Genomics and Proteomics

239. The Ashbya gossypii genome: lessons learned by comparison to the Saccharomyces cerevisiae genome. S. Brachat1, F. S. Dietrich1,3, S. Voegeli1, A. Lerch1, T. Gaffney2 and P. Philippsen1. 1Biozentrum der Universität Basel, Basel, CH. 2Syngenta, Research Triangle Park, NC, USA. 3Current Address: Department of Molecular Genetics and Microbiology, Duke University Medical Centre, Durham, NC, USA.

We completed the sequencing of the 9Mb genome the filamentous fungus Ashbya gossypii which encodes 4720 protein coding genes, 190 tRNA genes, 50 small RNA genes and 40-50 copies of the rRNA genes. With respect to the size and the number of genes, this represents presently the smallest genome of a free living eukaryot. Surprisingly, the protein coding genes revealed striking similarities to that of S. cerevisiae with over 95% of the A. gossypii genes sharing significant homology to S. cerevisiae genes. In addition, 90% of A. gossypii genes show both, homology and synteny, with the genome of the baker's yeast. The synteny can be described as "relaxed synteny" since the gene order in any A. gossypii chromosomal region reflects the sum of the gene order of 2 different regions in the S. cerevisiae genome. This observation can only be explained by a whole genome duplication in the S. cerevisiae lineage followed by frequent deletion of one of the two gene copies. Thus, the A. gossypii genome becomes the strongest evidence for the yeast genome doubling hypothesis. Careful alignment of both genomes lead to the identification of most translocation and inversion events in the evolutionary history of both organisms (500 rearrangements) and of the complete set of still duplicated genes in S. cerevisiae. It also made possible the detection of potential annotation or sequencing errors in the yeast genome. 5% of the A. gossypii genes were not found in S. cerevisiae (NOHBYs NO Homologues in Baker's Yeast). Nearly 60% of the NOHBYs have homologues in other species. Functional assessment based on homology and domain searches demonstrated the presence of many transcription factors among NOHBYs as well as the presence of new gene families. Similarly, over 400 confirmed S. cerevisiae genes could not be found in the A. gossypii genome. Analysis of this difference in gene pool is likely to corroborate the difference in life style between the two organisms.

240. Analysis of Thiol Peroxidases in Cryptococcus neoformans. Tricia Missall, Mary Ellen Pusateri, and Jennifer Lodge. Biochemistry, Saint Louis University School of Medicine, St. Louis, MO.

Cryptococcus neoformans is a human fungal pathogen found worldwide, most commonly causing meningitis in immunocompromised patients. There are many factors that affect the virulence of C. neoformans including its ability to grow at 37̊C. Two-dimensional gel electrophoretic analysis illustrates a difference in the protein expression of C. neoformansgrown at 37̊C versus 25̊C. MALDI-TOF analysis and sequence homology to other species, including Saccharomyces cerevisiae, identify these proteins as peroxidases that belong to the thiol specific antioxidant (TSA) family. Both of these enzymes, Tsa1 and Tsa3, are shown to be induced when C. neoformans is exposed to the oxidative stress of hydrogen peroxide. To determine the function of Tsa1 and Tsa3 in cryptococcal pathogenesis, deletion mutants of each and both of these enzymes were generated through homologous recombination at theTSA1 and TSA3 loci. The contributions of these thiol peroxidases to the virulence of C. neoformans were then analyzed by comparing the in vitro andin vivo growth of these mutants to that of the wild type strain. The tsa1 mutant and tsa1,tsa3 double mutant show sensitivity to both hydrogen peroxide and t-butyl hydroperoxide as well as significant growth retardation at high temperatures. In mice, the tsa1 and tsa1, tsa3 mutants showed significantly less growth in the brain and lung (p tsa3 mutant grew only moderately less in the brain (p <0.05) than the wild type strain. This is the first example showing the importance of a thiol peroxidase to the virulence of an organism.

241. Characterisation of appressoria specific proteins from Phytophthora infestans. Laura J. Grenville1, Catherine R. Taylor1, Alison Williams1, Paul Birch2, and Pieter van West1.1 University of Aberdeen, Department of Molecular and Cell Biology, Foresterhill, Aberdeen, AB25 2ZD. 2 Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, Scotland UK.

A thorough understanding of the molecular events taking place during early interactions between P. infestans and host and non-host plants is crucial for developing new control measures. The appressorial stage of the interaction is the first point in which direct contact between the pathogen and the plant occurs via the formation of highly specialised infection structures, such as the appressorium, penetration peg, and the infection vesicle. Moreover, it is during this phase that plant defence responses are initiated. Resistance is based, principally, on recognition of a particular elicitor component from the pathogen. Therefore, we anticipate that secreted and cell wall proteins from the appressorial infection stage of P. infestans are likely to be rich in important signalling molecules involved in disease resistance or establishing a successful infection process. A proteomic approach is employed to accelerate the discovery of novel extra-cellular and appressorial stage-specific proteins. Here we present our latest results.

242. A proteomic approach to identify secreted proteins from Phytophthora infestans relevant to the host-pathogen interaction. Shuang Li1, Pieter van West1, Sophien Kamoun2, Neil A.R. Gow1. 1 University of Aberdeen, Department of Molecular and Cell Biology, Foresterhill, Aberdeen, AB25 2ZD, Scotland UK. 2 The Ohio State University, Department of Plant Pathology, Ohio Agricultural Research and Development Center, Wooster, Ohio 44691, USA.

Microbial pathogens of plants secrete avirulence factors that are recognised by the products of plant resistance genes and trigger host resistance responses. In order to identify potential avirulence factors from Phytophthora infestans, secreted protein profiles of several strains with different avirulence phenotypes were analysed by proteomics. About 500 proteins were identified from culture filtrates of each strain. The most abundant 50 protein spots from one strain were analysed by peptide fingerprinting using MALDI-TOF mass spectrometry. Comparative analysis of the profiles of strains with known avirulence characteristics identified several proteins that were expressed differentially. The possible function of the corresponding genes could not be determined from the DNA sequences. Northern analysis showed that some of the genes were differentially expressed in the tested strains under the same or different culture conditions. Functional analysis of these genes will establish their potential as avirulence determinants of P. infestans.

243. Differential Gene Expression in Germinating Basidiospores. Lori G. Baker1, Sarah F. Covert2, and Paula Spaine3. 1Department of Genetics, 2Warnell School of Forest Resources, 3Forestry Sciences Laboratory, Southeastern Forest Experiment Station, USDA Forest Services. The University of Georgia, Athens, GA, U.S.A

Cronartium quercuum f.sp. fusiforme is the causative agent of the southern pine disease known as fusiform rust. Depending upon the environment, C. q. fusiforme basidiospores can germinate either directly or indirectly. When C .q. fusiforme basidiospores are cast upon pine needles or polystyrene they germinate directly by sending out a thin long germ tube. In contrast on non-host surfaces or glass, C. q. fusiforme basidiospores germinate indirectly by sending out a very short, wide germ tube that leads to the formation of a secondary basidiospore. The first part of this study is a comparison of gene expression patterns between the two germination fates. Suppression subtraction hybridization was used to make two cDNA libraries and a total of 1920 clones from them were sequenced. Sixty-eight of the clones are unique to the direct germination library, 82 are unique to the indirect germination library, and 15 are common to both libraries. Array hybridizations will be used to identify genes that are differentially expressed between directly and indirectly germinating basidiospores, germinating aeciospores, and pycniospore and aeciospore formation. By identifying genes that are differentially expressed in these distinct germination events, we will expand our understanding of C. q. fusiforme's germination, sporulation, and infection process as well as contribute to our general understanding of the same events in other rust/pathogenic fungi. The second part of this project tests the hypothesis that substrate hydrophobicity/wettability regulates basidiospore germination fate. The substrate surface wettabilities were altered as follows: polystyrene was exposed to ultra violet light to make it more hydraphilic and glass was treated with silane to make it more hydraphobic. Basidiospore germination experiments on the altered surfaces are in progress.

244. The asexual pathogen Aspergillus fumigatus has the coding capacity for mating-type proteins, pheromones and pheromone receptors. Stefanie Poeggeler and Ulrich Kuck. Botany, Ruhr-University Bochum, Bochum, Germany.

The filamentous fungus Aspergillus fumigatus is one of the causes of invasive lung disease in immunocompromised individuals. It has been classified as asexual because no direct observation of mating or meiosis has been reported. Sequencing of the complete genome by an international collaboration including the Wellcome Trust Sanger Institute (UK) and The Institute for Genomic Research (TIGR, USA) has made most of the genomic sequence information fromA. fumigatus publicly available. By searching the incomplete genome sequence of A. fumigatus, I have identified the coding capacity for a set of proteins that could be involved in mating and the pheromone response pathway. These include one putative mating-type gene, one gene encoding a pheromone and two pheromone receptor genes. The mating-type gene encodes a HMG domain protein exhibiting significant similarity with mating-type proteins from sexually reproducing filamentous ascomycetes. The pheromone gene is predicted to encode a precursor pheromone that is processed by a KEX2-like protease to yield a pheromone that is structurally similar to the alpha-factor of the yeast Saccharomyces cerevisiae. In addition, the deduced gene products of the receptor genes are putative seven-transmembrane proteins, which displays a high-level amino acid identity with the a-factor receptor Ste3p and the alpha-receptor Ste2p of S. cerevisiae, respectively. The identification of these homologues suggests the existence of a sexual cycle in A. fumigatus.


245. Identification of Perennial Ryegrass and Endophyte Proteins Involved in Symbiosis. Shalome Campbell1,2, Mike Christensen1, Richard Johnson1, T. William Jordan2 and Gregory Bryan1. 1AgResearch Limited, Tennent Drive, Private Bag 11008, Palmerston North, New Zealand; 2Victoria University of Wellington, PO Box 600, Wellington, New Zealand

Perennial ryegrass (Lolium perenne) is one of New Zealand's most important pasture species. Most New Zealand ryegrass pastures are infected with Neotyphodium lolii, an endophytic fungus which grows intercellularly within the host thereby obtaining nutrients. In return the fungus produces a range of secondary metabolites which confer host resistance to a number of insect pests. We are developing 2D electrophoresis and proteomic techniques to identify components of this symbiotic relationship. Initial approaches focused on comparison of isogenic ryegrass lines with and without endophyte infection. We are targeting enriched protein fractions from plant tissues, including intercellular fluid from the apoplastic space of leaf and stem tissue. Using a combination of 2D electrophoresis and polyclonal antibodies to N. lolii, we have obtained mass fingerprint analysis for nine protein spots. Four of the nine spots were matched to plant proteins that are either up or down regulated during pathogenesis in other plant-microbe interactions. A further three protein spots have been identified by mass spectrometry after narrowing the pH range in the first dimension. We are currently using degenerate PCR to clone these gene products, two of which may be of fungal origin. Future work will focus on gene silencing to elucidate the role of these genes in symbiosis.

246. Expression Analysis of Aspergillus oryzae in Solid-state Fermentations. Rob te Biesebeke, Peter Punt, Ana Levin, Margreet van Heerikhuisen, Arjen Rinzema, Kees van den Hondel. Wageningen Centre for Food Sciences, The Netherlands, TNO Nutrition and Food Research Institute, The Netherlands, Food and Bioprocess engineering group, Wageningen University, The Netherlands

Fungal solid-state fermentations (SSF) are applied in many traditional food fermentation processes. There is abundant proof in literature that the product spectrum from SSF is very different from that obtained in submerged fermentation (SmF). However, the mechanisms underlying these differences are not at all understood. Therefore rational new design of SSF processes to make new products and optimise the production of existing products is not possible. Only recently, significant advances have been made in understanding the physical (process engineering) aspects of SSF but the information on physiology and molecular genetics is limited. We have initiated a comparative analysis of gene expression of the model organism Aspergillus oryzae in SSF and SmF. With the development of fungal genomics technology a genome-wide approach can be followed to study SSF specific gene expression. Our results show a large difference in the spectrum of expressed genes in SSF and SmF as confirmed by Northern analysis. Our results indicate the presence of gene regulatory mechanisms specific for surface grown Aspergillus oryzae not observed in liquid cultures.

