Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSthe media and weight of the mycelium were measured in nit-10 (KO) and nit-3 (KO) strains in comparison to a wild type strain. The nit-10 (KO) mutant wasnot able to grow on nitrate as a sole nitrogen source, and no nitrate depletion from the media was observed. Therefore, it was concluded that NIT10 is theonly active transporter. The accumulation of nitrate in the mycelium was measured and it was found that in the nit-3 (KO) mutant it was 9 times higherand in the nit-10 (KO) mutant 3 times higher than in the wild type strain. Therefore, we concluded that nitrate reductase is not required for the nitratetransport.42. Protein kinases affecting glycogen accumulation and likely regulating the glycogen synthase phosphorylation status in Neurospora crassa. T.Candido 1 , A. P. Felício 2 , R. Gonçalves 1 , F. Cupertino 1 , F. Freitas 1 , M. C. Bertolini 1 . 1) UNESP - IQ - Araraquara, Araraquara, SP., Brazil; 2) Departamento deGenética e Evolução, UFSCar, São Carlos, SP, Brazil.The ability to sense and respond appropriately to environmental changes is required for all living organisms and reversible phosphorylation of proteinsmediated by protein kinases plays a key role in this aspect. In this work we describe the results of a screen aimed to identify protein kinases regulatingglycogen metabolism in Neurospora crassa. The glycogen synthase (GS) and glycogen phosphorylase (GP), the regulatory enzymes in glycogen synthesisand degradation processes, respectively, are highly regulated by phosphorylation, however the protein quinases that phosphorylate them in N. crassahave not been identified. In this work, a set of mutant strains individually knocked-out in genes encoding proteins kinases was used. The glycogen levelswere quantified under normal growth temperature (30°C) and under heat stress (45°C). From 84 mutant strains, 37 strains presented glycogenaccumulation profile different from the wild-type strain suggesting that the missing protein kinase is implicated in glycogen metabolism control. Amongthe protein kinases selected most are Ser/Thr protein kinases, and it is important to mention proteins already characterized as regulators of glycogenmetabolism, such as the Saccharomyces cerevisiae Pho85 and Snif1 proteins. The GSN activity was quantified in the selected strains grown under normaltemperature (30°C) and under heat stress (45°C) in the presence and absence of the allosteric activator glucose-6-phosphate (G6P). The ratio -/+ G6P isconsidered as an index of phosphorylation, lower levels correlating with higher phosphorylation. Some protein kinases were implicated in glycogenmetabolism control by likely influencing the GSN phosphorylation status. The GSN phosphorylation profile in the mutant strains were analyzed in 2D-PAGEfollowed by Western blot using polyclonal GSN antibody. Some mutant strains showed phosphorylation profile different from the wild-type strain and theresults revealed putative proteins kinases not yet described as able to phosphorylate GSN. The expression of glycogen synthase (gsn) and glycogenphosphorylase (gpn) genes was analyzed by qRT-PCR in the mutant strains and the results showed that some protein kinases regulate the expression ofboth genes. Supported by FAPESP and CNPq.43. Endogenous ergothioneine is required for wild type levels of Neurospora crassa conidiogenesis and conidial survival, but does not protect againstuv-induced kill or mutagenesis. Lynn Epstein, Marco Bello, John Mogannam. Plant Pathology, University of California, Davis, CA. 95616-8680.Ergothioneine (EGT) is a histidine derivative that apparently is only synthesized by fungi (except in the Saccharomycotina), and by some bacteria in theCyanophyta and Actinomycetales. Although plants and animals do not synthesize EGT, they acquire it from the environment; EGT is concentrated in animalcells with an EGT-specific transporter. Bello et al. (2012, Fungal Genet Biol 49:160) showed that the concentration of EGT is 5x greater in Neurosporacrassa conidia than in mycelia, and that growth of strain NcDEgt-1 with a knockout in gene NCU04343 is indistinguishable from the wild type. Toinvestigate the function of EGT, wild type (Egt+) and NcDEgt-1 were crossed and six Egt+ and six Egt- sib strains were analyzed. Compared to the Egt+ sibs,Egt- sibs had a highly significant reduction (59 + 6%, + SE) in the number of conidia produced on Vogel’s agar; the detransformed mean of the