulfur-regulated structural genes (e.g., arylsulfatase) are under the coordinate control of the CYS3 positive regulator and several SCON (sulfur controller) negative regulators. The negative regulator SCON2 encodes a protein containing the F-box motif; a motif involved inprotein-protein interactions. In S. cerevisiae, and other organisms, an F-box protein along with Cdc53p (cullin), and Skp1p form a complex, referred to as the SCF, which recruits specific substrates for ubiquitin-mediated proteolysis. The F-box protein provides target specificity for these complexes and the F-box motif is required for interaction with Skp1p, and therefore complex formation. SCON2's regulatory role may be attributed to its ability to form an SCF-complex and target specific substrates (e.g., CYS3) for rapid degradation. We have cloned the N. crassa homolog to SKP1, which we have designated scon-3⁺. The scon-3⁺ gene encodes a polypeptide of 171 amino acids interrupted by two introns of 80 bp and 98 bp respectively. Through yeast two-hybrid experiments, we demonstrate that SCON3 interacts with SCON2 in a manner dependent upon the F-box motif of SCON2. Further, Northern blot analysis provides evidence that expression of scon-3⁺ is regulated by sulfur availability.

14
Antioxidant defence systems of Blakeslea trispora and Neurospora crassa.
Tatyana Belozerskaya, Alexander Sokolov, Natalya Gessler. A.N.Bach Institute of Biochemistry, Moscow, Russia.

  Comparative investigation of antioxidant defence mechanisms of B.trispora and N.crassa has been performed. Activation of constitutive SOD was observed in the mycelium of N.crassa in the dark in presence of trace amounts of carotenoids upon introduction of menadione. Neurosporaxanthin increase was found in N.crassa under the influence of light together with activation of SOD and CAT. SOD activity was significantly lower in mixed (+/-) cultures of B.trispora able to synthesize b-carotene in the dark. Further decline in SOD activity, and a decrease in CAT activity together with an increase of b-carotene was observed in B.trispora under oxidative stress. Thus, in N.crassa neurosporaxanthin acted mainly as photoprotector whereas in B.trispora b-carotene functioned as the main antioxidant on the background of inactivation of enzymes detoxifying active oxygen species. In the nap strain of N.crassa with increased level of carotenoids SOD activity was much lower and varied from 11 to 17% of the wild type.

15
Construction of Neurospora microarrays and its application for evolutionary study
Takao Kasuga¹, Betty Gilbert¹, David Jacobson², Don Natvig³, Louise Glass¹ and John Taylor¹. ¹Dept. Plant Biology, Univ. California, Berkeley, ²Dept. Biological Sciences, Stanford Univ., ³Dept Biology, Univ. New Mexico.

Rates of adaptation and speciation are generally not correlated with rates of molecular evolution at the genome level. A resolution to this paradox is that in order to adapt to new environments, organisms may modify a small number of regulatory genes, which consequently alter the timing of expression of a number of key genes, rather than de novo adaptive mutations. Until recently, however, it was virtually impossible to identify and quantify which genes in the genome are responsible for changes in fitness and speciation. Microarray technology enables us to assess the expression of thousands of genes simultaneously, and thereby potentially reveals the differences in gene expression between closely related species or subpopulations. A cDNA library of N. crassa is being constructed at the UNM. Using a portion of this library we have generated a preliminary cDNA microarray of approximately 550 genes. Natural isolates of Neurospora may be found growing on burned substrates over a wide geographic range. We are currently analyzing the differences between N. crassa isolates from different geographical regions and different Neurospora species in response to a particular stress or growth condition. These results will allow us to assess how genetic differences contribute to gene expression differences under identical laboratory conditions. Eventually, we intend to assess gene expression patterns of different Neurospora isolates in nature. Such information will help to understand the mode of evolution of Neurospora in the environment.

