Kevin McCluskey, Aric Wiest, Michael Plamann

As we approach the post genomics age the number of fungal organisms which can call themselves genetic systems continues to grow. Because of this, the number of organisms in the Fungal Genetics Stock Center collection has expanded to include most strains that have had their genomes sequenced as well as genome associated libraries for a number of organisms.

As more fungal systems are amenable to genetic research new researchers have come into the field. This has led to an increase in the number of requests for materials from the FGSC including knock-out strains of Neurospora, cloning vectors and strains of sequenced organisms. These increases include numerous people who previously did not use filamentous fungi in their research. Overall we have seen over thirty seven percent increase in orders in 2007 as compared to the average of the eight previous years. This number does not include the nearly 32,000 knockout strains sent in 96-well arrays. Including these, the increase is on the order of 2,300 percent. While most of these are Neurospora, the FGSC has also received knockouts of Cryptococcus and tagged integration mutants of Magnaporthe. In total, we hold over 53,000 knockout or tagged mutants in 96-well format.

Beyond fungal strains, the FGSC holds and distributes gene libraries and individual clones from genome-mapped libraries for many of the main organisms used in genetic research. Using these resources, we have identified the underlying mutation for several temperature sensitive mutants in Neurospora crassa. We are endeavoring to develop useful applications of these findings in Neurospora and Aspergillus.

Minou Nowrousian, Ulrich Kück

During fungal fruiting body development, vegetative hyphae aggregate to form complex multicellular structures. Within the mature fruiting body, the sexual spores are formed. Using microarray analysis, we have identified a cluster of genes that are strongly upregulated during fruiting body development in the filamentous ascomycete Sordaria macrospora. Further analysis by quantitative real time PCR showed that the genes from the orthologous cluster in Neurospora crassa are upregulated during development, too. The genes occupy a region of ~50 kb in the genomes of both fungi and encode enzymes that are predicted to participate in polyketide biosynthesis, including a putative polyketide synthase. A partly conserved orthologous cluster is present in the genome of the rice pathogen Magnaporthe grisea. However, there are no functionally characterized orthologs from other organisms, thus, the product of the putative biosynthetic pathway remains to be elucidated. Analysis of both a N. crassa knockout strain as well as the corresponding S. macrospora knockout of one of the predicted dehydrogenase genes of the cluster demonstrated that this gene is involved fruiting body formation in both fungi. These data indicate that polyketides may play a role in sexual development in filamentous ascomycetes.

Andriy Kovalchuk, Marta Woszczynska, Arnold Driessen

The filamentous fungus Penicillium chrysogenum is well known as a major producer of β-lactam antibiotics, first of all penicillin G and V. Metabolic engineering approaches now open new ways for the industrial use of P. chrysogenum, as it allows efficient production of unnatural compounds not synthesized by natural strains. The availability of the genome sequence (van den Berg, M.A. et al., submitted) will stimulate the further improvement of P. chrysogenum as a versatile cellular factory. An important prerequisite for the successful introduction of novel metabolic pathway, however, is a characterization of transport processes and capacities within the cell.

One of the most prominent classes of transporter proteins are the ATP binding cassette (ABC) transporters. These systems use the energy of ATP to transport molecules across biological membranes, many of which are responsible for multidrug resistance. Some ABC transporters are also known to be involved in the secretion of secondary metabolites. Despite their importance, our knowledge about fungal ABC transporters is limited.

This project is focused on a genome-wide characterization of ABC transporters in P. chrysogenum. We use a combination of bioinformatics tools, localization studies and functional analyses to characterize several selected ABC transporters. GFP fusions were used to determine their intracellular localization. Analyzed transporters localized to the plasma membrane, vacuolar membrane, mitochondria, ER and microbodies. Further characterization of the expressed ABC transporters includes the construction of knock-out strains. The data obtained in this project will contribute to the better understanding of transport processes in P. chrysogenum cells.

This project is financially supported by the Netherlands Ministry of Economic Affairs and the B-Basic partner organizations (www.b-basic.nl) through B-Basic, a public-private NWO-ACTS programme (ACTS = Advanced Chemical Technologies for Sustainability).

Mikko Arvas¹, Teemu Kivioja², Alex Mitchell³, Markku Saloheimo¹, David Ussery⁴, Merja Penttilä¹, Stephen Oliver⁵

1VTT, Espoo, Finland, ²University of Helsinki, Helsinki, Finland, ³EBI, Cambridge, Finland, ⁴CBS, Lyngby, Denmark, ⁵University of Manchester, Manchester, United Kingdom

Several dozens of fungi encompassing traditional model organisms, industrial production organisms and human and plant pathogens have been sequenced recently and their particular genomic features analysed in detail. In addition comparative genomics has been applied to make analyses within sub groups of fungi. Notably, analysis of the subphylum Saccharomycotina has revealed major events of evolution such as the recent genome duplication and subsequent gene loss. However, little has been done to gain a comprehensive comparative view to the fungal kingdom. We have carried out a computational genome wide comparison of protein coding gene content of the subphyla Saccharomycotina and Pezizomycotina, which include industrially important yeasts and filamentous fungi, respectively.

Our analysis shows that based on genome redundancy the traditional model organisms Saccharomyces cerevisiae and Neurospora crassa are exceptional among fungi. This can be explained by the recent genome duplication in S. cerevisiae and the repeat induced point mutation mechanism (RIP) in N. crassa. Interestingly, in Pezizomycotina a subset of protein families related to plant biomass degradation and secondary metabolism are the only ones showing signs of recent expansion. In addition, Pezizomycotina have a wealth of phylum-specific poorly characterised genes with a wide variety of predicted functions. These genes are well conserved in Pezizomycotina, but show no signs of recent expansion. The genes found in all fungi except Saccharomycotina are slightly better characterised and predicted to encode mainly enzymes. The gene classes specifically diverged in Saccharomycotina are enriched in transcription and mitochondrion related functions. Especially mitochondrial ribosomal proteins seem to have evolved from those of Pezizomycotina.

Our analysis predicts that all Pezizomycotina unlike Saccharomycotina can potentially produce a wide variety of secondary metabolites and secreted enzymes and that the respective genetic systems are likely to evolve fast. These systems allow Pezizomycotina to interact with their environment and therefore they are directly subjected to various evolutionary pressures due to changing environments.

Birgit Gruben¹, Evy Battaglia¹, Han A.B. Wösten¹, Pedro Coutinho², Etienne Danchin², Bernard Henrissat², Ronald P. de Vries¹

1Microbiology, Utrecht University, Utrecht, Netherlands, ²Architecture et Fonction des Macromolecules Biologiques, Universites Aix-Marseille I & II, Marseille, France

Plant polysaccharides are the most abundant carbon source in nature and are therefore commonly utilized by saprophytic and phytopathogenic fungi. These fungi produce a broad spectrum of extracellular enzymes to degrade the polysaccharides to monomeric components that can be taken up into the cell and used for carbon metabolism. The different enzyme activities can be divided into families based on their amino acid sequence and modular structure. A comprehensive database of these enzyme activities is available at the CAZy website (www.cazy.org). The availability of fungal genome sequences has resulted in an exponential increase in the number of putative enzymes in the CAZy database and allows for fungal comparison with respect to these functions. We have initiated a study to link the genome content with respect to polysaccharide degrading functions to the growth profile of fungi. To this end we have obtained growth profiled of 15 fungi on 37 carbon sources (crude substrates, polysaccharides, oligosaccharides, monosaccharides). Improved or reduced growth on specific substrates has been linked to enriched or reduced numbers of relevant enzymes in the genome of these fungi. Initial results demonstrate a nearly perfect correlation between the genome content with respect to polysaccharide degrading enzymes and the ability to grow on specific sets of substrates.

Ronald P. de Vries¹, Loek Visser¹, Evy Battaglia¹, Birgit Gruben¹, Han A.B. Wösten¹, Richard B. Todd²

1Microbiology, Utrecht University, Utrecht, Netherlands, ²Department of Genetics, The University of Melbourne, Melbourne, Australia

Transcriptional regulators are the major control points for all aspects of fungal life. Over the years, many transcriptional regulators have been identified and their biological function has been studied. Many of these regulators have been mainly studied in a relatively small number of model fungi, although some have also only been described for less commonly used fungal species. The availability of more than 40 fungal genomes allows an analysis on the prevalence of these regulators. We have selected 60 characterized fungal transcriptional regulators and analyzed their presence in the available fungal genomes. This demonstrated that some regulators are commonly found throughout the fungal kingdom (e.g. CreA), while others are restricted to a class (e.g. PrnA in the ascomycetes) or a genus (e.g. AraR). The presence or absence of a regulator has been linked to the natural habitat of the fungi to find correlations with respect to fungal physiology.

Behrouz Vazir, Nahid Mortazavi, Bahareh Azarian, Vahid Kahl

Annexin C4 (ANXC4), the third member of fungal annexins, has been identified as a novel annexin with significant differences compare to other fungal and human annexins. To understand the ANXC4 function in human pathogen, A. fumigatus, a knock out strain has been developed using homologous recombination technique. Preliminary data on growth phenotype of anxC4 deletant confirmed a non-essential role in viability or sporulation of this fungus. For detailed analysis of ANXC4 loss effects and possible compensatory mechanisms, a proteomics approach was considered. In this sense, two dimensional protein profiles of anxC4 mutant and wild type strain were prepared. Comparison analysis of 1469 spots detected in the protein maps, showed expressional differences with nine over expressed and six under expressed spots in mutant type. Protein translation elongation factors were among those over expressed spots which identified by MALDI-TOF/TOF mass spectrometry. Further analysis of the proteomes is underway.

Marika Vitikainen, Mikko Arvas, Merja Penttilä, Markku Saloheimo

Comparative Genomic Hybridization (CGH) allows detection of mutations and copy number differences between two genomic DNA samples. In high-density microarray CGH (array CGH), genomic DNA is hybridised to oligonucleotide arrays where the oligonucleotides cover the whole genomic sequence of the organism. Test and reference genomic DNA samples are independently labelled with fluorescent dyes and co-hybridized to a chip followed by scanning the spots and further data analysis, enabling detection of small genomic alterations.

Methodology has been developed for comparative genomic analysis of Trichoderma reesei, a fungus used widely in industrial protein production. The strains analyzed are high-cellulase producing strains generated by conventional mutagenesis and selection programs. The unique feature is that all the T. reesei strains used by industry and academic research groups are members of the same pedigree of cellulase production strains, each derived from a single natural isolate. Custom array design with 2,1 million isothermal overlapping oligonucleotide probes covering the entire T. reesei genome was made, and a number of strains from the T. reesei pedigree have been analysed.

