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

79FUP004Next-generation genome sequencing, assembly, annotation andanalysis of a marine isolate of Scopulariopsis brevicaulis.A. Kumar*, F. KempkenBotanisches Institut und Botanischer Garten Christian-Albrechts-Universität zu Kiel, Abteilung für Botanische Genetik undMolekularbiologie, Kiel, GermanyThe kingdom fungi constitute the largest branch in the tree of life.However, a very little is known about fungal genomics, although someprogress have been made using Sanger sequencing in last two decades.Recently, the Sordaria macrospora genome became available using nextgenerationsequencing (1). To further explore fungal diversity, we set outto sequence marine isolates of fungi. Here we report the first example,Scopulariopsis brevicaulis, which has previously been known as acommon soil saprophyte and has been isolated from a wide variety ofsubstrates. Some species ofScopulariopsisare reported to cause humandiseases (2).S. brevicaulisis also known to produce cyclic peptidesscopularide A and B (3).We have established the genomic sequence of a marine isolate ofS.brevicaulisusing three different next-generation sequencing methodsnamely, roche 454, illumina and ion-torrent. Herein, we present ourcurrent results ofS. brevicaulisassembled genome of about 32 Mb sizeusing 726,314, 247,824,350 and 2,556,553 reads from roche 454, illuminaand ion-torrent, respectively. We found the contig length is large for roche454 (935 contigs/N50 - 88 kb) in comparison to contigs of illumina (29330contigs/N50 - 1.7 kb) and ion-torrent (32008 contigs/N50 - 1.6 kb).Furthermore, we will provide complete annotation of individual assembliesusing each sequencing method and also a hybrid assembly achieved usingpublically and commercially available next-generation sequence assemblyand annotation tools. This genome characterization assists fungal biologistto further carry out research with this species, which largely hindered dueto unavailability of the genome.1. Nowrousian et al. (2010). De novo Assembly of a 40 Mb Eukaryotic Genome from ShortSequence Reads:Sordaria macrospora, a Model Organism for Fungal Morphogenesis. PLoS Genet6(4): e1000891.2. Cuenca-Estrella, et al. (2003). Scopulariopsis brevicaulis, a Fungal Pathogen Resistant to Broad-Spectrum Antifungal Agents. Antimicrobial Agents and Chemotherapy 47, 2339-41.3. Zhiguo et al. (2008). Scopularides A and B, Cyclodepsipeptides from a Marine Sponge-DerivedFungus, Scopulariopsis brevicaulis.Journal of Natural Products71 (6), 1052-1054FUP005Establishment of an appropriate transformation model for therock inhabiting fungi Sarcinomyces petricola (A95)S. Noack* 1 , W.J. Broughton 1 , T. Bus 2 , C. Nai 1,3 , L. Schneider 1 ,R. Banasiak 1 , A.A. Gorbushina 11 Federal Institute for Materials Research and Testing, Materials andEnvironment (IV), Berlin, Germany2 University of Applied Sciences Jena, Department of Medical Engineeringand Biotechnology, Jena, Germany3 Free University of Berlin, Institute of Geological Sciences, DivisionGeochemistry, Hydrogeologie, Mineralogy, Berlin, GermanyMelanised micro-colonial fungi (MCF) colonize bare rock surfaces indeserts and other arid areas and are unequalled among eukaryoticorganisms in their ability to withstand extreme heat, desiccation and UVradiation. These organisms are crucial in the establishment of subaerialrock biofilms and, as such, set the stage for a variety of interactionsimportant for mineral/material stability and rock weathering. MCF are ataxonomically diverse group of ascomycetes and are characterised bysimplified stress-protective morphologies including a peculiar compactcolonial structure, protective cell walls and multiple secondary metabolicproducts supporting their stress tolerance - melanins, carotenoids,mycosporines and compatible solutes. A meristematic black yeastspecies,Sarcinomyces petricola (A95), was isolated from a sun exposedmarble monument in Athens (Greece). Different methods have been testedto establish a transformation protocol for A95. A common method usingthe binary Ti vector system of Agrobacterium tumefaciens was employed(De Groot et al., 1998). The stress-tolerant morphology of black yeasts,especially their thick cell walls and melanisation complicates the transferof DNA from A. tumefaciens to A95 however. Several methods tocircumvent this problem were tested, including DNA transfer by microprojectilebombardment and chemical weakening of the cell wall bytreatment with DMSO. Different protoplasts isolation protocols based onenzymes with chitinase and ß-glucanase activity were also tested. Anefficient protocol yielded sufficient protoplasts for transformation withpolyethylenglycol. All that remains is to find an appropriate vector systemthat allows integration of the gene of interest and its translation into thefugal genome.De Groot MJA, Bundock P, Hooykaas PJJ, Beijersbergen AGM (1998). Agrobacterium tumefaciens- mediated transformation of filamentous fungi. Nature Biotechnology 16: 839-842FUP006Influence of microclimatic conditions on fungal diversity inbiofilms from the facades of buildings.S. Noack* 1 , M. Adler 2 , F. Seiffert 3,4 , W.J. Broughton 3 , A.A. Gorbushina 3,41 Federal Institute for Materials Research and Testing, Materials andEnvironment (4), Berlin, Germany2 Free University of Berlin, Institute of Biology, Berlin, Germany3 Federal Institute for Materials Research and Testing, Materials andEnvironment (IV), Berlin, Germany4 Free University of Berlin, Institute of Geological Sciences, DivisionGeochemistry, Hydrogeologie, Mineralogy, Berlin, GermanyThe facades of buildings and their structural elements are colonized bydiverse microbes including algae, bacteria and fungi. On older buildingsand monuments, these biofilms contribute to the general appearance.Because they cause surface discoloration and material damage,microorganisms that live on the facade of buildings have been the subjectof intense interest.Our research concerns the interaction of subaerial biofilms (SAB) and theunderlying substrates. Important components of SAB include melanisedmicro-colonial fungi (MCF) and phototrophic micro-organisms. Highlymelanised MCF are well adapted to extreme environments and thus arestable partners in weathering processes. Currently we are investigating theinfluence of different microclimatic conditions and seasonal fluctuationson fungal diversity in natural biofilms. A public building in Berlin waschosen for this purpose. Seasonal variations in the composition of thebiofilms on the shaded and damp northwest side of the building werecompared with those on the sunny and dry southeast side. DGGE analysesbased on sequence differences in the 18S rDNA and the ITS rDNA regionfrom different fungi were used to compare the populations. In this wayfingerprints of fungal diversity can be generated and compared to othercharacteristics of biofilm such as chlorophyll contents, spectral propertiesand other with biofilm partners.FUP007Differential 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. Braus-Stromeyer 1 , G. Braus 11 Georg-August-Universität Göttingen, Institut für Mikrobiologie undGenetik, Göttingen, Germany2 Georg-August Universität Göttingen, Albrecht-von-Haller-Institut fürPflanzenwissenschaften, 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.BIOspektrum | Tagungsband 2012