16
Sterol Fingerprints of pk a complementary tool to Genetic Analysis.
Eduardo Jovel¹ and Anthony G. F. Griffith². Department of Botany The Univeristy of British Columbia, B.C. Canada. ^1,2

Among the fungi Neurospora crassa is one of the best-characterized genetic model. It is also one of the fastest growing fungi with a lateral branching system. Tip growth and branching in N. crassa remain poorly understood. Although molecular and morphological characterization of this fungus has been well researched, few studies have been devoted to biochemical analyses. Sterol glycosides and cerebrosides are an important component of the fungal cell wall, and may be involved in morphogenesis and branching. These compounds have been associated with the biological processes of growth, development, and signal transmission in many organisms. We are developing chemical fingerprints to determine whether mutant strain pk and similar strains are deficient in certain steps in ergosterol biosynthesis. Chemical analyses will include TLC, HPLC, GC, and other physico-chemical methods. Mutant strains deficient in ergosterol biosynthesis share morphological characteristics with pk. The deficiencies include slow growth, sterile females, and poor conidia formation. Backcrossing and examination of double mutants obtained form pk and similar strains, may provide insight on the metabolic blocks affecting pk branching.

17
Interactions of genes during Neurospora crassa development
O. Gavric and A.G.F.Griffiths, Botany Department, University of British Columbia, B.C. Canada.

 There are more than 100 loci that encode products that can affect tip growth and branching in Neurospora crassa. In order to dissect the genetics of branching we have examined the functional relationship between 35 of these loci. By using epistasis analysis some of these genes were grouped into common developmental pathways. We obtained double mutants through ascus analysis. In most cases,double mutants show a phenotype more severe than either single mutant alone, indicating that each mutant probably affects separate pathways involved in growth and branching. However, several double mutants were found to closely resemble one of the parental strains in morphology. This suggests both genes are part of a single developmental pathway, with the epistatic gene acting before the hypostatic one. The presented analysis gave a rudimentary characterization of a pathway involved in tip growth and branching.

18
The identification of vib-1, a gene involved in vegetative incompatibility mediated by het-c locus in Neurospora crassa.
Qijun Xiang, and N. Louise Glass. Department of Plant and Microbial Biology University of California, Berkeley CA 94720

Vegetative incompatibility is ubiquitous in filamentous fungi. It is controlled by het loci. One way to explore the molecular mechanisms controlling vegetative incompatibility is to isolate suppressors. In this study, three suppressor deletion mutants, ahc (Aerial hyphae, Hyphae fusion and Conidiation), vc1 (Vegetative incompatibility and Conidiation) and vc2 were identified from the strains that "escaped" from het-c vegetative incompatibility. The deletions in ahc, vc1 andvc2 mutants are ~26kpb, 19442bp and ~7kbp long, respectively. There are also unknown insertional fragments in ahc and vc2 deletion regions. The three deletions map to chromosome V, between lys-2 and ilv-2. They overlap each other in an ORF named as vib-1 (Vegetative Incompatibility Blocked). Single vib-1 mutants were also generated by RIP mutagenesis. Mutations in vib-1 fully relieve inhibition of mycelial growth and suppression of conidiation conferred by het-c vegetative incompatibility and significantly reduce the rates of hyphal compartmentation and death. The vib-1 mutants have a copious conidiation pattern, suggesting that VIB-1 is a conidiation suppressor. VIB-1 has a region sharing high similarity to PHOG, a possible phosphate non-repressible acid phosphatase in Aspergillus nidulans. Our native gel analysis, however, shows that vib-1 is not a structural gene for non-repressive acid phosphatase. It may be, however, involved in the regulation of the activity of phosphate non-repressible acid phosphatase. VIB-1 is a putative nuclear protein due to the presence of a bipartite NLS (nuclear localization signal). We are conducting experiments to determine whether or not VIB-1 acts at a transcriptional level to mediate vegetative incompatibility, suppress conidiation and activate phosphate non-repressible acid phosphatase.

19 Neurospora G gamma subunit (GNG-1) identification and characterization.
S. Krystofova, K. A. Borkovich. University of California Riverside, Dept. Plant Pathology, California.