Dozens of genomic changes, both intragenic and intergenic, in the T. reesei strains have been detected by array CGH. The number of changes in individual strains was proportional to the number of mutagenesis rounds to which each strain has been subjected. Most of the detected changes were deletions, yet some duplications were detected as well. Changes as small as two nucleotide substitution could be observed. Our results show that high-density array CGH is a very useful tool for genome-wide analysis of fungal strains to identify novel genes involved in industrially relevant strain qualities such as the efficacy in protein production.

Ilka Braumann¹, Marco van-den-Berg², Frank Kempken¹

1Botanisches Institut und Botanischer Garten, Christian-Albrechts-Universität zu Kiel, Olshausenstraße 40, 24098 Kiel, Germany, ²DSM Anti-Infectives (624-0270), Alexander Fleminglaan 1, 2613 AX, Delft, Netherlands

Transposons are mobile and mostly also repetitive sequences, which are found in all eukaryotic genomes. Basically transposons can move around their host genome in two different ways: via true transposition using a "copy and paste" or a "cut and paste" mechanism depending on which type of transposon is considered. But due to their repetitive nature transposons can also change their position via recombination resulting in genomic rearrangements of various extent.

We have analyzed transposon mobility in the A. niger CBS513.88 strain lineage both experimentally and in silico. A comparison of transposon distribution in different A. niger strains of this strain lineage showed that most transposons have remained at their genomic positions during the classical strain improvement programs. Still one non-autonomous element, the transposon Vader, was shown to be active. Vader mobility could also be shown in a transposon trap experiment. Due to its obvious activity during classical strain improvement and to its ability to insert into genes Vader can be used as a gene tagging tool. In addition to the described transposition events, recombination between two copies of the A. niger retrotransposon ANiTa1, resulting in the genomic rearrangement of an approximately 45 kb region, was observed. This genomic region contains nine hypothetical and furthermore eleven annotated genes. Many of them could be involved in plant cell wall degradation.

Maria Cervantes, Juan P. de Haro, Santiago Torres-Martinez, Rosa M. Ruiz Vazquez

RNA silencing is a regulatory mechanism that involves the suppression of gene expression through the sequence-specific degradation of the target mRNA, the repression of its translation or the inhibition of its transcription. This mechanism is triggered by dsRNA molecules, which are processed into small RNAs of 21-26 nt by the Dicer enzyme. The small RNAs are incorporated into RISC complexes (RNA-induced silencing complex) containing a protein from the Argonaute-Piwi family, which uses the antisense strand of the small RNAs to scan for complementary sequences in the mRNA, causing its degradation or preventing its translation. Although the machinery of RNA silencing is well conserved throughout the evolutionary scale, the number of paralogous silencing proteins differs considerably among species, as it does the specificity of each protein in different RNA silencing pathways. We have shown the existence of a silencing mechanism in the zygomycete Mucor circinelloides. Two dicer-like genes (dcl) have been identified, although only dcl-2 is essential for efficient gene silencing and production of the two size classes of siRNAs, 21- and 25-nt long, which are accumulated in silenced strains.

In order to characterize components of the RISC complex, we have cloned the M. circinelloides argonaute (ago) genes. A DNA fragment amplified with degenerated oligonucleotides was used to screen a lambda genomic library, and three different ago genes, ago-1, ago-2 and ago-3, were isolated. Genomic analysis of these genes demonstrated the presence of a premature stop codon in the ago-2 sequence, which would produce a truncated protein lacking the PIWI domain. This mutation is fixed in the parental wild type strain. However, the high level of similarity between the amino acid sequences of Ago-2 and the ago-1 and ago-3 gene products, together with the presence of all the conserved residues of the PAZ and PIWI domains in Ago-2 suggest that the mutation event occurred recently. Alternatively, a ribosome frameshift might occur during Ago-2 translation. Expression analysis indicated the three genes were expressed at different levels in the wild type strain and none of them increased their expression by induction of silencing by dsRNA. Construction of null mutants in each ago gene and analysis of their phenotype would allow us to clarify the role of these genes in different silencing pathways.

Jan Stenlid, Åke Olson, Kerstin Dalman, Magnus Karlsson, Mikael Brandström, Karl Lundén, Carl-Gunnar Fossdal, Matteo Garbelotto, Fred Asiegbu

Heterobasidion annosum s.l. causes a devastating root rot in conifer plantations and natural forests throughout the northern hemisphere. The genome sequencing of H. annosum will provide the first comprehensive genetic information on a plant pathogenic homobasidiomycete allowing for new insights into plant-microbe interactions with trees, in particular conifers. It is important to broaden the taxonomic base for understanding the mechanisms of plant-microbe interactions, studying genes and proteins involved, and identifying pathogenicity determinants. Comparative genomics of plant pathogens with a gradient of taxonomic relatedness to H. annosum will help to understand the evolution of such factors. The project involves resequencing of several species in the H. annosum species complex including pathogenic and non-pathogenic species. Comparisons can also be made with fungi with different nutritional relations to plants; necrotrophic, biotrophic and mycorrhizal mutualist. The project integrates genetic approaches e.g QTL mapping with comparative genomics. Data bases generated on genes under positive selection in the sequenced genomes will be compared with candidate genes derived from published QTL mapping and EST-projects but also known pathogenicity factors in other pathosystems.

Jiujiang Yu¹, William Nierman², Natalie Fedorova², Masayuki Machida³, Joan Bennett⁴, Bruce Campbell⁵, Deepak Bhatnagar¹, Thomas Cleveland¹, Gary Payne⁶

1USDA/ARS, Southern Regional Research Center, New Orleans, LA, United States, ²J. Craig Venter Institute, Rockville, MD, United States, ³National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan, ⁴Rutgers University, New Brunswick, NJ, United States, ⁵USDA/ARS, Western Regional Research Center, Albany, CA, United States, ⁶North Carolina State University, Raleigh, NC, United States

Aflatoxins produced by Aspergillus flavus and A. parasiticus are the most toxic and potent natural carcinogens known to date. A. flavus is also an opportunistic pathogen that is capable of infecting plants, animals and humans. Infection in preharvest crops such as corn, cotton, peanut and tree nuts by A. flavus not only reduces yield but also renders them unsafe for human and animal consumption due to aflatoxin contamination. The economic losses due to the contamination are staggering. The primary objectives of our A. flavus genomics program are to reduce and eliminate aflatoxin contamination in food and feed through validation of known genes involved in aflatoxin formation and fungal infection and identification of novel targets. To this end, we performed functional genomics studies in A. flavus and the non-toxigenic and non-pathogenic A. oryzae strain using three different types of microarrays (amplicon, oligo and Affymetrix arrays). Gene expression profiling demonstrated that genes involved in aflatoxin production are differentially expressed in A. flavus compared to A. oryzae under conditions that support aflatoxin formation and mimic infection. In addition, genes encoding cellulosic degrading enzymes and spore surface proteins are differentially expressed in A. flavus, but not in A. oryzae. This suggests that these proteins could play a key role in fungal infection. A. flavus is a saprobe having the ability to survive in the natural environment by extracting nutrition from plant debris and dead insects. The nature of its survivability could be related to the mechanisms of fungal pathogenicity. Functional characterization of these enzymes can help devise strategies to control fungal infection of agricultural crops.

Simona Abba, Marta Vallino, Roberto Borriello, Silvia Perotto

In the past 15 years, a large number class I and class II transposons were identified in filamentous fungi. These classes contain retrotransposons and DNA transposons, respectively, some of which have been described on the molecular level almost three decades ago. However, in recent years new types of transposons have been discovered exhibiting clear differences to those previously known. A third class of transposons is called politons. These elements are widespread among protists, fungi and animals. They encode up to ten proteins and are the most complex transposable elements known. Transposition is coupled with their own DNA synthesis. Interestingly, politons share some characteristics with mitochondrial linear plasmids in fungi and plants. The fourth class of eukaryotic transposons consists of helitrons, which were first found in Arabidopsis thaliana and Caenorhabditis elegans. These elements are characterized by rolling-circle replication initiator and DNA helicase domains, which are part of a protein of about 1000-3000 amino acids.

Several years ago a repetitive sequence was discovered in the genome of the filamentous fungus Tolypocladium inflatum. This element was called CPA. When first detected it was found to be strain-specific, but no evidence for belonging to either transposon class I or class II was determined. Here the sequence of a large open reading frame encoded by the CPA element is described, which is most similar to a RecQ helicase-like protein from Metarhizium anisophila. As a consequence, the repetitive strain-specific CPA element appears to be a fungal member of the eukaryotic helitron class of transposable elements.

Peter van de Vondervoort, Robbert Damveld, Noël van Peij, Hans Roubos, Denise Jacobs, Maurien Olsthoorn, Stefaan Breestraat, Hein Stam

For many decades, A. niger has been safely used in the commercial production of various food enzymes, such as glucose oxidase, pectinase, alpha-amylase and glucoamylase. DSM and the A. niger consortium published the genome of CBS 513.88, the ancestor of currently used enzyme production strains. A high level of synteny was observed with other aspergilli sequenced. Based on the sequence, Affymetrix TX chips were developed and used to study enzyme production in production strains. The knowledge obtained from this genomics effort enabled us to improve our "PluGbug" in a generic way, for example by using codon-optimization and to further develop proteomics. We will present important findings that led to a rational improvement of protein production in Aspergillus niger.

