Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSdiploids are more virulent than the parental haploids. Our results suggest that conidial populations of A. flavus are predominantly homokaryotic but asmall percentage of conidia are heterokaryotic. Within a heterokaryon, a diploid nucleus could be formed by fusion of two haploid nuclei, which may allowthe generation of a pathogenic strain.70. Inhibition of appressorium formation of Magnaporthe oryzae by roxithromycin and its possible molecular target. Akira Ishii, Mayu Kumasaka,Megumi Narukawa, Takashi Kamakura. Applied Biological Science, Tokyo Univ. of Science, Noda, Chiba, Japan.Roxithromycin (RXM), a 14-membered macrolide which was originally active against prokaryote, has beneficial side effects such as anti-inflammatoryactivities were reported and actually applied to human. However, the mechanisms underlying these side effects are still unclear. In this study, we foundthat RXM inhibited appressorium formation of rice blast fungus Magnaporthe oryzae (M. oryzae). These results suggest that there are alternative targetsin broad eukaryotic organisms and it is interesting to identify the molecular target of the secondary effect on human using M. oryzae. Magnaporthe oryzaeis the causal agent of rice-blast disease. M. oryzae enters its host plant using a specialized infection structure known as an appressorium. Thedevelopmental stage of appressorium is sensitive to various chemical inhibitors, because large numbers of genes are involved in cellular differentiation.Since appressorium formation by M. oryzae can be observed on artificial surfaces, it can be a useful tool to search new activity of various chemicals. Weperformed phage display to search novel molecular target(s) of the antibiotic. We found that one candidate gene 32-11 may play important roles inappressorium formation. Expression of 32-11 gene, in a 32-11 mutant, was lower than wild type during developing infection structure, and the mutant wasless affected by RXM. Although germinate and formation of appressoria were normal. Over expression of 32-11 gene caused no effect to RXM sensitivity,germination nor appressorium formation compared with the wild type. Over expression of 32-11 caused no effect to RXM activity, germination orappressorium formation compare to the wild type. To investigate whether lower expression of 32-11 causes the less sensitivity to RXM, we introduced 32-11 over expression vector into 32-11 reduced mutant. These mutants restored their wild type phenotype. These results possibly suggest that the complexof 32-11 product and RXM affects another molecule which plays an important role in appressorium formation at M. oryzae.71. Identification of novel genes involved in induction of appressorium development triggered by plant-derived signals in Colletotrichum orbiculare.Sayo Kodama 1 , Ayumu Sakaguchi 2 , Yasuyuki Kubo 1 . 1) Laboratory of Plant Pathology, Graduate School of Life and Environmental Science, Kyoto PrefecturalUniversity, Kyoto, Japan; 2) National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan.Many plant pathogenic fungi initiate infection of host leaves by the germination of conidia and differentiation of appressoria at the tip of germ tubes.These morphological changes are triggered by various external signals such as physical or chemical signals from the plant surface. In our previous study,cucumber anthracnose fungus Colletotrichum orbiculare CoKEL2, a Schizosaccharomyces pombe tea1 homologue, encoding a kelch repeat protein wasidentified. The cokel2 mutants formed abnormal appressoria on glass slides, and those appressoria were defective in penetration hyphae developmentinto cellulose membranes, an artificial model substrate for fungal infection. In contrast, the cokel2 mutants formed normal appressoria on the hostcucumber plant and retained penetration ability. Moreover, when conidia were incubated in the presence of exudates from cucumber cotyledon, normalappressorium formation on the artificial substrate by the cokel2 mutants was restored. These results suggest that CoKEL2 is essential for normalmorphogenesis of appressoria and that there is a bypass pathway that transduces plant-derived signals for appressorium formation independent ofCoKEL2. These plant-derived signaling pathways for appressorium formation have not been characterized in fungal pathogens including C. orbiculare. Todetermine specific components of the plant-derived