The G-protein-linked pathways evolved to respond to extracellular agonists in cells ranging from yeast, filamentous fungi and slime molds to mammals. In filamentous fungus Neurospora crassa genetic studies and genomic sequencing revealed at least 7 G-protein coupled receptors (GPCRs), three G alpha subunits (GNA-1, GNA-2, GNA-3), one G beta subunit (GNB-1), and one G gamma subunit (GNG-1). The previous genetic and biochemical studies of all three G alpha subunits, and the G beta indicate their roles in apical growth rate, aerial hyphae formation, nutritional sensing, female fertility and mating. The putative protein sequence of GNG-1 shows a COOH-terminal CAAX motif of isoprenylation commonly found in G gamma subunits (CVVM). GNG-1 is closely related to Saccharomyces cerevisiae, STE18 (35% identity) and non-visual mammalian G gamma subunits (33% identity). A delta gng-1 mutant has been constructed and characterized. Phenotypic analysis of gng-1 mutant shows a slow apical growth rate on solid medium, inappropriate conidiation in submerged culture, short aerial hyphae formation in standing liquid cultures and inability to develop female reproduction structures. The gng-1 deletion impacts levels of all G alpha proteins and G beta protein in plasma membrane fraction, and causes reduced intracellular cAMP levels in cultures grown on solid medium.

20
Identification and characterization of a crnA related nitrate transporter gene in Neurospora crassa.
Fei G Rubinelli, Peter M Rubinelli, Bo Feng, and George A. Marzluf; The Ohio State University, Department of Biochemistry, Columbus, OH 43210

Neurospora crassa genome database was searched for sequence similarity to crnA, a nitrate transporter in Aspergillus nidulans. A 4 kb fragment (contig 2.585, subsequence [183125, 187125]) was cloned by PCR and was termed nit-10. In Aspergillus nidulans, crnA is clustered together with niaD, encoding nitrate reductase, and niiA, encoding nitrite reductase. However, our RFLP mapping results indicated that nit-10 is not linked to any genes known so far involved in nitrate assimilation such as nit-3 (nitrate reductase), nit-6 (nitrite reductase), nit-2, nit-4 (both are positive transcription regulators of nit-3), and nmr (negative regulator of nit-3). A nit-10 rip mutant was generated by crossing a normal wild type strain with a derived wild type strain carrying two copies of nit-10 the endogenous copy and a second one obtained by transformation. The mutant showed poor growth when nitrate was used as the sole nitrogen source. In addition, it showed strong sensitivity to cesium in the presence of nitrate and resistance to chlorate in the presence of nitrate, alanine, proline, or hypoxanthine. The transcripts of nit-10 were analyzed by Northern blots. The expression of nit-10 requires nitrate induction and is subject to feedback repression by nitrogen metabolites such as glutamine or ammonium. The transcripts of nit-10 could not be detected in either nit-2 or nit-4 mutants. This suggested that the products of nit-2 and nit-4 mediate the expression of nit-10. The half-life of nit-10 mRNA was determined by Northern blots after transferring wild type cells from an induction (nitrate) to a repression (glutamine) environment. It was found that half-life of nit-10 mRNA is approximately 4 minutes. In a similar experiment, except order of incubation was reversed (repression to induction), we found that nit-10 transcripts were induced after 30 minutes exposure to nitrate.

21
Hyphal fusion pathway of Neurospora crassa
Carolyn Rasmussen, Qijun Xiang, Louise Glass. Department of Plant and Microbial Biology, University of California, Berkeley CA 94720

Cell fusion is presumably an important factor in the growth and development of multicellular organisms. During the vegetative growth of filamentous fungi, there are many cell (or hyphal) fusion events within a colony or between colonies. Because filamentous fungi grow rapidly and extensively, hyphal fusion may be necessary to facilitate the sharing of resources or serve as a means of communication within the colony. In the model filamentous fungus, Neurospora crassa, the processes of hyphal fusion have been described microscopically. It is apparent that the hyphae respond and grow towards each other before hyphal fusion occurs. However, the molecular mechanism has not been characterized. Recently, a hyphal fusion mutant gene, ham-2 (for hyphal anastomosis) has been cloned. The mutant has a greatly reduced ability to fuse with itself or with wild type strains. The ability of the ham-2 mutant to fuse is reduced by a factor of a thousand fold when compared to wild type. Other phenotypes of the ham-2 mutant strains include a reduced growth rate, short aerial hyphae and female sterility. These phenotypes may be caused by a defect in hyphal fusion. Currently, we are working to understand how HAM-2 is involved in hyphal fusion by looking at the localization of the protein and the mRNA expression pattern. Computer prediction models predict that HAM-2 is localized to the plasma membrane. We hypothesize that HAM-2 could be involved in the signaling pathway of hyphal fusion. Genetic analysis has indicated that a MAP kinase pathway mediates the process of hyphal fusion. We are assessing whether or not HAM-2 is regulated by or regulates a MAP kinase signaling cascade.