Thierry Rouxel¹, Victoria Dominguez², Stefano Torriani³, James Hane⁴, Barbara Howlett⁵, Steve Goodwin⁶, Richard Oliver⁴, Isabelle Fudal¹, Michel Meyer¹, Marie-Line Kuhn¹, Maria Eckert¹, Bruce McDonald³, Joelle Amselem², Hadi Quesneville², Marc-Henri Lebrun¹, Patrick Wincker⁷, Arnaud Couloux⁷, Marie-Hélène Balesdent¹

1INRA-Bioger, Versailles, France, ²INRA-URGI, Evry, France, ³ETH, Zurich, Switzerland, ⁴Murdoch University, Murdoch, WA, Australia, ⁵School of Botany, Melbourne University, Australia, ⁶USDA-ARS, West Lafayette, IN, United States, ⁷Genoscope, Evry, France

The genome of the Dothideomycete Leptosphaeria maculans ‘brassicae’ (Lmb), a pathogen of oilseed rape, was recently sequenced by Genoscope and a final assembly was provided in August 2007. Lmb estimated genome size is bigger (44.9 Mb) than those of the closely related species Stagonospora nodorum (37 Mb), or the related species Mycosphaerella graminicola (41.2 Mb). This increase in size is most likely due to an abundance of retrotransposons in the genome of Lmb, as compared to the other two species. These repeated elements were estimated to represent more than 30% of the Lmb genome, as compared to 4.25% for S. nodorum and less than 5% for M. graminicola. Nine of the Lmb retrotransposons already characterized are present in high copy number with the very abundant Pholy (Copia-like), Polly and Olly (Gypsy-like) being present in up to 1400 copies each. Retrotransposons consistently occurred as large clusters of nested repeats with many Repeat Induced Point mutation-degenerated and truncated intermingled copies. These regions are A+T-rich and mostly devoid of coding sequences, thus conferring to the genome of Lmb an isochore-like structure reminiscent of the heterochromatin organisation in upper Eukaryotes. Such an isochore-like genome structure was not observed in the related genomes of S. nodorum or M. graminicola, nor in other filamentous ascomycetes that have been sequenced, but may also occur in the 74 Mb genome of Mycosphaerella fijiensis. Surprisingly, the mitochondrial genome of Lmb (160 kb) is also larger than that of other filamentous fungi including M. graminicola (44 kb), S. nodorum (50 kb) and M. fijiensis (73 kb). Apart from a few small-sized duplications, this was attributable to the invasion of mitochondrial genes by mobile group I introns containing homing endonuclease genes of the LAGLIDADG and GIY-YIG families.

Compared to Lmb, other members of the L. maculans-L. biglobosa species complex have small genome sizes, e.g., L. maculans ‘lepidii’ (20-25 Mb) and L. biglobosa ‘brassicae’ (25-29 Mb), that both seem to lack the four most abundant Lmb retrotransposons. These two sub-species are weakly or non pathogenic on oilseed rape. These genomic data thus suggest that the retrotransposon invasion may be correlated with an increased efficiency of Lmb as a successful pathogen of oilseed rape and its world-wide geographic expansion.

Jose Luis Lavin, Lucia Ramirez, Jose Antonio Oguiza, Antonio Gerardo Pisabarro

Mitochondria are the energy producing organelles of the eukaryotic cell. The mitochondrial oxidative phosphorylation (OXPHOS) pathway is the primary energy-producing biological process in all aerobic organisms. In order to study the evolution of the OXPHOS pathway, we have carried out a detailed comparative analysis of proteins involved in OXPHOS among the mitochondrial and/or nuclear genome sequences of representative fungal species corresponding to the major taxonomic groups and exhibiting different lifestyles and evolutionary relationship. To identify OXPHOS proteins in the selected fungal genomes, the deduced proteomes were compared using BlastP to a set of Saccharomyces cerevisiae, Neurospora crassa, Cryptococcus neoformans and Yarrowia lipolytica OXPHOS proteins. Each fungal OXPHOS pathway was reconstructed, and their components and structures were compared. The comparative analysis revealed a high conservation of the OXPHOS pathway between fungal species, but significant differences were also observed in OXPHOS proteins suggesting that certain features of the OXPHOS pathway are not identical across fungi.

Ralph Dean⁵, Yeon Yee Oh⁵, Nicole Donofrio¹, Hauqin Pan², Sean Coughlan³, Douglas Brown⁵, Shaowu Meng⁵, Thomas Mitchell⁴

1University of Delaware, Newark, NJ, United States, ²RTI International, Research Triangle Park, NC, United States, ³Agilent Technologies, Little Falls, Delaware, United States, ⁴Ohio State University, Columbus, OH, United States, ⁵North Carolina State University, Raleigh, NC, United States

Rice blast disease is caused by the filamentous Ascomycetous fungus Magnaporthe oryzae and results in significant annual rice yield losses worldwide. Infection by M. oryzae and several other fungal plant pathogens requires the development of a specialized infection cell called an appressorium. To further investigate the molecular processes regulating appressorium formation, we analyzed genome-wide gene expression changes during spore germination and appressorium formation on a hydrophobic surface compared to induction by cAMP. During spore germination, 2,154 (~21%) genes showed differential expression, with the majority being up-regulated. Three hundred fifty seven genes were differentially expressed during appressorium formation in response to both induction stimuli. These genes, which we refer to as appressorium consensus genes, were functionally grouped into GO categories. Overall, we found a significant decrease in expression of genes involved in protein synthesis. Conversely, expression of genes associated with protein and amino acid degradation, lipid metabolism, secondary metabolism and cellular transportation exhibited a dramatic increase. We functionally characterized several differentially regulated genes, including a subtilisin protease (SPM1) and a NAD specific glutamate dehydrogenase (Mgd1), by targeted gene disruption. These studies revealed hitherto unknown findings that protein degradation and amino acid metabolism are essential for appressorium formation and subsequent infection.

Francisco Luna-Martínez, Mariela Elizabeth Álvarez-Cruz, Raúl Rodríguez-Guerra, June Simpson

Most heterothallic Ascomycetes carry a single mating-type (MAT) locus with two alternate idiomorphs (MAT1-1 and MAT1-2) and sexual reproduction results when strains carrying different idiomorphs come into contact. Homothallic strains where a single individual carries both MAT1-1 and MAT 1-2 have also been described. In the genus Glomerella mating pairs in which both individuals carry MAT 1-2 have been characterized but to date the corresponding MAT1-1 idiomorph has not been identified for this genus. Both heterothallic and homothallic isolates of Glomerella have been reported, with heterothallic strains showing significant differences in fertility levels during sexual reproduction. Several models have been proposed to explain the mating system in Glomerella including: a. unbalanced heterothallism, b. the presence of multiple alleles at MAT1-2, c. the presence of more than 1 MAT locus. Although many reports exist on classical genetic analysis of mating in Glomerella, little is known in regard to the genes involved and the regulation of their expression.

In order to identify and characterize genes involved in sexual reproduction in Glomerella we have generated mutants in this process for the phytopathogenic fungus Colletotrichum gloeosporioides (teleomorph, Glomerella cingulata). The mutants were obtained via Agrobacterium tumefaciens-mediated-transformation (ATMT) using ascospores of a homothallic strain of C. gloeosporioides and strain LBA4404 of A. tumefaciens carrying the binary vector pGFP-HPH (whose T-DNA contains both the green fluorescent protein (GFP) and the hygromycin B phosphotransferase (HPT) genes under the control of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase promoter). ATMT of C. gloeosporioides resulted in a high transformation efficiency (1000 transformants per 10⁶ ascospores). Putative transformants were confirmed by the detection of GFP and/or the presence of the HPT gene. This result is superior or similar to transformation efficiencies previously reported for conidia or mycelia in other Glomerella species. Screening of candidates affected in their capacity to undergo sexual reproduction is underway.

PR1.20

Masafumi Tokuoka¹, Masahiro Ogawa¹, Tadashi Takahashi¹, Toshi Furukido¹, Hideaki Koike², Yasunobu Terabayashi², Noriko Yamane², Masayuki Machida², Yasuji Koyama¹

1Noda Institute for Scientific Research, Noda, Chiba, Japan, ²National Institute Of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan

Aspergillus oryzae has been used as an advantageous fungus for the production of foods and enzymes because of its safety. The recent completion of A. oryzae genome sequencing revealed the presence of numerous genes for secondary metabolite. However, studies on metabolome of A. oryzae are scarce so far. Therefore, in this study, using liquid chromatography/mass spectrometry (LC/MS), we analyzed the metabolites of A. oryzae wild-type or mutant strains grown under various conditions to understand the effect of environmental conditions and genetic background on the metabolite profiles of A. oryzae.

At first, we investigated the metabolites of A. oryzae RIB40 strain cultured in various conditions including plate culture, submerged culture, and solid state culture for 0 to 7 days. Metabolite profiles obtained by LC/MS showed that the most of the metabolites produced by A. oryzae could be classified according to their temporal pattern. We also searched stage-specific metabolites by which we could evaluate the physiological state of A. oryzae. Next, we analyzed strains of gene-disruption library of A. oryzae. Metabolome analysis showed that the amount of metabolites was significantly decreased in the strains exhibiting aberrant conidiation, suggesting that a number of metabolites are related with conidiation process. In addition, using statistical analysis such as principal component analysis, we found that disruptants exhibiting aberrant conidiation could be grouped into at least three groups based on their metabolite profiles. Our study indicated that metabolome analysis has a high potential to find out a gene function related to not only metabolism but also morphological differentiation. Further analysis is in progress, and results will be discussed.

Ilka Braumann¹, Marco van den Berg², Frank Kempken¹

1Botanisches Institut und Botanischer Garten, Christian-Albrechts-Universität zu Kiel, Olshausenstraße 40, 24098 Kiel, Germany, ²2DSM Anti-Infectives, Alexander Fleminglaan 1, 2613 AX, Delft, Netherlands

Despite of the relatively late discovery of fungal transposons and despite of the small fraction of repetitive DNA in fungal genomes (1.4%, 2.9%, and 3% in the genomes of Aspergillus oryzae, A. fumigatus and A. nidulans (Galagan et al., 2005)), today virtually all types of eukaryotic transposons have been identified in fungi (reviewed in Pöggeler and Kempken 2004). However, the impact of transposons on their fungal hosts’ genomic development is still a matter of debate. Transposition might play a role in enhancing recombination in asexual fungi (Rep et al., 2006). However, it often failed to identify actively transposing mobile elements in the genomes of fungal laboratory strains, possibly because of ongoing selection for phenotypic stability (Daboussi and Capy, 2003) .

It is particularly interesting that relatively little is known about the role transposons have in industrial producing strains. We set out to investigate the transposon content in the genomes of biotechnologically important strains of the two fungi Aspergillus niger and Penicillium chrysogenum using the annotated genome sequences of strains CBS 513.88 and ATCC 28089.

A compilation of transposon-like sequences identified in the two genomes is given. Single sequence sets have been analysed in more detail: For example the P. chrysogenum class II element PeTraII was identified, to our knowledge the first transposon known in this fungus. We further compared the distribution of selected elements in a set of different fungal strains, all emanating from each other, to investigate transposon mobility in classical strain improvement programs. As an additional approach to test for transposon activity we performed transposon trap analysis for both fungi. Moreover we are currently developing a system which allows us to test the activity of putative transposable elements identified during database analysis.

