Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSH 2O 2 was analyzed. Expression profiles of genes encoding oxidative stress response enzymes potentially controlled by Fgap1p and of genes involved in thebiosynthesis of type B trichothecenes were analyzed by Q-RT-PCR. Trichothecene accumulation is strongly enhanced in the deleted strain, with an increasein Tri genes expression. On the other hand, Tri genes expression and toxin accumulation are drastically repressed in the mutant in which Fgap1p isconstitutively activated. Moreover, the level of expression of two genes encoding catalases is modulated in both mutants. The involvement of Fgap1 inother types of stress has also been investigated. In particular, cadmium and osmotic stress affect growth in the deleted strain.411. Functional analyses of FgLaeA in Fusarium graminearum. Hee-Kyoung Kim, Seong-mi Jo, Seunghoon Lee, Sung-Hwan Yun. Dept Med Biotech,Soonchunhyang Univ, Asan, Chungnam 336-745, South Korea.Fusarium graminearum (telomorph: Gibberella zeae) is the casual agent of the head blight of cereal crops and produces mycotoxins such astrichothecenes and zearalenone in infected plants. The expression of genes involved in biosyntheses of these mycotoxins are controlled at the differentlevels ranging from by a pathway-specific transcription regulator (encoded by TRI6 or ZEB2) to by a global regulator involved in chromatin remodeling.Here we focused on the function of FgLaeA in F. graminearum, which is an ortholog of the Aspergillus nidulans LaeA, encoding the global regulator forboth secondary metabolism and sexual development. For functional analysis of FgLaeA in mycotoxin production, we used a transgenic F. graminearumstrain expressing a firefly luciferase gene under control of TRI6 or ZEB2 promoter as a reporter system. Targeted deletion of FgLaeA led to a dramaticreduction of luminescence in the reporter strain, indicating that FgLaeA controls the expression of both TRI6 and ZEB2 in F. graminearum; the reducedtoxin accumulation was further confirmed by HPLC analysis. In addition, the FgLaeA deletion strains exhibited not only albino phenotype on CM mediumbut also earlier formation of sexual fruiting bodies (perithecia) on carrot agar than its wild-type progenitor, the latter indicating that FgLaeA seems tonegatively control the perithecial induction. Quantitative real-time PCR revealed that FgLaeA was expressed constitutively under both mycotoxinproduction and sexual development. Overexpression of a GFP-FgLaeA fusion construct in a FgLaeA-deletion strain recovered all the phenotypic changes tothe wild-type levels, and led to constitutive expression of GFP in the entire cells at different developmental stages. A split luciferase assay for in vivoprotein-protein interaction demonstrated that FgLaeA could not interact with FgveA, an ortholog of A. nidulans veA. Taken together, it is likely that FgLaeAcontrols both secondary metabolism and sexual development in F. graminearum, but the regulation pattern operated by FgLaeA is somewhat differentfrom that by LaeA in A. nidulans.412. Molecular cloning and differential expression of two novel Family 1 b-glucosidases genes from the rare fungus Stachybotrys microspora. SalmaAbdeljalil, Houcine Lazzez, Ali Gargouri. Centre of Biotechnology of Sfax, Sfax-Tunisia.The cellulolytic system of the fungus Stacchybotrys microspora is characterized by the existence of several b-glucosidases. From a compilation of fungalb-glucosidases belonging to family GH1, we designed primers to isolate b-glucosidases by PCR. Using different primers combination, three differentfragments genes were firstly obtained. Two of them are overlapping and constitute a novel gene named Smbgl1A while the third one is a part of a secondgene named Smbgl1B. RT-PCR analysis showed the first gene is induced by cellulose and repressed by glucose while Smbgl1B is equally expressed on bothconditions The identification of putative catalytic residues as well as the conserved glycone and aglycone binding sites was performed on SmBgl1Adeduced aminoacid sequence. The predicted secondary structure of Smbgl1 confirmed its appurtenance to GHI family: the presence of a classical (b/a)8barrel and all the characteristic of subsite -1 (glycone site).413. The