247. Genomic analysis, deletion and the characterization of Multidrug tranporters of a fungal plant pathogen, Cochliobolus heterostrophus. Uvini Gunawardena1, Olen Yoder1 and Gillian Turgeon2 1 Torrey Mesa Research Institute, San Diego, CA2 Cornell University, Ithaca, NY

Eukaryotic membrane transporters include two major families of proteins that are involved in multidrug transport – ABC (A TP Binding Cassette) and MFS (Major Facilitator Superfamily) transporters. In the context of plant pathogen interactions, these multidrug resistance proteins are of interest for their potential to mediate interactions between hosts and pathogens, as well as for the opportunities they offer to genetically engineer plants for resistance to microbial virulence factors such as toxins. We have mined the genomic sequence ofCochliobolus heterostrophus, a pathogen of corn, and found 41 putative ABC and 51 putative MFS proteins. All the ABC transporters of C. heterostrophus have been targeted for deletion and null mutants of 38 genes have been collected. Deletions of two ABC transporters are believed to cause lethality because of repetitive failures to obtain gene deletants. Interestingly, one of these transporters resides close to NPS9 (Non Ribosomal PeptideSynthetase encoding gene) suggesting a role in the transport of a small peptide with cytotoxic properties, whose synthesis is controlled by NPS9. Deletion of two other ABC transporters resulted in reduced virulence of C. heterostrophus on corn. One is presumed to be involved in the efflux of phytoalexins from fungal cells. The other is hypothesized to have a role in the transport of NPS6, a virulence factor, thus mediating disease establishment. Characterization of these transporters including their role in fungal mating and resistance to cytotoxic compounds will be presented.

248. Conservation of Active Site and Essential Structural Components Encoded by a Putative Neurospora crassa Phytase Gene. Edward J. Mullaney and Catherine B. Daly. SRRC ARS USDA New Orleans, LA 70124

The Neurospora crassa sequencing project has identified many hypothetical proteins. One such protein, Locus NCU06351.1, has been reported as having the conserved sequence unique to the histidine acid phosphatase class of enzyme. Aspergillus niger phytase is a member of this class of enzyme. The gene for this enzyme has been cloned, over-expressed, and its product is now commercialized as an animal feed additive with an annual market value of over $$500 million. A study of the amino acid sequence of NCU06351.1 reveals additional evidence that it is analogous to the Aspergillus phytase. All the essential components of the active site and the substrate specificity site are present in this putative Neurospora protein. Remarkably, it also appears that all the constituents of the five disulfide bridges found in Aspergillus phytase have been conserved in this NCU06351.1. These disulfide bridges seem to have a vital role not only in the folding pathway, but also in maintaining the proper configuration of the molecule for catalytic activity. As more is learned about the structure and function of other fungal phytases, ascribing characteristics to this putative Neurospora phytase will become more precise.

249. Gene Ontology Tools at SGD. Chandra Theesfeld, Shuai Weng, Dianna Fisk, Rama Balakrishnan, Karen Christie, Kara Dolinski, Selina Dwight, Eurie Hong, Laurie Issel-Tarver, Anand Sethuraman, Gavin Sherlock, David Botstein and Michael Cherry. Department of Genetics, Stanford University

Every Saccharomyces cerevisiae protein is now associated with a biological process term(s) from the Gene Ontology (GO). In order to help biologists understand and utilize the ontologies created by the GO Consortium, the Saccharomyces Genome Database (SGD) has created two new web-based tools and a graphic display: the GO Term Mapper, the GO Term Finder and the GO Tree View. The GO Term Mapper and GO Term Finder tools were designed as aids to researchers employing large-scale methods of analysis. The GO Term Mapper allows users to input a list of genes and determine the upper level GO terms associated with those genes by tracing the ontologies from the granular, specific term (associated directly with a gene) to an upper level GO term. The upper level GO terms used by the GO Term Mapper are pre-defined by SGD and represent a low-granularity slice of the ontology (GO Slim). The GO Term Finder also allows users to input a list of genes, but rather than mapping the genes to a defined set of terms, this tool searches the GO structure to find significant GO terms shared among the genes in the list. The GO Tree View helps users place selected GO terms into context by graphically illustrating the terms within the GO structure; genes directly or indirectly associated with each term are also shown. To help familiarize users with GO resources at SGD we have created a GO tutorial. The GO tutorial is available here: The GO tools are available at the following URLs:

The SGD home page is:

250. Prediction of Questionable Open Reading Frames. Hong, E.L., Dolinski, K., Balakrishnan, R., Christie, K. R., Costanzo, M. C., Dwight, S. S., Engel, S. R., Fisk, D. G., Sethuraman, A., Theesfeld, C. L., Weng, S., Botstein, D., and Cherry, J. M. Dept. of Genetics, Stanford University School of Medicine, Stanford, CA 94305

Several years after the completion of the systematic sequencing of the yeast genome, researchers are still questioning the total number of biologically significant open reading frames (biological ORFs) in S. cerevisiae. Recently, Jason Lieb and Patrick O. Brown (Dept. of Biochemistry, SUMC) proposed a scoring method involving multiple criteria such as ORF length, codon probability, and microarray data to discriminate between biological ORFs and those occurring by chance (questionable ORFs). We have expanded this idea to include other criteria and propose here a method for a more complete evaluation of questionable ORFs within the yeast genome. Our method of discrimination between biological and questionable ORFs included the presence or absence of S. cerevisiae ORFs in the genomes of other related species such as S. paradoxus, S. mikatae, S. bayanus, Kluyveromyces yarowii, Schizosaccharomyces pombe, and Candida albicans. ORFs were also validated based on the Gene Ontology (GO) function annotations described in the Saccharomyces Genome Database (SGD). Published data from large-scale analysis of S. cerevisiae genome were also given due considerations. ORFs were ranked for each criteria and all failure candidates were flagged questionable. Our ranking procedure to validate ORFs as well as the conclusions of our data analysis will be presented. SGD is supported by NHGRI.

251. Proteome analysis of extracellular proteins from solid-state culture of Aspergillus oryzae. Ken Oda, Kazuhiro Iwashita*, Dararat Kakizono, Haruyuki Iefuji, and Osamu Akita National research institute of brewing, 3-7-1, Kagamiyama, Higashihiroshima, Hiroshima, 739-0046, Japan

 A. oryzae is an important microorganism for food industries, such as beverages and seasonings, and enzyme industries. The high productivity of various enzymes is the most important character of this industrial mold. For this importance, about 50 of genes encoding extracellular enzymes have been cloned and characterized. However, by the observation of 2D-electrophoresis, it is known there are still many kinds of proteins remain uncharacterized. Recently, more than 15,000 clones of EST sequence were reported and genome DNA sequencing project have been carried out by the Japanese consortium including universities, companies and national research industries. Using this DNA sequence database and mass spectrometry, we performed the identification of secreted proteins. The identification of proteins by mass spectrometry is based on the protease hydrolysis and released peptide mass. However, some extracellular proteins are highly glycosylated and resistant to protease hydrolysis. To overcome this problem, we developed in-gel deglycosylation method for increasing the efficiency of protease hydrolysis and decreasing the heterogeneity of peptide by the modification of oligosaccharide. Numbers of the detected peptides and the coverage of amino acid sequences were increased in several proteins by this treatment. Using this method, 41 proteins from extract of solid-culture were identified, in which 35 proteins have not been identified before.

252. Gene hunting in Mycosphaerella graminicola. Gert Kema1, Els Verstappen1, Theo van der Lee1, Odette Mendes1, Hans Sandbrink2, Rene Klein-Lankhorst2, Lute-Harm Zwiers3, Mike Csukai4, Ken Baker4 and Cees Waalwijk1. 1Biointeractions and Plant Health and 2Greenomics, Plant Research International B.V., 3Laboratory of Phytopathology, Wageningen University vand Research Center, P.O. Box 16, 6700 AA Wageningen, The Netherlands 4Syngenta, Jealott's Hill, Berkshire, England

Septoria tritici leaf blotch of wheat, caused by Mycosphaerella graminicola, is a wheat disease of global importance. Approximately € 400 million which equals 30% of the total fungicide input in Western Europe, is used to control this disease. Although the interaction between M. graminicola and its host is microscopically well described, knowledge of M. graminicola genes involved in basic cellular functions as well as pathogenesis and stress responses is limited. We aimed to fill this gap by a large EST project, providing sequence information of M. graminicola to underpin future control strategies. We produced EST libraries from seven in vitro conditions, representing various morphological forms and stress conditions, as well as three different interaction libraries to obtain the widest possible range of genes. Fungal clones were selected from the interaction libraries using a proprietary capture technology. In total, we generated 30.137 high quality reads of which 3894 were derived from in planta libraries. Based on the BLAST homology, clones selected from these libraries are largely of fungal origin. EST reads were assembled into 10.976 unigenes of which a large number were unique to the interaction libraries. A large proportion of the clones appeared to be full length, as exemplified for the elongation factor 1. We identified several novel ABC and MFS transporters as well as a comprehensive set of homologs of the various signal transduction pathways. In the in planta libraries a range of cellulytic enzymes was found. Further analysis of these genes by targeted disruption, expression analysis and comparative genomics is currently undertaken. Together with the wide body of sequence data currently available from related fungi this will boost our knowledge on the infection process of this important pathogen.

253. Diverse LTR-retrotransposons of the cultivated basidiomycete Agaricus bisporus. Richard W. Kerrigan1 and Anton Sonnenberg2. 1Sylvan Research, Kittanning, PA, USA. 2 Applied Plant Research BV, Horst, the Netherlands.

Screening of cosmid and lambda libraries of Agaricus bisporus (Lange) Imbach have revealed a diverse assemblage of LTR-retrotransposons, often with affinities to gypsy or copia elements. Based on the deduced amino acid sequences of ORFs corresponding to gag and pol genes, more than 10 distinct LTR-retrotransposons, either fragmentary or intact, are present in the genome of this mushroom. These elements are often present in high copy numbers and appear to be dispersed throughout the genome. It is common to find multiple elements represented within relatively short segments of chromosomal DNA. Fragments of elements also commonly disrupt the organization of other elements, making reconstruction of full-length sequences challenging. RT-PCR indicates that some of these retrotransposon genes are transcribed under certain conditions. Although we have no direct evidence for active transposition, some retroelements are associated with occasional alterations of the genome, based on data from Southern hybridizations to genomic DNA.

254. Deletion of Aspergillus nidulans aroC by a Novel Blaster Module Combining ET Cloning and Marker Rescue. Kerstin Helmstaedt, Sven Krappmann, Verena Grosse, and Gerhard H. Braus Institute of Microbiology and Genetics, Department of Molecular Microbiology, Georg-August University Goettingen, Germany

Blaster cassettes are of significant value in functional genomics, as they represent tools to inactivate duplicated or homologous genes in an individual organism subsequently. We have constructed a novel blaster module for the potential use of repeated gene deletion in the filamentous fungus Aspergillus nidulans. Due to the employment of bacterial resistance marker cassettes as direct flanking repeats, the blaster cassette is suited for recombinogenic engineering by ET cloning in E. coli. Functionality of the blaster module was demonstrated by deleting the chorismate mutase-encoding gene aroC of A. nidulans followed by marker rescue based on mitotic recombination. The resulting aroC Delta strains are auxotroph for phenylalanine but not tyrosine and display limited capacities of fruit body formation and ascosporogenesis, dependent on the phenylalanine/tyrosine supply. The data support the strong impact of the amino acid status on cleistothecia development in A. nidulans.

255. The role of lipid metabolism in virulence of Candida albicans. Katarzyna Piekarska and Marlene van den Berg, Guy Hardy and Ben Distel. Department of Biochemistry, Academic Medical Center, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.

One typical characteristic of the pathogenic yeast C.albicans is its ability to grow in two different cell shapes, namely a yeast-form or a hyphal-form. Conclusive evidence has been provided that the ability to switch between these two morphologies is essential for its pathogenesis (1). Once initiated, the hyphal program not only results in the switch to the hyphal morphology to allow penetration of host tissues, but it also facilitates adaptation to the new environmental conditions, such as the host's internal lipid environment (2). Expression analysis showed that some peroxisomal enzymes are upregulated under those conditions notably two glyoxylate cycle enzymes: isocitrate lyase and malate synthase. Using a PCR based disruption technique we have constructed deletion strains of PEX5 and PEX13 genes, both of which are essential for peroxisome formation. The phenotypic characterization of these C.albicans mutants will be presented. The second approach we are following is the application of DNA microarrays to identify hyphal specific genes encoding proteins involved in lipid metabolism. As a pilot for the expression analysis of the whole genome of C.albicans a set of genes was selected and used to generate submacroarrays to monitor differential gene expression during the yeast-to-hyphal transition in wild type and mutant C.albicans strains. 1. Lo, H.J. et al., Cell 90: 939-949, 1997 2. Lorenz, M.C. and Fink, G.R., Nature 412: 83-86, 2001

256. An ordered collection of expressed sequences from Cryphonectria parasitica. Angus L. Dawe, Vanessa C. McMains, Maria Panglao, Shin Kasahara, Bao Chen and Donald L. Nuss. Center for Biosystems Research, University of Maryland Biotechnology Inst, College Park, MD.

