Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSidentified close to the glutathione binding site and allows the binding of 8-anilino-1-naphtalene sulfonic acid (ANS). Competition experiments betweenANS, which has fluorescent properties, and various molecules, showed that this GST binds glutathionylated and sulfated compounds, but also woodextractive molecules such as vanillin, chloro nitrobenzoic acid, hydroxyacetophenone, catechins and aldehydes in the glutathione pocket. This enzymecould thus function as a classical GST through the addition of glutathione mainly to phenethyl isothiocyanate but alternatively and in a competitive way, itcould also act as a ligandin of wood extractive compounds. These new structural and functional properties, lead us to propose that this GST belongs to anew class that we name GSTFuA for Fungal specific GST class A.46. Secretome analysis of Trichoderma harzianum cultivated in the presence of Fusarium solani cell wall or glucose. Marcelo HS Ramada 1,3 , Andrei SSteindorff 1 , Carlos Bloch Jr. 3 , Cirano J Ulhoa 2 . 1) Brasilia University, Cell Biology Department, Brasilia, DF, Brazil; 2) Federal University of Goias, BiochemistryDepartment, Goiania, GO, Brazil; 3) EMBRAPA CENARGEN, Mass Spectrometry Laboratory, Brasilia, DF, Brazil.Trichoderma harzianum is a fungus well known for its potential as a biocontrol agent of many fungal phytopathogens. The aim of this study was toevaluate the potential of T. harzianum ALL42 to control Fusarium solani, a phytopathogen fungus that causes several losses in common bean and soy cropsin Brazil and to evaluate the secreted proteins of T. harzianum ALL42 when its spores were inoculated and incubated in culture media supplemented (TLE)or not (MM) with nitrogen sources and in the presence or not of F. solani cell walls (FsCW). In the absence of FsCW, the media were supplemented withglucose (GLU). T. harzianum was able to control the phytopathogen growth and started to sporulate in its area after 7 days in a dual culture assay,indicating that it had successfully parasitized the host. T. harzianum was able to grow in TLE+FsCW, MM+FsCW, TLE+GLU, but unable to grow in MM+GLU.Protein quantification showed that TLE+FsCW and MM+FsCW had 45 and 30 fold, respectively, more proteins than TLE+GLU, and this difference wasobserved in the bidimensional gels, as the two supernatants from media supplemented with FsCW had around 200 spots and the one supplemented withglucose only had 18. TLE+FsCW and MM+FsCW had above 80% of spot similarity. A total of 100 proteins were excised from all three conditions andsubmitted to mass spectrometry analysis. 85 out of 100 proteins were identified. The only protein observed in all three conditions is a small protein, calledepl1, involved in eliciting plant-response against phytopathogens. An aspartic protease, previously described as related to mycoparasitism, was only foundwhen T. harzianum was grown with glucose. Gene expression was evaluated and confirmed the gel results. In the media supplemented with FsCW,different hydrolases like chitinases, beta-1,3-glucanases, glucoamylases, alpha-1,3-glucanases, and proteases were identified. Some proteins like a smallcystein-rich, alpha-L-arabinofuranosidase and NPP1, with no known function in mycoparasitism were also identified. T. harzianum ALL42 is able to inhibitthe growth and parasitize F. solani and showed a complex and diverse arsenal of proteins that are secreted in response to the presence of the cell walls,with novel proteins not previously described in mycoparasitism studies.47. Analysis of carbon catabolite repression (CCR) during cellulase formation by Trichoderma reesei (Hypocrea jecorina) using two-dimensionaldifferential gel electrophoresis (2D-DIGE). Wellington Pedersoli, Lilian Castro, Amanda Antonieto, Vitor M. Faça, Roberto N. Silva. Biochemistry andImmunology, University of São Paulo, Ribeirao Preto, Sao Paulo, Brazil.The production of cellulases by Trichoderma reesei (Hypocrea jecorina) is fundamental for the production of second generation biofuels from cellulosicwastes. The complex of cellulases of fungi T. reesei is strongly induced by sophorose and cellulose and also antagonized by glucose. Thus, the objective ofthis study was to compare the differential secretome using 2D-DIGE of T. reesei at induction and repression conditions. The fungus T. reesei (QM9414)were grown in media containing 1% cellulose for 