Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSbiochemical distinction is in agreement with the taxonomic grouping based on molecular markers of the species studied. The 3 cell wall types aredistinguishable by their cellulose content and the fine structure of their 1,3-b-glucans. Furthermore, unique features were found in each case. Type I cellwalls (e.g. Phytophthora) are devoid of N-acetylglucosamine (GlcNAc) but contain glucuronic acid and mannose; type II (e.g. Achlya, Dictyuchus,Leptolegnia and Saprolegnia) contain up to 5% GlcNAc and residues indicative of cross-links between cellulose and 1,3-b-glucans; type III (e.g.Aphanomyces) are characterized by the highest GlcNAc content (> 5%) and the occurrence of unusual carbohydrates that consist of 1,6-linked GlcNAcresidues. Analysis of the recently sequenced genome of S. parasitica was combined with quantitative mass spectrometry-based proteomics (label-free andiTRAQ) to characterize the plasma membrane proteome of hyphal cells. This strategy allowed us to experimentally identify a total of 677 plasmamembrane proteins, including several key cell wall polysaccharide synthases, e.g. cellulose, 1,3-b-glucan and chitin synthases, some of which arespecifically enriched in plasma membrane microdomains similar to lipid rafts in animal cells.22. Identification and characterization of the chitin synthase genes in the fish pathogen Saprolegnia parasitica. Elzbieta Rzeszutek, Sara Diaz, VincentBulone. School of Biotechnology, Division of Glycoscience, Royal Institute of Technology (KTH), Stockholm, Sweden.The oomycete Saprolegnia parasitica is a fungus-like microorganism responsible for fish diseases and huge losses in aquaculture. The analysis of the cellwall composition of the microorganism and the characterization of key enzymes involved in cell wall biosynthesis may facilitate the identification of newtarget proteins for disease control. The cell wall of hyphal cells of S. parasitica consists mainly of cellulose, b-(1®3)- and b-(1®6) glucans, whereas chitin ispresent in minute amounts only. The main objective of this work was to test the effect of nikkomycin Z, a competitive inhibitor of chitin synthase (CHS), onthe growth of S. parasitica. Genome mining allowed the identification of six different putative chs genes whose actual occurrence in the genomic DNA ofthe microorganism was confirmed by Southern blot analysis. The expression of the chs genes in the mycelium was analyzed using Real-Time PCR. Theresults revealed a higher expression level of four of the six genes while the two others exhibited undetectable levels of expression in the mycelium. Thissuggests that the latter genes are most likely primarily involved in chitin formation at a different developmental stage. The presence of nikkomycin Zincreased the expression level of one of the genes, chs3, suggesting that the corresponding product is involved in forming the abnormal branchingstructures in the hyphae exposed to the inhibitor. The capacity of the mycelium to synthesize chitin was demonstrated by performing in vitro synthesisreactions using cell-free extracts. CHS activity was measured in intact cell membranes as well as in detergent-extract of membranes. The polysaccharidesynthesized in vitro was characterized by enzymatic hydrolysis with a specific chitinase. Our data demonstrate that CHS represent promising targets ofanti-oomycete drugs, even though the amount of chitin in the cell wall of S. parasitica does not exceed a few percent.23. Role of Ccr4-mediated mRNA turnover in nucleotide/deoxynucleotide homeostasis and Amphotericin B susceptibility in Cryptococcus neoformans.D. Banerjee, J. Panepinto. Department of Microbiology and Immunology.University at Buffalo, SUNY, Buffalo, NY.Ccr4 mediated deadenylation is the first and rate limiting step in eukaryotic mRNA decay. The end products of mRNA degradation are nucleosidemonophosphates (NMPs) which are then converted to nucleotides (NDPs and NTPs) and deoxynucleotides (dNTPs) in downstream reactions. A C.neoformans degradation deficient ccr4D mutant exhibits replication stress sensitivity and stabilizes ribosomal protein (RP) transcripts during carbonstarvation, suggesting that ccr4D mutant is deficient in intracellular nucleotide stores. Analysis of gene expression showed an up-regulation of thenucleotide synthesis machinery in ccr4D mutant even under