Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSis fzo1. Attempting to disruption the fzo1 gene was failed, so the fzo1 gene were suggested to be essential for viability as showing in almost all eukaryotesexcept for yeast. Therefore the mutation of uvs-5 may be a useful model for studying the relation between mitochondrial fusion and early senescence inhigher eukaryotes.189. Localization of EGL-1 and EGL-2, two GPI anchored cell wall b (1-3) endoglucanases, at hyphal apices and septa, and in interconidial septa inNeurospora crassa. Leonora Martinez, Meritxell Riquelme. Microbiology Department, Center for Scientific Research and Higher Education at Ensenada,Baja California.The unitary model of cell wall growth suggests that the polarized extension of hyphae in filamentous fungi results from the coordinated synthesis anddischarge of new cell wall polymers, the action of hydrolytic enzymes that provide plasticity to the wall and turgor pressure to drive cell expansion.Currently, there is limited information on enzymes capable of hydrolyzing cell wall polymers and that could be contributing to cell wall remodeling. EGL-1and EGL-2 are putative b (1-3) endoglucanases in Neurospora crassa, with potential binding sites for a glucosyl phosphatidylinositol group (GPI), whichwould allow them to get anchored into the plasma membrane. To investigate whether these proteins participate in key morphogenetic events during thedevelopment of N. crassa, EGL-1 and EGL-2 were labeled with the green fluorescent protein (GFP). For egl-1, the gfp gene was inserted within the egl-1encoding sequence, just after the signal peptide sequence. For egl-2, the insertion took place right before the GPI-binding site. Both endoglucanases werelocalized in the hyphal apical plasma membrane and in septa, however, EGL-2-GFP was strongly and more definite localized at the apical dome and EGL-1-GFP showed less intensity with increasing fluorescence from the subapex to the tip. EGL-1-GFP was mostly found at hyphal septa and interconidial septaand EGL-2-GFP was faintly present in a few old septa. Our results suggest that lytic activity of enzymes, such as the endoglucanases EGL-1 and EGL-2 in N.crassa, is present in critical areas during vegetative morphogenesis, where these enzymes probably play a role in cell wall remodeling, as postulated by theunitary model of cell wall growth.190. Stability of a G protein alpha subunit in genetic backgrounds lacking the G beta subunit or a cytosolic guanine nucleotide exchange factor.Alexander V. Michkov, Katherine A. Borkovich. Plant Pathology and Microbiology, University of California, Riverside, Riverside, CA.Heterotrimeric G proteins consist of alpha, beta and gamma subunits. Regulation is accomplished through the alternation between binding of GDP(inactive form) and GTP (active form) by the alpha subunit and dissociation of the alpha subunit and beta-gamma dimer. GDP/GTP exchange is facilitatedby both cell surface G protein coupled receptors and cytosolic guanine nucleotide exchange factors (GEFs), such as RIC8. Neurospora crassa has three Galpha subunits (GNA-1, GNA-2 and GNA-3), one G beta (GNB-1), and one G gamma (GNG-1). Interestingly, mutants lacking gnb-1 or the cytosolic GEF ric8exhibit some defects in common with the gna-1 deletion mutant, which may be explained by the reduced GNA-1 protein levels observed in these mutants.Previous studies in our laboratory showed that levels of gna-1 mRNA are similar in wild type and mutants lacking gnb-1 or ric8, consistent with a posttranscriptionalmechanism. Using genetic and biochemical approaches, this study investigated the mechanism underlying regulation of GNA-1 stability inregards to GTP/GDP bound state and amount of protein (normal or overexpressed). The results demonstrate that levels of GNA-1 protein are not visiblyreduced over 36 hours in a wild-type background after halting translation using cycloheximide, suggesting GNA-1 is very stable in wild type. To checkstability of GDP or GTP bound GNA-1 in different backgrounds, we transformed mutants lacking the gna-1 gene and gnb-1 or ric8 with a wild type (gna-1 WT ) or constitutively active, GTPase-deficient gna-1 allele (gna-1 Q204L ). Overexpressing gna-1 WT (GDP bound) in a wild-type background increased the levelof GNA-1 protein ~ 3 fold, while