VAAM-Jahrestagung 2012 18.–21. März in Tübingen

76[3]. In summary, hypoxia has a strong influence on the metabolicregulation of A. fumigatus and the character of the long- and short-termresponse to hypoxia differs only partly. In future experiments, we willanalyze the function of the alcohol dehydrogenase in the adaptationprocess of A. fumigatus to hypoxia in more detail.[1] SD. Willger, S. Puttikamonkul and R.A. Cramer, PLOS Pathogens 4 (2008), p. 680-685[2] M. Vödisch, K. Scherlach and O. Kniemeyer, Journal of Proteome Research 10, (2011), p. 2508-2524[3] C.H. Doy, J.A. Pateman and E.H. Creaser, DNA 4 (1985), p. 105-114FBV5-FGDifferential analysis of intra- and extra-cellular proteomes ofVerticillium longisporum during biotrophic and saprophyticgrowthA. Kühn* 1 , H. Kusch 1 , C. Hoppenau 1 , K. Michels 2 , I. Feussner 2 , B. Voigt 3 ,D. Becher 3 , M. Hecker 3 , S.A. Braus-Stromeyer 1 , G.H. Braus 11 Georg-August Universität Göttingen, Institut für Mikrobiologie und Genetik,Göttingen, Germany2 Georg-August Universität Göttingen, Abteilung Biochemie der Pflanze,Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Göttingen, Germany3 Ernst-Moritz-Arndt-Universität Greifswald, Institut für Mikrobiologie,Greifswald, GermanyThe soil-born, hemibiotrophic plant pathogenic fungus Verticilliumlongisporum causes premature senescence and flowering in oilseed rape(Brassica napus), which results in immense agricultural yield reduction. Inspite of the significant economical importance of this pathogen, the factorsfor host specificity are still unknown and the network of virulence factors(effectors) is poorly analyzed. The focus of this study is to identify fungalproteins expressed during plant infection. Therefore we investigated theextra- and intracellular changes of the V. longisporum proteome inducedby oilseed rape xylem sap (biotrophic model) versus conventionalsaprophytic growth media. Procedures for the isolation and purification ofproteins were optimized for Verticillium samples. Protein extracts wereseparated by one- and two-dimensional gel electrophoresis and peptidesamples were analyzed by MALDI-TOF and LC-MSMS. The resultingspectra were searched against peptide data derived of the draft genomesequence of V. longisporum 43 we are currently assembling andannotating. Exoproteomes vary to a great extent depending on growthmedium, growth phase and light conditions. The identified proteins andtheir functional categories may represent the different phases of theinfection cycle. We identified adhesins and many different groups ofcarbohydrate-active enzymes like polysaccharide lyases and glycosylhydrolases, which could be important for penetration and degradation ofstructurally complex pectin molecules of the plant. Additionally severalmembers of peptidase families were detected, which might be importantfor proteolysis of host substrates or host defense proteins. Furthermoremany small cysteine-rich proteins and necrosis and ethylene-inducing-likeproteins (NLP) were identified, which are potential effectors inpathogenicity. Candidate genes and proteins are currently analyzedregarding their importance during plant infection.FBV6-FGRegulation of fruiting body formation in Coprinopsis cinereaM. Navarro-Gonzalez*, M. Arndt, M. Zomorrodi, A. Majcherczyk, U. KüesGeorg-August-Universität Göttingen, Molekulare Holzbiotechnologie undtechnische Mykologie, Göttingen, GermanyFruiting body formation in the edible dung fungus Coprinopsis cinerea isregulated by environmental cues (nutrients, temperature, light, humidity),physiological conditions (mycelial status, age) and genetic controlelements (e.g. A and B mating type genes, dst1 and dst2 genes for lightreceptors). Fruiting body development consists of a series of defined stepsoccurring in a concerted process over seven days. Following hyphalaggregation, stipe and cap tissues differentiate controlled by light and darkphases. Once light-induced karyogamy takes place in basidia present at thesurface of the gills in the cap of mature primordia, stipe elongation and capexpansion start parallel to meiosis in the basidia and subsequentbasidiospore formation. All these processes are expected to appointnumerous intracellular as well as extracellular protein functions, many ofwhich might be specific to steps in fruiting. Since the genome of thefungus is available, identification of proteins can now be addressed bylarge scale proteomic techniques. Stipe and cap fractions from immaturefruiting bodies at meiosis are shown to differ from each other, both in theintracellular and the extracellular proteome.FBV7-FGThe stress-activated protein kinase FgOS-2 is a key regulatorin the life cycle of the cereal pathogen