Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSDNA is nearly completely assembled, allowing ChIP-seq reads to be mapped unambiguously [4]. Here, we report on our investigations of the NeurosporaCENP-T-W-S-X complex, including its interactions with centromeric DNA and canonical centromeric nucleosomes.[1] Nishino, T. et al. 2012. CENP-T-W-S-X Forms a Unique Centromeric Chromatin Structure with a Histone-like Fold. Cell 148, 487-501.[2] Schleiffer, A. et al. 2012. CENP-T proteins are conserved centromere receptors of the Ndc80 complex. Nat. Cell Biol. 14, 604-613.[3] Smith, K. M. et al. 2012. Centromeres of filamentous fungi. Chrom. Res. 20, 635-656.[4] Smith, K. M. et al. 2011. Heterochromatin is required for normal distribution of Neurospora crassa CenH3. Mol. Cell. Biol. 31, 2528-2542.176. Proper actin ring formation and septum constriction requires coordination of SIN and MOR pathways through the germinal centre kinase MST1.Yvonne Heilig, Anne Dettmann, Stephan Seiler. Institute for Biology II, Molecular Plant Physiology, Freiburg, Germany.The highly conserved nuclear Dbf2p-related (NDR) kinases control polar morphogenesis and cell proliferation. In fungi, NDR kinases function as effectorsof the morphogenesis (MOR) and septation initiation (SIN) networks and are activated by germinal centre (GC) kinases. The Neurospora crassa SIN kinasesSID1 and DBF2 are essential for septum formation. In contrast, the MOR kinases POD6 and COT1 promote apical tip growth and function as negativeregulators of septation. We identified a third GC kinase MST1 that functions as promiscuous enzyme, activating DBF2 and COT1. As typical for SINcomponents, MST1 localized to spindle pole bodies and constricting septa. Moreover, Dmst-1 displayed synthetic interactions with sin, but not mormutants, placing MST1 in parallel to the central SIN kinase cascade CDC7-SID1-DBF2. Consistent with these genetic data, we determined that the two GCkinases MST1 and SID1 are regulated by CDC7 in an opposite manner. Lifeact- and formin-GFP reporter constructs revealed the formation of aberrantcortical actin rings in Dmst-1, which resulted in mispositioned septa and irregular spirals in the mutant. In summary, our data identify an antagonisticrelationship between the SIN and MOR during septum formation that is, at least in parts, coordinated through the GC kinase MST1.177. Regulatation of the BUD3-BUD4 landmark complex by the NDR kinases DBF2 and COT1 during septum formation in Neurospora crassa. YvonneHeilig, Stephan Seiler. Institute for Biology II, Molecular Plant Physiology, Freiburg, Germany.Cytokinesis is essential for cell proliferation, yet the mechanisms for determining the site of cell division are poorly understood. Our data indicate thatthe anillin BUD4 marks septum placement by organizing the RHO4-BUD3-BUD4 GTPase module and that this complex is controlled through two NDRkinase signaling cascades, the septation initiation network (SIN) and the morphogenesis network (MOR). Epistasis analysis of sin and mor mutants placesthe SIN upstream of the MOR. DBF2 functions as competitive inhibitor of COT1 by forming hetero-dimers, thereby replacing the COT1 co-activatorsMOB2A/B. In turn, COT1 functions as negative regulator of septum formation. We demonstrate that COT1, but not DBF2, binds to and phosphorylatesBUD3 and BUD4. Mutational analysis of BUD3 identifies Ser798, located within an amphiphatic helix of BUD3 that is phosphorylated by COT1. Localizationof this amphiphatic helix at septa is only possible in its nonphosphorylated form. In summary, our data suggest a model, in which the MOR kinase COT1phosphorylates BUD3 and BUD4 and that this modification inhibits cortical localization and function of the BUD complex. Interference of the SIN with MORactivity at the septum relieves this inhibition and allows initiation of septation.178. Development of a Protein-Protein Interaction Platform in Neurospora Crassa. Shouqiang Ouyang, Katherine Borkovich. Plantn Pathology andMicrobiology, University of California, Riverside, Riverside, CA.The objective of this study is to generate a protein-protein interaction platform for Neurospora crassa. We have constructed Dmus-51::nat and Dmus-52::nat strains