Program Book - 27th Fungal Genetics Conference

CONCURRENT SESSION ABSTRACTSRegulating the Aspergillus nidulans global nitrogen transcription factor AreA. Richard B. Todd. Department of Plant Pathology, Kansas State University,Manhattan, KS.Nitrogen nutrient utilization genes are regulated in Aspergillus nidulans by the GATA DNA-binding transcription activator AreA. The transcriptionalactivity of AreA is highly regulated by multiple mechanisms including autogenous transcriptional regulation, differential areA transcript stability,interaction of AreA with the corepressor NmrA, and repression by the negative-acting GATA factor AreB. In addition, AreA shows regulated nuclearaccumulation. AreA accumulates in the nucleus specifically during nitrogen starvation, and is rapidly exported to the cytoplasm upon addition of nitrogennutrients to nitrogen-starved cells. I will focus on recent developments in our understanding of AreA nuclear import and nuclear export, the key controlpoints of regulated AreA nuclear accumulation. We have shown that the six conserved nuclear localization signals (NLSs) in AreA show redundancy andcollaborate to mediate nuclear import. In contrast, a single CrmA exportin-dependent nuclear export signal (NES) in AreA is required for nuclear export.We have shown that fusion of the AreA NES to a constitutively nuclear protein confers nucleocytoplasmic localization and a loss of function phenotype.We have exploited this phenotype to select mutants defective in the AreA-CrmA interaction.Transcriptional analysis of oxalate degradation in the white rot basidiomycete Dichomitus squalens. Miia R. Mäkelä, Johanna Rytioja, Outi-Maaria Sietiö,Sari Timonen, Annele Hatakka, Kristiina Hildén. Department of Food and Environmental Sciences, University of Helsinki, Helsinki, Finland.Basidiomycetous white rot fungi are the most efficient degraders of lignocellulose with a unique ability to mineralize the recalcitrant lignin polymer.Lignocellulose decay involves a complex enzymatic system, but is also suggested to be promoted by the fungal secretion of oxalic acid. White rot fungisynthesize oxalate as a metabolic waste compound and typically secrete it to their environment in millimolar quantities. As oxalate is a toxic compound,regulation of its intra- and extracellular concentration is extremely crucial for fungi and also for lignocellulose degradation since high oxalate levels areshown to inhibit the decomposition reactions. Therefore, specific oxalate-converting enzymes, namely oxalate decarboxylases (ODCs) that work inconjunction with formate-degrading formate dehydrogenases (FDHs), are recognized as key fungal enzymes in lignocellulose decay. Dichomitus squalens isa white rot fungus that degrades effectively all the wood polymers, i.e. cellulose, hemicelluloses and lignin, and secretes oxalic acid during its growth onwood. The genome of D. squalens harbours 5 putative ODC and 3 putative FDH encoding genes, while these numbers differ in other fungi based oncomparative genomics. In order to enlighten the roles of the multiple oxalic-acid catabolising enzymes of D. squalens, the expression of the odc and fdhgenes was followed with quantitative real-time RT-PCR when the fungus was grown on its natural substrate, i.e. Norway spruce (Picea abies) wood. Inaddition, the effect of organic acid (oxalic acid) and inorganic acid (HCl) supplementation on the relative transcript levels of the oxalate-catabolizing geneswas examined in the submerged liquid cultures of D. squalens. The results show for the first time the sequential action of ODC and FDH at the transcriptlevel in a white rot fungal species. The constitutive expression of odc1 suggests the pivotal role of the corresponding enzyme during the growth of D.squalens on wood. In addition, the strong upregulation of the transcription of odc2 in oxalic-acid amended cultures indicates the distinct roles of individualODC isoenzymes.TOR-mediated control of virulence functions in the trans-kingdom pathogen Fusarium oxysporum. Gesabel Y. Navarro Velasco, Antonio Di Pietro.Departamento de Genética, Universidad de Córdoba, 14071 Córdoba, Spain.Infectious growth of fungal pathogens is controlled by environmental cues, including nutrient status. The soilborne fungus Fusarium oxysporum producesvascular wilt disease in more than a hundred different crop species and can cause lethal systemic infections in immunodepressed humans. Previous workshowed that the preferred nitrogen source ammonium causes repression of infection-related processes in F. oxysporum that could be reversed byrapamycin, a specific inhibitor of the conserved protein kinase TOR. Here we generated mutations in upstream components that should result inconstitutive activation of TOR, including null mutants in tuberous sclerosis complex 2 (TSC2), a small GTPase that represses TOR activity, as well as strainsexpressing a dominant activating allele of the small GTPase Rag (ragA Q86L ), an activator of TOR. The Dtsc2 mutants and, to a minor extent, the ragA Q86Lstrains showed defects in hyphal growth and colony morphology on several amino acids, as well as decreased efficiency in cellophane penetration andvegetative hyphal fusion. These phenotypes were exacerbated in Dtsc2ragA Q86L double mutants and could be reversed by rapamycin, suggesting that theyare caused by hyperactivation of TOR. The mutants caused significantly lower mortality on tomato plants and on larvae of the animal model host Galleriamellonella. These results suggest that TOR functions as a negative regulator of fungal virulence on plant and animal hosts.Transcriptional regulation of peptidases and nitrogen transporters during the assimilation of organic nitrogen by the ectomycorrhizal fungi Paxillusinvolutus. Firoz Shah 1 , Francois Rineau 2 , Tomas Johansson 1 , Anders Tunlid 1 . 1) Microbial Ecology Group, Department of Biology, Lund University, SE-22362,Lund, Sweden; 2) Centre for Environmental Sciences, Hasselt University, Building D, Agoralaan, 3590 Diepenbeek, Limburg, Belgium.Proteins and amino acids form a major part of the organic nitrogen (N) sources in soils. Though a poorly characterized process, this N is mobilized andbecomes available to plants due to the activity of ectomycorrhizal (ECM) fungi. We have examined the role of ectomycorrhizal extracellular peptidases andamino acid transporters in the degradation, uptake and transfer of various protein sources (BSA, Gliadin and pollen) as well as of plant litter material usingthe ECM model fungus Paxillus involutus. During N-deprived conditions, all substrates induced secretion of peptidase activities. The activity had acidic pHoptimum (2.3-3.0), and it was mainly due to aspartic peptidases and with minor contribution of metallo and serine peptidases. The activity was partly andtemporarily repressed by low concentrations of ammonium (1mg/L). Transcriptional analysis showed that P. involutus expressed a large array of proteinsand enzymes involved in the assimilation of organic N including peptidases, N-transporters and enzymes of the N-metabolism. Extensive in-silico analysisrevealed the presence of genes encoding 312 peptidases, 129 N transporters and 284 enzymes involved in amino acid metabolism. Out of these, 89peptidases and 37 N-transporters and 109 amino acid metabolism enzymes encoding genes were significantly upregulated during organic N assimilation.The genes were encoding a variety of secreted (23) and non-secreted (20) peptidases which were differentially expressed depending on the medium withthe highest expression of the aspartic and metallo peptidases. Apart from the YAAH/ATO family, upregulated genes were found in all the other families oftransporters for amino acids, oligopeptides, ammonium, urea and allantoate/allantoin. The results shows that the expression levels of peptidases andtransporters in P. involutus are coordinately regulated during the assimilation of organic N sources.27th Fungal Genetics Conference | 43