22
Genetic analysis of temperature-sensitive mutant for het-c mediated vegetative incompatibility in Neurospora crassa
Isao Kaneko, Qijun Xiang, Magdalen Barton and N. Louise Glass. Plant and Microbial Biology Department, University of California, Berkeley, CA, USA

 Different fungal individuals are able to undergo hyphal fusion with each other. This event results in the vegetative heterokaryons containing two different nuclei in a common cytoplasm. The stability of heterokaryotic cell is controlled by het loci. When individuals have one or more different specificity at het loci, these heterokaryotic cells are unstable and usually undergo hyphal compartmentation and cell death. This phenomenon is called vegetative incompatibility. In forced heterokaryons and partial diploids at het loci, suppressor mutants of vegetative incompatibility are frequently generated by spontaneous mutation called "escape". One suppressor mutant of het-c mediated vegetative incompatibility was identified from incompatible transformants of a het-c^PA strain transformed with a het-c^OR allele. This escape strain showed temperature-dependent vegetative incompatibility with het-c^OR strains in heterokaryon tests. Heterokaryons were incompatible at 20^oC, but compatible at 34^oC. Heterokaryon tests with het-c^PA strains showed incompatibility at both temperatures. Progeny containing the het-c^PA allele were isolated from a cross with a wild-type het-c^OR strain. These progeny had no functional het-c^OR allele and maintained temperature-dependent vegetative incompatibility with het-c^OR strains. When these progeny were crossed with wild-type het-c^OR strains, temperature-dependent phenotype co-segregated with het-c^PA phenotype. This phenotype was not complemented by introduction of a wild-type het-c^PA allele. These results indicate that the locus associated with temperature-dependent vegetative incompatibility is closely linked to the het-c locus.

23
Comparing biological and phylogenetic species recognition in Neurospora.
David J. Jacobson, Jeremy R. Dettman, Elizabeth Turner, Ann J. Pringle, Takao Kasuga and John W. Taylor. University of California, Plant and Microbial Biology, Berkeley, CA 94720

We are using Neurospora to provide a benchmark for recognizing fungal species. Under Simpson's Evolutionary Species Concept, we have compared Biological Species Recognition using mating tests with Phylogenetic Species Recognition using concordance of gene genealogies. The foundation for recognizing phylogenetic species is a comprehensive phylogeny of 147 isolates of all the conidiating Neurospora species (presented separately at this meeting; Dettman, et al.). Simultaneously, Biological Species Recognition was examined by crossing a subset of 73 strains of N. crassa, N. intermedia, and putative N. crassa/N. intermedia hybrids in an experiment designed to be independent of any phylogenetic results. Mating success from 894 crosses was used to circumscribe reproductively isolated groups. Statistical comparisons showed a generally strong correlation between species recognized by biological and phylogenetic criteria with at least five species detected within the N. crassa/N. intermedia complex. However, two important areas of incongruence should be noted. First, in both N. crassa and N. intermedia, two or more phylogenetic species were recognized in a single biological species, indicating that genetic isolation can precede reproductive isolation. Second, two biologically recognized species were found in a single N. crassa phylogenetic species, indicating that reproductive isolation can occur before genetic isolation can be detected, even though the marker loci were polymorphic within each biologically recognized species. When reproductive isolation occurs in a distinct geographic region, it provides support for the controversial concept of sympatric speciation. Moreover, the incongruence of phylogenetically and biologically recognized species suggests that speciation mechanisms within Neurospora are complex. While neither method of species recognition is superior to the other, when combined results suggest that species recognized by either methodare, in nature, on a trajectory to genetic and reproductive isolation and deserving of the species rank.