Cryphonectria parasitica is the causative agent of the chestnut blight disease. Earlier studies have demonstrated that C. parasitica represents a tractable model plant-pathogenic system. The availability of naturally occurring dsRNA elements (hypoviruses) that are capable of reducing fungal virulence provides the opportunity to examine molecular aspects of fungal plant pathogenesis in the context of biological control. In order to establish a genomic base for future studies of C. parasitica, we have analyzed a collection of expressed sequences (ESTs). A mixed cDNA library was prepared from RNA isolated from wild-type (virus free) and virus containing strains. Plasmid DNA was recovered from individual transformants and sequenced from the 5-prime end of the insert. Sequence data was trimmed of vector and poor quality information before being used to create a BLAST-searchable database of the complete EST dataset. Contig analysis of the collected sequences revealed that they represented approximately 2200 individual open reading frames. An assessment of functional diversity present in this collection was achieved by comparing the new sequence information against local (downloaded) copies of the NCBI protein database using the BLAST software utilities. The output was analyzed according to the significance likelihood of the hits returned for each clone. Using those hits deemed to be of greatest significance, this information was then classified according to the Gene Ontology Consortium guidelines for both molecular function and biological process. This data represents the largest collection of sequence information currently available for C. parasitica and is now forming the basis of further studies using microarray analyses to determine global changes in transcription that occur in response to hypovirus infection.

257. Exploring the pathogenesis of Cryptococcus neoformans using serial analysis of gene expression. Steen, B.R., Zuyderduyn, S., Lesiuk, J., Toffaletti, J., Rusaw, S., Marra, M., Jones, S., Perfect, J. and Jim Kronstad. University of British Columbia, Vancouver BC. Canada

Cryptococcus neoformans is a basidiomycete fungus that has emerged as an important opportunistic human pathogen of immunocompromised patients. C. neoformans, if inhaled, can disseminate from the lung to the blood stream where it is capable of crossing the blood-brain barrier. This often leads to fatal meningoencephalitis. To begin to explore transcription during growth of the fungus in the brain, C. neoformans H99 cells were harvested from the cerebral spinal fluid of twelve New Zealand white rabbits following intrathecal injection with 1.3x109 H99 cells. Cells were harvested on days five (2.6x107 cells), seven (1.1x107 cells) and nine (2.0x106 cells) post infection. mRNA was isolated from a pooled collection of 3.9x107 cells and the micro serial analysis of gene expression protocol (microSAGE) was used to construct a SAGE library. SAGE tags were collected, the most highly expressed tags were analyzed and the corresponding cDNAs or genomic contigs were identified. BLASTx was used to find similarities in the non-redundant database at the National Centre for Biotechnology Information (NCBI) website for each sequence. The most abundant tags in vivo were compared with their corresponding abundance profiles in vitro. The in vitro SAGE libraries were constructed from cells that were grown in minimal media at 25 and 37 degrees. In addition, tags that matched known genes were compared with the expression of these same genes in a serotype D strain of C. neoformans. Several genes that were highly expressed in vivo appear to be regulated differently between strains. It is possible that this may contribute to the higher degree of virulence for the serotype A strain relative to the serotype D strain. Several genes, four of which are HSP60, HSP90, HSP12 and the novel gene designated HOT1, have been chosen for further studies. These genes are being characterized in both serotypes to determine the role that these genes may play in the pathogenesis of C. neoformans and how differential regulation between serotypes may account for the differences in pathogenesis.

258. Development of expression systems for use in Aspergillus fumigatus. Michael J Bromley, Caroline L Gordon, Catherine E Rycroft, Peter M Hey and Jayne L Brookman. F2G Ltd., Manchester Incubator Building, Manchester, England.

Aspergillus fumigatus is an opportunistic fungal pathogen of man causing a wide spectrum of diseases some of which are fatal. To facilitate the discovery of efficient anti-fungal drugs we have identified genes that are required for fungal growth in vitro. To verify the potential of putative targets we have developed a conditional gene expression system in A. fumigatus. An important consideration for development was the ability of the expression system to act in any host models of fungal infection. We have previously demonstrated the use of wax moth larvae Galleria mellonella as a model for gene expression studies during A. fumigatus infection. The development of expression systems that can be manipulated in this and a more standard mouse model host are discussed in this study. The expression of a selection of A. fumigatus genes has been assessed in vitro. Two genes were identified (cbaA and cbhB) which were strongly expressed in the presence of an inducer (CMC) but repressed in the presence of glucose. Expression of cbaA and cbhB was not detected during infection of G. mellonella. Data will be presented on the expression of these genes during infection in the presence of the inducer. The promoter regions of these genes have been used as the basis for construction of expression cassettes in A. fumigatus. Data will be presented on the expression of a reporter gene in the cassette.

259. Microarray profiling of Hypovirus-infected Chestnut Blight Fungus, Cryphonectria parasitica. Todd D. Allen, Angus L. Dawe, Donald L. Nuss. Center for Biosystems Research, University of Maryland Biotechnology Institute, College Park, MD 20742.

Members of the RNA virus family Hypoviridae persistently alter phenotypic traits, modulate gene expression and attenuate virulence of their fungal host, the chestnut blight fungusCryphonectria parasitica. Creation of an ordered C. parasitica EST library/database consisting of 4200 sequences and representing approximately 2200 genes has facilitated the creation of a spotted cDNA microarray chip to examine global transcriptional responses to hypovirus infection. Infection by the severe hypovirus isolate CHV1-EP713 resulted in a significant alteration in transcript accumulation for approximately 10% of the arrayed ESTs, confirming and extending the results obtained from previous differential mRNA display analysis. In contrast, transcripts corresponding to only 5% of the spotted ESTs were found to be significantly altered in accumulation following infection by the mild hypovirus CHV1-Euro7. Of these genes, less than half were also altered in expression as a result of infection by the severe hypovirus CHV1-EP713. Thus, the changes in cellular transcriptional profiles caused by mild and severe hypoviruses differ considerably in both magnitude and composition. These results are consistent with previous observations of differential modulation of cellular signaling pathways by the mild and severe hypovirus isolates based on single gene readouts. Moreover, these surprising differences in the transcriptional profiles suggest a basis for differences in phenotypic changes caused by the two hypoviruses. The unique capabilities provided by the hypovirus/C. parasitica system for functional characterization of differentially expressed genes identified by microarray analysis will be discussed.

260. Functional genomics in Magnaporthe grisea: a strategic evolution in fungicide discovery. Anne Lappartient1, François Villalba1, Philippe Perret1, Anne Marie Dechampesme1, Christophe Fargeix1, Mathieu Gourgues1, Andy Tag2, Sophie Rozenfeld1, Jean Luc Zundel1, Alain Villier1, Terry Thomas2, Marc Henri Lebrun1, Roland Beffa1. 1 CNRS-UMR 1932 and Bayer CropScience, 14 rue P. Baizet, 69009 Lyon, France. 2 Texas A&M University, College Station, TX 77843-3258, USA

Novel modes of action are needed to overcome resistance to existing fungicides. Combinations of classical biochemistry tests together with genomic analysis are required for their identification. Transcriptome, proteome and metabolite analysis of fungi treated with antifungal compounds with unknown mode of action revealed the pathway inhibited. The validation of these results by classical biochemistry (e.g. enzyme activity test) as well as by functional genomic led to the enzyme inhibited by the tested compounds. Unraveling functions implicated in the infection process of plant or fungal growth is an important challenge for crop protection. Availability of the fungal genome sequences, such as Magnaporthe grisea, will facilitate the discovery of such genes. Engineered fungal transposons such as impala, an autonomous TC1/mariner element from Fusarium oxysporum; or Agrobacterium mediated transformation are versatile tools for insertion mutagenesis and gene replacement. Alternative methods using RNAi induced gene silencing were developed to inactivate rapidly genes of interest. Mutants expression profiles highlight pathways controlled by genes required for pathogenicity or growth and can be compared with those obtained with known inhibitors.

261. Identifying protein interaction networks in signaling pathways that regulate appressorium development in the rice blast pathogen Magnaporthe grisea. Resham Kulkarni and Ralph Dean, Fungal Genomics Laboratory, North Carolina State University.

Magnaporthe grisea, the causative agent of the rice blast disease, produces a specialized infection structure the appressorium to penetrate plant tissues. The cAMP signal transduction pathway is one of the signaling pathways that controls appressorium development. To identify new components within this pathway, we used the yeast two-hybrid system to screen adenylate cyclase (MAC1) and the catalytic subunit of cAMP-dependent protein kinase A (CPKA), against an appressorial cDNA library. The appressorial cDNA library was constructed in the GATEWAY donor vector (Invitrogen), enabling its transfer to different vectors by recombinational cloning. The protein phosphatase domain in MAC1, unique to fungal adenylate cyclases, interacted with a MAP kinase kinase and a Ser/Thr protein kinase. These interactions could be a part of feedback between the different pathways. A putative extracellular membrane protein ACI-1, which may serve as a cell receptor or an adhesion molecule, was also isolated as a MAC1 interacting protein. This protein contained an extracellular cysteine-rich domain, which also appears in a number of other fungal membrane proteins including M. grisea Pth11. The glutamate-rich N-terminus of CPKA interacted with a putative transcriptional regulator and two different glycosyl hydrolases. Phosphorylation motifs in these sequences suggest that they could be CPKA substrates. We are now scaling up the protein interaction assays to delineate interactions both in and between signaling pathways that are implicated in fungal development.

262. SAGE analysis of iron-regulated transcription in the human pathogenic fungus Cryptococcus neoformans. T. Lian, M., Zuyderduyn, S., Marra, S. Jones and J. Kronstad. Biotechnology laboratory, Department of Microbiology and Immunology, and Faculty of Agricultural Science, The University of British Columbia, Vancouver, B.C V6T 1Z3, Canada. Genome Sciences Center, B.C Cancer Agency, Vancouver, B.C. V5Z 4E6, Canada

The basidiomycete fungus Cryptococcus neoformans is an opportunistic pathogen that causes meningioencephalitis in immunocompromised individuals. The extracellular polysaccharide capsule produced by C. neoformans is essential for virulence and capsule production can be induced by iron starvation. We used the technique of serial analysis of gene expression (SAGE) to analyze iron-regulated gene expression in C. neoformans. Specifically, we constructed and characterized SAGE libraries from C. neoformansserotype D cells grown in low iron and iron replete media. This work provides the first analysis of the most abundantly transcribed genes for two different iron levels in this fungus. In addition, sets of genes that are implicated in iron metabolism were identified, including those encoding an iron permease (Ftr), a multicopper oxidase (Fet3), a ferric reductase, and an aconitase. Northern blot analysis confirmed that transcript levels for these genes were increased or decreased in response to iron deprivation. From the analysis of the most highly expressed tags under iron deprivation, a gene for a novel iron regulatory protein (IRP) was identified and analyzed. The disruption of the IRP gene in the strains B3501 and ATCC24067 resulted in cell growth arrest and loss of the ability to form enlarged capsule in iron-limiting conditions. These results suggest that IRP acts to facilitate or regulate the uptake of iron from the environment, perhaps acting as a sensor of iron availability. Further functional analysis of this gene and virulence tests in animals are in progress. These studies will contribute to an understanding the iron regulation of capsule synthesis and identify potential targets for anti-fungal therapy.

263. Characterization of the genetic interval spanning the Avr1a locus fromPhytophthora sojae. D. Qutob1, T. MacGregor1, Y. Cui1, T. Sharifian1, and M. Gijzen1. 1Agriculture and Agri-Food Canada, 1391 Sandford Street, London, ON N5V 4T3, Canada.