overexpression in a gnb-1 mutant gave a nominal increase (~ 1.6x). Overexpressing gna-1 Q204L (GTP bound) in the Dgnb-1or Dric8 backgrounds led to ~ 2 fold higher levels of GNA-1 compared to wild type. In summary, GNA-1 is very stable in wild type, but stability decreasesdramatically in gnb-1 and ric8 deletion mutants. The GTP-bound G alpha protein is more stable in a gnb-1 mutant background than GDP-bound GNA-1protein.191. Functional analysis the Saccharomyces cerevisiae Ste20, Cla4 homologue in Neurospora crassa. Yuhei Nogami, Makoto Fujimura, Akihiko Ichiishi.Faculty of Life Sciences, Toyo University, ORA-GUN, GUNMA, Japan.Signal transduction pathways are important for a variety of features of fungal development. Small GTPases of Rho family act as molecular switchesregulating cell signalling, cytoskeletal organization and vesicle trafficking in eukaryotic cells. The Rho family GTPase Cdc42 was first identified in the yeastSaccharomyces cerevisiae, where it is essential for initiation of bud formation and the subsequent switch from apical to isotropic growth. The activation ofCdc42 is catalyzed by Cdc24 guanine nucleotide exchange factors (GEFs), which convert Cdc42 from an inactive GDP-bound form to the active GTP boundform. Bem1 functions as a scaffold connecting Cdc42 with its activator Cdc24. The GTP-bound Cdc42 can activate p21-activated kinase (PAK), Ste20 andCla4. Neurospora crassa has two PAK family kinases Cla4 and Ste20 homologs. We have few knowledge of the function of their PAK kinases in N. crassa. Inthis study, we performed functional analysis of stk-4 (Ste20 homolog) and vel (Cla4 homolog) in N. crassa. The stk-4 deletion mutant showed slow growththan wild type strain, and vel deletion mutant showed more severe growth defects. To determine the subcellular localization and dynamics of STK-4protein, we constructed GFP-STK-4 fusion constructs. The gfp encoding sequence was fused to the 3’ end of the stk-4 open reading frame. We alsoconstructed the GFP-BEM-1 fusion protein. These constructs were introduced into his-3 locus, and observed using confocal fluorescence microscopy (LSM-510). Both fusion proteins were accumulated at growing hyphal tips and septa. From there results, we consider that STK-4 and BEM-1 are function at thesame site.192. Dissecting the Pathway of Cellulase Secretion in Neurospora crassa. Trevor Starr, Timo Schuerg, Louise Glass. Plant and Microbial Biology, UCBerkeley, Berkeley, CA.Due to their capacity to secrete large amounts of proteins, particularly hydrolytic enzymes, filamentous fungi are of great interest for high-level proteinproduction in various industries, such as the textile, pharmaceutical, and biofuels industries. Although the basic components of the eukaryotic secretionpathway characterized in yeast and higher organisms are also conserved in filamentous fungi, the highly polarized and compartmentalized growth mode offilamentous fungal hyphae mandates pathways of secretion that are specific to these fungi. While certain aspects of filamentous fungal secretion areunder active study, a basic characterization of the entire pathway from start to finish remains to be performed. Such a characterization may provideinsights into how filamentous fungi are able to secrete large amounts of enzymes and how these fungi can be engineered to produce even more enzymesin the future. This is particularly of interest to the process of biofuels production, in which the inexpensive production of large amounts of cellulases is amajor bottleneck to the efficient and cost-effective production of cellulosic biofuels. In nature the model fungus Neurospora crassa secretes a host ofcellulases to allow it to grow on burnt vegetation. The tractability of N. crassa makes it an excellent model to characterize protein secretion in filamentousfungi, particularly the secretion of industrially relevant cellulases. To achieve this goal we are characterizing the cellulase secretion pathway in N. crassa byfollowing the trafficking of fluorescently tagged Endoglucanase 2 (EG-2), a major secreted endocellulase. To determine the compartments through whichcellulases traffic we are co-localizing EG-2-GFP with fluorescently-tagged markers of the ER, Golgi, endosomes, and the Spitzenkorper and are assaying the27th Fungal Genetics Conference | 167