signaling pathway that leads to appressorium formation, we screened six cokel2 double mutants thatformed abnormal appressoria not only on artificial substrates but also on the host plant surface. Furthermore, reintroduction of CoKEL2 into those cokel2double mutants restored normal appressorium formation on artificial substrates, suggesting that cokel2 double mutants have defects in CoKEL2-independent and plant-derived specific signaling pathway for appressorium formation. We identified and characterized candidate mutated genes by wholegenome sequencing of the six cokel2 double mutants. To define the involvement of those candidate mutated genes in appressorium formation, weobserved the phenotypes of candidate geneD single mutants, cokel2D candidate geneD double mutants, and complementation strains. As expected,candidate geneD mutants in cokel2D back ground showed same phenotypes as those of screened cokel2 double mutants.72. Unique protein domains regulate Aspergillus fumigatus RasA localization and signaling during invasive growth. Rachel V. Lovingood 1 , Praveen R.Juvvadi 2 , William J. Steinbach 2 , Jarrod R. Fortwendel 1 . 1) Microbiology and Immunology, University of South Alabama, Mobile AL, USA; 2) PediatricInfectious Diseases, Duke University, Durham NC, USA.Invasive pulmonary aspergillosis (IPA) is propagated by inhalation of A. fumigatus spores that germinate and invade the lung tissue in search of nutrients.We have shown that the A. fumigatus RasA GTPase protein is necessary for hyphal morphogenesis, cell wall integrity, and virulence during IPA. Ourprevious studies focused on conserved protein domains regulating RasA localization and signaling. These studies revealed the requirement for plasmamembrane (PM)-localized Ras for proper signaling and regulation of A. fumigatus growth and virulence. Therefore, mechanisms controlling Ras localizationare of interest in designing novel antifungal Ras inhibitors. Although Ras pathways may represent valid antifungal targets, the importance of fungal-specificRas protein domains to Ras function in fungal pathogenesis remains unexplored. To address this important knowledge gap, we identified fungal-specificRas protein domains by comparing fungal Ras sequences to their human counterpart, H-ras. We hypothesized that such domains could serve as targetableareas to selectively inhibit the fungal Ras protein. This analysis revealed two areas of significant divergence with H-ras: i) the Invariant Arginine Domain(IRD), a novel domain conserved in the RasA homologs of every available fungal genome but not present in H-ras and ii) an extended hypervariable region(HVR). Truncation analysis of the HVR identified a serine-rich region that is necessary for localization to the PM and for RasA signaling during hyphalmorphogenesis. Interestingly, mutational analysis of the IRD produced a properly localized yet non-functional RasA protein. However, activation of the IRDRasA mutant was not altered suggesting a role for the IRD during interactions of RasA with downstream effectors. Further characterization of the IRD andHVR, and the protein interactions to which they contribute, will reveal fungal-specific aspects of Ras function and may define a new paradigm for Rassignal transduction in fungal organisms.73. Light regulates growth, stress resistance and metabolism in the fungal pathogen Aspergillus fumigatus. Kevin K. Fuller, Carol S. Ringleberg, Jennifer J.Loros, Jay C. Dunlap. Genetics, Geisel School of Medicine at Darmouth, Hanover, NH.Light serves as an important environmental cue that influences developmental and metabolic pathways in a variety of fungi. Interestingly, orthologs of aconserved blue light receptor, WC-1, promote virulence in two divergently related pathogenic species, Cryptococcus neoformans and Fusarium oxysporum,suggesting that photosensory systems may be conservatively linked to fungal pathogenesis. Aspergillus fumigatus is the predominant mold pathogen ofimmunocompromised patients, but if and how the organism responds to light has not been described. In this report, we demonstrate that the fungus canindeed sense and distinctly respond to both blue and red portions of the visible spectrum. Included in the A. fumigatus photoresponse is a reduction inconidial germination kinetics, increased hyphal pigmentation, enhanced resistance to acute ultra-violet and oxidative stresses, and an increased27th Fungal Genetics Conference | 139