Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSgrowth. Exploring in vivo responses during infection of susceptible (S) and resistant (R gene) host plants reveals that pathogen penetration andproliferation is hugely restricted in the R plants, but surprisingly, there is no change in the roGFP ratio in planta. Thus the sparse infection hyphae within Rplants maintain a highly reduced cytoplasm at all times. This questions whether production of ROS by the host is the primary mechanism responsible forrestricting pathogen growth in resistant plants.197. Dimorphism and virulence in pathogenic zygomycetes. Soo Chan Lee, Alicia Li, Joseph Heitman. Molec Gen & Microbiol, Duke Med Ctr, Durham, NC.Fungal dimorphism evolved in multiple fungal lineages. Many pathogenic fungi are dimorphic, for example, switching between yeast and filamentousstates. This switch alters host-pathogen interactions and is critical for pathogenicity. However, in pathogenic zygomycetes, whether dimorphismcontributes to pathogenesis is a central unanswered question. The pathogenic zygomycete Mucor circinelloides exhibits multi-budded yeast growth underanaerobic/high CO 2 growth conditions, which Louis Pasteur discovered (Etudes sur la Biere. 1876). Interestingly, we found that in the presence of thecalcineurin inhibitor FK506, Mucor exhibits multi-budded yeast growth. We discovered that Mucor encodes three calcineurin catalytic A subunits (CnaA,CnaB, and CnaC) and one calcineurin regulatory B subunit (CnbR). Disruption of the cnbR gene results in mutants locked in yeast phase growth. Theseresults reveal that the calcineurin pathway governs the dimorphic transition from yeast to hyphae. In virulence tests, we found that the cnbR yeast-lockedmutants are less virulent than wild-type in a heterologous host system, providing evidence that hyphae are a more virulent form of this fungus. Proteinkinase A activity was elevated during yeast growth under anaerobic conditions, in the presence of FK506, or in the yeast-locked cnbR mutants, indicating anovel connection between PKA and calcineurin. The cnaA mutants are hypersensitive to calcineurin inhibitors and display a hyphal polarity defect. Themutants produce spores that are larger than wild-type. Notably, we found spore size is linked to virulence in previous studies (Li et al. PLoS Pathogens.2011). Interestingly, the cnaA mutants were found to be more virulent than wild-type. We also observed that the cnaA mutants germinate earlier insidemacrophages, providing a possible explanation for the greater virulence of the mutants. Another pathogenic zygomycete, Rhizopus delemar has three cnagenes. Phylogenetic analysis revealed that the triplicated cna genes might result from a whole genome and/or segmental gene duplications. Our resultsdemonstrate that the calcineurin pathway orchestrates the dimorphic transition, spore size dimorphism, virulence, and hyphal polarity in Mucor, and thecalcineurin pathway elements have been adapted in zygomycetes via variation in their evolutionary trajectory.198. Genetic analysis of the components of the ime-2 mediated signaling events during nonself recognition and programmed cell death (PCD) inNeurospora crassa. Joanna A. Bueche 1 , Elizabeth A. Hutchison1 1,2 , N. Louise Glass 1 . 1) Plant and Microbial Biology, UC Berkeley, Berekeley, CA, 94720; 2)Cornell University Microbiology Department, Ithaca, NY 14853.Recently, we revealed genetic and functional differences in meiotic initiation machinery between Neurospora crassa and Saccharomyces cerevisiae.While N. crassa is missing some meiotic genes identified in yeast, it has three homologs of the middle meiotic transcriptional regulator, Ndt80. None of theNDT80 homologs are required for meiosis in N. crassa. One of the NDT80 homologs, vib-1 is essential for heterokaryon incompatibility (HI) in N. crassa, anonself recognition mechanism in filamentous fungi. Mutations in vib-1 suppress cell death caused by HI as well as secretion of the extracellular proteasesduring the nitrogen starvation. Furthermore, deletion of a IME2 (a kinase involved in initiation of meiosis in S. cerevisiae) homolog in N. crassa, ime-2, doesnot