References: Daboussi MJ, Capy P (2003) Ann Rev Microbiol 57, 275-99, Galagan JE, Calvo SE, Cuomo C et al. (2005), Nature 438, 1105-15, Pöggeler S, Kempken F (2004) In: The Mycota II, Genetics and Biotechnology, 2^nd edition (ed. Kück U). Springer Verlag, Heidelberg, New York, Tokyo, Rep M, van der Does HC, Cornelissen BJC (2005) Fungal Genet Biol 42, 546-553

Brisa Ramos², José J. de Vega-Bartol¹, María A. García-Sánchez¹, Noemí Martín-Rodrigues³, Arturo P. Eslava¹, José M. Díaz-Mínguez¹

1Centro Hispano-Luso de Investigaciones Agrarias (CIALE), Universidad de Salamanca, Salamanca, Spain, ²Centro de Biotecnología y Genómica de Plantas (CBGP), Universidad Politécnica de Madrid, Madrid, Spain, ³Instituto NEIKER, Centro de Arkaute, Vitoria, Spain

We have described a new transcription factor, called ftf1, which is likely a virulence factor in F. oxysporum, as it is found only in highly virulent strains and is drastically upregulated during infection of the host plant. We have also identified a highly homologous gene to ftf1, named ftf2, which can be found in F. oxysporum strains, either pathogenic and nonpathogenic.

Experimental results obtained in F. oxysporum f.sp. phaseoli and analyses of the genome sequences of F. graminearum, F. verticillioides and the recently available F. oxysporum f.sp. lycopersicy, show that ftf1 is a multiple copy gene only present in F. oxysporum. The four copies in F. oxysporum f.sp. phaseoli are located in a small chromosome, closely linked to copies of transposon marsu, while the nine copies found in the genome of the lycopersicy strain are scattered over different chromosomes of the optic map, but also linked to different kinds of transposons, including marsu and Fot types. On the other hand, ftf2 is a single copy gene present in the genomes of the three fusaria, the rice pathogen Magnaporthe grisea and the nonpathogenic fungi Neurospora crassa and Aspergillus nidulans. The genomic architecture of the ftf2 region is very similar in the three fusaria and shows syntheny with the M. grisea genome, but syntheny is lost in the nonpathogenic fungi A. nidulans and N. crassa.

The three Fusarium genus members show different host ranges, being F. oxysporum a very versatile pathogen with dozens of formae speciales, while F. verticillioides is restricted to maize and F. graminearum can only infect barley and wheat. The analyses of the genome sequences have the potential to enlighten our understanding of the genetic basis of host specificity. The results here presented show that the, comparatively, larger genome of F. oxysporum contains species specific genes (such as ftf1) involved in virulence. The linkage between ftf1 and transposable sequences and the high homology of this gene to ftf2 suggest that it may have evolved by gene duplication and the resulting copies become dispersed as part of genome reorganizations.

Jongsun Park¹, Jaejin Park¹, Suwang Jang¹, Seryun Kim¹, Sunghyung Kong¹, Jaeyoung Choi¹, Kyohun Ahn¹, Juhyeon Kim¹, Seungmin Lee¹, Sunggon Kim¹, Bongsoo Park², Kyoungyong Jung¹, Soonok Kim¹, Seogchan Kang², Yong-Hwan Lee¹

1Fungal Bioinformatics Laboratory, Department of Agricultural Biotechnology, Center for Fungal Genetic Resources and Center for Agricultural Biomaterials, Seoul National University, Seoul, Korea, Republic of, ²Penn State University, University Park, PA, United States

Transcription factors (TF) are proteins that regulate the activity of RNA polymerase by assembling on promoter regions and consequently modulate gene expression in organisms. In the fungal kingdom, TFs modulate not only conserved regulatory networks but also specialized functions like pathogenicity in certain fungi. Genomes of more than 60 fungal species have been sequenced to date, yet there has been no systematic approach to identify and analyze fungal TFs kingdom-wide. We developed a standardized two pipelines for annotating TFs in fungal genome sequences using the InterPro database. Resulting data have been archived in a new database termed the Fungal Transcription Factor Database (FTFD; http://ftfd.snu.ac.kr/). In the FTFD, 31,175 putative fungal TFs, archived from 61 fungal and 3 Oomycete species, were classified into 61 TF families and phylogenetically analyzed. The proportions of TFs in the total number of proteins in fungi ranged from 2.29% to 7.15%, which were slightly lower than those in human and mouse. Via hierarchical clustering analysis, these TF families were grouped into seven clusters, one of which exhibited fungal-specific distribution. The FTFD will serve as a community resource supporting comparative analyses of the distribution of TFs and phylogenomic analyses of TFs within and across species.

Andrew Beacham, John Antoniw, Martin Urban, Kim Hammond-Kosack

Using a novel bioinformatics approach, we have identified a micro-region of verified pathogenicity gene homologues in the important crop pathogen Fusarium graminearum. This region is now being analysed by a combination of bioinformatic and reverse genetics approaches to ascertain its role in the pathogenicity of this species.

Comparative genomics using FASTA alignment, protein identity and property prediction, together with nucleotide repeated element and gene expression analyses have been used to investigate the conservation and properties of the micro-region in other closely and less closely related species. Targeted deletion of single genes using the split marker technique has allowed the determination of the role of each micro-region gene in F. graminearum pathogenicity.

The micro-region, which contains 15 genes and spans 25kb in F. graminearum, appears highly conserved in the other Fusarium species but less so in more distantly related species. It is present in a region of the genome with a low recombination frequency and has a low content of repetitive sequence. The region appears, unlike other fungal gene clusters, not to exhibit coordinated gene expression either during in vitro growth or during plant infection. Deletion of the neutral trehalase gene NTH1 appears to slow infection of wheat ears, while deletion of the SNF1 kinase gene inhibits spread of the pathogen in wheat.

The micro-region is beginning to appear as a novel type of pathogenicity gene cluster, differing from the virulence-associated biosynthetic and secreted protein clusters identified so far in pathogenic fungi. Further investigation will reveal more about the nature of this unusual pathogenicity region.

Elissavet Nikolaou¹, Ian Stansfield¹, Janet Quinn², Alistair Brown¹

1University of Aberdeen, Aberdeen, United Kingdom, ²University of Newcastle upon Tyne, Newcastle, United Kingdom

Microbes require robust stress responses to survive changing environments, and in particular pathogenic microbes need such responses to counter host defences. Cells sense changes in their environment (detection of external signals), activate signal transduction pathways (information routes) to trigger an appropriate response. These responses protect cells against the stress and repair damage caused by the stress. We have explored the sequence conservation of specific stress regulatory proteins across the fungal kingdom, including pathogenic and benign species that have evolved in divergent niches. These species are likely to have evolved stress responses appropriate to their diverse niches. The degree of conservation is measured relative to Saccharomyces cerevisiae because signalling pathways have been studied in detail in this organism using combination of genetic, molecular, biochemical and genomic tools. Comparative genomic analysis from our laboratory has been focused on the osmotic, oxidative and cell wall stress responses of pathogenic and benign fungi because, at least in the pathogen C. albicans, these pathways contribute to virulence, and they affect the sensitivity of cells to antifungal drugs. In parallel we have compared the resistance of the 18 fungal species under analysis to osmotic, oxidative and cell wall stresses. We have extended this work by developing a statistically robust approach to define the degree of conservation amongst stress regulators in the target fungi. Our approach, which allows direct analyses of proteins in functional peer groups, considers both the percentage identity between fungal orthologues and the percentage length of their sequence alignments. Using this approach we have shown significant differences in the rate of evolution of fungal MAPKs, MAPKKs and MAPKKKs, and in the rate of evolution of specific MAPKs. The implications of these data will be discussed.

Neuza Carvalho¹, Theo Goosen², Cees van den Hondel¹, Arthur Ram¹

1Institute of Biology Leiden, Leiden, Netherlands, ²Biocentre, HAN University, Nijmegen, Netherlands

The presence of unfolded proteins in the ER induces a cellular response called the Unfolded Protein Response (UPR). The HacA transcription factor plays a central role in the UPR pathway, resulting on the induction of a large number of genes, among them chaperones and foldases that assist in the protein folding in the ER.

We were interested in studying the effects of the hacA and ireA deletions, as well as the constitutive hacA expression on the growth and morphology of A. niger. For the knock-out mutants, deletion cassettes were made containing 500bp of the 5’ and 3’ flanking regions of the respective gene, and pyrG to be used as a selection marker. For generating the constitutive hacA mutant, a hacA gene construct was made that lacked the 20 nt intron sequence and transformed to replace the wild type hacA gene in the genome. For the construction of the deletion strains, an A. niger strain with kusA (ku70) gene deleted was used. In this background, not only the targeting efficiency is high, but also in the case of essential genes, heterokaryons are obtained as primary transformants, as ireA turn out to be. In S. cerevisiae, ire1 deletion is not lethal and results in inositol auxotrophy. However, supplementing growth media with inositol did not allow the growth of A. niger ireA deletion strains. Southern blot analysis confirmed the heterokaryotic nature of ireA transformants obtained. In A. niger, ΔhacA is not lethal, but the mutant shows a severe growth defect leading to a small and compact colony phenotype. Primary transformants displaying the hacA growth phenotype were never observed, but hacA deletion strains were efficiently obtained after purification of spores from primary transformants. These results show that gene deletions mutants with a strong defect in growth can easily be obtained via heterokaryons in the kusA background. The constitutive hacA mutant also shows a growth defect when compared with the wild type. Both on solid or liquid medium, this mutant grows slower then the wt. Morphologically, hyphae of the constitutive hacA mutant grow on a curly way, resembling mutants with defect in the tubulin cytoskeleton. The effect of constitutive hacA expression on tubulins is currently investigated.