Phytophthora sojae is an aggressive soil-borne pathogen that causes damping-off diseases of soybean. In this patho-system, host resistant (Rps) genes and pathogen avirulence (Avr) genes determine race-cultivar specificity. Here we report progress on a map-based cloning study to isolate avirulence gene, Avr1a. Previously, a high-resolution genetic map of the region was constructed using DNA markers. A marker co-segregating withAvr1a was used to screen a bacterial artificial chromosome (BAC) library. Subsequent steps to chromosome walk resulted in an assembly of eight overlapping BAC clones spanning 170 kb and encompassing the Avr1a locus. A total of 119 kb of this contig has been fully sequenced and examined for predicted open reading frames (ORFs) and matches to expressed sequence tags. Among the 25-30 ORFs identified, there are few that show any similarity to known proteins. Three of the ORFs match expressed sequence tags from zoospore or infected plant cDNA libraries. At least four other ORFs appear to represent expressed genes, by RT-PCR analysis. A total of 10 of the potential ORFs share sequence similarities with one another, and constitute part of a large gene family that is clustered in the region. Currently, we are developing more markers along the contig and creating F3 populations from recombinant F2's to delineate the exact boundries of Avr1a. This information will facilitate identification of candidate genes to be screened for functional analysis and transformation.

264. Proteomics Of Magnaporthe grisea: Liquid Chromatography Mass Spectrometry For The Identification Of Extracellular Proteins. Sheng-Cheng Wu, Jeremi Johnson, Kumar Kolli, Peter Albersheim, Alan Darvill and Ron Orlando. Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, USA

Extracellular proteins (ECPs) serve diverse and essential biological functions in various cell types at different physiological states. In fungi, ECPs are required for uptake of molecules for nutrition, growth, cell sensing and communication. Perhaps most importantly, many ECPs play crucial roles in fungal pathogenicity and fungus-host interactions. Despite their biological significance, very little is known about fungal ECPs. As fungal genomics enters a new era, it has become apparent that development of high-throughput proteomics technologies for systemic identification and profiling of fungal ECPs and other network proteins is required. We will report the use of 2-dimensional gel electrophoresis (2DE) to display, and liquid chromatography tandem mass spectrometry (LC MS/MS) to identify and archive Magnaporthe griseaECPs from various culture conditions. [This research was supported by U.S. Department of Energy (DOE) grant DE-FG05-93ER20221, the DOE-funded (DE-FG02-93ER20097) Center for Plant and Microbial Complex Carbohydrates, The National Science Foundation (NSF grant number 9626835), and the National Institutes of Health (NIH grant number P41RRR05351).]

265. Gene expression studies in the soybean rust pathogen Phakopsora pachyrhizi. Martha L. Posada1, Laura J. Ewing2 and Reid D. Frederick3. 1DOE Joint Genome Institute. 2800 Mitchell Drive, Walnut Creek, CA 94598. 2Hood College, 401 Rosemont Ave, Frederick, MD.321701USDA-ARS Foreign Disease-Weed Science Research Unit, 1301 Ditto Avenue, Fort Detrick, MD 21702.

Soybean rust is caused by the obligate fungal pathogen Phakopsora pachyrhizi. To understand gene expression during spore germination, a unidirectional cDNA library was constructed using mRNA isolated from P. pachyrhizi urediniospores germinating on a water surface. Sequence analysis of 908 clones revealed that 404 Expressed Sequence Tags (ESTs) displayed significant similarities to sequences in the "non-redundant" protein database. 56.5 percent of the identified ESTs were either hypothetical proteins or proteins of unknown function. The remaining 43.5 percent of the 404 ESTs were arranged using the Expressed Gene Anatomy Classification (EGAD) scheme. Approximately 20 percent of these genes were involved in metabolism, 7.5 percent in gene/protein expression, 5.5 percent in cell structure/motility, 4.5 percent in cell division, 3.5 percent in cell/organism defense, and 3.0 percent in cell signaling/cell communication. 488 unique ESTs were identified from the 908 clones, of which 380 ESTs appeared as a single copy. Several ESTs were identified as potential gene family members (i.e. gEgh16, DAHP synthase and non-histone chromosomal proteins). Expression of selected ESTs was measured during the infection process using real time RT-PCR.

266. Proteomic analysis of the Neurospora crassa cell wall. C. Alexander Valencia1, John F. Kelly2 and P. John Vierula1. 1Department of Biology, Carleton University, Ottawa, ON, Canada, K1S5B6. 2Institute for Biological Sciences, National Research Council, Ottawa, ON, Canada, K1A0R6.

The Neurospora crassa cell wall has a complex, laminate structure composed primarily of polymeric, mixed glucans and chitin, as well as a minor fraction of proteins which is poorly understood. A large number of proteins were extracted from purified cell walls by various chemical treatments, including urea and 2-mercaptoethanol, and separated by two-dimensional polyacrylamide gel electrophoresis. Altogether 89 proteins were examined by on-line liquid chromatography tandem mass spectrometry (LC-MS/MS) and amino acid sequences were obtained for 56 proteins. Fifty six proteins were identified by MASCOT searches against the NCBI and Whitehead Institute Neurospora protein databases, including known cell wall proteins, glycolytic enzymes, heat shock proteins and proteins involved in several other processes. Examples of identified proteins include enolase, glyceraldehyde-3-phosphate dehydrogenase, heat shock 70 kDa protein, elongation factor 1-alpha, outer mitochondrial membrane porin, alcohol dehydrogenase and NMT1. Moreover, this approach identified several novel proteins which could be involved in cell wall biogenesis.

267. EST analysis of the human pathogen Paracoccidioides brasiliensis yeast phase: identification of putative homologues of Candida albicans virulence/pathogenicity genes. Gustavo H. Goldman1, Regina C. de Oliveira3, Luiz R. Nunes3, Luiz R. Travassos4, Rosana Puccia4, Fredj Tekaia5, Marina P. Nobrega6, Francisco G. Nobrega6 and Maria Helena S. Goldman2.1FCFRP and 2FFCLRP, Universidade de São Paulo, Brazil;3Univ.Mogi Cruzes, Brazil; 4UNIFESP, Brazil;5Institut Pasteur, France; 6UNIVAP

Paracoccidioides brasiliensis is the causative agent of the prevalent systemic mycosis in Latin America, paracoccidioidomycosis (PCM). We present a survey of ESTs in the yeast pathogenic phase. We obtained 13,490 ESTs from both 5' and 3'-ends. Clustering analysis yielded the partial sequences of 4,692 genes. We have identified several Candida albicans virulence/pathogenicity homologues in P. brasiliensis. We have analyzed the expression of some of these genes during the transition yeast-mycelium-yeast (Y-M-Y) by real-time quantitative RT-PCR. Clustering analysis showed that mycelium-yeast transition revealed three groups: (i) RBT, hydrophobin, and isocitrate lyase; (ii) malate dehydrogenase, Contigs Pb1067 and Pb1145, GPI, and alternative oxidase; and (iii) ubiquitin, delta-9-desaturase,HSP70, -82, and -104. The first two groups displayed high mRNA expression in the mycelial phase, while the third group had higher mRNA expression in the yeast phase. Our results suggest the possible conservation of pathogenicity/virulence mechanisms among fungi.

Financial support: PRONEX-MCT, CNPq, and FAPESP, Brazil

268. Sequencing the Podospora anserina genome. Philippe Silar1, Christian Barreau2,Véronique Berteaux-Lecellier1, Michèle Chablat1, Véronique Contamine1, Evelyne Coppin1, Corinne Clavé2, Fons Debets3, Robert Debuchy1, Rolf Hoekstra3, Marguerite Picard1, Annie Sainsard-Chanet4, Sven J. Saupe2, Carole H. Sellem4, Béatrice Segurens5 and Jean Weissenbach5.1Institut de Génétique et Microbiologie, UMR CNRS-Université 8621, Bâtiment 400, Université Paris-Sud, 91405 Orsay, France. 2Institut de Biochimie et Génétique Cellulaire, UMR CNRS-Université 5095, Université de Bordeaux 2, 33077 Bordeaux, France. 3Laboratory of Genetics, Wageningen University, arboretumlaan 4, 6703 BD Wageningen, The Netherlands.4Centre de Génétique Moléculaire, UPR CNRS 2167, 91198 Gif sur Yvette, France. 5Génoscope, Centre National de Sequençage, 2 rue Gaston Crémieux, 91057 Evry, France.

P. anserina is used as a model in studies on prion propagation, cell degeneration, vegetative incompatibility, translation, sexual development and stability of mitochondrial DNA. We have started a 3-fold genome sequencing project. Our approach is a whole genome shotgun strategy combined with the sequencing of 5000 BAC extremities. Sequencing should be completed within one year. This work was supported by the Ministère de la Recherche et des Nouvelles Technologies and the Centre National de la Recherche Scientifique (ACI Séquençage à Grande Echelle) and by the Fondation pour la Recherche Médicale (Grant n̊ EXC2001215009/1).

269. Transcript Profiling of Shiitake Mushroom Lentinula edodes revealed by Differential Display, cDNA Microarray and Serial Analysis of Gene Expression (SAGE). H. S. Kwan, W. Y. Chum, X. L. Bian, W. J. Xie, T. P. Ng, W. L. Ng, M. D. Zhang, G. S. W. Leung, S.M. Shih. Department of Biology, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR, PRC

Development of mushrooms is an important area in mushroom biology being studied at the molecular level.We aim to characterize gene expression profiles during development of Shiitake mushroom Lentinula edodes.First, we used RNA fingerprinting with arbitrarily primed polymerase chain reaction (RAP) to isolate genes differentially expressed in fruiting. Over 100 genes were sequenced and about 15 genes were analysed. Second, RAP products were used as probes to hybridize cDNA macroarray membranes to identify differentially expressed clones. More than 100 genes were analysed. Third,500 unique Expressed Sequence Tags (ESTs) were generated from a primordium cDNA library. Differential expression of the ESTs were analyzed by cDNA macroarray and microarray analysis.To determine the proportions of each mRNA transcript among total transcripts, we used Serial Analysis of Gene Expression (SAGE).About 7,000 transcripts were counted from the mycelium and primordium and 110 genes couldbe identified.The genes abundantly and differentially expressed in primordium indicated that at the initiation of fruit body:(1)Specific sets of genes expressed in primordium,(2)mycelium-specific genes suppressed,(3)different sets of structural proteins appeared in different stages, (4) protein turn-over increased, (5) protein synthesis increased,and (6) signal tansduction increased. Differentilly expressed genes in the dikaryotic comparing to monokaryotic mycelia has also been studied using a cDNA microarray printed with 2000 cDNA clones from a subtraction cDNA library.

270. Identification and Characterization of Differentially Expressed Genes in Dikaryons of Lentinula edodes by cDNA Microaray. S.M. Shih and H.S. Kwan Department of Biology, the Chinese University of Hong Kong, Shatin, N.T., Hong Kong Sar, PRC

Lentinula edodes (Shiitake mushroom) is a popular edible mushroom for its high nutrition and medical values. Fruiting process can only evolve from the dikaryons. Morphological and biochemical differences among dikaryons and monokaryons which relate to fruiting process can be analysed by generating the gene expression profiles of dikaryons. A subtraction cDNA library was constructed by multiple subtractions of a dikaryon, L54, with RNA from its two monokaryotic parents, A and B. Over 9000 cDNA clones were randomly picked and dotted on nylon membranes with a robot. The membranes were screened with probes of PCR products of total RNA of L54, A, and B to obtain a total of 1800 cDNA clones. These clones were printed onto glass chips to produce m microarrays. Fluorescent labeled probes were synthesized from total RNA of L54, A, and B, and hybridized to the cDNA microarray. Signal was amplified with tyramide and scanned and analysed. After normalization, among the 1800 dikaryotic clones, 20.3% was up-regulated and 16.6% down-regulated, and 23.6% was up-regulated and 13.2% down-regulated clones in L54 when compared with A and B respectively. The up-regulated clones were selected and sequenced. The genes sequenced were analysed with BlastX and matched with known structural proteins such as hydrophobin 2 and 3, extensin, and genes related to specific biochemical processes such as calcium binding protein, glutamate decarboxylase, heme-binding protein and pyruvate dehydrogenase.

