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

81system represents an attractive alternative to siRNA-based screeningsystems in mammalian cells.Kindly supported by a grant from the Deutsche Forschungsgemeinschaft.B. Becker and M.J. Schmitt (2011). Toxins 7, 834-847.FUP013Molecular mechanism of light repression of sexual sporeformation in the filamentous fungus Aspergillus nidulansK. Seither*, R. FischerKarlsruhe Institute of Technology (KIT), Microbiology, Karlsruhe, GermanyThe filamentous ascomycete Aspergillus nidulans is able to perceive lightdue to a repertoire of light sensors, among which is a pyhtochrome (FphA)and a flavin-containing transcription factor (LreA) (1,2,3). Light triggersmany physiological processes and morphogenetic pathways in fungi. Forinstance in Aspergillus nidulans, light induces the development of asexualspores whereas the sexual cycle is preferred in darkness. Whereas lightinductionis studied quite well, repression of sexual genes in light has notbeen studied yet. NosA (numberof sexual spores) and NsdD (neverinsexualdevelopment) are both important activators of sexual development(4,5). Both proteins localize to the nucleus in all stages of development,which correlates with their function as putative transcription factors andthe fact that they both harbor a NLS.In order to understand the effect of light on these transcription factors,direct interaction between them and light regulator proteins has beenstudied. Indeed, NosA interacted with FphA in the nucleus as shown bybimolecular fluorescence complementation. This could indicate negativeregulation of the NosA activity. In addition, it was found that FphA bindsto the promoters of nosA and nsdD as shown by ChIP (Chromatin-Immunoprecipitation). This suggests transcriptional control of theirexpression. LreA also bound to the promoter of nsdD, but this bindingoccurred only in light. As the expression of nsdD was lower in light than inthe dark, LreA appears to repress nsdD. As NsdD and NosA are bothputative transcription factors, they also activate or repress other genes.CpeA, a catalase-peroxidase, was found to be regulated by NosA. It wasshown that NosA binds to the promoter of cpeA and that in the nosAstrainthe expression of CpeA was drastically reduced (4). In addition, agene with a WSC-domain and a putative FAD-dependent oxidoreductaseappeared to be regulated by NsdD.(1) Purschwitz J. et al., (2008) Curr. Biol. 18(4):255-9(2) Blumenstein A. et al., (2005) Curr. Biol. 15(20):1833-8(3) Rodriguez-Romero J. et al., (2010) Annu. Rev. Microbiol. 64:585-610(4) Vienken, K. & Fischer, R. (2006) Mol. Microbiol. 61:544-554(5) Han, K. et al., (2001) Mol. Microbiol. 41:299-309FUP014g1i-diagnosis: development of a strain-specific diagnostic toolfor the entomopathogenic fungus Beauveria brongniartiiA.-C. Fatu 1,2 , V. Fatu 1,2 , A.-M. Andrei 2 , C. Ciornei 3 , D. Lupastean 4 ,A. Leclerque* 11 Julius Kühn-Institut (JKI), Institut für Biologischen Pflanzenschutz,Darmstadt, Germany2 Research-Development Institute for Plant Protection (ICDPP),Bucharest, Romania3 Forest Research and Management Institute (ICAS), Forest ResearchStation Bacau, Bacau, Romania4 Stefan cel Mare University of Suceava, Faculty of Forestry, Suceava, RomaniaMitosporic fungi e.g. of the genus Beauveria are of considerable economicand ecological interest as insect biocontrol agents. Strains of the speciesBeauveria brongniartii have been found particularly promising for thecontrol of scarabaeid pests as the European cock-chafer, Melolontha sp.,and to date several B. brongniartii formulations, e.g. “Melocont ® ”, areregistered mycoinsecticides. Beyond immediate control efficiencies,parameters as the persistence of fungal spores in the environment, thepossible build-up of a residual insecticidal activity, and the long-termimpact of biocontrol fungi upon ecosystem biodiversity are of relevancefor mycoinsecticide evaluation and registration. Therefore, diagnostic toolsfor the assessment of these parameters are highly solicited.In several mitosporic fungi including Beauveria brongniartii, nuclearrRNA encoding genes have previously been found interrupted bysequences homologous to self-splicing group 1 introns. We have made useof the presence of these genetic elements to develop a PCR-basedapproach to strain-specific diagnosis.Within the framework of research activities aiming towards thedevelopment of a Melolontha biocontrol strategy based upon endemicfungal isolates from Romania, the Romanian B. brongniartii isolateICDPP#1a was genetically compared to the “Melocont” producer strain.Amplified 18S rRNA encoding sequences from both strains were found tobe 100% identical, and strains clustered tightly within the B. brongniartiiclade of a phylogenetic tree reconstructed from a second, independentmarker, namely elongation factor 1 alpha, that is currently the marker