Claire Burns¹, J.E. Stajich², J.D. Lieb³, S.E. Hanlon³, W.W. Lilly⁴, A.C. Gathman⁴, S.K. Wilke³, M.E. Zolan¹, P.J. Pukkila³

1Indiana University, Bloomington, IN, United States, ²UC Berkeley, Berkeley, CA, United States, ³University of North Carolina, Chapel Hill, NC, United States, ⁴SE Missouri State University, Cape Girardeau, MO, United States

Coprinus cinereus, a basidiomycete mushroom fungus ideally suited to meiotic studies due to its synchronous development, has a near-completed genome. The 13 chromosomes show an unusual distribution of repeated elements and regions of elevated recombination. Gene prediction algorithms indicate a total of approximately 12,500 genes. To analyze meiotic development at the transcriptional level, we designed and fabricated a 13,230 element 70-mer oligonucleotide microarray using ArrayOligoSelector. The tightly regulated light and temperature- dependent life cycle of the mushroom allows sampling of gill tissue in which cells are at a specific meiotic stage at a certain time. Meiosis occurs synchronously and takes ca. 12 hours. Our experiments concern a broad 15-hour timecourse through the meiotic process, describing events prior to fusion of dikaryotic nuclei, nuclear fusion, condensation and alignment of meiotic chromosomes, first meiotic division, and completion of the second division. Replicate microarrays were hybridized with cDNA from 3 hours prior to karyogamy, karyogamy, and 3, 6, 9 and 12 hours post-karyogamy, using a pooled mixture of cDNAs as the reference sample for each array. Analysis using materials from 4 independent fruitbody collections revealed that ~ 3000 genes were significantly differentially regulated during this timecourse, at a false discovery rate of 10%. Using a bifurcation strategy with K-means data clustering, we were able to identify temporally regulated waves of transcription throughout meiosis and mushroom development. Furthermore, clusters containing meiotic genes and predicted genes of meiotic function were found. These clusters also exhibit strong enrichment for Gene Ontology classes associated with meiosis. Array data concur with existing northern data. We conclude that our annotation pipeline produced informative gene predictions, which allowed design of informative 70-mer oligonucleotides, and our resultant microarrays provide an important tool for analysis of synchronous meiotic development in C. cinereus.

Supported by NSF, NIH, and by the Indiana METACyt Initiative of Indiana University, which is funded in part through a major grant from the Lilly Endowment, Inc.

Martin Vödisch, Olaf Kniemeyer, Franziska Leßing, Daniela Albrecht, Robert Winkler, Axel A. Brakhage

Aspergillus fumigatus is a ubiquitously distributed saprophytic mould. In the last decade, A. fumigatus has become the most important airborne fungal pathogen causing life-threatening infections in immunosuppressed patients. Little is known about the pathogenicity determinants of A. fumigatus which enable this fungus to survive and grow within the host. In comparison to other fungi, A. fumigatus possesses obviously higher stress tolerance and better mechanisms to adapt to the host environment. As a basis for comparative proteomic studies, we established a reference proteome map. Using MALDI-TOF-MS/MS we identified 392 protein spots representing 344 proteins separated on 2D-gels. Proteins involved in primary metabolism, protein synthesis, cell cycle regulation, DNA processing and transcription, transport, cellular organisation and stress response were most abundant. Since mitochondria are important organelles involved in energy production, metabolism of amino acids, lipids and iron, we also established a protocol for the isolation of mitochondria of A. fumigatus. The mitochondrial protein fraction was separated by 2D-gel electrophoresis and a partially annotated mitochondrial proteome reference map was developed. 126 different proteins spots have been identified so far.

Luis M. Corrochano¹, Alan Kuo², Asaf Salamov², Jasmyn Pangilinan², Erika Lindquist², Harris Shapiro², Scott Baker³, Jan-Fang Cheng², Igor Grigoriev², Phycomyces Genome Group¹

1Universidad de Sevilla, Sevilla, Spain, ²DOE Joint Genome Institute, Walnut Creek, CA, United States, ³Pacific Northwest National Laboratory, Richland, WA, United States

The zygomycete Phycomyces blakesleeanus is a model microorganism for research on sensory biology. The giant fruiting bodies, sporangiophores, of Phycomyces change their speed and direction of growth in response to a variety of environmental signals, including light, gravity, wind, touch, and the presence of nearby objects. Additionally, light regulates sporangiophore development and induces the synthesis of the pigment beta-carotene. Phycomyces is an intensively studied, experimentally tractable model organism, and whole-genome analysis is expected to further elucidate the signaling pathways underlying its photoregulation. To this end the genome was sequenced to 7.49X depth and assembled into 475 scaffolds totaling 56Mbp, and 47847 ESTs were assembled from cDNAs of light and dark cultures. We combined into a single annotation pipeline a variety of gene modeling methods (homology-based, EST-based, and ab initio), and predicted 14792 protein-coding genes. Many of these gene predictions are supported by homology in nr (68%), by Pfam domains (44%), or by ESTs (35%). We next assigned GO terms to 41% of the proteins and EC numbers to 16%. We then distributed these annotations to the Phycomyces consortium, along with tools to curate them manually. We expect that the annotation will provide a solid platform for expression analysis. The initial characterization of the Phycomyces protein set has uncovered the presence of multiple photoreceptor proteins and large families of proteins involved in signal transduction. In addition, several new sets of repetitive DNA, including transposable elements, have been described in the genome of Phycomyces. Comparisons with genomes from Ascomycete, Basidiomycete, and Zygomycete fungi will help us to unravel the evolution of fungal photoreceptors and other sensory transduction proteins. We expect that the Phycomyces genome will help to understand the molecular mechanisms involved in the sensing of light, gravity and other environmental signals by this model fungus.

André D. Schmidt¹, Olaf Kniemeyer¹, Hubertus Haas², Axel A. Brakhage¹

1Leibniz Institute for Natural Product Research and Infection Biology (HKI) / Friedrich-Schiller-University Jena, Jena, Germany, ²Division of Molecular Biology/Biocenter, Innsbruck Medical University, Innsbruck, Austria

The acquisition of iron is known to be an essential step in any microbial infection process due to iron-limiting conditions in the human host. This iron limitation is caused by high-affinity iron-binding proteins like transferrin or lactoferrin in the host. Since iron plays an essential role in key metabolic processes like DNA synthesis, oxidative phosphorylation or electron transport A. fumigatus has to overcome the iron deficiency by the synthesis of siderophores, which chelate iron. It was shown that an A. fumigatus strain unable to synthesise siderophores was attenuated in virulence in a murine infection model (Schrettl et al. 2004). Moreover, regulation of iron homeostasis via two transcription factors was recently described in filamentous fungi (Hortschansky et al. 2007). To understand the cellular processes induced by iron starvation, we analysed the proteome of A. fumigatus strain ATCC 46645 grown under iron-deficiency conditions. In total 98 spots, representing 88 different proteins, were upregulated under -Fe conditions and 88 spots, representing 78 different proteins, were downregulated under iron deficiency conditions. The major parts of the downregulated proteins belonged to proteins involved in primary metabolism (31 %), ribosomal protein synthesis (13 %) and chaperone activity (9 %). Many iron-sulphur cluster and heme-containing enzymes such as aconitase and cytochrome c peroxidase were found to be downregulated. On the other hand, proteins involved in the amino acid metabolism (16 %) represented the major group of upregulated proteins under iron starvation. In addition, proteins, which were related to siderophore biosynthesis (3%) or protein degradation (5 %) or proteins involved in antioxidative response like Cu, Zn superoxide dismutases (4%) that lack iron, showed also an upregulation under iron deficiency. Further proteins analysed under different non-linear pH-scales will be presented and their putative roles during iron depletion will be discussed.

Schrettl et al. (2004) J. Exp. Med. 200: 1213-1219, Hortschansky et al. (2007) EMBO J. 26, 3157-3168

Gumer Pérez, Anotnio G. Pisabarro, Lucía Ramírez

The white rot fungus P. ostreatus is an edible basidiomycete with increasing biotechnological interest and applications. Telomeres are specialized structures at the end of all eukaryotic chromosomes; they ensure chromosome stability and protect their ends from degradation and from fusing with other chromosomes. Telomeres sequences are extraordinary highly conserved in evolution. The loss of telomeric repeats triggers replicative senescence in cells. Telomeric DNA from several organisms has been identified and cloned by using telomeric repeats from non-related organisms as probes. Similarly, we have identified, mapped and cloned the P. ostreatus telomeres using the human telomeric repeat as probe. For mapping the telomeric, the genomic DNAs isolated form individuals of a segregating population was digested with restriction enzymes, electrophoresed and transferred to nylon membranes. The use of the heterologous human telomeric probe (TTAGGG)₁₃₂ revealed numerous polymorphic bands. Some of them were mapped to the genetic linkage map using the segregation of the RFLP markers identified. The segregation of each telomeric restriction fragment was recorded as the presence vs. absence of a hibridizing band. Segregation data for seventy-three telomeric restriction fragments were used as an input table to be analysed using the MAPRF program software. Eighteen out of twenty two telomeres were identified and assigned to linkage map of this fungus. P. ostreatus telomere and subtelomere sequences were isolated using a modified version of single-specific-primer polymerase chain reaction technique. The results showed that P. ostreatus telomeres contain at least fifty-five tandem copies of the TTAGGG sequence. The analysis of their terminal restriction fragment showed that the range of variation in P. ostreatus telomere length ranges between 150 and 1500 bp. These results indicate that the telomeric hexanucleotide copy number ranges from 25 to 250 repetitions. We used fragments subtelomeric sequences identified with the SSP-PCR technique as probes to isolate the telomeric sequence of P. ostreatus in a lambda phage genomic library from vegetative mycelium of the dikaryotic strain N001. The results show that P. ostreatus contains a subtelomeric sequence in which it has found homology to a recQ helicase gene family, an ABC sugar transporter, and a short chain dehydrogenase.

Julian Christians¹, Cortney Watt²

1Simon Fraser University, Burnaby, BC, Canada, ²St. Francis Xavier University, Antigonish, NS, Canada

Introduction: Studying natural phenotypic variation within species will contribute to our understanding of the genes underlying traits of medical, industrial and evolutionary importance. Approaches for studying such variation, including quantitative trait loci (QTL) mapping, require many molecular markers. Microsatellites are a commonly-used marker in other organisms, but are relatively difficult to develop in fungi.

Methods: This study examined the relative polymorphism rates of mononucleotide, dinucleotide and trinucleotide repeats among four wild-type strains of Aspergillus nidulans.

Results: The probability of polymorphism increased with number of repeating units. Di- and trinucleotide repeats had higher polymorphism rates than mononucleotide repeats, but this was offset by the presence of numerous long mononucleotide repeats.

Discussion: The use of mononucleotide repeats will substantially increase the number of potential markers available for studies of Aspergillus nidulans. Given that mononucleotide repeats are more abundant than other repeats in fungal genomes in general, mononucleotide repeats are likely to be an important resource in other species as well.

