Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSallele isolated two NPC proteins, which were named SONA and SONB for Suppressors Of NimA1. Although NIMA is essential for mitotic entry there is alsoevidence that NIMA and conserved related kinases have functions later in mitosis and in the DNA damage response. To further characterize the roles ofNIMA we designed a genetic screen to isolate additional suppressors of nimA1 that also cause conditional temperature-dependent DNA damagesensitivity. Our expectation was the identification of additional genes involved in NIMA regulation and in the DNA damage response. Here we describe onesuch gene, which we have named sonC. SonC contains a unique Zn(II)Cys6 binuclear DNA binding domain, which is highly conserved among theAscomycota. Deletion of sonC results in swollen, ungerminated spores, suggesting it is essential for a core growth process. As expected for a DNA bindingprotein, SonC localizes to nuclei during interphase. Interestingly, dual fluorescence imaging of SonC with histone H1 during mitosis revealed that a portionof SonC localizes with histone H1 along a distinct projection of chromatin that juts away from the main, condensed chromatin mass, which we hypothesizemay be the NOR. Supporting this hypothesis, the region of DNA that likely forms the projection is cradled by the nucleolus prior to mitosis as seen bycolocalization studies of SonC with the nucleolar protein Bop1. As mitosis proceeds, the H1 histones are evicted from the middle region of this projectionbut not at its distal end. This indicates that the chromatin in this region of the genome is altered during mitotic progression and we are testing the ideathat SonC might be important for NOR condensation and/or nucleolar disassembly during its mitotic segregation. Because SonC was identified as asuppressor of NIMA we propose that NIMA may have a function in regulating nucleolar disassembly during mitosis.116. Investigating Cell Cycle-Regulated Control of Appressorium Morphogenesis in the Rice Blast Fungus Magnaporthe oryzae. Wasin Sakulkoo, NicholasJ. Talbot. School of Biosciences, University of Exeter, Exeter EX4 4QD, United Kingdom.The rice blast fungus Magnaporthe oryzae elaborates specialized infection structures called appressoria to gain entry into rice plant tissue. The initiationof appresssorium morphogenesis has previously been shown to require a single round of mitosis in the germ tube, shortly after spore germination. Ondaughter nucleus migrates to the incipient appressorium at the germ tube tip and the other daughter nucleus moves back to the conidial cell from whichthe germ tube originates. We reasoned that an S-phase checkpoint mediates the apical-isotropic switch leading to swelling of the germ tube tip.Perturbation of DNA synthesis by hydroxyurea (HU) blocks the initiation of appressorium formation, but only when applied within 3-4h of sporegermination, prior to S-phase. Here, we report investigations regarding the interplay between cell cycle control and operation of the Pmk1 Mitogenactivatedprotein kinase cascade, which is essential for appressorium morphogenesis in M. oryzae. Furthermore we report changes in the global pattern ofgene expression of HU-treated conidia which has been carried out in order to determine the identity of morphogenetic genes that are controlled by the S-phase checkpoint. Progress on understanding the genetic control of early appressorium development will be presented.117. THE ROLE AND TRAFFIC OF CHITIN SYNTHASES IN Neurospora crassa. R. Fajardo 1 , R. Roberson 2 , B. Jöhnk 3 , Ö. Bayram 3 , G.H. Braus 3 , M. Riquelme 1 . 1)Department of Microbiology, CICESE, Ensenada, Mexico; 2) School of Life Sciences, Arizona State University, Arizona, USA; 3) Molecular Microbiology andGenetics, Georg-August University, Göttingen, Germany.Chitin is one of the most important carbohydrates in the cell wall in filamentous fungi. Chitin synthases (CHS) are involved in the addition of N-acetylglucosamine monomers to form chitin microfibrils. The filamentous fungus Neurospora crassa has one representative for each of the seven CHSclasses described. Previous studies have shown that in N. crassa, CHS-1, CHS-3 and CHS-6, are concentrated at the core of the Spitzenkörper and informing septa and seem to be transported in different populations of chitosomes. In this study we have endogenously tagged chs-2, chs-4, chs-5 and chs-7with gfp to study their distribution in living hyphae of N. crassa. CHS-5 