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

84defence enzymes, were found to be induced by both fungi. Altogether, thesimilar responses of Lycopersicum esculentum to P. fortinii and A. solaniindicatea classification of the endophyte P. fortinii as a pathogen bytomato plants.Schneider, T., Vieira de Castro Junior, J., Schmid, E., Cardinale, M., Eberl, L., Grube, M., Berg, G.,Riedel K. (2011). Structure and function of the symbiosis partners of the lung lichen (LobariapulmonariaL. Hoffm.) analyzed by metaproteomics. Proteomics11, 2752-2756FUP028Protein-protein interaction studies to decipher fungal sexualdevelopmentI. Teichert*, E. Steffens, S. Nordzieke, A. Beier, U. KückRuhr-University Bochum, General and Molecular Botany, Bochum, GermanyFungi are able to produce a number of different cell types and multicellularstructures during their life cycle. One prominent example is the formationof fruiting bodies to propagate sexually. Our studies focused on thefilamentous fungus Sordaria macrospora which produces fruiting bodieswithin seven days under laboratory conditions. By analyzing sterilemutants, several proteins required for sexual development were identified,the so-called “PRO” proteins [1]. To gain more insight into the regulationof fruiting body differentiation, we performed protein-protein interactionstudies with several PRO proteins using yeast-two-hybrid, TAP-tagpurification and subsequent mass spectrometry, co-immunoprecipitation,and fluorescence microscopy. We have further tested different fungalpromoters for expression of recombinant proteins. Interestingly, we wereable to link several developmental proteins via shared interaction partnersand propose the formation of multi-protein complexes containingdevelopmental and signaling proteins. In conclusion, our data hint to anextensive network regulating cellular differentiation in a fungal modelsystem.[1] Engh I, Nowrousian M, Kück U (2010) Sordaria macrospora, a model organism to study fungalcellular development. Europ J Cell Biol 89: 864-872FUP029Optimisation of vectors for transformations in Coprinopsis cinereaB. Dörnte*, U. KüesUniversität Göttingen, Molekulare Holzbiotechnologie und technischeMykologie, Göttingen, GermanyThe genetic transformation of the model fungus Coprinopsis cinereaallows the genomic analysis and manipulation of this organism. Initially,transformations were used to study the structure; functions and regulationof expression of genes; in recent years usage for overexpression ofindustrially important enzymes are also emerging. For the transfer ofgenetic material, chromosomal integrative vectors are used. These vectorscontain a selectable marker gene and/or a gene of interest under the controlof regulatory sequences such as promoter or terminator. Due to lack ofsystematic experimental data, little is known about the influence of vectorson transformation frequencies. This work targets at improvement of thetransformation vector pCc1001 (1). This pUC9-based vector contains a 6.5kb PstI genomic fragment of C. cinerea with the tryptophan synthetasegene (trp1) that can be used to complement trp1- defects. The vectorhowever shows a surprising phenomenon. In single transformation it givesonly low numbers of transformants whereas efficiencies in cotransformationraise by factors of >100%, yielding several hundreds oftransformants per experiment. To investigate this phenomenon further, thevector was modified in length and fragments with thetrp1gene weresubcloned into pBluescriptKS-. The effects on the transformationefficiency were investigated by using several co-transformationexperiments.(1) Binninger DM et al. (1987)DNA-mediated transformation of the basidiomycete Coprinuscinereus. EMBO J 6:835-840FUP030A mating loci in Coprinopsis cinerea differ in the numbers ofHD1 and HD2 homeodomain transcription factor genesU. Kües*, Y. Yu, M. Navarro-GonzalézUniversität Göttingen, Molekulare Holzbiotechnologie und technischeMykologie, Göttingen, GermanyThe 25 kb-long A mating type locus in the mushroom Coprinopsis cinereacontrols defined steps in the formation of a dikaryotic mycelium aftermating of two compatible monokaryons, as well as the formation of thefruiting bodies on the established dikaryon. Usually, three paralogous pairsof divergently transcribed genes for two distinct types of homeodomaintranscription factors (termed HD1 and HD2 after distinguishedhomeodomain sequences) are found in the multiple alleles of the A locus.For dikaryon formation and regulation of sexual development,heterodimerization of HD1 and HD2 proteins from allelic pairs fromdifferent A loci is required. In some A loci found in nature, alleles of genepairs are not complete or one of two genes have been made in-active.Functional redundancy allows