Cora van Zeijl¹, Marian van Muijlwijk¹, Margreet Heerikhuisen¹, Jan Verdoes², Peter Jan Punt¹

1TNO Quality of Life, Zeist, Netherlands, ²Dyadic Nederland BV, Wageningen, Netherlands

Current approaches for the development of fungal industrial host strains have long time-lines and the resulting strains are genetically ill-defined. Moreover, available approaches to resolve the latter bottleneck with targeted gene replacement have even longer timelines. A new approach based on a KU70 mutant strain resulting in a non-homologous recombination deficient mutant strain is reported for several non-industrial host strains. We used the KU70 approach for actual commercial strains of Chrysosporium lucknowense. For industrial applications the presence of a KU70 mutation in a final production host strain is unwanted as the recombination deficient KU70 phenotype may have adverse effects in large scale fermentation. Therefore, we designed two different KU70 disruption vectors. One type of KU70 disruption vector was designed for strain development approaches, involving multiple gene disruptions at different loci. In the primary KU70::amdS disruption strain the amdS selection marker can easily be removed by fluoroacetamide selection leaving a KU70 mutant phenotype. The subsequent gene disruption could be carried out using a reusable amdS selection marker. The other KU70 disruption vector was designed to disrupt the KU70 gene in a strain background to be used for construction of genetically defined production strains, by introducing a desired expression cassette e.g. at the cbh1 locus. The wild type KU70 gene can easily be regenerated by fluroacetamide selection. The latter type of KU70 disruption vector can also be used to regenerate the WT KU70 locus in a multiple disrupted KU70 mutant host strain.

The integration properties of the Chrysosporium lucknowense KU70 mutant strains will be presented.

Barbara Reithner, Enrique Ibarra-Laclette, Alfredo Herrera-Estrella

The present study displays an extensive analysis of genes involved in the crosstalk between T. atroviride and it's hosts.

Alberto Flores Martínez¹, Sandra González Hernández¹, Patricia Ponce Noyola¹, Alfredo Herrera Estrella²

1Universidad de Guanajuato, IIBE Facultad de Química, Guanajuato, Guanajuato, Mexico, ²Langebio CINVESTAV Campus Guanajuato, Irapuato, Guanajuato, Mexico

Trichoderma atroviride, a fungus used in biological control, sporulates in a synchronized manner following a brief pulse of blue light. BLR-1 and BLR-2 are key regulatory elements of photoconidiation and mycelial growth in T. atroviride and T. reesei. We are using a proteomic approach, to study and analyze differences between wild type and brl1^- and blr 2^- mutants during the light response.

As a preliminiray step, the protein profile of soluble cellular extracts from three strains of Trichoderma reesei, wild type, brl 1and blr 2 mutants, exposed to light pulse has been compared by using two-dimensional gel electrophoresis. Up to 500 individual spots can be resolved in the 4 to 7 pI and 14 to 200 kDa MW ranges. The biological variability for 197 major spots was determined for each isolate. The isolates showed a similar 2-DE map, with both qualitative as well as quantitative differences in quite a number of spots, In silico analysis of some of the differential expressed spots (27 for wt, 7 for blr1 and 1 for blr2) have been identified. Such as Ornithine decarboxylase antizyme, Protein cwf17, Enolase, Serine/threonine phosphatase.

This study was supported by grant from the DINPO, Universidad de Guanajuato.

Patricia Ponce-Noyola, Jorge Alegría-Torres, Edgar Alfaro-Rangel, Sandra González-Hernández, Arturo Flores-Carreón, Alberto Flores-Martínez

Sclerotium cepivorum Berk is the etiological agent of the garlic disease known as "white rot", which is one of the major causes the low production of garlic in The Bajio region in Central Mexico. We are interested in learning more about the physiology and biochemistry of this microorganism, in order to define molecular markers that can be used to detect the fungus.

In this work, purification, location and molecular characterization of a protein (Sc1) from sclerotia that may be used as molecular markers to detect the fungus in soil and seed samples is presented. We have found that the Sc1 is the predominant protein representing up to 70% of the total Sclerotium protein. It exhibits a molecular weight of 36 kDa, a pI close to 5.0 and displays similar characteristics to other developmental proteins described in other fungi. Fungal growth kinetics and formation of sclerotia showed that this protein is sclerotia specific, i.e., it is not detected in mycelia but only at the onset of sclerotia formation where it can be immunodetected using specific antibodies. Reverse genetics based on the amino terminal sequence of the protein demonstrated that the Sc1 RNA expression starts 5 days before sclerotia formation during the growth period. The estimated mRNA size was near 1000b.

The study was supported by grant CONACyT-SAGARPA-CO1-11919 from Consejo Nacional de Ciencia y Tecnología, México.

Marie Foulongne-Oriol, Jugurta Chedded, Cathy Spataro, Jean-Michel Savoie

Transposable elements constitute a significant fraction of fungal genome and contribute to genome plasticity and evolution. Each transposition event generates new variability and this property could be exploited in several molecular marker systems. We discuss here the identification of transposable elements in Agaricus bisporus and their potential use as tools for genetic analysis. Two different strategies were used. Firstly we performed PCR with degenerated primers designed in conserved domain of reverse transcriptase, using previously available sequence information from other fungi in order to isolate homologous genes in A. bisporus. From the sequences obtained we developed IRAP marker analysis, based on PCR amplification of genomic DNA fragments between two transposon insertion sites. Secondly, we exploited the SSAP technique. This approach similar to AFLP system required primer designed in sequence from terminal region. We used primers derived from LTR of other fungi retrotransposon such as Tricholoma matsutake and we used also A. bisporus. LTR sequence isolated in our lab. Either IRAP or SSAP techniques were tested on thirty genotypes of A. bisporus chosen in cultivated or wild strains. The usefulness of these marker systems is compared to others already used for this mushroom, and their potential application for genetic studies such as linkage mapping or diversity analysis is discussed.

Jaehee Jung¹, Gangman Yi¹, Serenella Sukno², Michael Thon²

1Universidad de Salamanca, Centro Hispano-Luso de Investigaciones Agrarías (CIALE), Salamanca, Spain, ²Texas A&M University, College Station, TX, United States

Our ability to predict gene function for newly sequenced fungal genomes is based to a large extent on comparisons to other organisms. It is important when making these comparisons to take into account the large evolutionary distances, sometimes hundreds of millions of years, that separate many of the fungal taxa for which whole genome sequences exist. It is commonly accepted that orthologous proteins tend to have conserved function in different species, however numerous examples that demonstrate functional diversification among orthologous genes exist. The phylogenetic context of a gene family must be taken into account when transferring knowledge of gene function from other species. The Fungal Protein Cluster Database (FPC-DB) is designed to enable users to quickly locate information about the evolutionary history and the functions of fungal proteins and gene families and present the information in a number of comparative views. The proteins from fungal whole-genome sequencing projects are annotated with InterPro terms, SwissProt keywords, and Gene Ontology terms using an automated functional classification server previously developed in our group. The proteins are then clustered into putative gene families using the MCL application. Each protein cluster includes multiple sequence alignments, phylogenetic trees and a summary of functional categories found in the cluster. Users can perform queries using proteins IDs, protein functional categories, and species names as search terms. The results are displayed in a variety of forms that allow users to compare the occurrence of gene and their functional categories between species. Users can also view a detailed page for each protein cluster and simultaneously view a phylogenetic tree and a matrix of functional categories, enabling them to identify cases of lineage specific gains and losses of functional categories.

Pietro Spanu¹, Sarah Butcher¹, James Abbot¹, Timothy Burgis¹, Marc-Henri Lebrun², Joelle Amselem², James Brown³, Christopher Ridout³, Nicholas Talbot⁴, Darren Soanes⁴, Sarah Gurr⁵

1Imperial College London, London, United Kingdom, ²INRA/CNRS, Paris, France, ³John Innes Centre, Norwich, United Kingdom, ⁴University of Exeter, Exeter, United Kingdom, ⁵University of Oxford, Oxford, United Kingdom

We have sequenced the genome of Blumeria graminis f sp hordei, strain DH14. A shotgun sequencing approach was employed using short insert (~2kb) plasmid libraries and large insert (~38kb) fosmid libraries made from purified conidial DNA. The resulting 460Mb have been assembled (Arachne) and are currently being analysed. Preliminary projections of genome size estimate a value of around 120Mb which is much higher than expected and needs to be taken with caution do to the possibility of a skew in the models due to the presence of large amounts of repetitive DNA (>60%) largely unique to Blumeria. We are currently analysing the repeat structure to evaluate this possibility. In collaboration with others we have access to 620Mb of Solexa generated sequence and we are combining the datasets by using de-novo and scaffold-based methodologies in the expectation of providing a reference genome for powdery mildews sequenced by high-throughput platforms. Comparison with existing published mapped genetic markers in Blumeria show that the two datasets are consistent and co-linear; we have been able to draw some of the linkage groups together and we expect many more to do so once the genomic assembly is improved.We have extended the Blumeria EST dataset by sequencing cDNA libraries from conidia, germlings isolated from infected barley (20hpi, 3dpi and 6dpi), cleistothecia and infected barley epidermis. The ESTs cluster into 3549 unisequences, 99% of these map to the current genome assembly. This data provides us with the training set for the gene finding software that will serve for the initial machine-based annotation of the genomic sequence. Of note is the fact that the cleistothecial cDNAs have an exceptionally high level of novel/unique sequences, indicating a highly differentiated state in which gene expression is very different from the fungal tissues analysed so far.

All this data is deposited in GenBank and publicly available through the website www.blugen.org . In addition to sequence data it is intended that the site will be a general repository for information about powdery mildews in general and Blumeria in particular with links and access to relevant publications. All clones are also available for anyone requesting them through this website.

