Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSthen quantified by determining parameters such as germination time and growth speed from the movies. The experiments show that the germinationrate, growth speed and growth density of P. rubens on gypsum increase with q while aw is constant, and increase with a w while q is constant. We concludefrom this that q and aw have separate effects on growth on porous substrates. An explanation for the effect of q could be that it limits a fungus’s access toboth water and nutrients. Follow up research will focus on modeling and explaining these effects.162. Localization of Ga proteins during germination in the filamentous fungus, Neurospora crassa. Ilva Esther Cabrera 1 , Carla Eaton 2 , Jacqueline Servin 1 ,Katherine Borkovich 1 . 1) Plant Pathology and Microbiology, University of California, Riverside, Riverside, CA; 2) Institute of Molecular BioSciences, MasseyUniversity, Palmerston North, New Zealand.Heterotrimeric G protein signaling is essential for normal hyphal growth in the filamentous fungus Neurospora crassa. We have previously demonstratedthat the non-receptor guanine nucleotide exchange factor RIC8 acts upstream of the Ga proteins GNA-1 and GNA-3 to regulate hyphal extension.Germination assays revealed essential roles for RIC8 and GNA-3 during this crucial developmental process. Localization of the three Ga proteins duringconidial germination was probed through analysis of cells expressing fluorescently tagged proteins. Functional TagRFP fusions of each of the three Gasubunits were constructed through insertion of TagRFP in a conserved loop region of the Ga subunits. The results demonstrated that GNA-1 localizes tothe plasma membrane and vacuoles, and also to septa throughout conidial germination. GNA-2 localizes to both the plasma membrane and vacuolesduring early germination, but is then found in vacuoles later during hyphal outgrowth. Interestingly, in addition to y plasma membrane and vacuolarlocalization, GNA-3 was found in distinct patches on the plasma membrane of the original conidium during early germination. This distinct localization ofGNA-3 supports the hypothesis that GNA-3 is needed for proper conidial germination, and this specific localization may be required for development.Further investigation is under way to determine the consequence of this localization. Colocalization of RIC8-GFP with GNA-1-TagRFP or GNA-3-TagRFP wasnot detected in cells expressing two fluorescent proteins. This finding suggests that their interaction may be transient not able to be captured via thismethod. A more sensitive microscopic approach is being implemented to better test for colocalization.163. Deciphering the roles of the secretory pathway key regulators YPT-1 and SEC-4 in the filamentous fungus Neurospora crassa. E. Sanchez, M.Riquelme. Center for Scientific Research and Higher Education of Ensenada (CICESE). Carretera Ensenada-Tijuana No. 3918, Zona Playitas, C.P. 28860,Ensenada-B.C.-Mexico.The transport of proteins through different compartments of the secretory pathway is mediated by vesicles. It is well known that vesicular trafficking isregulated by Rab GTPases, which in their active state interact with the membrane of the vesicles. Subsequently, through protein-protein interactions, theycoordinately associate with factors involved in transport and/or tethering to the receptor organelle. In contrast to other eukaryotic model systems, mostfilamentous fungi contain a Spitzenkörper (Spk), which is a multi-vesicular complex found at the hyphal apex to which cargo-carrying vesicles arrive beforebeing redirected to specific cell sites. The exact regulatory mechanisms utilized by the hyphae to ensure the directionality of the secretory vesicles thatreach the Spk are still unknown. Hence, we have analyzed the N. crassa Rab-GTPases YPT-1 and SEC-4, key regulators of the secretory pathway rather wellcharacterized in S. cerevisiae. YPT-1 regulates ER-Golgi and late endosome-Golgi traffic steps, while SEC-4 regulates post-Golgi vesicle traffic en route tothe plasma membrane. Laser scanning confocal microscopy of strains expressing fluorescently tagged versions of the proteins revealed that YPT-1 localizesat the Spk microvesicular core and at cytoplasmic pleomorphic punctate structures, suggesting its participation in different traffic steps. YPT-1accumulation at the Spk might suggest its function in mediating the traffic