Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSexpression) strains in mnps and studied their degradation capacity. The compounds studied were: azo-dyes such as orange II and reactive black,recalcitrant pharmaceutical compounds found in treated waste water such as Carbamazepine and lignocellulosic agricultural waste. We engineered atransformant, constitutively expressing mnp4 a VP naturally repressed by Mn (designated OEmnp4) under the control of the b-tubulin promoter. Now,despite the presence of Mn in the medium, OEmnp4 produced mnp4 transcript as well as VP activity as soon as four days after inoculation. OEmnp4decolorized the azo-dyes two days earlier relative to the wild type in Mn amended medium. RNAi silencing targeting mnp3 resulted in a delay in thedecolorization capacity which occurred concomitantly along with a marked reduction of the expression level of all mnps, particularly mnp3 and mnp9. Thisobservation supported the conclusion that MnPs are involved in the process but could not determine the specific contribution of the different genes to theoutcome. Therefore we produced a Dku80 strain, exhibiting a 100% homologous DNA recombination rate, to enable specific gene replacement.Subsequently, homokaryon mnp2, 3, 4 and 9 knockout strains were produced. In Mn amended GP, orange II decolorization was not significantly inhibitedby any of these strains, indicating on functional redundancy. In Mn deficient GP, inactivation of mnp4 proved that it encodes the key VP responsible forMn dependent and Mn independent peroxidase activity, as well as resulted in reduction of the azo dye reactive black 5 decolorization capacity. The toolsand protocols developed increase the amenability of P. ostreatus to genetic manipulations and expand options for gene function analyses.712. Temperature- and pH characteristics of endo-cellulases in Rhizophlyctis rosea. Bo Pilgaard 1 , Frank Gleason 2 , Peter Busk 1 , Lene Lange 1 . 1) Section forsustainable biotechnology, Aalborg University- Copenhagen, Denmark; 2) School of Biological Sciences, University of Sydney, New South Wales, Australia.The zoosporic true fungi of the order Chytridiales are of special interest since they constitute a central root position of the entire fungal kingdom.Enzymes of the anaerobic zoosporic rumen fungi have been studied quite extensively, but only scarce information is available about enzymes from aerobiczoosporic true fungi from soil. We have previously confirmed the presence of cellulases in Rhizophlyctis rosea (AUS13), which was isolated from soils of theSydney Basin. Other studies have shown that this fungus can survive and grow within a wide pH-range (Gleason et. al 2010) and high temperatures(Gleason et. al 2005). We investigate the cellulolytic potential of Rhizophlyctis rosea in several cellulase assays and characterize the enzymatic propertieswith respect to temperature and pH stability of the enzymes. Moreover, we are sequencing the Rhizophlyctis rosea genome in order to find the genes forcellulose-degrading enzymes (GH6, GH7, GH45, GH6) that are present in Chytrids as compared to the cellulases of these families found in other fungalgroups.713. Applying unconventional secretion of the endochitinase Cts1 to export heterologous proteins in Ustilago maydis . J. Stock, P. Sarkari, M. Knopp, S.Jankowski, S. Bergmann, M. Feldbrügge, K. Schipper. Institute for Microbiology, Heinrich-Heine University Düsseldorf, 40204 Düsseldorf, Germany.The demand on the biotechnological production of proteins for pharmaceutical, medical and industrial applications is steadily growing. Not every proteincan easily be produced by the existing platforms. For the production of such challenging proteins, we aim to establish a novel expression system in the wellcharacterized eukaryotic microorganism Ustilago maydis. In this fungus, secretion of the endochitinase Cts1 depends on mRNA transport alongmicrotubules, which is mediated by the key RNA-binding protein Rrm4. We recently demonstrated that Cts1 secretion occurs via a novel unconventionalroute. We used b-glucuronidase (Gus) as a reporter for unconventional secretion. This bacterial enzyme is inactivated by N-glycosylation during its passagethrough the conventional eukaryotic secretory pathway. By contrast, in our system Gus was exported in its active form by fusion to