M Carmo Basilio¹, Andreia Santos¹, Susana Marcelino¹, Rob Samson², Rogério Tenreiro³, Vitória San Romão¹, Vitória San Romão⁴

11 Instituto de Biologia Experimental e Tecnológica/Instituto de Tecnologia Química e Biológica,Universidade Nova de Lisboa, Oeiras, Portugal, ²Centraalbureau voor Schimmelcultures, P.O. Box 85167, NL-3508 AD, Utrecht, Netherlands, ³Universidade de Lisboa, Faculdade de Ciências, Centro de Genética e Biologia Molecular and Instituto de Ciência Aplicada e Tecnologia, Edificio ICAT, Campus da FCUL, Campo Grande, Lisboa, Portugal, ⁴LNRB/L-INIA (Ex-Estação Vitivinícola Nacional), Dois Portos, Portugal

Cork is the best sealing material for wine, champagne and sparkling drinks. 80 Penicillium strains were isolated from factory environments and from cork slabs in different stages of the manufacturing process of cork. More than half of those isolates belonging to the genus Penicillium were identified phenotypically as members of the "Glabra group". To study if members of the group, namely in P. glabrum and P. spinulosum, could be separated by molecular methods two approaches were established. The first fingerprinting method used was amplification of DNA using four different microsatellite primers. Clustering of profiles obtained with all results was performed using software that analyses gel band profiles and intensity. Strain/group discriminative bands were selected as potential molecular marker for species definition. Furthermore, restriction analysis of amplified ITS region (ITS-ARDRA) was also used. The combination of the two techniques contributes to distinguish the taxonomical position of P. glabrum and P. spinulosum.

Acknowledgments: FCT for PhD grant BD/19264/2004. Amorim & Irmãos (Coruche, Portugal). Jos Houbraken for helping in the phenotypical identification of the Glabra group.Paula Alves for technical analysis.

Gethin Allen, David Keszenman-Pereyra, Geoff Turner

Many secondary metabolism pathways of A. nidulans and A. fumigatus are dependent on the pptA gene product for post-translational modification of non-ribosomal peptide synthetases or polyketide synthases by addition of a 4’-phosphopantetheine cofactor. Inspection of the genome sequences of A. nidulans and A. fumigatus identified a second putative pantothenyl transferase termed pptB. Deletion of pptB from A. fumigatus resulted in stable heterokaryons, and permitted the identification of a deletion phenotype as microcolonies, capable of only very slow growth. When the gene was placed under control of the PalcA promoter, repression led to slow growing colonies. GFP localisation showed the presence of the gene product within the mitochondria.

Taken together, our data suggest that pptB is an orthologue of the yeast gene PPT2. PPT2 is known to encode a PPTase specific for a small mitochondrial acyltransferase Acp1p.

An orthologue of Acp1p has been previously investigated in N. crassa, and is a component of mitochondrial complex I. While yeast lacks complex I, Acp1p is involved in lipoic acid biosynthesis within the mitochondria. Putative orthologues of Acp1p are also present in A. fumigatus and A. nidulans.

Tjasa Gril, Branka Javornik, Franci Celar, Jernej Jakse

Brown rot fungi are found in most temperate regions, causing considerable losses to apples, pears and stone fruits. The group of rot fungi includes three species: Monilinia laxa (Aderh. & Ruhl.) Honey, Monilinia fructigena (Aderh. & Ruhl.) Honey and Monilinia fructicola (Wint.) Honey. A special form of the fungus, M. laxa f. sp. mali, is found only in apple, in which it causes blossom wilt, spur-kill and canker. It has not yet been clearly confirmed whether this is a specialized form of the fungus or merely a race.

The purpose of the present study was to analyse isolates of Monilinia laxa and Monilinia laxa f. sp. mali from different hosts by the AFLP method, in order to evaluate their genetic diversity and relationships and to detect possible intraspecific variability.

A total of sixty-seven fungi isolates of Monilinia laxa were acquired from a wide range of hosts comprising pome and stone fruit plants from different fruit growing areas in Slovenia and from abroad (Japan and three reference isolates from the Centraalbureau voor Schimmelcultures) were analysed. Genomic DNA from fungal mycelium was isolated with the standard CTAB procedure and fluorescent AFLP analysis was carried out using 20 different EcoRI/MspI primer combinations with two selective bases; electrophoregrams were analyzed by AlleleLocator 1.03. Distance-based clustering (Dice's coefficient of similarity, UPGMA and PCA method) and model-based clustering (Structure) were used to evaluate genetic similarities and relationships among the analysed samples.

A total of 1089 AFLP bands were scored, of which 354 or 32.5% were polymorphic. AFLP polymorphism thus showed high intraspecific variability. On average, 54 AFLP markers, ranging from 44 to 745 bp in fragment length, were amplified per combination. The highest number of fragments (96) was amplified with primer combination E-GA+M-AT and the lowest number of bands (33) with two different M-selective nucleotides (CG, GA). Some specific markers within the fingerprints of the individual isolates were also discovered. Clustering data analysis showed that Monilinia laxa isolates collected from apple trees form a well separated group of fungal isolates, which is clearly distinguished from the other group of host plant isolates.

Jose Miguel Oliveira, Peter J Schaap, Leo H de Graaff

Aspergillus niger is among the most important micro-organisms for the industrial production of organic acids and of extracellular enzymes used in the food and feed industry. Until recently, the most common approach for the study of gene function in A. niger was the construction of gene knock-out mutants. Despite its effectiveness, gene knock-out is a time-consuming and laborious method for silencing genes. Furthermore, this method cannot be applied to the study of essential genes. To address this problem, a system for gene knock-down through RNA interference (RNAi) in A. niger was created. As a first step, a destination vector for generation of RNAi clones by recombination reaction was constructed. To test this system, a segment of sequence coding for the xylanolytic regulator XlnR was recombined with the constructed destination vector to yield the corresponding silencing vector. After co-transformation in A. niger, strains were further selected by plate screening on the basis of low xylan-degrading activities. Compared to the wild type, these strains showed lower levels of xlnR transcripts and furthermore two tested genes regulated by XlnR (xyrA and xynB) also presented decreased transcript levels in these co-transformants. Since RNAi has not been applied or described in A. niger, these data show that RNAi constructs effectively work in this fungus. The newly developed system for RNA-mediated gene silencing in A. niger could therefore be efficiently used in functional genomics studies in this fungus.

Frederique Bidard¹, Sandrine Imbeaud², Nancie Reymond², Olivier Lespinet¹, Philippe Silar¹, Corinne Clave³, Veronique Berteaux-Lecellier¹, Herve Delacroix², Robert Debuchy¹

1Institut de Genetique et Microbiologie, Univ. Paris-Sud, Orsay, France, ²Centre de Genetique Moleculaire, Godmap, Gif Sur Yvette, France, ³Institut de Biochimie et Genetique cellulaire, Univ. Bordeaux2, Bordeaux, France

The sexual development of the heterothallic euascomycete Podospora anserina has been analyzed for many years in terms of signal transduction pathways controlling the switch from vegetative to reproductive state, autophagy requirement for fruiting-body maturation, functions of MAT proteins and the developmental role of peroxisomal proteins. The completion of the P. anserina genome sequence along with developmental mutants disposal allow all these research areas to benefit now from a microarray approach.

A genome wide microarray, covering the 10546 presently known and predicted CDS, has been constructed with the Agilent in-situ synthesized 60-mers technology. A library of 10 oligonucleotides per CDS has been design by the manufacturer. To select only one oligonucleotide per CDS, a screening strategy has then been elaborated. 1) A computational step has been performed to determine 3’ position, intron position and cross-hybridization rate of all probes. These data have been compiled to attribute a score to each probe and thus, to select a first set of 4 oligonucleotides per CDS. 2) Microarrays, manufactured with this design, have been experimentally validated. 3) The last step of the selection procedure has been based on the use of transcriptomic experimental data generated with diverse culture conditions. An oligonucleotide (flag, signal on noise, coefficient of variation) and CDS (expression, intensity) scoring have been settled to select the best oligonucleotide per CDS.

To conclude :

1) A Podospora whole genome microarray containing (one 60-mers per CDS) X4 of high quality has been constructed.

2) A catalog of 10 probes per CDS with computational information and a sub-catalog of 4 probes per CDS with experimental data have been settled. This information would be useful, in the future, to create new microarray design in function of genome annotation evolution.

3) The powerful and highly stringent selection strategy elaborated in this work can be applied for any sequenced organism.

This project is funded by Agence Nationale de la Recherche, n° ANR-05-BLAN-0385-01.

Sarrah Ben M'Barek¹, Theo A.J Van der Lee¹, Alexander H.J Wittenberg¹, Henk J. Schouten¹, Manoel Souza², Gert H.J Kema¹

1Plant Research International B.V., Wageningen University and Research Centre (WUR), P.O. Box 16, 6700 AA, Wageningen, Netherlands, ²Embrapa-LABEX Europe - Wageningen University and Research Centre (WUR), P.O. Box 16, 6700 AA, Wageningen, Netherlands

Comparative Genomic Hybridization (CGH) arrays using a high density whole genome tilling NimbleGen platform (http://www.nimblegen.com/) were employed to further understand the genome plasticity of Mycosphaerella graminicola, the causal agent of septoria tritici blotch of wheat. The availability of the finished genome of M. graminicola IPO323 (http://genome.jgi-psf.org/Mycgr1/Mycgr1.home), detailed genetic maps and karyotypes enables genome wide analysis for variation in DNA copy number and provides a new window to study chromosome polymorphism and translocations. In total, 387000 probes based on the IPO323 genome, were spotted on the array. These microarrays consist of 50 to 75–mer probes with a mean probe spacing of 118bp and a maximum stringency of 2 covering therefore the whole genome.

We have analyzed the parents and progeny isolates of the M. graminicola mapping population IPO94269 and the sequenced isolate IPO323. Hybridization of M. graminicola IPO94269 displayed substantial aneuploidy against the IPO323 reference genome.

Our results reveal widespread deletions between these two parental isolates and confirm the absence of two complete chromosomes in isolate IPO94269 previously demonstrated by karyotyping (Mehrabi et al. 2007) and genetic linkage analysis. Our data indicate CGH arrays can be used to detect aneuploidy, to predict the sites of chromosome breaks, and to identify recombination breakpoints. We currently exploit CGH to identify large genome differences such as deletions or translocations between isolates to understand genome plasticity and its relation with virulence, host specificity and speciation.

Mehrabi R., Masatoki T., Kema G.H.J. 2007. Electrophoretic and cytological karyotyping of the foliar wheat pathogen Mycosphaerella graminicola reveals many chromosomes with a large size range. Mycologia 99 (6): 804-812.

The authors thank the Joint Genome Institute (JGI) and the Stanford Human Genome Center for expert genome sequencing, and the members of the International Mycosphaerella Genomics consortium for discussions.