and CHS-7 both have a myosin motor-like domain at their amino termini, suggestingthat they interact with the actin cytoskeleton. CHS-2 and CHS-7, appeared solely involved in septum formation. As the septum ring developed, CHS-2-GFPmoved centripetally until it localized exclusively around the septal pore. CHS-4 and CHS-5 were localized both at nascent septa and in the core of the Spk.We observed a partial colocalization of CHS-1-mCherry and CHS-5-GFP in the Spk. Total internal reflection fluorescence microscopy (TIRFM) analysisrevealed putative chitosomes containing CHS-5-GFP moving along wavy tracks, presumably actin cables. Collectively our results suggest that there aredifferent populations of chitosomes, each containing a class of CHS. Mutants with single gene deletions of chs-1, chs-3, chs-5, chs-6, or chs-7 grew slightlyslower than the control strain (FGSC#9718 and FGSC#988); only chs-6D displayed a marked reduction in growth. Both chs-5D and chs-7D strains producedless aerial hyphae and conidia. The double mutant chs-5D; chs-7D showed less growth, aerial hyphae production and conidiation than the single mutantchs-5D, but not than the chs-7D single mutant. A synergic effect was observed in double mutant chs-1D; chs-3D, in which growth, aerial hyphae productionand conidiation were significantly decreased. During the sexual cycle, after homozygous crosses, chs-3D and chs-7D strains did not produce perithecia andchs-5D produced less perithecia. We are analyzing chitin and glucan synthase activities in these single and double mutants. Additionally, we are conductingpulldown assays, and mass spectrometry to identify putative proteins that are interacting with CHS.118. DFG5 and DCW1 cross-link Cell Wall Proteins into the Cell Wall Matrix. Abhiram Maddi, Jie Ao, Stephen J. Free. Dept Biological Sci, SUNY Univ,Buffalo, Buffalo, NY.The cell wall is an essential organelle for the growth and survival of a fungus. The cell wall structure consists of matrix of cross-linked chitin, glucans, andcell wall glycoproteins. In Neurospora crassa, we have shown that the DFG5 and DCW1 proteins function in cross-linking the cell wall proteins into the cellwall matrix. We have also shown that the Candida albicans DFG5 and DCW1 proteins are required for the cross-linking of cell wall proteins into the cellwall. The DFG5 and DCW1 proteins are predicted to have a-1,6-mannanase activity. Our results suggest that they function in transglycosylation reactionsbetween a-1,6-mannans, which are found in galactomannan and the outer chain mannan structures present as modifications on cell wall proteins, and cellwall glucans. These galactomannans and outer chain mannans are modifications to the N-linked oligosaccharides attached to cell wall glycoproteins. As aresult of these transglycosylation reactions, the cell wall proteins are effectively cross-linked into the cell wall. The DFG5 and DCW1 enzymes are excellenttargets for the development of anti-fungal agents that could disrupt cell wall biosynthesis.119. Cell wall biology to illuminate mechanisms of pathogenicity in Phytophthora infestans. Laura Grenville-Briggs, Stefan Klinter, Francisco Vilaplana,Annie Inman, Hugo Mélida, Osei Ampomah, Vincent Bulone. Division of Glycoscience, Royal Institute of Technology, (KTH), Stockholm, Sweden.The cell wall is a dynamic extracellular compartment protecting the cell, providing rigidity, and playing an essential role in the uptake of molecules andsignalling. In pathogenic organisms, the cell wall is at the forefront of disease, providing contact between the pathogen and host. Using a multidisciplinaryapproach, we seek to understand the role of the cell wall in oomycete disease, both as a communication centre with the host organism and as acompartment that is continually reshaped and strengthened throughout the lifecycle, to penetrate and colonise the host. Understanding thesemechanisms in more detail will pave the way for better control of oomycete diseases. We are combining novel chemical genomics approaches with stateof-the-artbiochemistry and biophysics to study the cell wall and to develop new anti-oomycete drugs. P. infestans produces a variety of spores andinfection structures that are essential for disease development throughout its lifecycle. In particular thick-walled sporangia release wall-less motilezoospores that rapidly synthesise a cell wall upon contact with host plant cells. These cysts further differentiates to produce appressoria which build up150