271. Fungal proteomics: initial mapping of biological control strain Trichoderma harzianum. Jasmine Grinyer1, Ben R. Herbert1, Helena Nevalainen2. 1 Proteome Systems, North Ryde, Sydney, NSW, Australia. 2 Department of Biological Sciences, Macquarie University, Sydney, New South Wales, Australia

Trichoderma harzianum is a biological control agent applied against plant pathogenic fungi causing economical losses worldwide. We are using proteomics to identify key proteins involved in host recognition and biological control in a strain of T. harzianum with well-established biocontrol properties. Isolation of the corresponding genes will become a vital tool for developing genetically improved strains. One of the challenges with filamentous fungi is the solubilisation of proteins especially from the fungal cell envelope. We have found that an alkaline extraction pH does not favour the solubility of alkaline proteins, because they are almost uncharged at the extraction solution pH. Therefore, we have developed a method of treating a sample to extract proteins under acidic conditions. The method increases the solubilisation of alkaline proteins and eliminates acidic cell wall artifacts from microorganisms in general. Using this technique, combined with the use of protease inhibitors during sample preparation, thousands of proteins from T. harzianum have been extracted and separated using two-dimensional gel electrophoresis (2D PAGE). These proteins were identified using a combination of MALDI MS (matrix assisted laser desorption ionization mass spectrometry) and LC MS/MS (liquid chromatography mass spectrometry). Manual de novo sequencing was conducted to obtain sequence tags on unidentified proteins. Using these techniques, 24 protein spots were positively identified derived from 17 gene products.

272. Fusarium virulence and plant resistance mechanisms: a project within the Austrian genome programme GEN-AU. Gerhard Adam and Josef Glössl. Center of Applied Genetics, University of Agricultural Sciences, Vienna, Austria,

In 2002 the Austrian Federal Ministry for Education, Science and Culture has established the national genome programme GEN-AU ( In a small pilot project researchers from the Center of Applied Genetics of the University of Agricultural Sciences, Vienna (CAG, coordinator), the Technical University Vienna (TU), the Institute for Agrobiotechnology in Tulln (IFA), the Austrian Research Center Seibersdorf (ARCS), and from the wheat breeding company Saatzucht Donau (SZD) will collaborate on several aspects. A prime goal is the development of efficient gene disruption methods for F. graminearum (CAG, TU). Mutants will be tested for altered virulence and altered metabolite production by LC-MS-MS (IFA). Also analytical techniques and reference materials for "masked mycotoxins" will be developed. Arabidopsis thaliana genes encoding mycotoxin inactivating enzymes will be characterized, and the role of zearalenone in plants will be investigated (CAG). The group from ARCS will work on the identification of differentially expressed genes in wheat and the development of DNA arrays. The genetic basis of so far uncharacterized highly F. graminearum resistant wheat genetic resources will be determined (IFA), the knowledge gained about resistance QTLs will be utilized by the commercial partner (SZD). The aim of the GEN-AU program is "to secure and expand Austria's competitiveness and ability to cooperate on an international level". Collabrations are welcome!

273. Construction and validation of Aspergillus nidulans microarrays. Manda E. Gent1, Andrew Sims1, Geoffrey Robson1, Geoffrey Turner2, Rolf Prade3, Hugh Russell3, Nigel Dunn-Coleman4, Stephen G. Oliver1 . 1School of Biological Sciences, Stopford Building, University of Manchester, Manchester, UK. 2Department of Molecular Biology & Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK. 3Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK 74078, USA. 4Genencor International Inc, 925 Page Mill Road, Palo Alto, CA 94304, USA.

The use of microarrays in the analysis of gene expression is becoming widespread for many organisms including yeast. However, although a number of filamentous fungi have been fully or partially sequenced, microarray analysis is still in its infancy in these organisms. Here we describe the construction of microarrays for the fungus Aspergillus nidulans. A set of 4100 ESTs were isolated from conidial cDNA libraries and amplified by PCR. PCR products in 50% (v/v) DMSO were spotted onto Corning CMT-GAPS II slides using a Biorobotics Total Array System robot in 16 blocks of 17x16 spots 200mm apart. An experiment was designed to validate these arrays by monitoring the expression profile of known genes following the addition of 1% (w/v) glucose to wild-type A.nidulans cultures grown to mid-log phase in Vogel's minimal medium with ethanol as sole carbon source. Biomass samples for RNA extraction were taken from a 2 litre fermenter immediately before, (t=0) and at 1 hour, 2 hours and 4 hours after the addition of the glucose. RNA was extracted from biomass samples flash frozen in liquid nitrogen, labelled with cy3 or cy5 fluorescent dyes and hybridised to the arrays. Hybridisation profiles were analysed and quantified using Genepix software. The expression profiles following the glucose upshift will be presented and an assessment of the quality and reproducibility of the A.nidulans arrays discussed.

274. The Paxillus involutus / Betula pendula symbiosis: Gene expression in ectomycorrhizal root tissue. Tomas Johansson, Antoine Le Quéré, Derek Wright, Dag Ahrén, Bengt Söderström, and Anders Tunlid. Microbial Ecology, Lund University, Sweden

Ectomycorrhizas (ECM) are symbiotic associations formed between plants and soil fungi. In order to obtain information on genes specifically expressed during ECM symbiosis, transcript profiling in mycorrhizal root tissue was conducted. By Expressed Sequence Tag (EST) analyses 3555 clones were sequenced from a cDNA library constructed from ECM formed between the basidiomycete Paxillus involutus and birch (Betula pendula). In parallel, cDNA libraries from saprophytically growing fungus (3964 ESTs) and from axenic plants (2532 ESTs), respectively, were analysed. By clustering of all ESTs obtained (10,051), 2284 unique transcripts, either of fungal or plant origin, were discovered. From sequence homology analyses (GenBank (nr) protein database) a majority of these transcripts could be assigned putative functional and metabolic roles. This unique set of genes was further analyzed using cDNA microarrays on which a RNA target from the mycorrhizal root tissue was compared to RNA isolated either from the free-living fungus or the plant, respectively. From these analyses we found 170 fungal and 230 plant genes significantly up or down regulated (>2-fold) during mycorrhizal conditions. By comparison of EST and microarray data, we found a 78% agreement in results using the two different methods. Among regulated genes there was a significant portion being putatively involved in cell rescue and defence, metabolism and protein synthesis. Nevertheless, 59% of the fungal and 47% of the plant transcripts, respectively, were orphans without any recognized cellular function and could represent a rich source of information for future exploration on mycorrhizal symbiosis.

275. Identification and functional analysis of secreted proteins from Magnaporthe grisea. Cristina Filippi, Guodong Lu, Kiran Bhatterai, Elena Kolomiets and Daniel J. Ebbole. Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77843, USA

Secreted proteins are candidates for eliciting defense responses or acting as virulence/aggressiveness factors in fungus/plant interactions. Virtually nothing is known about the range of secreted proteins that may function in such capacities. Using the availableMagnaporthe genome sequence and an extensive collection of EST sequences we used available computer algorithms to make genome-wide predictions of secreted proteins in the genome. Magnaporthe is predicted to have twice as many secreted proteins as its close relative, Neurospora crassa (~740 vs ~370). A comparison of N. crassa and M. grisea predicted secreted proteins reveals interesting differences (gain/loss, family expansion), but no clear means of distinguishing pathogenicity-related secreted protein factors from ‘saprophytic' secreted proteins. As part of a major functional genomics project funded by NSF, we are cloning and expressing ~300 proteins in Magnaporthe to functionally test computer algorithm predictions for secretion of fungal proteins. The proteins are being directly tested for the ability to evoke a reaction on plants. We present a progress update as well as our current annotation of secreted proteins in N. crassa and M. grisea.

276. A Second Generation Genetic Map of Gibberella zeae. R.L. Bowden1, J.E. Jurgenson2, J.K. Lee3, Y.-W. Lee4, S-H Yun4, K.A. Zeller3, & J.F. Leslie3. 1USDA-ARS Plant Science & Entomology Res. Unit, 4008 Throckmorton Hall, Manhattan, KS, 2Dept. of Biology, Univ. of Northern Iowa, Cedar Falls, IA, 3 Dept. of Plant Pathol., Kansas State Univ., Manhattan, KS,4School of Agric. Biotechnol., Seoul National Univ., Su-won, Korea.

We have reported a genetic map of G. zeae made by crossing nitrate non-utilizing (nit) mutants of phylogenetic lineages 6 and 7, which contains 1048 AFLP markers assigned to nine linkage groups, but which contains regions with distorted segregation ratios and two possible chromosome rearrangements. We have now constructed a second generation genetic map by crossing two lineage 7 strains (Z-3639 & PH-1). The Z-3639 derivative strain used was heterothallic due to deletion of the MAT2 gene. Thus, this cross avoids the segregation distortion accompanying selection for prototrophic nit progeny, and permits mapping of the MAT locus in G. zeae. AFLP marker polymorphism in this cross is lower, but no segregation distortion is occurring. At present the second map has 236 markers in 16 linkage groups, with more markers needed to saturate the map and consolidate linkage groups. Common AFLP loci can be used to synonimize linkage groups and for studies of the putative chromosome rearrangements and previous segregation distortion. The maps can be used for "forward genetics," e.g., QTL analysis of aggressiveness in natural variants.

277. Proteomics studies in Trichoderma reesei. Tiina Pakula, Markku Saloheimo and Merja Penttilä. VTT Biotechnology, P.O. Box 1500, 02044 VTT, Finland

Proteome analysis is the simultaneous examination of a large subset of the protein species in a given cell. Traditionally proteomics is performed by two-dimensional gel electrophoresis combined with protein identification by mass spectrometry. We have set up proteomics methodology for the analysis of both extracellular and intracellular proteins of Trichoderma reesei. We use 2-D gel electrophoresis at different pI ranges accompanied with staining with silver stain or the fluorescent dye Sybro ruby. Also protein samples from in vivo labelling with 35S-methionine have been analysed to obtain more sensitivity and to study the synthesis rates of different proteins. Moreover, we have used antibodies against phosphoserine, phosphothreonine and phosphotyrosine to reveal changes in protein phosphorylation. The major cellulases produced by T. reesei can be readily recognised in 2-D gels run from both extracellular and intracellular samples. The cellulases, as most extracellular proteins are secreted from T. reesei as multiple pI forms. Deglycosylation of the proteins, e.g. the major cellulase CBHI, turns the protein into a single pI form, showing that the pI heterogeneity resides in the glycans. Our proteomics studies from intracellular samples have mostly been focused on analysis of secretion stress, i.e. the effects of compromised protein folding, processing or transport in cells producing heterologous protein or cells exposed to inhibitors of the secretory functions. These studies have pointed out a large number of protein spots affected by secretion stress. Major differences in the protein phosphorylation patterns also occur in these experimental conditions.

278. Identification of essential genes in the human fungal pathogen Aspergillus fumigatus by transposon mutagenesis. Arnaud Firon (1), François Villalba (2), Roland Beffa (2), and Christophe d'Enfert (1). (1) Institut Pasteur, Unité Postulante Biologie et Pathogénicité Fongiques, Paris, France, (2) Bayer CropScience, Biochemistry and Molecular Biology Department, Lyon, France

The opportunistic pathogen Aspergillus fumigatus is the cause of the most frequent deadly airborne fungal infection in developed countries. In order to identify novel antifungal drug targets, we investigated the genome of A. fumigatus for genes that are necessary for efficient fungal growth. An artificial A. fumigatus diploid strain with one copy of an engineered impala160 transposon from Fusarium oxysporum integrated into its genome was used to generate a library of diploid strains by random in vivo transposon mutagenesis. Among 2,386 heterozygous diploid strains screened by parasexual genetics, 1.2% had a copy of the transposable element integrated into a locus essential for A. fumigatus growth. Comparison of genomic sequences flanking impala160 in these mutants with that of the genome of A. fumigatus allowed the characterization of 20 previously uncharacterized A. fumigatus genes. Among these, homologues of genes essential for Saccharomyces cerevisiae growth have been identified, as well as genes that do not share homologues in other fungal species. These results confirm that heterologous transposition using the transposable element impala is a powerful tool for functional genomics in ascomycota and pave the way for defining the complete set of essential genes in A. fumigatus, the first step towards a target-based development of new antifungal drugs.