24, 48 and 72 hours and 1% glucose for 24 and 48 hours in a orbital shaker at 180 rpm at 28°C. Afterfiltration of each medium the supernatant was used for protein determination. Samples from each time in the different conditions were mixed inproportional amounts and submitted to isoelectric focusing in 18 cm strip of pH 4.0 - 7.0 and subsequently subjected to electrophoresis on twodimensionalgel - differential (2D-DIGE) for the separation of proteins. The comparative analysis PDQuest software (BioRad) 130 selected spotsdifferentially expressed, wherein the inhibitor glucose showed 41 distinct protein spots and inductor cellulose 89 spots. However, only the 34 spots and 63spots from glucose and cellulose respectively could be spotted. Identification of these spots was carried out using the mass spectrometer XEVO TQ-S(Waters). A total of 57 proteins were identified, 30 and 27 proteins from cellulose and glucose conditions respectively. The proteins identified from glucosecondition were amidases, proteases, isoamyl alcohol oxidases and protein belonging to Glycosyl Hydrolases 16, 17, 43 and 54. On the other hand, theproteins identified from cellulose condition were Glycosyl Hydrolases 3, 7, 54, 55, 72, two Cip 1 (envolved in the degradation of cellulose, but with nofunction described), two proteases and a protein responsible for the regulation of stress. Two other proteins, one of each condition were identified asunknown. Thus, the identification of these proteins will allow a better understanding of the mechanism formation of cellulases by T reesei and thuscontribute to improve the production of second generation ethanol. Supported by: FAPESP, CAPES and FAEPA-HC/USP-RP.48. Transposon-associated evolution of a fungal NRPS. Daniel Berry 1 , Carolyn Young 2 , Paul Dijkwel 1 , Barry Scott 1 . 1) Massey University, Palmerston North,New Zealand; 2) The Samuel Roberts Nobel Foundation, Ardmore, Oklahoma.Epichloë endophytes systemically colonise the aerial tissues of cool season grasses to form mutually beneficial symbiotic associations. A defining featureof these associations is fungal synthesis of a range of secondary metabolites that protect the host from biotic and abiotic stress. One key bioprotectivemetabolite is the insect feeding deterrent, peramine, which is synthesized by the two-module non-ribosomal peptide synthetase (NRPS) PerA. This NRPShas an A-T-C-A-M-T-R domain structure where the Adenylation-domains provide specificity for and activation of the two amino acid substrates, theThiolation-domains bind the reaction intermediates, the Condensation-domain catalyzes formation of the peptide bond, the Methylation-domainmethylates the substrate amino acid, and the Reductase-domain cyclizes and releases the finished product from the NRPS. The perA gene is foundexclusively within the Epichloë, where it is widespread among the different species, but peramine production is somewhat more discontinuous. We showthat transposon-mediated deletion of the R-domain present in some E. typhina and E. festucae peramine deficient isolates is associated with a change inthe predicted substrate specificity code of the first A-domain. Phylogenetic analysis of this domain groups the peramine negative isolates togetherwhereas the phylogeny based on the whole gene matches the species phylogeny. The recombination mechanism responsible for the evolution of thisnovel NRPS is still to be resolved.49. Velvet family control of penicillin production in Penicillium chrysogenum: PcVelB binding to isopenicillin N synthase suggests a novel regulatorymechanism. Sandra Bloemendal, Katarina Kopke, Birgit Hoff, Sarah Milbredt, Alexandra Katschorowski, Ulrich Kück. Christian Doppler Laboratory forFungal Biotechnology, Ruhr-University Bochum, Universitätsstr. 150, 44780 Bochum, Germany.The filamentous fungus Penicillium chrysogenum is the main industrial producer of the pharmaceutically relevant beta-lactam antibiotic penicillin. Allthree biosynthesis genes are found in a single cluster and the expression of these genes is known to be controlled by a complex network of globalregulators. It is supposed that subunits of the velvet complex, which were recently detected for P. chrysogenum, function as such global regulators,although the exact regulatory mechanism still have to be elucidated. Core components of this complex are PcVelA and PcLaeA, which regulate secondary27th Fungal Genetics Conference | 133