Program Book - 27th Fungal Genetics Conference

CONCURRENT SESSION ABSTRACTSCo-expression analysis of Phanerochaete carnosa during growth on hardwood and softwood species to predict proteins with unknown functionrelevant to biomass conversion. Hitoshi Suzuki 1 , Chi Yip Ho 2 , Kin Chan 2 , Philip Wong 1 , Yunchen Gong 1 , Elisabeth Tillier 1 , Emma Master 1 . 1) University ofToronto, Toronto, Ontario, Canada; 2) Mount Sinai Hospital, Toronto, Ontario, Canada.Softwood is the predominant form of land plant biomass in the Northern hemisphere and is among the most recalcitrant biomass resource to bioprocesstechnologies. The white rot fungus Phanerochaete carnosa has been isolated almost exclusively from softwoods, while most known white-rot species,including the model fungus Phanerochaete chrysosporium, were mainly isolated from hardwoods. Growth studies of P. carnosa and P. chrysosporium onsapwood and heartwood from deciduous and coniferous species revealed comparable growth of P.carnosa on all wood samples, while P. chrysosporiumgrew poorly on heartwood from conifers. A contributing factor to growth on extractive-rich heartwood samples could be the comparatively high numberof P450 monooxygenases encoded by P. carnosa. Notably, genome sequencing revealed that P. carnosa possesses one of the largest P450 contingents(239 P450s) among the sequenced and annotated wood-rotting basidiomycetes. However, like most sequencing efforts, a significant fraction of the P.carnosa genome comprises genes that encode proteins with unknown function. Moreover, transcripts from several of these genes were identified inmycelia collected at a single time point from P. carnosa cultivations growing on woody biomass. Accordingly, the aim of the current study was to analyzeco-expression patterns of known and unknown genes to identify those with unknown function that might be most relevant to biomass conversion. Ourapproach was to separately cultivate P. carnosa on ball-milled trembling aspen (Populus tremuloides) and ball-milled white spruce (Picea glauca) and tocollect mycelia at five time points over a one-month cultivation period. RNA collected from all cultures at each time point was sequenced separately usingthe Illumina HiSeq platform. Co-expression patterns will be described and used to predict new gene products that are particularly interesting to target fordetailed biochemical characterization.Functional Analysis of the Pleurotus ostreatus Manganese-Peroxidase Gene Family. Tomer Salame, Doriv Knop, Dana Levinson, Oded Yarden, YitzhakHadar. Microbiology and Plat Pathology, Hebrew Unversity, Rehovot, Israel.Mn amendment to P. ostreatus cultures enhances degradation of recalcitrant aromatic compounds. Manganese peroxidase (MnP) isoenzymes are keyplayers in these processes. The MnP gene family is comprised of five Mn -dependent peroxidases (mnp3, 6, 7, 8 and 9) and four versatile-peroxidases(mnp1, 2, 4 and 5; VPs). In liquid medium, Mn amendment resulted in a drastic up-regulation of the predominantly expressed mnp3 and mnp9, and downregulationof mnp4. To obtain direct evidence for the role of these enzymes, we produced genetically-modified (knockout, knockdown and/or overexpression)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.Carbon source and light dependent regulation of gene clusters in Trichoderma reesei (Hypocrea jecorina). Doris Tisch 2 , Monika Schmoll 1 . 1) Health andEnvironment, Bioresources, Austrian Institute of Technology AIT, Tulln, Austria; 2) Vienna University of Technology, Institute of Chemical Engineering,Vienna, Austria.Trichoderma reesei (anamorph of Hypocrea jecorina) is one of the most prolific producers of plant cell wall degrading enzymes. Regulation of the genesencoding these enzymes occurs in response to the nutrient sources available in the environment and many of them are responsive to light as well.Cellulose as the natural substrate induces the most complete enzyme set, while induction of cellulases also occurs on sophorose and lactose. In contrast,no cellulases are induced on glycerol and the respective genes are repressed on glucose. We therefore investigated the transcriptome on these five carbonsources in light and darkness and aimed to identify genes specifically expressed under cellulase inducing conditions. These conditions are characterized bya significant enrichment of genes involved in C-compound and carbohydrate degradation and transport among the upregulated gene set. Genes downregulatedunder inducing conditions show a significant enrichment in amino acid metabolism and energy metabolism. We were further interested whetherlight dependent regulation is clustered in the genome and if the carbon source is relevant for activation of light dependent clusters. We found that lightdependent clustering predominantly occurs upon growth on cellulose, with the most significant regulation in a gene cluster comprising env1. This clusterappears on glucose as well, but is not down regulated in mutants of blr1 or blr2. Also cbh2, the arabinofuranosidase gene abf2 and the histoneacetyltransferase gene gcn5 are part of light dependent clusters. Hierarchical clustering of gene expression patterns was performed to reveal functionaldivergence of gene regulation with respect to light response or carbon specific regulation. Glycoside hydrolase genes follow the whole transcriptomepattern with carbon source being superior to light in terms of regulation. ENV1 in in part the G-protein beta subunit GNB1 were found to be crucial forcarbon source specific regulation of G-protein coupled receptors, genes involved in secretion, sulphur metabolism and oxidative processes as well astransporters. We conclude that clustered regulation of light responsive genes preferentially occurs upon growth cellulose and that ENV1 and to a lesserextent GNB1 play a role in carbon source dependent regulation of specific gene groups in light.62