Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTS30. Metabolic adaptations in Phytophthora infestans and the role of a phosphagen kinase system in energy metabolism. Meenakshi Kagda, HowardJudelson. Plant Pathology and Microbiology, University of California, Riverside, CA 92521.Nutrient acquisition and metabolic adaptation to host-derived nutrients is an important aspect of pathogen biology. An understanding of the metabolicadaptations made by Phytophthora infestans, an important pathogen of potato and tomato, to optimize nutrient uptake from diverse host tissues andwithin the microenvironments of the host will lead to a better understanding of host-pathogen relationships. In order to study metabolic adaptations of P.infestans, transcriptional profiling and live cell imaging using promoter-fluorescent protein fusions will be used. Preliminary results demonstrated thedifferential gene expression of many metabolic genes of P. infestans grown on different natural hosts and that grown on rich media. The next step involvesanswering the question: Are some metabolic genes expressed in a stage-specific or time-dependent manner? In addition, the role of enzymes involved inenergy homeostasis and metabolite channeling are being studied. The roles of two such genes encoding putative creatine kinases are being elucidatedusing subcellular localization, substrate utilization and loss of function studies.31. Platforms for secondary metabolite analysis in filamentous fungi. Uffe H. Mortensen 1 , Jakob B. Nielsen 1 , Diana C. Anyaogu 1 , Dorte K. Holm 1 , Lene M.Petersen 1 , Morten T. Nielsen 1 , Mikael S. Joergensen 1,2 , Kristian F. Nielsen 1 , Pia F. Johannesen 2 , Dominique A. Skovlund 2 , Thomas O. Larsen 1 . 1) DTU SystemsBiology, Technical University of Denmark, Kgs. Lyngby, Denmark; 2) Department 463, Fungal Gene Technology, Novozymes, Denmark.We are developing versatile methods that allows for rapid and simple genetic manipulation of filamentous fungi. Currently, we use our methods forelucidation of pathways for secondary metabolite production in a number of different species. The platform includes simple systems for gene targetingand defined expression platforms for pathway reconstitution. Alternatively, if few or no genetic tools are available for the fungus, we use AMA1 basedplasmids for transformation. All DNA handling prior to fungal transformation is based on assembly by efficient USER cloning that allows for many DNAfragments to be merged in a single cloning step. Examples of pathway reconstitution will be presented including functional transfer of the entire geodinproducing gene cluster from Aspergillus terreus into A. nidulans. In an attempt to map the first intermediates of polyketide pathways in a fungal species,we have individually expressed all PKS genes from A. niger as a starting point for pathway elucidation. Using this approach we identified a PKS generesponsible for production of 6-MSA. Next, we individually deleted all genes in the corresponding gene cluster in A. niger to further map the pathway.Theses analyses suggest that 6-MSA is a precursor of Yanuthone D/E. In a similar study, we have identified a related PKS gene in A. aculeatus that alsoproduces 6-MSA when expressed in A. nidulans. The corresponding gene cluster in A. aculeatus contains a gene encoding a transcription factor. Using ourAMA1 based expression system, this gene has been overexpressed in A.aculeatus. As a result a new 6-MSA based compound has been identified. Lastly,using a knock-in/knock-out platform in Trichoderma reesei we use the same principles to uncover a gene cluster that is responsible for a very complexfamily of sorbicillinoids.32. Transcriptional analysis of oxalate degradation in the white rot basidiomycete Dichomitus squalens. Miia R. Mäkelä, Johanna Rytioja, Outi-MaariaSietiö, 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.33. Creation of temperature-influenced hyphal growth mutants in a basidiomycete fungus through the use of UV mutagenesis. Stephen J. Horton, CarlyWender, Suhasini Padhi. Dept Biological Sci, Union Col, Schenectady, NY.Filamentous fungi have been used extensively in industry for decades, most prominently for the purposes of protein expression. An emerging technologyis the use of fungi in the production of ecologically-friendly materials used in packaging and insulation, materials presently manufactured using nonrenewablepetroleum-based technologies. The growth characteristics of the mycelia used in these manufacturing processes play a pivotal role in theproperties of the final product. We decided to utilize the classical approach of UV mutagenesis to create new strains of a basidiomycete fungus that wouldpotentially have growth characteristics more suited than the wild type to particular industrial applications. One example of this would be the production offungal strains with a wider temperature spectrum for growth, a factor applicable to the industrial reality of the fluctuating ambient temperature found innon-laboratory conditions. Protoplasts from our wild-type strain were subjected to 70,000 microjoules/cm2 of UV irradiation at a wavelength of 254 nm.Survivors were allowed to recover overnight at 28°C, and then plated at a selective temperature of 40°C. Out of an estimated 6400 protoplasts irradiated,we observed 48 colonies of various sizes after 11 days growth at the selective temperature of 40°C. This temperature was chosen because it approachesthe maximum permissible growth temperature for this fungus. We further characterized 22 of these presumed mutants and were able to sort them intobroad categories based upon their growth rates over the range of 28°C to 37°C. The categories were: (1) strains that grew well at the normal laboratorytemperature (28°C), but less well at elevated temperatures (33.5°C and 37°C), (2) strains that grew best at elevated temperature (33.5°C), and (3) strainsthat grew at relatively similar rates at all three temperatures. The morphology of the hyphae (density, branching pattern) was also found to differ betweenthe growth mutants. Selected mutants will be analyzed by both RNA seq and genomic sequencing approaches in an effort to identify any common genesthat may have been altered as a result of the mutagenesis regime.27th Fungal Genetics Conference | 129