Cts1 confirming itssecretion by an unconventional route. Furthermore, we showed that this secretory mechanism can be exploited for the export of active heterologousproteins. As a proof-of-principle for economically important biopharmaceuticals we expressed an active single-chain antibody. Importantly, the novelprotein export pathway circumvents N-glycosylation which is advantageous in many applications, for example to avoid undesired immune reactions inhumans. Currently, the system is optimized with respect to product yield by e.g., reducing the proteolytic activity in the culture supernatants. Thus, theunconventional Cts1 secretion machinery has a high potential for the production of biotechnologically relevant proteins.714. A new method for fungal genetics: flow cytometry of microencapsulated filamentous microcolonies. L. Delgado-Ramos 1 , A. T. Marcos 1 , X. Peñate 1 ,M. S. Ramos-Guelfo 1 , L. Sánchez-Barrionuevo 1 , F. Smet 2 , D. Canovas 1 , S. Chávez 1 . 1) Dept of Genetics, Univ of Sevilla, Spain; 2) Union Biometrica, Geel,Belgium.Genetic analysis of non-filamentous microorganisms is facilitated by the isolation of consistent, well-defined colonies on solid media and the handling ofindividual cells by flow cytometry. In contrast, some filamentous fungi are hard to be analyzed using these procedures; in particular by flow cytometry. Thecombination of single spores microencapsulation and large particle flow cytometry is a possible alternative for the analysis of filamentous fungi.Microencapsulation allows the early detection of fungal growth by monitoring the development of hyphae from encapsulated individual spores. Myceliumproliferation inside the microcapsules can be detected using COPAS large particle flow cytometry. Here we show the successful application of the FlowFocusing® technology to the microencapsulation of filamentous fungi in monodisperse alginate microspheres, using Aspergillus and Trychoderma as modelsystems. Using a Cellena® Flow Focusing microencapsulator, we managed to produce monodisperse microparticles containing individual spores and todevelop microcolonies of these fungi upon germination in the appropriate conditions. Proliferation inside the particles was monitored by microscopy andlarge particle flow cytometry without requiring fluorescent labeling. Sterility was preserved during the microencapsulation procedure, preventingundesired contaminations. Conditional mutants were utilized to demonstrate the feasibility of the method. This procedure allows for the handling,screening and analysis of clonal colonies in liquid culture. Examples of applications will be provided.715. DNA methylation dynamics during development in the rice blast fungus. Junhyun Jeon 1 , Jaeyoung Choi 2 , Gir-Won Lee 2 , Sook-Young Park 3 , AramHuh 1 , Ralph Dean 4 , Yong-Hwan Lee 1,2,3,5 . 1) Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, South Korea; 2) FungalBioinformatics Laboratory, Seoul National University, Seoul 151-921, Korea; 3) Center for Fungal Pathogenesis, Seoul National University, Seoul 151-921,Korea; 4) Center for Integrated Fungal Research, Department of Plant Pathology, North Carolina State University, Raleigh, NC 27607, USA; 5) Center forFungal Genetic Resources, Seoul National University, Seoul 151-921, Korea.Cytosine methylation is an important epigenetic modification of DNA that is involved in genome defense and transcriptional regulation in eukaryotes. Inmammals and plants, many roles of DNA methylation depend on dynamic changes of DNA methylation pattern. In fungi, DNA methylation is consideredprimarily as a stable mark for silencing transposable elements. Here we used genetic manipulations and high-throughput bisulphite sequencing on themodel plant pathogenic fungus, Magnaporthe oryzae to elucidate the dynamics and mechanics of DNA methylation during pathogenic development. Wefound that genome-wide reprogramming of DNA methylation in and around genes occurs during progression of fungal development and that suchreprogramming is important for normal development. RNA-seq analysis showed that DNA methylation is associated with transcript abundance of genes incontext-dependent manner. Our study reveals that DNA methylation in fungi could be a dynamic epigenetic entity that has assumed new roles indevelopmental processes other than genome defense.296