24
Spore killer sensitive isolates become the killers in heterokaryons.
Edward G. Barry. Biology, University of North Carolina, Chapel Hill, NC, USA.

Meiotic drive acts during or after meiotic divisions to diminish the transfer of a sensitive gene or chromosome region to the progeny. In Neurospora the Spore killer variants perform this activity. In matings between Spore killer and Spore killer sensitive strains, the progeny are almost exclusively Spore killers. The four ascospores with sensitive linkage group III regions abort after the second mitotic division and the four ascospores with the killer chromosome region survive (Turner and Perkins, Genetics 93: 587-606, 1979). When heterokaryons of Spore killer-2 (Sk-2K) and sensitive (Sk-2S) are formed using forcing auxotrophic markers, the conidia which germinate and form colonies are almost exclusively Sk-2S. This is the reverse of the mating challenge. Spore killer-3 and Sk-3S do not have this interaction. Equal types of both Sk-3K and Sk-3S heterokaryon constituents are observed as well as heterokaryons with both nuclear types if colonies are grown from plated conidia. When Spore killer-2 and Spore killer-3 are mated, there is almost exclusively mutual destruction with very few survivors and those are not either parental type. In contrast, when Sk-2K with Sk-3K heterokaryons are formed and conidiospores are plated, the Sk-3K class is almost exclusively the survivor and Sk-2K colonies are rare. The mechanism of killing in either ascospores or conidia is unknown.

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Phylogenetic species recognition in Neurospora.
Jeremy R. Dettman, David J. Jacobson, and John W. Taylor. University of California, Plant and Microbial Biology, Berkeley, CA, 94720-3102

Under an Evolutionary Species Concept, we compared phylogenetic species recognition with existing biological species recognition in the outcrossing, conidiating species of Neurospora. The five described biological species were represented by 147 strains from a wide geographic distribution; 128 of these were from the N. crassa/N. intermedia species complex, including nine strains described as putative N. crassa/N. intermedia hybrids. All strains previously had been identified by mating with designated tester strains. A comprehensive molecular phylogeny was constructed from four non-coding nuclear loci, and phylogenetic species were delineated by the concordance of gene genealogies. Several phylogenetic species were well supported, but the relationships among them were not. In general, the five described biological species corresponded well with phylogenetic species. The N. crassa/N. intermedia species complex, unresolved in previous phylogenetic studies, was resolved here into a large clade of each N. crassa and N. intermedia, and at least three additional phylogenetic species. None of the putative N. crassa/N. intermedia hybrids appeared to be true hybrids. While some clades showed geographic correlation, no major phylogeographic trends were consistent across all species. Simultaneously, biological species recognition within the N. crassa/N. intermedia complex was examined in a subset of 73 strains by nearly 900 mating tests (Jacobson et al. poster). Whereas phylogenetic and biological species recognition were generally congruent, there were several cases where phylogenetic methods recognized species in the N. crassa/N.intermedia complex not recognized by mating experiments, and one reverse case. These results also show that neither phylogenetic nor biological species recognition, alone, can uncover all the Neurospora species; both are needed, especially if isolates will be used in subsequent genetic and/or population studies.

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Neurospora in western North America.
David J. Jacobson^1,2, Amy J. Powell³, Jeremy R. Dettman², Gregory S. Saenz³, Magdalen M. Barton², Megan D. Hiltz², N. Louise Glass², John W. Taylor², and Donald O. Natvig³. ¹Stanford University, Biological Sciences, Stanford, CA; ²University of California, Plant and Microbial Biology, Berkeley, CA; ³University of New Mexico, Biology, Albuquerque, NM

