Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSas another Gb subunit in Neurospora.185. Communication Interference during Cell Fusion in Neurospora crassa is controlled by a Region under Balancing Selection in the HeterokaryonIncompatibility Locus het-c. Jens Heller, Javier Palma-Guerrero, N. Louise Glass. Department of Plant and Microbial Biology, University of California atBerkeley, Berkeley, CA.Vegetative hyphal fusion events are associated with establishment of a fungal colony. However, non-self recognition during fusion events is important toprevent hybrids between genetically dissimilar individuals that might spread mycoviruses, debilitated organelles, and others throughout a fungalpopulation. In filamentous fungi, the ability of two individuals to form a productive heterokaryon via hyphal fusion is controlled by specific loci termed hetloci. Stable heterokaryons will only form if the individuals involved have identical alleles at all het loci. Accordingly, heterokaryotic cells formed betweenstrains that differ in allelic specificity at one or more het loci are rapidly destroyed (programmed cell death) or strongly inhibited in their growth. InNeurospora crassa, three allelic specificity groups were identified for het-c, which is one of the eleven genetically identified het loci in this species. Weobserved that strains with different haplotypes at het-c not only show heterokaryon incompatibility (HI) after cell fusion, but also show reducedchemotrophic interactions and cell fusion between conidial germlings (communication interference). These data indicate that N. crassa germlings candistinguish both self and nonself at a distance, and which presumably involves diffusible ligands. Two regions of the glycine-rich single-pass plasmamembrane protein HET-C were shown to be under balancing selection and both have different functions. By analyzing different chimeras of het-c, wedemonstrate that the HET-C specificity domain (amino acids 194-236; region I), is required for inducing programmed cell death during HI, but does notaffect communication interference during germling fusion. In contrast, the second region of het-c that is also under balancing selection (amino acids 521-599; region II) is responsible for communication interference during germling fusion. To understand the mechanism underlying communicationinterference, we are identifying which amino acids in HET-C region II are responsible for this trait. In addition, we are determining the cellular localizationof HET-C during germling fusion and whether the HET-C region II is a processed form, resulting in a diffusible peptide that is responsible for communicationinterference during chemotropic interactions and cell fusion of conidial germlings.186. The N. crassa Bem46 protein: alternative splicing and eisosomal association. Krisztina Kolláth-Leib, Frank Kempken. Department of Botany,Christian-Albrechts University, Kiel, Germany.The bud emergence (BEM) 46 proteins are evolutionarily conserved members of the a/b-hydrolase super family. The exact function(s) of the proteinremain unknown. Vegetative hyphae, perithecia and ascospores of Neurospora crassa RNAi and over-expressing transformants develop normally, buthyphal germination from ascospores is impaired. These results indicate a role of BEM46 in maintaining cell type-specific polarity in N. crassa. In an attemptto further analyse BEM46 function, alternative splicing was observed in the bem46 RNAi line. We present evidence that alternative splice products impairascospore germination. The BEM46 protein is localized in the perinuclear endoplasmatic reticulum and also forms spots near to the plasma membrane(Mercker et al. 2009). The use of Lifeact-TagRFP (Lichius & Read pers. comm.) and Bem46-eGFP in heterokaryons of N. crassa indicated that the Bem46protein is not interacting with actin. Likewise, the use of the lipid raft-stainer TexasRedTM showed no co-localization with Bem46-eGFP. We analyzed thepotential co-localization of Bem46 with the eisosomal protein LSP1. To that end we cloned the corresponding N. crassa ortholog of lsp1 and fused it toRFP. Indeed we were able to demonstrate a co-localization of LSP1 and BEM46. A yeast two-hybrid approach was undertaken using a previouslyestablished N. crassa two-hybrid library (Seiler