of vesicles from early endosomes as a recycling process. The pleomorphicstructures could correspond to late Golgi equivalents. The localization of SEC-4 at the Spk, suggests the participation of this Rab in late traffic steps ofGolgi-derived vesicles previous to exocytic events. The relative distribution of both Rabs compared to the molecular motor MYO-2 (presumably involved insecretory vesicle transport), the long coiled-coil protein USO-1 (tethering factor), the secreted protein INV-1, and proteins involved in cell wall biosynthesisis being analyzed and will provide better clues on the nature of the identified compartments.164. Functional characterization of CBM18 proteins, an expanded family of chitin binding genes in the Batrachochytrium dendrobatidis genome. PengLiu, Jason Stajich. Plant Pathology & Microbiology, Univ California, Riverside, Riverside, CA.Batrachochytrium dendrobatidis (Bd) is the causative agent of chytridiomycosis, one of the major causes of worldwide decline in amphibian populations.Little is known about the molecular mechanisms of its pathogenicity. Our previous work 1 from the initial analysis of the Bd genome revealed a uniqueexpansion 18 copies of the carbohydrate-binding module family 18 (CBM18), specific to Bd, and evolving under positive directional selection. CBM18 ispredicted to be a sub-class of chitin recognition domains. Our hypothesis is that some of these copies of CBM18 can bind chitin, a major component offungal cell walls, in vitro. In order to investigate CBM18’s intracellular localization, four CBM18 genes, representing tyrosinase-like, deacetylase-like andlectin-like groups, were cloned into a yeast GFP expression vector. Only two genes from lectin-like group fused with GFP, showing cell boundarylocalization. Furthermore, intracellular signals were observed on both GFP fusion proteins. According to the TargetP database, both proteins are predictedto have the secretion signal peptide. When co-stained with FM4-64, a dye to label vacuole membranes, the FM4-64 and GFP signals were mutuallyexclusive, indicating that the GFP fusion proteins were not destined for degradation. Expression of the proteins from the pHIL-S1 vector in the Pichiasystem will enable purification and characterization of binding properties of these molecules and affinity for chitin and other substrates. 1. Abramyan andStajich, mBio 2012; 3(3): e00150-12.165. The exocyst complex is necessary for secretion of effector proteins during plant infection by Magnaporthe oryzae. Yogesh K. Gupta 1 , MarthaGiraldo 2 , Yasin Dagdas 1 , Barbara Valent 2 , Nicholas J. Talbot 1 . 1) School of Biosciences, University of Exeter, EX4 4QD, UK; 2) Department of Plant Pathology,Kansas State University, Manhattan, Kansas, USA.Magnaporthe oryzae is a devastating plant pathogenic fungus, which causes blast disease in a broad range of cereals and grasses. A specialized infectionstructure called the appressorium breaches the leaf cuticle and subsequently the fungus colonizes host epidermal cells. Colonization of host tissue isfacilitated by small secreted proteins called effectors, that suppress plant immunity responses and may also mediate invasive growth. Some of theseeffectors have been shown to localize at the appressorium pore prior to plant infection, at the tips of primary invasive hyphae and in a specialized plantderived,membrane-rich structure called the Biotrophic Interfacial Complex (BIC). However the underlying mechanism controlling polarized secretion is notwell defined in M. oryzae. The exocyst is an octameric protein complex (composed of Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84) that appearsto be evolutionary conserved in fungi and to play a crucial role in vesicle tethering to the plasma-membrane. The exocyst plays an important role inpolarized exocytosis and interacts with various signaling pathways at the apex of fungal cells. We are currently characterizing components of exocystcomplex during infection related development of M. oryzae. We have shown that the exocyst localizes to hyphal tips as in other fungi during hyphalgrowth in culture. Interestingly, exocyst components also localize around the appressorium pore, which suggests the pore is an active site for secretion atthe point of plant infection. We have recently shown that organization of the appressorium pore requires a hetero polymeric septin network and we show27th Fungal Genetics Conference | 161