279. Simple Sequence Repeat (SSR) Markers for Magnaporthe grisea and Their Integration into an Established Molecular Linkage Map. Claudia Kaye1 , Joëlle Milazzo1, Amandine Bordat1, Sophie Rozenfeld2, Marc-Henri Lebrun3 and Didier Tharreau1.1UMR 385, Montpellier France, 2Bayer CropScience, Evry, France, 3UMR 1932 INRA-CNRS-Bayer CropScience, Lyon, Fance

Although initially time consuming and expensive to develop, microsatellite markers are robust, easy to assay and easily transferable between different research groups making them excellent markers to act as anchors to integrate maps constructed from different crosses and for locating genes of interest in the genome. We have identified and characterized SSR containing loci in the filamentous ascomycete Magnaporthe grisea, the causal agent of rice blast disease. Construction and screening of an SSR enriched library and searching databases were useful tools for finding microsatellites. A total of 49 SSR were tested for their ability to detect polymorphism among six isolates of M. grisea. The number of alleles at each locus ranged from one to six when assayed on 3% agarose gels. Inheritance and linkage were determined for 23 polymorphic sites using 58 F1 progeny from the above reference cross. Genetic analysis showed that all the markers segregated in the expected 1:1 ratio. Map positions were determined for all 23 loci. SSR were dispersed on all seven of the chromosomes. New microsatellite markers are now being developed using the sequence of the entire M. grisea genome with a goal of saturating the reference map. All possible di, tri and tetra repeats will be used to search the genome for candidate sequences.

280. New modules for versatile PCR-based gene disruption and modification in Candida albicans. Susanne Gola, Ronny Martin, Diana Schade, Andrea Walter and Juergen Wendland. Junior Research Group "Growth control of fungal pathogens", Hans-Knoell-Institute and Friedrich-Schiller-University, Jena, Germany.

Gene knockout in the diploid fungus Candida albicans requires the disruption of both alleles of the target gene. Recently, an important advance in the functional analysis of C. albicans genes was reported by the development of PCR-mediated techniques for gene deletion and GFP-tagging of chromosomal genes (Wilson et al., 1999; Gerami-Nejad et al., 2001). Here, we present a new set of modules that can serve as templates for the PCR-mediated synthesis of fragments that allow (i) gene disruption (ii) XFP-tagging to the 3`end of the gene and (iii) promotor exchange using regulatable or strong promotors. Due to the modular nature of the plasmids a minimal set of primers is required e.g. for successive gene disruptions. We hope that these plasmids will further facilitate functional gene analysis inC. albicans.

281. FRQ-based and FRQ-less oscillators regulate output from the Neurospora circadian clock. Zachary Lewis, Alejandro Correa, Andrea Cass, and Deborah Bell-Pedersen. Dept of Biology, Texas A&M Unversity, Texas

Circadian clocks have been examined at the molecular level in organisms ranging from cyanobacteria to humans. These organisms possess endogenous clocks that are synchronized to local time through environmental signals, such as changes in light or temperature. Circadian clocks enable organisms to anticipate changes in the environment and temporally partition biochemical, physiological, and behavioral processes to the appropriate time of the day. In eukaryotic cells, the core clock mechanism involves one or more transcription/translation feedback loops. In the filamentous fungus Neurospora crassa, the frequency (frq), white collar–1 (wc–1), and wc–2 genes comprise one feedback loop. While the contribution of these genes to the time-keeping process is well understood, very little is known about how this molecular clock functions to control the output processes. To identify components of the output pathways in N. crassa, we first constructed cDNA microarrays to identify genes that accumulate rhythmic mRNA. We demonstrate that 20% of the genes are clock-controlled, indicating the importance of the clock in the life of the fungus. In addition, we have identified genes that show a circadian oscillation of steady-state mRNA levels in a frq-null(frq10) strain. These data suggest that output pathways from the Neurospora circadian clock are regulated by both FRQ-based and FRQ-less oscillators.

282. Genomic analysis of calcium signalling proteins in Neurospora crassa. Alex Zelter1,2, Oded Yarden2 and Nick D. Read1. 1Fungal Cell Biology Group, Institute of Cell and Molecular Biology, University of Edinburgh, Scotland. 2Dept. of Plant Pathology and Microbiology, Hebrew University of Jerusalem, Israel

A considerable body of evidence, primarily from pharmacological studies, indicates that Ca2+-signalling regulates numerous processes in Neurospora. These processes include the control of conidial germination, circadian rhythms, tip growth and hyphal branching. Examples include the involvement of Ca2+ in suppression of the hyperbranching phenotypes of the mutants frost and spray, and the fact that genetic or chemical impairment of the catalytic or regulatory subunit of calcineurin (a Ca2+-dependent phosphatase) causes loss of apical dominance and hyperbranching followed by growth arrest. Despite the obvious importance of Ca2+-signalling in Neurospora, and in contrast to the situation in budding yeast, only a handful of Ca2+-signalling related genes have been cloned or characterised in Neurospora to date. We have made a thorough BLAST analysis of the Ca2+-signalling related proteins encoded in the recently published genome sequence of Neurospora crassa. This analysis has identified many of the proteins likely to be necessary for Ca2+-signaling in Neurospora. These proteins include previously unknown Ca2+-permiable channels, Ca2+-ATPases, Ca2+/H+-exchangers, Ca2+/Na+-exchangers, phospholipase C proteins and Ca2+/calmodulin binding proteins. Our analysis suggests that Neurosporahas over 50% more Ca2+-signalling related proteins than budding yeast. These results highlight both the potential importance and apparent complexity of Ca2+-signalling in Neurospora. A major challenge for the future will be to develop and adapt the tools necessary to investigate these important proteins experimentally.

283. Functional genomics of Aspergillus niger strains. Noël van Peij1, Hildegard Menke1, Stefaan Breestraat1, Rogier Meulenberg1, Herman Pel1, Hans van den Hombergh1, Marco van den Berg2, Rutger van Rooijen3, Albert van Ooyen1, Alard van Dijk1, Hein Stam1. 1DSM Food Specialties, 2DSM Anti-Infectives, 3DSM Bakery-Ingredients, P.O. Box 1, 2600 MA Delft, The Netherlands

The filamentous fungus Aspergillus niger is a main microorganism used for enzyme production. Wild-type strains of A. niger have the capacity of secreting large amounts of various enzymes and are potentially suitable host strains for homologous and heterologous gene expression. Classical strain improvement programs as well as defined genetic modifications have been used successfully to improve enzyme productivity. In order to rationalize and speed up the ongoing strain- and process-improvement program, as well as to identify potential new products, DSM sequenced the complete 35.9 Mb genome of A. niger. The 7.5-fold coverage random sequencing of carefully selected large insert BACs allowed the assembly of the 8 linkage groups into 19 large so-called supercontigs, each supercontig containing only small sequence gaps. Over 14,000 open reading frames (ORFs) were identified and functionally classified using a combination of specifically trained computer algorithms and manual ORF verification and annotation. This program was one of the largest commercial sequencing projects in Europe. Recently, A. niger GeneChips were designed allowing the analysis of the genome-wide transcriptome. The dynamics of A. niger strains at the mRNA- and also at the DNA-level can be followed using these DNA chips. Examples of Comparative Genomic Hybridisation and controlled fermentations followed by whole genome transcriptomic analysis will be shown. Especially, the importance of a highly reproducible chip protocol including fermentation sampling, RNA- and chip-processing will be discussed. The A. niger genome was sequenced by Gene Alliance ( The genome was annotated by Biomax Informatics ( The GeneChips experiments were performed in collaboration with the Microarray Department (

284. Global gene deletion project in Cryptococcus neoformans. Jennifer K. Lodge, Maureen J. Donlin and Kim J. Gerik, Saint Louis University, St. Louis, MO USA

Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningioencephalitis in immunocompromised patients, particularly patients with AIDS. Current antifungal agents are inadequate for safe and effective therapy. Over the past three years, a genome project conducted jointly by Stanford University, TIGR and University of Oklahoma has produced >10X coverage of the genome for C. neoformans var. neoformans as well as cDNA sequences through an EST project. Functional genomics has the potential to accelerate our understanding of pathogenesis in complex organisms. The opportunity exists now to efficiently test the contribution of C. neoformans genes to pathogenesis using a mass screening approach employing signature tagged mutagenesis. Each insertion mutant is being tagged with a unique sequence that will allow identification of that mutant within a large pool of mutants. Groups of 48 mutants will be screened together for growth in a mouse model, and mutations in genes that affect virulence in this competitive assay will be rapidly identified. We have begun a large-scale gene deletion project for C. neoformans var. grubii. Var. grubii is more virulent than var. neoformans and therefore is more suitable for virulence screen. In addition, there is limited sequence available for var. grubii through Duke University. We have developed bioinformatics tools to help identify genes. We currently have deletions in 14 genes that are homologs of S. cerevisiae cell wall genes, including ALG1, CDA1, CHS1, CRR1, CWH8,DAN4, SMI1, YUR1, UTR2, CHS5, ECM4,EXG1, HOC1, SSD1. These mutants are being analyzed for their in vitro and in vivo phenotypes.

285. Exploring gene expression in Ustilago maydis: EST libraries, comparative genomics and microarray hybridization. B.Saville, R. Austin, M. Babu, K. Nugent, N. Sacadura, K. Choffe, N. Provart. Botany, University of Toronto at Mississauga, Mississauga, ON, Canada.

We have created two EST libraries from the causal agent of common smut of corn, U. maydis. These libraries are from two cellular stages that vary greatly in physiology and structure, a forced diploid growing as a mycelia on charcoal media plates and the germinating teliospore growing in liquid 1% sucrose and 0.2% casamino acids. Combining the libraries and selecting for contigs followed by the combination of contigs with identical top matches in BLASTX searches reveals 3918 unique contiguous sequences. From sequencing and analysis of the U. maydis genome by Bayer a coding capacity of 6700 ORFs was indicated. Thus our EST contigs represent 59% of the coding capacity of the U. maydis genome. We have used this substantial coverage in a species by species comparative analysis with genomic and EST sequence databases of pathogenic and non-pathogenic fungi. The results of this analysis will be presented along with a break down of the functional categories of genes discovered in U. maydis. The EST identified clones are also being used in the construction of cDNA microarrays for the comparison of gene expression between different cellular states. Data from initial hybridizations with the first generation low-density microarrays will be presented, as well as an update on the construction of higher density arrays.

286. Expression profiling of b-mediated gene expression in Ustilago maydis. Mario Scherer and Joerg Kaemper, MPI terrestrial Microbiology, Marburg, Germany

The phytopathogenic fungus Ustilago maydis has a dimorphic life cycle. For successful infection of its host plant maize two compatible haploid sporidia have to fuse on a leaf surface and form a filamentous dikaryon, which is able to penetrate the plant cuticula and subsequently leads to the fungal proliferation in planta. Filament formation and the subsequent steps in pathogenic development are controlled by the multiallelic b-mating type locus encoding the homeodomain proteins bE and bW. bE and bW proteins expressed from different alleles can form a heterodimer, which is thought to regulate the b-dependent processes via its function as a transcriptional regulator. Several approaches helped to identify direct and indirect b-target genes during the past years, but yet did not reveal a complete view of the biological processes regulated by b. Taking advantage of the U. maydis genome sequence, a gene chip was designed that allows parallel expression analysis of about 6300 U. maydis genes. The gene chip technology was used to compare expression profiles of haploid cells expressing either compatible or incompatible combinations of bE and bW genes under control of two different inducible promoters during an 12 hours time course. We will present data on this genome wide expression analysis of b-dependent developmental regulation.

287. BAC by BAC sequencing of the rice blast fungus (Magnaporthe grisea) chromosome 7. Michael Thon, Huaqin Pan, Audrey Taro, Douglas Brown, Thomas Mitchell, and Ralph A. Dean. Center for Integrated Fungal Research, Plant Pathology Department, Box 7251, North Carolina State University, Raleigh, NC 27695-7251 USA

Magnaporthe grisea, the causal agent of rice blast disease, is a serious pathological threat to food supplies worldwide.  Recent studies have increased our understanding of the biology and molecular determinants of pathogenesis for M. grisea and related fungi.  These activities provide a starting point and the necessary tools to more thoroughly elucidate the mechanisms involved in host pathogen interactions.  Previously, a physical map of the M. grisea genome was constructed using a 25X BAC library.  Using this BAC library, we are performing large-scale sequence analysis of chromosome 7 using a BAC-by-BAC approach.  Selected BAC clones are sequenced to 5X coverage giving an average of 11 contigs per clone.  The contigs are being combined with the whole genome shotgun sequence of M. grisea in order to reduce the number of gaps and resolve assembly discrepancies.  By combining the BAC sequences with the shotgun sequence, we closed approximately 100 gaps.  The remaining gaps in chromosome 7 are being closed using a variety of sequencing techniques.  We will present results from BAC sequencing including analyses of gene content and order, EST location, and chromosome-wide synteny with Neurospora crassa.