that also carry the Drid::nat mutation to eliminate RIP. These strains are used as recipients for transformation. Ten genes were solicited ascandidates from the N. crassa community, including SAD-1/SAD-2, WC-1/WC-2, FRQ/FRH, OS-4/ RRG-1and GNB-1/GNG-1. We construct vectors for eachprotein by amplifying the ORFs from wild type N. crassa genomic DNA using gene-specific primers. Protein constructs are expressed with a V5-GFP or S-tag-RFP tag from the pan-2 or inl locus, respectively in N. crassa. Protein complexes can be isolated by immunoprecipitation using antibody to the GFP/V5or RFP/S-tag epitope. Both immunoprecipitation and the overlap localization of fluorescent proteins (GFP and RFP) data will streamline our ability tomonitor protein-protein interactions and co-localization in vivo in N. crassa.179. Specific Structural Features of Sterols Affect Cell-Cell Signaling and Fusion in Neurospora crassa. Martin Weichert 1 , Ewald Priegnitz 1 , RaphaelBrandt 1 , Thorben Nawrath 2 , Stefan Schulz 2 , André Fleissner 1 . 1) Institut für Genetik, Technische Universität Braunschweig, Spielmannstrasse 7, 38106Braunschweig, Germany; 2) Institut für Organische Chemie, Technische Universität Braunschweig, Hagenring 30, 38106 Braunschweig, Germany.Sterols are major constituents in the plasma membrane of eukaryotic cells. They modulate the physical properties of the lipid bilayer, e.g. fluidity. Byinteracting with certain lipids and proteins in the plasma membrane, sterols cluster into microdomains which might act as platforms for many biologicalfunctions, such as signal transduction. In the early stages of colony formation in Neurospora crassa, germinating spores direct their growth towards eachother, establish physical contact, and fuse. Cell-to-cell signaling requires the coordinated dynamic recruitment of the MAP kinase MAK-2 and thecytoplasmic protein SO to the tips of interacting cells. Subsequent plasma membrane fusion is facilitated by the transmembrane protein PRM1. Here, wereport that mutants affected in the biosynthesis of ergosterol, the major sterol in most fungal species, show distinct defects during germling fusion.Deletion of erg-2, which encodes an enzyme mediating the last step in the pathway, strongly impairs both directed growth and cell fusion. Interestingly,both MAK-2 and SO mislocalize at the tips of interacting Derg-2 germlings. In contrast, the absence of ERG-10a and ERG-10b, two enzymes with redundantfunction that act upstream of ERG-2, does not affect cell-to-cell communication. However, Derg-10a Derg-10b germling pairs show DPrm1-like deficienciesin plasma membrane merger. By relating the sterol composition and fusion competence of several erg mutants, we find that not the absence of ergosterolbut the accumulation of sterol intermediates specifically impairs distinct steps of germling fusion. While the presence of two double bonds in the sterolside chain provokes Derg-2-like deficiencies, an altered double bond arrangement in the sterol ring system causes DPrm1-like defects. During sexualdevelopment, cell fusion precedes the fertilization of fruiting bodies. Unlike the defects during germling fusion, female and male mating partners of Derg-2and Derg-10a Derg-10b efficiently fuse, suggesting that alterations in the sterol composition specifically impair signaling mechanisms mediating vegetativecell fusion. These data suggest that specific structural features of sterols differentially affect membrane properties and functions, such as the membranerecruitment of proteins, the assembly of signaling complexes, and plasma membrane fusion.180. The role of NADPH oxidases in Neurospora crassa cell fusion. Nallely Cano-Dominguez 1 , Ernestina Casto-Longoria 1 , Jesus Aguirre 2 . 1) Departamentode Microbiologia, CICESE, Ensenada, Baja California, Mexico; 2) Departamento de Biologia Celular y Desarrollo. Instituto de Fisiologia Celular UNAM,Mexico City, D.F. Mexico.Hansberg and Aguirre proposed that reactive oxygen species (ROS) play essential roles in cell differentiation in microorganisms. ROS are generatedmainly during mitochondrial electron transport and by the action of certain enzymes. The NADPH oxidases (NOX) are enzymes that catalyze the production164