pers. comm.). We identified one interacting protein, the anthranylate synthase component II (Walker &DeMoss 1986). Further investigation showed that the BEM46 protein is likely to interact with the F domain of that protein, which is a N-(5’-phosphoribosyl) anthranylate isomerase. The interaction was confirmed in vivo by employing bimolecular fluorescence complementation assays.References:Mercker M, Kollath-Leib K, Allgaier S, Weiland N, Kempken F (2009) Curr Genet 55:151-161Margaret S. Walker & John A. DeMoss (1986) J Biol Chem 261:16073-16077.187. The alternative oxidase induction pathway is involved in senescence associated with over-replication of a mitochondrial plasmid in Neurosporacrassa. Nicolette Dutken, Jonathon Gutzeit, Maze Ndonwi, John Kennell. Biology, Saint Louis University, St Louis, MO.Senescence in Neurospora crassa is caused by dysfunctional mitochondria, which is most often due to the effects of mitochondrial plasmids. Variantforms of the Mauriceville plasmid cause senescence by integrating into the mitochondrial genome or by over-replicating, disrupting essentialmitochondrial genes or their synthesis. Genetic analysis of plasmid-containing strains that escape senescence indicate that two nuclear mutations arerequired for longevity. One of the mutations associated with a long lived (LL) strain involves the regulation of Alternative Oxidase (AOX). AOX is induced bymitochondrial dysfunction and is among several nuclear encoded genes involved in mitochondrial function and/or biogenesis that are upregulated duringsenescence. A model of senescence is proposed in which dysfunctional mitochondria stimulate mitochondrial biogenesis resulting in an accumulation ofdefective mitochondria. Here we show that the LL strain fails to induce AOX due to a mutation in aod-2 that encodes a zinc cluster transcriptional regulatorof the structural alternative oxidase gene, aod-1. Surprisingly, a functional AOX is not required for senescence. This implies that other genes controlled bythe AOX induction pathway play a critical role in mitochondrial function in N. crassa. Homologs of AOD2 in other fungal species have been shown toregulate gluconeogenesis as well as genes involved in mitochondrial function including subunits of the electron transport chain. Mutations in the AOXinduction pathway are not sufficient to overcome plasmid induced senescence and a second nuclear mutation is required. This mutation interferes withthe integrative form of senescence and is hypothesized to be associated with either mitochondrial recombination or the selection of mitochondrialrearrangements. The studies of senescence in N. crassa provide insights into how fungi respond to mitochondrial damage.188. Relationship among mutagen sensitivity, senescence and mitochondrial morphology in the ultraviolet sensitive-5 mutant of Neurospora crassa.Kiminori Kurashima, Michael Chae, Hirokazu Inoue, Shin Hatakeyama, Shuuitsu Tanaka. Laboratory of Genetics, Saitama Univercity, Saitama, Japan.The uvs-5 mutant of Neurospora crassa had been isolated that showed high sensitivity to mutagens (Schroeder, 1970 Mol. Gen. Genet. 107:291-304).This mutant also has phenotypes such as senescent, i.e. shortened life span, and progressive accumulation of mitochondrial DNA deletions (Hausner et al.,1995 Fungal Genet. Newsl. 42A: 59). These phenotypes were quite similar to the mus-10 mutant that we reported previously (Kato et al., 2010 Genetics185:1257-1269). Further, mus-10 and aged uvs-5 strains showed fragmented mitochondrial feature although tubular shape was observed in wild typestrain. Since we found that the uvs-5 mutation had been mapped very closely to fzo1, which encoded homologue of dynamin-like GTPase mitofusin, thesequence of the fzo1 gene in the uvs-5 mutant was determined. A single mutation was found as a deducing amino acid substitution of Gln to Arg in the386 th position locating in the conserved GTPase domain. Forced expression of wild-type FZO1 in the uvs-5 strain suppressed the defect in mitochondrialmorphology and the mutagen sensitivity, but did not in the case of expressing mutated FZO1. Moreover, introduction of this mutation into theendogenous fzo1 gene of the wild-type strain resulted in showing phenotypes of the uvs-5 mutant. Thus, we concluded that the responsible gene of uvs-5166