288. Viewing Aspergillus fumigatus genomic data via the Web. Jane E Mabey, Michael J Anderson, and David W Denning. Department of Medicine, University of Manchester, Manchester, UK.

The University of Manchester has been funded to manage and curate A. fumigatus genomic data. In addition, since the species within the genus Aspergillus have recently evolved from each other, this project will enable comparative studies by gathering other Aspergillus genomic data into a central data repository - CADRE (the CentralAspergillus Data REpository). Using Ensembl, a software system for maintaining and visualising annotated genomes, we have set up a database for housing the annotated A. fumigatus mitochondrial sequence received from The Institute for Genomic Research and provided several means of viewing the data. At present, using a Web browser, the sequence contig can be viewed alongside various features (e.g., protein-coding genes, RNA-coding genes and repeats) that have been mapped onto it. For each feature, links are provided to allow the user to retrieve further annotation. For genes, such annotation includes: the locus, a description of the encoded protein's function; transcripts and their sequence; and links to further information regarding translations. Next we will receive 922 kb of sequence centred around the niaDlocus from the Sanger Institute, which was generated as part of the A. fumigatus genome sequencing project. We aim to adapt Ensembl to enable viewing of this sequence and its associated annotation. We are also aiming to provide other web-based tools (e.g., pairwise sequence comparison and community-based annotation) in preparation for the complete A. fumigatus genome sequence, which is expected in late 2003.

289. Whole Genome Analysis of Pathogen-host Recognition and Subsequent Responses in the Rice Blast Patho-system. Thomas Mitchell*, Doug Brown*, Nicole Donofrio*, Daniel Ebbole&, Mark Farman$$, Yong-Hwan Lee+, Marc Orbach#, Cari Soderlund#, Guo-Liang Wang^, Rod Wing#, Jin-Rong Xu(), Ralph Dean*. *North Carolina State University Fungal Genomics Laboratory, &Texas A&M University, $$University of Kentucky, +Seoul National University Korea, #University of Arizona, ^Ohio State University, ()Purdue University.

Magnaporthe grisea is the causal agent for rice blast disease and is considered a leading model for studying fungal pathogenesis. A NSF funded project was launched to use genomic approaches to identify and characterize genes that contribute to pathogenicity in the fungus as well as resistance in the host. The goals of this initiative are to characterize putatively secreted proteins from the fungus and identify those that interact with the plant, generate 50,000 random integration fungal mutants to identify genes required for reproduction and pathogenicity, sequence 35,000 ESTs from infected and uninfected rice, analyze six different rice Long SAGE libraries, use over 400 oligo-based interactions microarray chips containing all predicted rice blast genes as well as rice genes identified in the unique set of ESTs, and perform targeted deletions of fungal genes predicted to be involved in pathogenicity. All data generated by this project is centralized in a database called MGOS, and is presented to the public via a web interface designed for researchers to query the data. Here we will present the current status of the project with particular consideration of two key elements. We will highlight the results of the mutant screen and characterization of genes found to be important in pathogenicity. We are also generating oligo-based microarray chips that will be made publicly available. We will present the chip design and status of generation as well as detail the results from chip validation experiments.

290. Fusarium graminearum genomics at AAFC. Audrey Saparno, Linda Harris, Robert Watson, Hélène Rocheleau, Anju Koul, Jiro Hattori, Anick DeMoors, Scott Kelso, Laurian Robert, Dave Sprott, Nick Tinker, and Thérèse Ouellet. Eastern Cereal & Oilseed Research Centre, Agriculture & Agri-Food Canada, Ottawa, Ontario, Canada.

We are using a functional genomics approach to explore, at the molecular level, the biological pathways involved in Fusarium graminearum (Fg) pathogenicity. A variety of Fg-tissues (macroconidia, mycelia, perithecia), challenged Fg-culture conditions (plant contact, high DON production, growth on cornmeal, starvation/stress treatment) have been used to generate a collection of >6,000 expressed sequence tags (ESTs) which group into ~2800 contigs or singletons. Preliminary data obtained from EST sequencing has already provided hints concerning the gene specificity of each Fg-tissue. We will present the main differences observed between Fg-libraries and some aspects of functional annotation. We will discuss how we have used datamining to select candidate genes suspected to be essential in the fungal infective process. A 2500 unigene Fg microarray has been produced and will be used for the high-throughput determination of transcriptional profiles.

291. Phytophthora Genome Sequence Resources. Brett M. Tyler 1, Felipe Arredondo 1, Howard S. Judelson2, Ralph A. Dean3, Mark E. Waugh 4, Jeffry L. Boore5, Sucheta Tripathy 1 and Bruno W.S. Sobral 1.1Virginia Bioinformatics Institute, 2University of California, Riverside, 3North Carolina State University, 4National Center for Genome Resources, NM, 5DOE Joint Genome Institute, CA.

Plant pathogens of the genus Phytophthora cause tens of billions of dollars of damage each year to a huge range of agriculturally and ornamentally important plants. The ornamental, nursery and forestry industries are also heavily affected. These pathogens outwardly resemble fungi, but are in fact more closely related to brown algae such as kelp. To create a resource to assist in identifying genes involved in pathogenicity and targets for control measures, we have developed a comprehensive survey of gene sequences of two key Phytophthora species that are expressed during growth, spread and infection. The species are P. sojae, which infects soybean, and P. infestans, which infects potato and tomato. We have cloned and sequenced over 30,000 messenger RNAs from P. sojae and over 10,000 from P. infestans from axenic culture and from infection sites. We have also commenced an 8X shotgun sequencing of the P. sojae genome (62 Mb) and a 4X sequence of the genome of the forest pathogen Phytophthora ramorum. Progress on these genome sequences will be reported. For further information see and

292. Fungal genes expressed during plant disease development in the Fusarium graminearum/wheat interaction. R.S. Goswami1, F. Trail2, J.R. Xu 3, H.C. Kistler 4. 1Dept. Plant Pathology, Univ. of Minnesota, St. Paul, MN 55108. 2 Dept. Botany and Plant Pathology, Michigan State Univ., E. Lansing, MI 48824. 3Dept. Botany and Plant Pathology, Purdue Univ., West Lafayette, IN. 4USDA-CDL, St. Paul, MN

Fusarium graminearum the causal agent of wheat scab disease is a species complex comprised of strains belonging to at least eight phylogenetically distinct lineages that can differ significantly in their aggressiveness on wheat and also in the type and amount of mycotoxin they produce. Based on differences in aggressiveness we selected two strains with high (NRRL 31084) and low (NRRL 28303) virulence for genomic studies. cDNA libraries were created by suppression subtractive hybridization to compare mRNA populations from wheat heads inoculated with them in order to identify genes specific to each interaction. EST sequences from both the forward and reverse libraries revealed marked differences in gene expression among strains during pathogenesis. Several of them had matches with sequences from other fungi indicating that they were Fusarium genes expressed in planta. Another subtracted cDNA library also has been constructed using wheat inoculated with NRRL 31084 and mock inoculated wheat heads to further characterize fungal genes expressed during the disease interaction. Ultimately, we anticipate that this information will be vital for identification, isolation and functional analysis of genes related to pathogenicity.

293. Analysis of telomere-associated sequences in Magnaporthe grisea. Cathyrn Richardson, Motoaki Kusaba, Venu Gopal-Puram, Weixi Li, Sherri Schwartz Chuck Staben, Chris Schardl and Mark Farman. Department of Plant Pathology, University of Kentucky, Lexington, KY

In many eukaryotic microbes, subtelomere regions harbor genes with important roles in niche adaptation, with frequent recombination in these locations producing variation that fuels adaptation. Some M. grisea telomeres are known to contain avirulence genes, suggesting that telomere-associated genes may contribute to pathogenic adaptation in this fungus. To test this hypothesis, we are cloning and sequencing the M. grisea telomeres. This will also enable extension of the recently-obtained genome sequence out to the chromosome tips. We have developed a ligation-anchored PCR-based method that targets amplification of subtelomeric regions of the genome. Our strategy effectively enriches for telomere-proximal sequences and promotes the rapid analysis of sequences associated with specific chromosome ends. Amplicons generated by this method have been used as probes to identify cosmids containing longer telomere-associated sequences in genomic libraries of M. grisea. These cosmids are currently being sequenced. In addition, we have isolated and sequenced cosmids containing members of a telomere-linked helicase gene family. We will present an overview of M. grisea telomere organization and will also demonstrate how the telomere sequences obtained using our targeted approach connect to the current M. grisea genome assembly.

294. Generation of a wheat leaf rust, Puccinia triticina, EST database from stage-specific cDNA libraries. G.G. Hu, R. Linning, B. McCallum, T. Banks, S. Cloutier, M. Jordan, C. Matsalla, J. Schein, Y. Butterfield, S. Jones, M. Marra and G. Bakkeren. Agriculture and Agri-Food Canada: G.G.H, R.L., G.B., Highway 97, Summerland, BC; B.M., T.B., S.C., M.J., 195 Dafoe Rd., Winnipeg, MB; C.M., 107 Science Place, Saskatoon, SK; J.S., Y.B., S.J., M.M., Genome Sciences Centre, 600 W. 10th Avenue, Vancouver, BC.

We have optimized methods for the construction of cDNA libraries from small amounts of mRNA representing several developmental stages of the wheat leaf rust fungus, Puccinia triticina. These stages included urediniospores, urediniospores germinated on water or plant extract, and interactive, appressorial and haustorial stages on a compatible host. These transcript populations were subjected to a variety of treatments such as full-length cDNA production, subtractive and normalizing hybridizations, and size selection methods combined with PCR amplification or traditional cloning using adapters. We sequenced 14,000 clones from which a non-redundant unigene set of over 3,000 ESTs was assembled. Ten percent exhibit homology via BLAST to known genes in public databases while an additional 10% match other ESTs of unknown function in a custom database of collected, public fungal sequences. Interactive libraries have been subtracted for wheat sequences in silico using a wheat EST collection and public sequences. Information on gene categories will be presented. This research has been funded by the Agriculture & Agri-Food Canada 'Crop Genomics Initiative'.

295. An ensemble method for identifying regulatory circuits with special reference to the qa gene cluster of Neurospora crassa. D. Battogtokh, D. K. Asch, M.E. Case, J. Arnold, and H.-B. Schuttler. Genetics, University of Georgia, Athens GA.

A chemical reaction network for the regulation of the quinic acid (qa) gene cluster of Neurospora crassa is proposed. An efficient Monte Carlo method for walking through the parameter space of possible chemical reaction networks is developed to identify an ensemble of deterministic kinetics models with rate constants consistent with RNA and protein profiling data. This method was successful in identifying a model ensemble fitting available RNA profiling data on the qa gene cluster.

296. Genome sequencing of Aspergillus oryzae. M. Machida1, M. Sano1, K. Asai1, T. Kin1, H. Nagasaki1, T. Komori2, T. Tanaka3, R. Igarashi3, O. Akita4, Y. Kashiwagi5, K. Gomi6, K. Abe6, M. Takeuchi7, T. Kobayashi8, H. Horiuchi9, K. Kitamoto9 (1Natl. Inst. Advanced Ind. Sci. Tech.,2INTEC Web and Genome Informatics, 3Natl. Inst. Tech. Eval,4Natl. Res. Inst. Brewing, 5Natl. Food Res. Inst.,6Tohoku Univ., 7Tokyo Univ. Agric. Technol.,8Nagoya Univ., 9Univ. Tokyo)

The whole genome sequencing project for Aspergillus oryzae launched in August 2001. Because there are no genetic maps of the A. oryzae availble, the sequencing is being done mainly by a whole genome shotgun sequencing approach in combination with some chromosome specific shotgun sequencing. A rough draft of the A. oryzae genome was completed in January 2002 by accumulating sequences of approximately 6X depth of coverage after 600,000 sequence reactions. Tentatively, the total genome size of A. oryzae is estimated to be 37 Mb with 954 contigs and 36 supercontigs. The computational gene finding by GeneDecoder (Asai et al., predicts presence of over 13,000 genes including ca. 1,700 for membrane proteins, ca. 380 for transcription factors, ca. 160 for translation factors, and ca. 70 for protein secretory pathways besides abundant genes for hydrolytic enzymes. Approximately 40% of genes are speculated to have introns. The completely fixed sequences will be published for each chromosome without significant delay.