Recent surveys have clearly established that species of Neurospora are common primary colonizers of trees and shrubs killed by wildfires in forests and woodlands of western North America. These studies substantially expand the known distribution and habitats of a genus best known from the tropics and subtropics. Neurospora species have been observed at 33 fire sites in habitats ranging from cottonwood stands along the Rio Grande to mountain forests in New Mexico, California, Nevada, Idaho, Montana and Alaska. Colonization occurs beneath the bark of diverse deciduous and conifer hosts. The combined 2000-2001 collection includes more than 500 isolates from 35 degrees to 63 degrees north latitude and from 515 m to >2400 m elevation. Approximately 95% of the collected isolates have been identified as N. discreta. Important questions remain regarding the reservoirs of inoculum, modes of colonization and dispersal. Perithecia have been observed at only one burn site, despite the fact that isolates can be recovered from soils within and adjacent to burn sites using plating procedures that select for ascospore-derived individuals. Within a site, mating type among individuals is sometimes significantly skewed from a 1:1 ratio. Nevertheless, evidence suggests substantial diversity on small and large spatial scales, as has been observed previously for isolates from Florida and Louisiana. Both mating types are present at western sites, ascospores can be obtained from crosses in the laboratory, and genetic variation independent of mating type exists within and between sites (at the het-c locus, for example).

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Molecular analysis of hyphal anastomosis.
Sovan Sarkar¹, Dave Jacobson ² and Louise Glass^{1, 1} PMB,University of Berkeley, ² Stanford University.

Growth in filamentous fungi occur through combinations of hyphal tip extension, branching and fusion( anastomosis). Initial live cell imaging as well as mutational analysis indicate the possible role for MAPK signaling pathway in hyphal fusion. We have four hyphal fusion defective mutants-so-1, ham-2 ,nrc-1 and mak-2. All show pleiotropic effects on growth and reproduction.These include slower growth rate, lack of aerial hyphae, female sterility and reduction or complete lack of self fusion or formation of a heterokaryon with a wild type strain. A wild type copy of nrc-1 is able to complement conidiation defect, growth rate, aerial hyphae production as well as restoration of self hyphal fusion and form heterokaryons with wild type strain. Two mutants, nrc-1 and mak-2 are known to have MAPKK and MAPK homologs respectively in yeast. We have created a dominant activated allele of NRC-1 by expressing only the carboxy terminal kinase domain. This allele will be introduced into wild type, nrc-1 null mutant , ham-2 and mak-2 to see what the effect(s) of expression of the activation domain is. Future work will also investigate the levels of phosphorylation of MAK-2 in a dominant activated nrc-1 mutant using phospho-specific antibodies to ERK-2. These experiments will indicate the relationship between nrc-1 and mak-2-whether they are involved in the same signaling pathway or whether these mutations affect different pathways but result in a similar mutant phenotypes.

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Comparative analysis of two-component histidine kinases in Neurospora crassa and the fungal pathogen Cochliobolus heterostrophus.
Natalie Catlett, Olen Yoder, and Gillian Turgeon. Torrey Mesa Research Institute, San Diego, CA

Two-component signal transduction systems have been found in bacteria, plants, fungi, and slime molds. These signaling systems often function to allow the organism to sense and adjust to changing environmental conditions. These systems consist of a histidine kinase component, that autophosphorylates in response to an environmental stimulus, and a response regulator component. Most eukaryotic histidine kinases are hybrid, containing both the histidine kinase and the response regulator within the same protein. N. crassa (WICGR 2nd assembly) contains at least eight putative hybrid histidine kinases while C. heterostrophus contains at least twelve. Only six of these histidine kinases appear to be orthologs common to both C. heterostrophus and N. crassa. The remaining six C. heterostrophus histidine kinases may be unique to Cochliobolus or to fungal pathogens. Deletions strains for each C. heterostrophus histidine kinase are under investigation.