Fusarium graminearumJ. Bormann*, N. Van Thuat, W. SchäferUniversity of Hamburg, Biocenter Klein Flottbek, Department ofMolecular Phytopathology and Genetics, Hamburg, GermanyFusarium graminearumis one of the most destructive pathogens of cerealsand a threat to food and feed production worldwide. It is an ascomycetousplant pathogen and the causal agent of Fusarium head blight disease insmall grain cereals and of cob rot disease in maize. Infection with F.graminearum leads to yield losses and mycotoxin contamination.Zearalenone (ZEA) and deoxynivalenol (DON) are hazardous mycotoxins;the latter is necessary for virulence towards wheat. Deletion mutants of theF. graminearum orthologue of the Saccharomyces cerevisiae Hog1 stressactivatedprotein kinase, FgOS-2 (FgOS-2), showed drastically reducedin planta DON and ZEA production. However, FgOS-2 produced evenmore DON than the wild type under in vitro conditions, whereas ZEAproduction was similar to that of the wild type. These deletion strainsshowed a dramatically reduced pathogenicity towards maize and wheat.We constitutively expressed the fluorescent protein dsRed in the deletionstrains and the wild type. Microscopic analysis revealed that FgOS-2 isunable to reach the rachis node at the base of wheat spikelets. Duringvegetative growth, FgOS-2 strains showed greater resistance againstphenylpyrrole and dicarboxymide fungicides. Growth was retarded uponosmotic treatment: the growth rate of mutant colonies on agar platessupplemented with NaCl was reduced but conidia formation remainedunchanged. However, the germination of mutant conidia on osmotic media wasseverely impaired. Germ tubes were swollen and contained multiple nuclei. Thedeletion mutants completely failed to produce perithecia and ascospores.Furthermore, FgOS-2 also plays a role in reactive oxygen species (ROS)-relatedsignalling. The transcription and activity of fungal catalases is modulated byFgOS-2. Among the genes regulated by FgOS-2 we found a putative calciumdependentNADPH-oxidase (noxC) and the transcriptional regulator of ROSmetabolism, atf1. The present study describes new aspects of stress-activatedprotein kinase signalling in F. graminearum.FUV001Genomics and transcriptomics based on next-generationsequencing techniques to characterize fungal developmentalgenesM. Nowrousian*, I. Teichert, G. Wolff, U. KückRuhr-Universität Bochum, Allgemeine und Molekulare Botanik, Bochum,GermanyNext-generation sequencing (NGS) techniques have revolutionized thefield of genomics/functional genomics. We have recently sequenced andassembled the genome of the filamentous ascomycete Sordariamacrospora, a model organism for fungal development, solely from NGSreads (PLoS Genet 6:e1000891). We are currently applying NGS in twoapproaches for the identification and characterization of developmentalgenes. (I) With laser capture microdissection, we can separateprotoperithecia from the surrounding hyphae. RNA isolation andamplification from 150 protoperithecia yields enough material for RNAseqanalysis. The resulting data were compared to RNA-seq data fromwhole mycelial exctracts to characterize the genome-wide spatialdistribution of gene expression during sexual development. Additionally,we used the RNA-seq information to improve the predicted S. macrosporagene models, and annotated UTRs for more than 50 % of the genes. (II)We sequenced the genomes from three mutants that were generated byconventional mutagenesis, and identified the three causative mutationsthrough bioinformatics analysis. One mutant carries a mutation in thedevelopmental gene pro41. The second, a spore color mutant, has a pointmutation in a gene that encodes an enzyme of the melanin biosynthesispathway. In the third mutant, a point mutation in the stop codon of aconserved fungal transcription factor causes the sterility of the mutant. Forall three mutants, transformation with a wild-type copy of the affectedgene restored the wild-type phenotype. These data show that wholegenome-sequencing of mutant strains is a rapid method for theidentification of developmental genes.FUV002VipA - a novel player in light sensing and development inAspergillus nidulansJ. Röhrig*, R. FischerKarlsruhe Institute of Technology (KIT), Microbiology, Karlsruhe, GermanyIn the filamentous ascomycete A. nidulans development and metabolismare strongly regulated by light. In light A. nidulans undergoes an asexuallife cycle with formation of conidiophores and conidiospores whereas inthe dark sexual development with ascospore formation and production ofsecondary metabolites takes place [1]. For light detection of severalwavelengths A. nidulans harbors different photosensors like theBIOspektrum | Tagungsband 2012