297. Population genetic variation in genome-wide gene expression in wine yeast. Jeffrey Townsend, Duccio Cavalieri and Daniel Hartl Plant and Microbial Bio, University of California, Berkeley, Berkeley, CA and Harvard University, Cambridge MA.

Genome-wide transcriptional profiling allows assessment of heterozygosity for alleles showing quantitative variation in gene expression in natural populations. We have used DNA microarray analysis to study global pattern of transcription in strains of Saccharomyces cerevisiae isolated from wine grapes in a Tuscan vineyard, along with the diploid progeny obtained after sporulation. One parental strain shows 2:2 segregation (heterozygosity) for a morphological phenotype observed as colonies with a ridged surface resembling a filigree. Global expression analysis of the progeny with the filigreed and smooth colony phenotypes revealed a greater than 2-fold difference in transcription for 378 genes (6% of the genome). A large number of the abundantly expressed genes function in pathways of amino acid biosynthesis (particularly methionine) and sulfur or nitrogen assimilation, whereas many of the meagerly expressed genes are amino acid permeases. These wholesale changes in amino acid metabolism segregate as a suite of traits resulting from a single gene or small number of genes. Other comparisons show consistent strain to strain variation in gene expression. We can conclude that natural vineyard populations of Saccharomyces can harbor alleles that cause massive alterations in the global patterns of gene expression. This work represents the first demonstration of global gene expression variation in natural populations of any organism, and argues for an enhanced consideration of the role of gene expression variation in the evolution of new traits.

298. Isolation and characterization of the methylated component of the Neurospora crassa genome. Eric U. Selker1, Nikolaos Tountas2, Sally Cross2, 3, Brian S. Margolin , Jonathan G. Murphy , Adrian P. Bird2 and Michael Freitag1 (1University of Oregon, Eugene, USA; 2Univ. of Edinburgh, UK; 3Western General Hospital, Edinburgh, UK; 4Univ. of California San Franciso, USA).

The filamentous fungus Neurospora crassa serves as a model organism to elucidate the function and control of eukaryotic DNA methylation. Few natural methylated sequences have been identified in Neurospora, however. We report the isolation and characterization of a representative sample of the methylated component of the Neurospora genome. We used the methylated-DNA-binding domain of mammalian MeCP2 as an affinity reagent to enrich for methylated DNA, cloned fragments from methylated fractions and characterized them by differential hybridization, sequence analyses and Southern hybridizations. All methylated fragments showed evidence of RIP, suggesting that most, if not all, vegetative DNA methylation in Neurospora occurs in regions previously subjected to RIP or in regions resembling RIP-mutated DNA. Most methylated fragments were related to mobile elements, which were found both clustered in repeat-rich centromeric regions or dispersed throughout euchromatin. In addition to homologues of four previously described transposon relics, we identified seven groups of transposon relics previously unknown from Neurospora, including gypsy-type retroelements and several DNA-type transposons of the Tc1, hAT and pogo superfamilies. Finally, we used the genome sequence of Neurospora to identify relics of RIP and tested them for DNA methylation. Almost all (19/20) of these sequences were found to be methylated. (Supported by NIH grant GM35690 and a Senior International Fellowship from the Fogarty International Center to E.U.S.).

299. Cloning of telomeric regions from Neurospora crassa wild-type strains Oak Ridge and Mauriceville. Cheng Wu, Julie Mitchell, Mark L. Farman* and Matthew S. Sachs. Oregon Health and Science University, Beaverton, OR 97006 *University of Kentucky, Lexington, KY 40546

Linear eukaryotic chromosomes terminate in simple sequence repeats called telomeres. New telomere repeats, typically 5' TTAGGG 3', are added to the existing chromosome ends to guard against the loss of DNA during replication. In fungi and other eukaryotic microbes, regions near telomeres are highly variable and may be rich in genes for ecological adaptation. Telomeres are poorly represented in the draft genome sequence database for Neurospora crassa. By characterizing clones for N. crassa telomeres and subtelomeric regions, we will be able to assess the functional and evolutionary significance of these regions and complete the genome sequence. To clone N. crassa telomeric regions, isolated chromosomal DNA fragments with polished ends were first ligated to linearized Bluescript vector. The ligated DNA was cut with a cohesive-end restriction enzyme, recircularized and transformed into Escherichia coli XL-10 cells. Recombinant colonies containing telomeres were then identified using a32P-labeled telomere probe. Our goal is to isolate and sequence the 14 telomeric regions of two different wild-type N. crassa strains Oak Ridge and Mauriceville. So far 10 apparently unique telomeres from the Oak Ridge strain and 5 apparently unique telomeres from the Mauriceville strain have been captured. Comparison of subtelomeric regions between strains will improve understanding of the pathways of genome evolution.

300. The Fungal Genome Initiative. Li-Jun Ma, James Galagan, Sarah Calvo, Cydney Nielsen, Tim Elkins, Robert Barrett, Mary Wong, Eric Lander, Chad Nusbaum, and Bruce Birren. Whitehead Institute/MIT Center for Genome Research, Cambridge, USA

The Fungal Genome Initiative was developed to sequence and analyze fungal genomes at various evolutionary distances within the Fungal Kingdom. The goal of the project is to utilize the power of comparative genomics to gain deeper understanding of fungal evolution, genetics, physiology, developmental regulation, and especially to understand the basis of fungal pathogenecity. Further, this kingdom-wide comparative study can lead us to understand the evolution of Eukaryotic genes, chromosomes, and regulatory and biochemical pathways. The FGI steering committee selected an initial list of important fungal sequencing targets. In 2002 a review by the National Human Genome Research Institute established the FGI organisms as a High Priority for genome sequencing. In the next year, seven genomes will be sequenced at the Whitehead for public release. These are Aspergillus nidulans, Cryptococcus neoformans,serotype A, Coccidiodes immitis, Pneumocystis carinii (human and murine), Rhizopus arrhizus, Coprinus cinereus, Ustilago maydis. We will describe the current status and plans for this project.

301. Improving the Neurospora crassa Genome Sequence. Cydney Nielsen1, James Galagan1, Li-Jun Ma1, Sarah Calvo1, Jonathan Butler1, Dayong Qui1, David Jaffe1, Seth Purcell1, William FitzHugh1, Robert Barrett1, Tony Rachupka1, Michael FitzGerald1, Nicole Stange-Thomann1, Xiaoping Yang1, Peter Ianakiev1, John Major1, Michael Frietag5 Asha Kamat1, Matthew Sachs2, Jak Kinsey3, Chuck Staben4, Eric Lander1, Chad Nusbaum1, and Bruce Birren1. 1Whitehead Institute Center for Genome Research, Nine Cambridge Center, Cambridge, MA, USA.2Department of Biochemistry and Molecular Biology, OGI School of Science and Engineering, Oregon Health & Science University, Beaverton, OR, USA.3Department of Microbiology, University of Kansas Medical School, Kansas City, KS, USA. 4T.H. Morgan School of Biological Sciences, University of Kentucky, Lexington, KY, USA. 5University of Oregon, Eugene, USA

Efforts are underway to produce a complete sequence of the Neurospora crassa genome. Working towards this goal, we generated a high-quality draft sequence of this >40 Mb genome that is available for download and query (Neurospora crassa Database Release 3). Here we report significant improvements to the draft sequence achieved through the addition of jumping clone sequences and the application of various gap closure techniques. The current assembly consists of 958 sequence contigs, comprising 38.9 Mb, ordered and oriented into 163 scaffolds by links arising from paired-end reads. Jumping clone links have improved the long-range contiguity of scaffolds, such that 50 % of all bases are contained in a scaffold of at least 1.5 Mb. Notably, the assembly now contains a >2 Mb scaffold spanning the centromere of chromosome VII. Placement of genetic markers has allowed us to anchor the vast majority of the assembly (95%) to the Neurospora genetic map. The majority of sequence gaps are present in sub-clones that provide the templates for additional sequencing needed for genome closure. Progress of ongoing finishing efforts will be reported.

302. Construction and preservation of Monascus purpureus genetic libraries. Li Ling Liaw; Gwo Fang Yuan; Wen Shen Chu; Chao Zong Lee and Chii Cherng. Liao Bioresource Collection and Research Center of Food Industry Research and Development Institute, Hsinchu, Taiwan

Genetic library is the basal of the genetic studies. Monascus purpureus BCRC 38072 is a strain with the ability of producing hypocholesterolemic agent –monacolin K. The genome size of M. purpureus BCRC 38072 was about 35 +1 mb according to PFGE analysis. We have constructed and preserved nine M. purpureus libraries including one BAC library, six cDNA libraries, one cosmid library, and one fosmid library. The BAC library has an average insert size of 131 kb and a total of 12,672 clones, which covered over ten times of the genome size. Average insert sizes of 41.5 and 39.3 kb respectively were constructed in the fosmid and cosmid libraries. The average insert size of six cDNA libraries ranged from 1.1 to 2.0 kb, and 6912, 4224, 11520, 11520, 18432, 19584 clones of these libraries were stored in the collection system of BCRC. The long-term and safe preservation method of the genetic libraries has been established. Duplicated copies of these cosmid, fosmid, cDNA and BAC libraries mentioned above were each prepared in 11 to 51 micro-plates of 384 wells. The standard operation practices of authentic methods for genetic resources such as DNA sequencing, restriction enzyme mapping and PCR fragment analysis were established. We performed the phage screen and survival test of ten 384-plates of BAC library. No phage contamination was found in BAC clones screened. After six months preservation, the survival rate of the tested BAC clones is 100%.

303. Whole Genome Annotation. Sarah Calvo, James Galagan, Bruce Birren. Whitehead Institute/MIT Center for Genome Research, Cambridge, MA.

With the annotated genomes of Neurospora and Magnaporthe currently available, and eight more whole genome sequence projects scheduled for this year, a whole new array of data is available for fungal genetics and molecular biology. Automated annotation produces a first pass set of gene predictions from the genome assembly. Since these genes are the basis for further investigation, it's important for researchers to understand the creation and validation processes that underlie these predictions.

We present the methodology of the Calhoun Automated Annotation System used to annotate the fungal genomes sequenced at the Whitehead Institute. We will discuss the different kinds of information available to aid in gene prediction, the problems that arise from conflicts between these data, and the power and limitations of the current state of the art in automated annotation.

497. Re-annotation of ORF start and stop sites in S. cerevisiae based on comparisons of orthologous ORFs from five other Saccharomyces species. Christie, K. R.1, Sethuraman, A.1, Balakrishnan, R.1, Dolinski, K.1, Dwight, S. S.1, Fisk, D. G.1, Hong, E. L.1, Theesfeld, C. L.1, Kamvysselis, M.2, Paul Cliften, P.3, Costanzo, M. C.1, Engel, S. R.1, Issel-Tarver, L.1, Dong, S.1, Weng, S.1, Johnston, M.3, Lander, E.2, Botstein, D.1, and Cherry, J. M.1. 1 Department of Genetics, Stanford University, Stanford, CA. 2 Whitehead Institute for Biomedical Research, MIT, Cambridge, MA. 3 Department of Genetics, Washington University School of Medicine, St. Louis, MO.

Since the publication of the genome sequence of S. cerevisiae in 1996, corrections have been ongoing to improve the accuracy of the sequence and the annotations of open reading frames (ORFs) and other sequence features. While most of these changes have been made on a gene by gene basis, the availability of genome sequences for five Saccharomyces species (S. paradoxus, S. mikatae, S. bayanus, S. kluyveri, and S. castelli) from groups at MIT and Washington University allowed the Saccharomyces Genome Database (SGD) to make a large scale comparison of orthologous ORFs in these species and re-evaluate ORF boundaries in Saccharomyces cerevisiae. Using the Fungal Alignment viewer and Synteny Viewer (links under Comparison Resources on SGD locus pages), users may view protein and DNA sequence from the other Saccharomyces species. For 402 of the ORFs considered, there were differences in the length of the one or more ORFs relative to other species at either or both the amino-terminal and carboxyl-terminal ends of the predicted protein sequence. Careful comparison of the DNA sequences, predicted protein sequences, and available literature for these ORFs has allowed us to conclude that changes should be made to the ORF start and/or stop sites for 104 ORFs. A further 161 changes were suggested, but require additional sequencing of S. cerevisiae S288C for confirmation. Any changes made will be documented in Locus History notes and a paper will be published detailing the changes to be made. Thus, comparison of S. cerevisiae with three closely related and two more distantly related Saccharomyces species has allowed us to improve the ORF annotations in SGD.