affect sexual development, results in a significant elevation of secreted proteases in response to nitrogen starvation. Morever, a Dvib-1 Dime-2 mutantrestored wild-type levels of cell death during the HI and normal production of extracellular proteases; a deletion of ime-2 suppressed these vib-1phenotypes. Based on the evidence, we hypothesize that IME-2 negatively regulates a cell death pathway that functions in parallel to the VIB-1 HI pathwayand a protease secretion pathway positively regulated by VIB-1. We used a slightly modified yeast consensus sequence for Ime2 phosphorylation to scan(Scansite) the entire N. crassa genome for possible targets and obtained a list of 30 candidates including VIB-1. All targets were assessed for secretion ofthe extracellular proteases in absence of nitrogen. Strains containing deletions of 13 of the 30 genes identified in the screen were significantly affected inprotease secretion. Mutations in these candidate genes will be tested for the ability to alleviate cell death and Heterokaryon Inocpatibilty (HI) in thepresence and absence of ime-2 and vib-1 hence assessing their role in the parallel HI/PCD pathway redundant with VIB-1.199. PRO45 is a component of the conserved STRIPAK complex in Sordaria macrospora. Steffen Nordzieke 1 , Benjamin Fränzel 2 , Sandra Bloemendal 1 , DirkWolters 2 , Ines Teichert 1 , Ulrich Kück 1 . 1) General and Molecular Botany, Ruhr-University Bochum; 2) Analytical Chemistry, Ruhr-University Bochum,Universitätsstr. 150, 44801 Bochum, Germany.The complex formation of three-dimensional fruiting bodies in Sordaria macrospora is mediated by an interaction between developmental proteins andconserved signaling cascades and thus an excellent experimental system for developmental biology.We recently have characterized a STRIPAK complex in Sordaria macrospora that is involved in the regulation of fruiting body development. This complexcontains striatin (PRO11), a striatin-interacting protein (PRO22), the scaffolding subunit of protein phosphatase 2A (SmPP2AA) and a phocein homologue(SmMOB3) [1, 2].Here we describe PRO45, a novel subunit of the STRIPAK complex in filamentous fungi which is a homolog of the human sarcolemmal membraneassociated protein (SLMAP). We also present the functional characterization of PRO45: Strains lacking the gene for PRO45 show sterility together with asevere defect in hyphal fusion and vegetative growth rate. The primary structure of PRO45 contains a forkhead-associated (FHA) as well as atransmembrane domain. Complementation studies showed that a lack of the FHA domain is responsible for the described defects, whereas a missingtransmembrane domain does not affect development.Tandem affinity purification (TAP) followed by mass spectrometry and coimmunoprecipitation (Co-IP) showed subunits of the STRIPAK-complex asinteraction partners, confirming the homology of human and fungal STRIPAK. Further microscopic studies provide evidence for a localization of PRO45 inthe ER as well as in the nuclear envelope. Integrating these observations, we propose that PRO45 has a function in the physical and signaling connection ofSTRIPAK-complex and nucleus.[1] Pöggeler S, Kück U 2004. Eukaryot. Cell 3: 232-240[2] Bloemendal S, Lord KM, Rech C, Hoff B, Engh I, Read ND, Kück U. 2010. Eukaryot. Cell 9: 1856-1866[3] Bloemendal S, Bernhards Y, Bartho K, Dettmann A, Voigt O, Teichert I, Seiler S, Wolters DA, Pöggeler S, Kück U. 2012. Mol. Microbiol. 84: 310-323.200. Molecular Determinants of Sporulation in Ashbya gossypii. Jurgen W. Wendland, Lisa Wasserstroem, Klaus Lengeler, Andrea Walther. YeastGenetics, Carlsberg Laboratory, Copenhagen V, Kopenhagen V, Denmark.Previously we have analysed the pheromone response MAPK signal transduction cascade in A. gossypii. The major findings were (i) deletion of bothpheromone receptor genes STE2 and STE3 did not inhibit sporulation whereas (ii) deletion of the transcription factor STE12 resulted in hypersporulation(Wendland et al. 2011). Here we present our analysis of key A. gossypii homologs of Saccharomyces cerevisiae sporulation specific genes. We show that27th Fungal Genetics Conference | 169