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A further study of the cr-5 mutant of Neurospora crassa.
*Heather Cathcart, Sara N. Bennett, and Wayne A. Krissinger. Georgia Southern University, Statesboro, Georgia 30461

The crisp-5 (cr-5) mutant (allele 123 CJ13-6A) of N. crassa was isolated in the Georgia Southern Neurospora Laboratory and was found to be linked to ad-4 in LG III. The present study mapped cr-5 to the right of ad-4 with 1.3% to 5% recombination. Both cr-5 and wild type 74A were grown on minimal medium in which the carbon source was 2% sucrose, 2% glucose, 2% fructose, 2% lactose, 2% mannitol, 2% glycerol, or 2% sorbose. The cr-5 mutant grew better on glucose and fructose separately than it did on sucrose. Both the mutant and wild type grew very poorly on lactose, mannitol, and glycerol. Neither strain tested grew on sorbose as the sole carbon source. Observations utilizing SEM indicated that, compared to wild type 74, cr-5 has a reduced mycelium containing clusters of conidia formed by highly branched conidiophores carried on short aerial hyphae. The mutant cr-5 conidiates prematurely and profusely. Conidia of cr-5 consistently germinated earlier than those of wild type, but the percent of conidia germinating one hour after the first observation of germination was higher for wild type.

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Characterization of osmotic sensitivity of os-8, os-9, os-10, and os-11.
DeWayne L. Adams, Sara Neville Bennett, and Wayne A Krissinger. Department of Biology, Georgia Southern University, Statesboro, GA 30460.

 Osmotic-sensitive mutants of N. crassa are identified by their failure to grow on medium with elevated concentrations (4% or 6%) of NaCl. A number of osmotic-sensitive mutants have been isolated in our laboratory. Two of these, os-9 (alleles SS-788 and SS-462) and os-11 (allele SS-18) had been found to fail to grow on medium with elevated NaCl, but to grow on medium with elevated KCl or glucose. This was in contrast to the first described osmotic mutant, os-1, which was sensitive to all three osmotica. In the present study two more of our mutants os-8 (allele SS-931) and os10 (allele SS-1018) were examined. The mutant os-8 failed to grow on medium with 2% NaCl, with 4% KCl or 6% glucose. The mutant os-10 failed to grow on medium with 5% NaCl, with 3% KCl or 3% glucose. To further characterize the osmotic sensitivity of os-9 and os-11, revertants of the mutants were obtained. Both mutants were crossed to the trp-1 tester strain and double mutants were recovered. Double mutants of os-9 and of os-11 were each subjected to UV irradiation. A total of 51 and 47 putative revertants of os-9 and os-11, respectively were recovered.

31
Neurospora proteins that bind methylated DNA and DNA mutated by RIP.
Gregory O. Kothe, Michael R. Rountree, Ashley McCormack, Larry David and Eric Selker. Institute of Molecular Biology, University of Oregon, Eugene, OR, 97403

 Using gel-mobility-shift assays we have detected proteins in Neurospora crassa that bind methylated DNA. One such factor binds most efficiently to DNA that is both methylated and contains mutations induced by RIP. We refer to this factor as MRBP-1 (Methyl/RIP Binding Protein 1). MRBP-1 was purified and peptide sequence data obtained using mass spectrometry. These data suggest that MRBP-1 might be a complex of several proteins. Methyl-DNA binding proteins may function "downstream", exerting their effects after methylation has been set up (eg. repressing gene expression). These factors may also be involved in maintaining methylation patterns in Neurospora. To test these possibilities we are introducing mutations in genes encoding the potential MRBP-1 complex components.

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Analysis of M-134, a crisp-like morphological mutant of Neurospora crassa.
Gregory Julian Digby, Wayne A. Krissinger, and Sara Neville Bennett. Georgia Southern University, Statesboro, GA 30460.

 M-134, a mutant strain of N.crassa, was isolated using ultraviolet irradiation in the Georgia Southern University Neurospora Laboratory. Preliminary mapping of M-134 indicated that the mutant is in Linkage Group I and linked to lys-4 by 9% recombination. Scanning electron microscopy was used to compare differences between M-134 and
wild type N. crassa. Both strains were grown on dialysis tubing coated with minimal medium, fixed, dehydrated, and sputter coated. Examination of the specimens showed that M-134 had reduced mycelium and highly developed conidiophores with primary, secondary, and tertiary branching when compared to the wild type N. crassa. Microscopy also revealed flask shaped clusters of conidia and moderate proconidial chains. Following 6 days of growth, clusters of densely packed conidia were observed on a reduced mycelial mat.