Egt- sibs was1.5 x 10 5 conidia/cm 2 with a detransformed 95% confidence interval (CI 95) from 1.2 x 10 5 to 1.8 x 10 5 conidia/cm 2 whereas the Egt+ sibs had a mean of 3.6 x10 5 conidia/cm 2 and a CI 95 from 2.9 x 10 5 to 4.6 x 10 5 conidia/cm 2 . The concentration of EGT in wild type conidia did not increase with increasing exposureto light during conidiogenesis. Seven-day-old conidia were stored at 30 °C at 97% and 51% relative humidity (RH) for a time course to either 17 or 98 days,respectively. Life expectancies (LE) were calculated from logistic curves fitted to percentage germination as a function of days in storage in two trials. At97% RH, Egt+ sibs had a LE = 11.0 + 0.2 days whereas Egt- sibs had a highly significantly lower LE = 8.4 + 0.2 days, a 23 + 8% reduction. At 51% RH, Egt+ sibshad a LE = 71 + 1 days whereas Egt- sibs had a highly significantly lower LE = 58 + 1 days, an 18 + 3% reduction. We tested the hypothesis that EGT protectsagainst uv-induced kill or mutagenesis. There were no significant differences between the germinability of Egt+ and Egt- sibs after exposure to 0 to 400Joules/m 2 of 254 nm light. There also were no significant differences between the Egt+ and Egt- sibs in the mtr mutation rate to fluorophenylalanineresistance after exposure of conidia to 0 to 400 Joules/m 2 of 254 nm light. Consequently, our in vivo analysis indicates that EGT does not protect againstuv-induced kill or mutagenesis.44. Thiolutin inhibits protein turnover in Neurospora and yeast. Linda Lauinger, Michael Brunner, Axel Diernfellner. BZH, Heidelberg, Germany.Proteasome inhibitors are a powerful tool for the characterization of proteins in vivo. In yeast as well as in filamentous fungi, however, the availableproteasome inhibitors, like e.g. MG132 do not function due to the barrier posed by the cell wall of the organisms and an efficient evacuation of themolecules out of the cells. The dithiole thiolutin has been shown to be a potent inhibitor of RNA polymerases in prokaryotes and fungi. In the filamentousfungus Neurospora crassa, thiolutin efficiently suppresses transcription, indicating that the drug is cell permeable and not subject to a significant efflux bythe multidrug resistance system. Our data indicate that thiolutin also significantly inhibits protein turnover. Concomitant with the increase in proteinstability after treatment with thiolutin, we observe an accumulation of ubiquitinated protein species. Thus, our findings suggest that thiolutin may be apleiotropic inhibitor suppressing both, RNA polymerase as well as the proteasomal activity.45. Characterization of a Phanerochaete chrysosporium glutathione transferase reveals a novel structural and functional class with ligandin propertiesfor wood extractive molecules. Yann Mathieu 1,2,6 , Pascalita Prosper 3,4 , Marc Buée 2 , Stéphane Dumarçay 5 , Frédérique Favier 3,4 , Eric Gelhaye 1,2 , PhilippeGérardin 5 , Luc Harvengt 6 , Jean-Pierre Jacquot 1,2 , Tiphaine Lamant 1,2 , Edgar Meux 1,2 , Sandrine Mathiot 3,4 , Claude Didierjean 3,4 , Melanie Morel 1,2 . 1)Université de Lorraine, IAM, UMR 1136, IFR 110 EFABA, Vandoeuvre-les-Nancy, F-54506, France; 2) INRA, IAM, UMR 1136, Vandoeuvre-les-Nancy, F-54506, France; 3) Université de Lorraine, CRM2, UMR 7036, Vandoeuvre-les-Nancy, F-54506, France; 4) CNRS, CRM2, UMR 7036, Vandoeuvre-les-Nancy, F-54506, France; 5) Université de Lorraine, LERMAB, EA 1093, Vandoeuvre-les-Nancy, F-54506, France; 6) Laboratoire de biotechnologie, Pole Biotechnologieet Sylviculture Avancée, FCBA, Campus Foret-Bois de Pierroton, 33610 Cestas, France.Glutathione transferases (GSTs) form a superfamily of multifunctional proteins with essential roles in cellular detoxification processes. A new fungalspecific class of GST has been highlighted by genomic approaches. The biochemical and structural characterization of one isoform of this class inPhanerochaete chrysosporium revealed original properties. The three-dimensional structure showed a new dimerization mode and specific features bycomparison with the canonical GST structure. An additional b-hairpin motif in the N-terminal domain prevents the formation of the regular GST dimer andacts as a lid, which closes upon glutathione binding. Moreover, this isoform is the first described GST that contains all secondary structural elements,including helix a4’ in the C-terminal domain, of the presumed common ancestor of cytosolic GSTs, i.e. glutaredoxin 2. A sulfate binding site has been132