the system still to work as long as an HD1gene in one and an HD2 in the other allelic gene pair are operative. In thisstudy, we present for the first time two completely sequenced A loci.Evidences for gene duplications, deletions and inactivations are found. Theloci differ in the number of potential gene pairs (five versus three), ingenes that have been duplicated in evolution, in genes that have been lostin evolution and in genes that are still present but have been made inactive.Kües U, James TY, Heitmann J (2011) Mating type in Basidiomycetes: Unipolar, bipolar, andtetrapolar patterns of sexuality. In: Pöggeler S, Wöstemeyer J (eds) Evolution of fungi and fungilikeorganisms. The mycota XIV. Springer, Berlin, pp 97-160FUP031Enzymatic oxidation of nitrophenols by a DyP-type peroxidaseF. Hahn*, R. Ullrich, M. Kluge, M. Hofrichter, C. LiersInternational Graduate School zittau, Environmental Biotechnology,Zittau, GermanyThe jelly fungus Auricularia auricula-judae produces a dye decolorizingperoxidase (DyP; EC 1.11.1.19) in plant-based complex media (e.g.tomato juice suspension). DyP-type peroxidases represent a separatesuperfamily of heme peroxidases and were first described forbasidiomycetous fungi and later also found in eubacteria. These enzymesoxidize diverse synthetic and natural dyes including recalcitrantanthraquinone derivatives (e.g. Reactive Blue 5), as well as typicalperoxidase substrates such as ABTS and 2,6-dimethoxyphenol. As ligninperoxidases (LiP; EC 1.11.1.14), some DyPs have been shown to oxidizemethoxylated aromatics with high-redox potential such as veratryl alcoholand a non-phenolic -O-4 lignin model dimer (Liers et al. 2010). Tosubstantiate this finding, the oxidation of several mononitrophenols weretested using the DyP of A. auricula-judae. For peroxidases, the conversionof these high-redox potential substrates has so far only been reported forLiP. The Auricularia DyP was found to act on i) ortho-nitrophenol (oNP),ii) meta-nitrophenol (mNP) and iii) para-nitrophenol (pNP). The pHdependency for pNP showed an oxidation optimum at pH 4.5, which istypical for phenol conversions by DyPs. In all cases, the correspondingquinones and dinitrophenols were identified as major products of NPoxidation; moreover, the formation of further unknown products wasobserved in the HPLC elution profiles. The mechanism of nitration wasexamined using 15 N-labeled pNP and an additional source of nitro-groups(sodium nitrite). Products were identified by HPLC-MS, and mass-tochargeratios evaluated to clarify the origin of nitro-groups. The additionalnitrogen in dinitrophenols formed during enzymatic conversion was foundto originate from both nitrophenol and sodium nitrite. Based on theseresults, a hypothetical reaction scheme has been postulated.FUP032Insights into gene regulation under hypoxia in the humanpathogenic fungus Aspergillus fumigatusF. Hillmann*, V. Pähtz, A.A. Brakhage, O. KniemeyerHans-Knöll-Institut, Molecular and Applied Microbiology, Jena, GermanyThe saprophytic mold Aspergillus fumigatus is the predominant airbornefungal pathogen causing locally restricted pulmonary diseases such asnoninvasive aspergilloma or systemic infections in immunocompromisedindividuals. A. fumigatus is usually acquired by the inhalation of fungalspores which, if not cleared by the immune system, germinate to formhyphae. At the site of infection very often inflammatory, necrotic lesionsoccur, which are in many cases associated with severe hypoxia. Only littleis known about the regulatory circuits involved in the adaptation of thisfungus to these low oxygen environments. Recently SrbA was identified asa homologue of the sterol regulatory element binding protein SREBP fromfission yeast, activating hypoxic gene expression in response to low sterollevels. This mutant was unable to grow in the presence of less than 5% O 2and attenuated in virulence in mouse infection models of invasiveaspergillosis (1). Our initial experiments analyzed the gene expression ofA. fumigatus during its long term response to hypoxia in a glucose limitedO 2-controlled fermenter (2). Differential mRNA levels of a number ofgenes during hypoxia were verified by Northern hybridization. Whenanalysing their expression in an srbA null mutant background we foundthat several of these genes were regulated independent from SrbA during ashort-term exposure to hypoxia. From microarray data and genome widesearches we are aiming to identify new regulatory proteins. Bycharacterizing the phenotypes of mutants in the respective genes theirputative roles in the response to low O 2 concentrations are discussed.(1) Willger, S.D. et al., 2008. PLoS Pathog. 4(11):e1000200.(2) Vödisch et al., 2011. J. Proteome Res. 10(5):2508-2524.BIOspektrum | Tagungsband 2012