transcriptional factors XYR1 and CRE1 regulate the expression of Cellulolytic and Xylanolytic genes at carbon source dependent-manner inHypocrea jecorina (Trichoderma reesei). Amanda C.C. Antoniêto, Lílian S. Castro, Wellington R. Pedersoli, Roberto N. Silva. Department of Biochemistryand Immunology, School , University of São Paulo, Ribeirão Preto-SP, São Paulo, Brazil.The ascomycete Hypocrea jecorina (anamorph of Trichderma reesei) is a one of the most well studied cellulolytic fungus and widely used in thebiotechnology industry, such as in the production of second generation ethanol, because it is a strong producer of hydrolytic enzymes such as cellulasesand xylanases. The objective of this study was evaluate the gene expression and enzymatic activity of cellulases and xylanases in the Dxyr1 and Dcre1mutants and compare with the parental T. reesei (QM9414), in three different carbon sources. The strains were grown in Mandels-Andreotti medium,supplemented with cellulose, sophorose or glucose. The expression of 22 set cellulases and xylanases genes were evaluated by real-time PCR (qRT-PCR)and cellulolytic and xylanolytic activities were observed using different substrates. The cel6a, cel3a, cel7b, cel3c, cel3e, xyn2 and swo genes showed asignificantly high expression in the mutant Dcre1 when compared with the parental QM9414 and low expression of the cel1a, cel3d and cel61b genes wasobserved when compared the mutant Dxyr1 with the QM9414 on cellulose, sophorose and glucose. Overall, all of cellulase and xylanase genes showedhigher expression in mutant Dcre1 and low expression in mutant Dxyr1 in all studied conditions, when compared to QM9414. Concerning to enzymaticprofiles, the activity of CMCase, b-glucosidase and Xylanases ranged also for the presence of specific carbon source. These results suggest that the deletionof the genes xyr1 and cre1 affects the formation of cellulases and xylanases directly at transcriptional level and shown to be specific and dependent of thecarbon source.414. Characterization of tannic acid-inducible and hypoviral-regulated CpsHsp1 expression level of the chestnut blight fungus Cryphonectria parasitica.J.-H. Baek 1 , J.-A. Park 1 , J.-M. Kim 2 , S.-M. Park 1 , D.-H. Kim 1 . 1) Institute for Molecular Biology and Genetics, Center for Fungal Pathogenesis, ChonbukNational University, Jeonju, Chonbuk, South Korea; 2) Department of Bio-Environmental Chemistry, Wonkwang University, Iksan, Chonbuk, South Korea.A small heat shock protein gene, CpsHsp1, a ubiquitous chaperone in Cryphonectria parasitica, was characterized. The predicted protein sequence ofCpsHsp1 gene contains a putative conserved domain, which is alpha crystallin domain (ACD) of alpha-crystallin-Hsps_p23-like superfamily. To characterizebiological functions of the CpsHsp1 gene in the C. parasitica, the replacement vector for CpsHsp1-null mutant was designed to favor double crossoverintegration events. Disruption of the CpsHsp1 protein resulted in retarded growth rate, approximately 78.5% of the radial growth observed in the virusfreestrain EP155/2. When the hypovirus CHV1 was transferred to the CpsHsp1-null mutant, all of the virus-containing CpsHsp1-null progeny displayedcharacteristics of invasive feeding hyphae, near absence of the typical mycelial mat on the surface, and sparse aerial hyphae. Northern blot analysisshowed little accumulation of the CpsHsp1 gene transcript under normal growth conditions. However, the accumulation of the CpsHsp1 gene transcriptwas induced in modified Bavendamm’s medium, which is a 0.7% tannic acid-.supplemented malt extract agar. To examine the viral regulation of theinduction, the CpsHsp1 induction pattern in the isogenic hypovirulent strain UEP1 was compared with that in the wild-type strain EP155/2. Northern blotanalysis of RNA from UEP1 cultured under induction conditions with tannic acid showed that hypoviral infection specifically reduced the level of CpsHsp1transcript induced by tannic acid. To determine whether CpsHsp1 is induced by cool or heat stress, we additionally observed difference in the expression,and induction pattern of CpsHsp1 between virus-free EP155/2 and virus-infected hypovirulent UEP1 strains by Northern blot analysis and Western blotanalysis.222