ofchoice for the infra-generic classification of Beauveria. However, adifference in the respective 18S rRNA gene exon-intron structures wasdetected. Based upon this genetic difference, a PCR-based diagnostic toolwas developed that renders the two-sided positive discrimination and thedifferential assessment of the environmental persistence of thesebiocontrol strains possible.However, as several conserved intron insertion sites that allow for aconsiderable number of different exon-intron structures have beenidentified throughout the 18S and 28S rRNA genes of Beauveria andrelated fungi, g1i-diagnosis clearly holds potential for application beyondthis specific context.Fatu A-C, Fatu V, Andrei A-M, Ciornei C, Lupastean D, Leclerque A (2011) Strain-specific PCRbaseddiagnosis for Beauveria brongniartii biocontrol strains. IOBC/wprs Bulletin 66: 213-216.FUP015Will be presented as FUV006!FUP016Deneddylation and fungal developmentJ. Schinke*, M. Christmann, G. BrausGeorg August Universität Göttingen, Mikrobiologie und Genetik,Göttingen, GermanyDeneddylation is the removal of the ubiquitin (Ub)-like protein Nedd8from cullins. Cullins are subunits of cullin-RING Ub ligases (CRL) whichare controlled in their activity and assembly/reassembly by neddylationand deneddylation. The most important eukaryotic deneddylases are theCOP9 signalosome (CSN) and the deneddylating enzyme 1 (DEN1).Mammalian Den1 has two functions: an isopeptidase activity removingNedd8 from cullins and other proteins and an additional linear peptidaseactivity processing Nedd8 from a precursor protein. Filamentous fungipossess an eight subunit COP9 signalosome (CSN) which is reminiscent tothe corresponding plant and vertebrate complex (Busch et al., 2007 PNAS104: 8089-8094; Braus et al., 2010 Curr Opin Microbiol 13: 672-676).Aspergillus nidulans requires CSN function to trigger development inresponse towards light, and for a coordinated secondary metabolism(Nahlik et al., 2010 Mol Microbiol 78: 964-79). We show here thecharacterization of the fungal Den1 ortholog DenA. The denA geneencodes a cysteine protease deneddylating enzyme. DenA is required forlight control and the asexual fungal development whereas CSN is requiredfor the sexual cycle. Processed Nedd8 is unable to rescue conidiaformation suggesting that the lack of the DenA deneddylase isopeptidaseactivity is responsible for the defect. Yeast-two-hybrid experimentssuggest a physical interaction between DenA and CSN which will befurther evaluated.FUP017Analysis of the F-box protein encoding genes of theopportunistic human pathogen Aspergillus fumigatusB. Joehnk* 1 , î Bayram 1 , T. Heinekamp 2 , A.A. Brakhage 2 , G.H. Braus 11 Georg-August-Universität, Department of molecular Microbiology andGenetics, Göttingen, Germany2 Leibniz Institute for Natural Product Research and Infection Biology –HKI, Department of Molecular and Applied Microbiology, Jena, GermanyA major virulence factor for the opportunistic human pathogen Aspergillusfumigatus is its ability to rapidly adapt to host conditions during infection.The rapid response to environmental changes in the host underlies a wellbalancedsystem of production and degradation of proteins. A highlyconserved mechanism for controlled protein degradation is the ubiquitinproteasome-system.Ubiquitin molecules are attached to the target proteinsby the ubiquitin-protein ligase (E3) and therefore polyubiquitnylatedproteins are destined for degradation via the 26S-proteasome. The largestgroup of E3-enzymes is the SCF Cullin1 Ring ligases (CRL), which aremultisubunit enzymes. The F-box subunit functions as a substrate adaptorand thus, is responsible for the substrate specificity of the E3 enzyme. Inthis study we have analyzed the genes, encoding the three F-box proteinsFbx15, Fbx23 and Fbx29 in the opportunistic pathogen Aspergillusfumigatus. Deletion of these genes results in growth defects under differentstress conditions including H 2O 2 mediated oxidative stress, and increasedtemperature, which are important parts of the innate immune response. Wecould further show that the gene for the F-box protein Fbx15 is essantialfor virulence of A. fumigatus in a murine model. In contrast to this thefbx15 deletion mutant displays a enhanced production of theimmunosupressive mycotoxin, gliotoxin compared to wt andcomplementation strain. In addition knock-out attemps of fbx25, another F-box encoding gene revealed that this is an essantial fbx-gene for A.fumigatus. Functional GFP-tagged versions of Fbx15 and Fbx25 could belocalized in the nucleus suggesting regulatory functions of these F-boxesfor certain transcription factors. Future studies aim to identify potentialtargets of these F-box proteins and their function in stress recognition andresponse.This work is supported by the Deutsche Forschungsgemeinschaft, DFGResearch Unit 1334.BIOspektrum | Tagungsband 2012