33
Characterization of cobalt-sensitive mutant of Neurospora crassa.
K.Rashmi and P.Maruthi Mohan. Department of Biochemistrty, Osmania University Hyderabad - 500 007 (A.P.), INDIA

Cobalt-sensitive mutants of N.crassa (CSM) were isolated by mutation enrichment protocols. CSM isolates showed differences in growth, morphology and sporulation. Single spore isolates of CSM showed that most of them to be 5-fold sensitive to cobalt. A CSM with good sporulation and growth was chosen for further experiments. CSM is 5-fold more sensitive to cobalt ions (I-50 = 0.1mM) on ammonium-N medium as compared to wild type N.crassa (I-50 = 0.5mM). Cobalt sensitivity was also observed on nitrate-N medium. Cross-sensitivity to Ni was also observed in CSM. Cobalt taken up by wild and CSM at their respective I-50 values was found to be similar, indicating that CSM accumulates more cobalt than wild type. Most of cobalt accumulated by wild type N.crassa was on surface (70%) while in case CSM it was in intracellular fraction. Fractionation of cell free extracts by DEAE-cellulose chromatography resolved cobalt in to protein-bound and free ionic fractions. No significant differences in cobalt distribution were observed between wild and CSM N.crassa. Mg ions that reverse the toxic effects of cobalt ions by suppressing transport in wild type were unable to do so in CSM. The above data suggest that the mechanism of increased toxicity of Co in CSM is due to hyperaccumulation of these ions resulting from modifications in cell wall and membrane transporter(s). In preliminary experiments some of the mutants showed features related to vacuolar mutants and hence N.crassa vma-1 was tested and found to be sensitive to cobalt, suggesting the role of Ca homeostasis in CSM. Mapping of CSM loci is in progress. The utility of CSM in bioremediation of toxic metal ions from low concentrations will be presented.
Acknowledgement: This work was supported by DAE grant No. 2000/37/20/BRNS

34
The search for roles in gene silencing and cell differentiation of the asd-2 gene, an essential gene for sexual development in Neurospora crassa.
Kelly A. Howe, Jeanne Louderbough and Mary Anne Nelson. University of New Mexico, USA.

Investigation into genes responsible for Neurospora crassa sexual development has led to the discovery of the asd-2 (ascus development) gene. The protein product of this gene has been found to be similar to the AGO/eIF2C2/Zwille family of proteins. This family has been implicated in two major processes: involvement in cell differentiation and involvement in posttranscriptional gene silencing. One member of this family, the N. crassa QDE-2 (quelling deficient) protein has been shown to be involved in quelling (posttranscriptional gene silencing). Initially, the asd-2 gene was identified as essential for sexual development; homozygous asd-2 crosses are blocked shortly after karyogamy. Ascospores are never produced and asci are short and few in number. Small perithecia are formed that lack perithecial beaks. Recently, we have shown that the asd-2 gene is not essential for quelling as quelling does occur in the asd-2 mutant strain. The mutant strain exhibits slow vegetative growth with an aberrant hyphal branching pattern as compared to wild-type. We are investigating the role of the asd-2 gene product in cell signaling and/or cell differentiation and the relationship of this role to sexual development.

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A report on the recent activites of the Fungal Genetics Stock Center.

Kevin McCluskey. FGSC, University of Kansas Medical Center, Kansas City, KS.

The Fungal Genetics Stock Center has continued to support both traditional genetic approaches and emerging molecular approaches towards working with Neurospora and other fungi. The FGSC has distributed thousands of fungal strains to researches around the world and continues to develop its holdings of Neurospora and related fungi. While our traditional focus has been on fungal strains, the inclusion of cloning vectors, gene libraries and cloned genes has enabled the FGSC to expand its reach into areas not served by its traditional materials. Beyond this, the FGSC has become the recognized home of fungal genetics on the internet. In our continuing effort to serve the fungal genetics research community, the FGSC always welcomes input and ideas from our constiuency.