Sarrah Ben M’Barek is sponsored by an UNESCO L’ Oreal fellowship

Libera Lo Presti¹, Lorenzo Cerutti², Michel Monod³, Philippe Hauser¹

1Institute of Microbiology, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland, ²Swiss Institute of Bioinformatics, Lausanne, Switzerland, ³Service of Dermatology, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland

Introduction: Functional complementation of deletion mutants of Saccharomyces cerevisiae by expression of a gene on a plasmid is a common procedure for a number of genetic analyses. We tried to determine the best approach for successful complementation with heterologous genes.

Methods: We attempted complementation of four S. cerevisiae deletion mutants of essential genes involved in various processes with the homologous S. cerevisiae gene.

Results: Two mutants were rescued by expression under the control of constitutive S. cerevisiae  promoters commonly used for complementation, whereas the other two could be rescued only using the natural promoter. However, the two latter mutants could be complemented by their orthologs from Candida glabrata under the control of the constitutive S. cerevisiae  promoters.

Discussion: The use of constitutive S. cerevisiae promoter can be deleterious for homologous but not for heterologous complementation in S. cerevisiae. Consequently, the use of the homologous gene is useless to set up the conditions for heterologous complementation. An algorithm for successful heterologous complementation in S. cerevisiae is proposed.

Theo A.J. Van der Lee¹, Larry D. Dunkle², Alice C.L. Churchill³, Jean Carlier⁴, Andy James⁵, Manoel T. Sousa Jr⁶, Pedro Crous⁷, Nicolas Roux⁸, Alexander Wittenberg¹, Erika Erika Lindquist⁹, Igor Grigoriev⁹, Jim Bristow⁹, Jane Grimwood⁹, Stephen B. Goodwin², Gerrit H.J. Kema¹

1Plant Research International B.V, Wageningen, Netherlands, ²USDA-ARS Purdue University, West Lafayette, IN, United States, ³3Department of Plant Pathology Cornell University, Ithaca, NY, United States, ⁴BGPI, CIRAD, Montpellier, France, ⁵CICY, Merida, Mexico, ⁶EMBRAPA Genetic Resources & Biotechnology, Brasília, Brazil, ⁷Fungal Biodiversity Centre, Utrecht, Netherlands, ⁸Bioversity, Montpellier, France, ⁹9DOE-Joint Genome Institute, Walnut Creek, CA, United States

Mycosphaerella is one of the largest genera of plant pathogenic fungi with more than 1,000 named species, many of which are important pathogens causing leaf spotting diseases in a wide variety of crops including cereals, citrus, banana, eucalypts, soft fruits, and horticultural crops. An international project was initiated to sequence the genomes of M. graminicola and M. fijiensis, two of the most economically important pathogens of wheat and banana, respectively, along with 40,000 ESTs from M. fijiensis and the related maize pathogen Cercospora zeae-maydis, through the Community Sequencing Program sponsored by the U.S. DOE-Joint Genome Institute. The 9x M. graminicola sequencing is complete and was made public November 1, 2006 following automated and manual annotation. Due to the very good assembly statistics as well as a >2000-marker DArT linkage map that was aligned to the genome, JGI decided to finish the M. graminicola genome at the Stanford Human Genome Center. The majority of chromosomes have been sequenced completely including both telomeres. These data indicate that M. graminicola has both the largest chromosome number and the smallest chromosome sizes recorded among filamentous ascomycetes. Detailed analyses of progeny isolates showed the plasticity of the M. graminicola genome, as a significant number of chromosomes can be missed and potentially contain redundant information for pathogenicity and mating. The repetitive content of the individual chromosomes is disproportionally larger on the <2Mb chromosomes indicating that at least some may be considered supernumerary. Major factors for pathogenicity, however, are located on the larger chromosomes and mostly towards the telomeres. More than 1200 genes have been annotated manually and indicate that this pathogen contains smaller gene families compared to other well-studied filamentous plant pathogenic fungi, which may reflect its incognito life style. The M. fijiensis EST sequencing has resulted in more than 30,000 ESTs and the genome sequencing resulted in an approximately 70Mb genome size that contains a substantial amount of repetitions. Interestingly, the genome sequence showed a double CG-peak that is absent in the M. graminicola genome and appears to be specific for Mycosphaerella pathogens of banana. The current status of both sequencing projects will be discussed.

Philippe Hauser¹, Libera Lo Presti¹, Moira Cockell¹, Lorenzo Cerutti², Viesturs Simanis³

1Institute of Microbiology, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland, ²Swiss Institute of Bioinformatics, Lausanne, Switzerland, ³Cell Cycle Control Laboratory, Swiss Institute for Experimental Cancer Research (ISREC), and School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne (EPFL), Epalinges, Switzerland

Pneumocystis jirovecii is a major cause of pneumonia in HIV infected patients. The only drugs available to manage infection target the folic acid biosynthetic pathway and selective pressure is giving rise to drug resistance. Development of new therapeutic agents is complicated by the fact that the organism cannot be cultured. The genome of the closely related species P. carinii has recently been sequenced. Approximately 4000 ORFs have been identified and ESTs corresponding to almost 1800 of them have been cloned. The fission yeast Schizosaccharomyces pombe is the closest relative of Pneumocystis that is amenable to classical and molecular analysis. To improve our understanding of Pneumocystis biology we use functional complementation of yeast mutants to study the function of P. carinii genes. We have focused upon genes for which there are sequence homologues in yeasts but not in higher eukaryotes, which may be useful drug targets. Our strategy is to transform a diploid heterozygous for a null mutation of the gene with a plasmid that expresses the P. carinii homologue. After meiosis, spores are germinated and colonies are analysed to determine whether cells carrying the null mutation can be rescued by the plasmid. We will present data for genes from S. pombe and Saccharomyces cerevisiae.

Joske Ruytinx, Marc Lambaerts, Jaco Vangronsveld, Jan Colpaert

Zinc is an essential micronutrient for all organisms. It is required for a wide range of cellular processes but becomes cytotoxic at elevated concentrations. Elevated concentrations of zinc in the environment may cause a selective pressure on exposed biota and can lead to the evolution of tolerant ecotypes. Zinc tolerant populations of the ectomycorrhizal basidiomycete Suillus luteus (L.:Fr.) have evolved in severely contaminated soils and have been shown to protect their host plant from zinc toxicity. Although the exact mechanism of zinc tolerance in S. luteus remains unclear, this trait is believed to be due to the adaptations of mechanisms involved in the general homeostasis of this essential element.

Zinc homeostasis in eukaryote cells is tightly controlled by transporters of different families: zinc-iron regulated protein (ZIP) family, cation diffusion facilitator (CDF) family, We isolated two S. luteus genes encoding transporters of the CDF-family by genome walking. Heterologous expression in yeast of the respective cDNA’s caused a slightly increased zinc tolerance and rescued the zinc sensitive zrt1 mutant. Both CDF-transporters seem to be zinc specific since Cd, Ni, Co and Mn sensitive strains could not be rescued by their expression. The increased zinc tolerance, zinc specificity and rescue of the zinc sensitive mutant indicate a role for the transporters in the zinc homeostasis of Suillus luteus. Further analysis (localisation, regulation of expression) of the transporters in the basidiomycete itself must reveal their involvement in the adaptive zinc tolerance.

Kazuhiro Iwashita, Yasuhide Kobayashi, Kazutoshi Sakamoto, Osamu Yamada, Shigeaki Mikami

Aspergillus oryzae has been used for the several foods and alcohol beverages industries, such as sake and soy sauce making, for supplying hydroritic enzymes and vitamins et. al.. In the history of these industries, various strains were isolated from wild and selected for efficient fermentation and improvement of the product quality. Then some strains were further improved by several mutation and selection procedure. The genome structure of these strains will affect their characteristics and important for such nice adaptation. Further more, genome wide analysis of A. oryzae strains will important to understand the nature of this species. However, only little is known about their whole genome evolution. Recently, whole genome sequence of A. oryzae RIB40 strain, which isolated from surface of broad bean, was published. Using this information, we prepared A. oryzae DNA microarray (AO DNAchip) for their genetic study. The 8 strains for sake making, 6 strains for soy sauce making, 5 strains for miso paste making, 3 strains for soy sauce and miso past making, and 12 strains from other source were randomly selected from our strain library and their genomes were examined by AO DNAchip. As the result, in all analysed strains, about 10-1,000 genes were absent and most of them are clustered in their genome. However, only little number of the gene duplications was found. Moreover, these strains were classified into 7 groups containing two major groups by their signal intensity of all ORFs. One major group contained most of sake and miso past related strains and another major group contained most of soy sauce related strains. Among each groups, the pattern of absent genes are similar. These results suggested that the loss of genes will one of important driven force for the genome evolution and nice adaptation of A. oryzae.

Annegret Kohler¹, Benoit Hilselberger¹, Frédéric Duchaussoy¹, Marie-Pierre Oudot-Le Secq¹, Erika Lindquist², Peter Brokstein², Igor Grigoriev², Arnaud Couloux³, Patrick Wincker³, Martina Peter⁴, Christine Delaruelle¹, Aurélie Deveau¹, Gopi Podila⁵, Francis Martin¹

1INRA UMR1136, INRA-Nancy Université, Interactions Arbres/Microorganismes, 54280 Champenoux, France, ²) US DOE Joint Genome Institute, Walnut Creek, CA 94598, United States, ³GENOSCOPE, Centre National de Séquençage, 91057 EVRY Cedex, France, ⁴Swiss Federal Research Institute WSL, 8903 Birmensdorf, Switzerland, ⁵Department of Biological Sciences, Huntsville, United States

In the genome sequence of Laccaria bicolor, the first ectomycorrhizal fungus sequenced so far, 20,614 protein-encoding genes were predicted. Using these gene models, we constructed a whole-genome expression array (NimbleGen) containing in duplicates eight independent, non-identical, 60-mer probes per whole gene model. This oligoarray was used to validate and correct the first generation of annotated gene models. Expression of nearly 80% of the predicted genes could be detected in either free-living mycelium, fruiting bodies or ectomycorrhizal root tips. Further, the gene expression in these different tissues was compared to identify tissue-specific expression pattern. For example, transcripts of several small secreted proteins and an ammonium transporter, which both could play a role in the establishment of the symbiosis, were highly up-regulated in mycorrhizal root tips. Our analysis of the Laccaria bicolor transcriptome was completed by the investigation of 38,901 expressed sequence tags (ESTs) generated from different cDNA libraries and 186k reads assembled into 10,598 contigs derived from a 454 sequencing run, which were used to identify previously not detected gene models and to evaluate the proportion and ways of alternative splicing occurring in Laccaria bicolor.