unstressed conditions consistent with our hypothesis. Time-kill assays in the presence ofmycophenolic acid (MPA), an inhibitor of guanine nucleotide de novo synthesis, showed a reduction in the viability of ccr4D mutant that was rescued bythe addition of exogenous guanine, suggesting that the salvage pathway is indeed functional. These results suggest that the degradation of mRNAtranscripts lead to the production of NMPs that replenish NTP/dNTP pools in C. neoformans during starvation stress. The fungicidal efficacy ofAmphotericin B (AmpB) is enhanced by the use of Flucytosine, a pyrimidine analog, suggesting a synergy between AmpB and nucleotide deficiency forcryptococcosis treatment. We compared the sensitivity of wild type (H99), ccr4D mutant and H99-FOA strain (de novo mutant of pyrimidine synthesis) to acombination of AmpB and NTP/dNTP inhibitors. Both mutants exhibited higher sensitivity to AmpB which was unaltered by additional stressors. H99exhibited an increased sensitivity to the combination of AmpB with both NTP and dNTP inhibitors, compared to AmpB alone. Taken together, these datasuggest that nucleotide depletion, either by a pharmacologic agent or a mutation predisposes the cells to enhanced AmpB mediated cell death.Thus ouroverall hypothesis is that Ccr4 mediated mRNA turnover results in the maintenance of intracellular NTP/dNTP pools to promote growth, virulence, stresstolerance and also modulates Amp B susceptibility in C. neoformans. Results from these studies will identify a novel role of the mRNA degradationmachinery in C. neoformans pathogenesis and stress tolerance and also aid in the identification of new anti-cryptococcal drug targets.24. WITHDRAWN25. Blue light induce Cordyceps militaris fruiting body formation and cordycepin production. Chun-Hsiang Yang 1 , Shun-Kuo Sun 2 , Su-Der Chen 1 . 1)Biotechnology and Animal science, National Ilan University, Ilan, Taiwan; 2) Bionin Biotechnology, INC.Cordyceps militaris is a very important fungal medicine in Chinese. The fruiting body of Cordyceps militaris has been described by many researcherscontaining biological activies, such as being able to inhibit cell proliferation, provide anti-ageing activity, inhibit protein synthesis and lowing cardiovascularrisk. Cordyceps militaris has been grown and harvested by many Chinese people, and were able to obtain its fruiting body with orange collar and barshape. Solid cultivation as carried out 3 to 4 days after mycelium seeding from liquid culture, then fruiting body formation can been induced by light( 12hours per day). In this research, the light-inducing mechanism of fruiting body formation was studied. The results showed the fruiting body was induced byblue light but not red light. Cordycepin, the most important compound with medical potential of Cordyceps militaris, is mainly stored in fruiting body,rather than in mycelium from liquid culture. Cordycepin production depends on various factors, including: wave length of light and culture in solid orliquid. The results also showed the relationship between cordycepin production and blue light sensor in Cordyceps militaris, which might contain LOVdomain.26. Insight into alkaloid diversity of the epichloae, protective symbionts of grasses. Carolyn A. Young 1 , Nikki D. Charlton 1 , Johanna E. Takach 1 , Ginger A.Swoboda 1 , Bradley A. Hall 1 , Kelly D. Craven 2 , Christopher L. Schardl 3 . 1) Forage Improvement Division, The Samuel Roberts Noble Foundation, Ardmore,OK; 2) Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, OK; 3) Plant Pathology, University of Kentucky, Lexington, KY.Cool season grasses from the subfamily Pooideae often form symbiotic associations with fungal endophytes known collectively as the epichloae (Epichloëand Neotyphodium species). The epichloae consist of both sexual (nonhybrid) and asexual (hybrid and nonhybrid) species that can produce the bioactiveanti-herbivore compounds, ergot alkaloids, indole-diterpenes, lolines and peramine. Epichloae can exhibit considerable chemotypic diversity within thepathways of these four alkaloid classes as well as the combination of alkaloids that can be produced by an individual, and as such, may equate to fitnessbenefits for the host. The current genome sequencing efforts, whereby at least 10 epichloae have been sequenced, now allows us to develop simple27th Fungal Genetics Conference | 127