Program Book - 27th Fungal Genetics Conference

CONCURRENT SESSION ABSTRACTSThe C 2H 2 transcription factor HgrA promotes hyphal growth in the dimorphic pathogen Penicillium marneffei. Hayley E. Bugeja, Michael J. Hynes, AlexAndrianopoulos. Department of Genetics, University of Melbourne, Parkville, VIC, Australia.Penicillium marneffei (recently renamed Talaromyces marneffei) is well placed as a model experimental system for investigating fungal growth processesand their contribution to pathogenicity. An opportunistic pathogen of humans, P. marneffei is a dimorphic fungus that displays multicellular hyphal growthand asexual development (conidiation) in the environment at 25°C and unicellular fission yeast growth in macrophages at 37°C. We have characterised thetranscription factor hgrA (hyphal growth regulator), which contains a C 2H 2 DNA binding domain closely related to that of the stress-response regulatorsMsn2/4 of Saccharomyces cerevisiae. HgrA is not required for controlling yeast growth in response to the host environment, nor does it appear to have akey role in response to stress agents, but is both necessary and sufficient to drive the hyphal growth program. hgrA expression is specific to hyphal growthand its deletion affects multiple aspects of hyphal morphogenesis and the dimorphic transition from yeast cells to hyphae. Loss of HgrA also causes cellwall defects, reduced expression of cell wall biosynthetic enzymes and increased sensitivity to cell wall, oxidative, but not osmotic stress agents. As well ascausing apical hyperbranching during hyphal growth, overexpression of hgrA prevents conidiation and yeast growth, even in the presence of inductivecues. HgrA is a strong inducer of hyphal growth and its activity must be appropriately regulated to allow alternative developmental programs to occur inthis dimorphic pathogen.A conserved splicing factor is required for vesicle transport in Ustilago maydis. Nikola Kellner 1 , Kai Heimel 1,2 , Florian Finkernagel 3 , Theresa Obhof 1 , JoergT. Kaemper 1 . 1) Dept. of Genetics, Karlsruhe Institute of Technology, Karlsruhe, Germany; 2) Dept. of Molecular Microbiology and Genetics, Georg-August-University Göttingen, Göttingen, Germany; 3) Institute for Molecular Biology and Tumor Research, Marburg, Germany.In the corn smut fungus Ustilago maydis, sexual development is initiated by the fusion of two yeast-like haploid sporidia, resulting in a filamentousdikaryon that is capable to infect the plant. Growth as a dikaryon requires an elaborate coordination of the cell cycle, the migration and distribution of thenuclei and polar hyphal growth. We have identified the U. maydis Num1 protein with a pivotal function during these processes. Num1 is homologous toSPF27, a core component of the evolutionary conserved Prp19/CDC5 complex (NTC). The NTC contributes to splicing efficiency and fidelity, but is alsoinvolved in cell cycle checkpoint control, response to DNA damage or formation and export of mRNP-particles. Deletion of num1 in U. maydis has noobvious phenotype in sporidia, however, hyphae exhibit polarity defects; in addition, the num1 mutation affects the cell cycle and cell division. Weidentified Cdc5 and Prp19, two conserved components of the Prp19/CDC5 complex, as Num1 interactors. In line with the function of the NTC, wedemonstrated by means of a genome-wide mRNA-Seq analysis that splicing in num1 deletion strains is impaired on a global level. In addition to the NTCcomponents, several proteins with putative functions during vesicle-mediated transport processes were identified as Num1 interactors; in particular theconventional kinesin 1 motor protein Kin1 was shown to physically interact with Num1. Both num1- and kin1-deletion strains exhibit identical phenotypeswith respect to filamentous and polar apical growth, the morphology of vacuoles, the subcellular distribution of the Dynein motor protein as well as themotility of early endosomes, strongly corroborating a genetic interaction between Num1 and Kin1. Our data implicate a previously unidentified connectionbetween a component of the splicing machinery and cytoplasmic transport processes. As the num1 mutation also affects cytoplasmic mRNA transport, theprotein might constitute a novel functional interconnection between the two disparate processes of splicing and trafficking.N-acetylglucosamine (GlcNAc) Triggers a Morphogenetic Program in Systemic Dimorphic Fungi. Sarah A. Gilmore 1 , Shamoon Naseem 2 , James B.Konopka 2 , Anita Sil 1 . 1) Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA; 2) Department ofMolecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY.Cellular differentiation is an essential process for the development and growth of multicellular eukaryotic organisms. Similarly, many unicellularorganisms undergo a program of cellular differentiation to produce a new cell type specialized for survival in a distinct environmental niche. Systemicdimorphic fungal pathogens, such as Histoplasma capsulatum (Hc) and Blastomyces dermatitidis (Bd), can switch between a unicellular parasitic yeast formadapted for growth within mammals and an infectious soil-growing filamentous form as part of their natural life cycles. Temperature is thought to be thepredominant environmental cue that promotes cellular differentiation of systemic dimorphic fungi; however, work with other fungi indicates thatadditional environmental cues including CO2, light, and nutrient availability can influence how an organism responds to its environment. Recent worksuggests that the ubiquitous monosaccharide N-acetylglucosamine (GlcNAc) can play a role in cell signaling in fungi. We identified GlcNAc as a potentinducer of the yeast-to-filament transition in Hc and Bd. Micromolar concentrations of exogenous GlcNAc were sufficient to induce a robust morphologicaltransition of Hc yeast cells to filamentous cells at room temperature, indicating that dimorphic fungal cells may be sensing GlcNAc, or one of its catabolicbyproducts, to promote filamentation. Using GlcNAc as a tool to induce a robust and more synchronous phase transition of Hc yeast cells to filaments, weexamined the temporal regulation of the Hc transcriptome during morphogenesis to reveal candidate genes involved in establishing the filamentousgrowth program. Two genes we identified during transcriptome analysis included NGT1 and NGT2, which encode GlcNAc major facilitator superfamilytransporters. RNAi depletion of NGT1 or NGT2 rendered Hc cells unable to respond to exogenous GlcNAc. Furthermore, wild type levels of NGT1 and NGT2transcripts were important for efficient Hc yeast-to-filament conversion even in the absence of exogenously added GlcNAc. These data suggest that Ngt1and Ngt2 may monitor endogenous GlcNAc as part of an autoregulatory system that allows Hc to regulate its filamentous growth.A GATA transcription factor encoded by SREB functions as a global regulator of transcription in Blastomyces dermatitidi. Amber Marty, Aimee T.Broman, Christina Kendziorski Gregory M. Gauthier. University of Wisconsin - Madison, 1550 Linden Drive, Microbial Sciences Building, Madison, WI,53706.The thermally dimorphic fungi infect several million people each year including those with normal immune defenses. These fungi grow as mold in the soil(22 o C) where they produce infectious conidia. Upon soil disruption, aerosolized conidia are inhaled into the lungs (37 o C) where they convert into yeast.This reversible, temperature-dependent phase transition defines the lifestyle of the dimorphic fungi. In Blastomyces dermatitidis, we discovered SREB(siderophore biosynthesis repressor in Blastomyces), which encodes a GATA transcription factor that promotes the conversion to mold at 22 o C and alsoregulates iron homeostasis. To begin to dissect how SREB affects the transcriptional response to temperature, we used gene expression microarrays andchromatin immunoprecipitation with quantitative real-time PCR (ChIP-qPCR). For microarrays, RNA was isolated from SREB and wild type (WT) isolates atbaseline (yeast) and 6, 24, and 48-hours after a drop in temperature to 22 o C. LIMMA and EBarrays were used to identify differentially expressed (DE)genes. For ChIP, we engineered SREB to contain an in-frame, C-terminal 3x-HA tag. SREB-3xHA was cross-linked to its DNA binding targets in vivo using 1%formaldehyde at 37 o C and 48-hours at 22 o C. Following chromatin shearing and reversal of cross-links, enrichment for SREB-3xHA binding (vs. isogeniccontrol) at GATA motifs was assessed by qPCR. Gene expression microarray analyses indicated that SREB was a global regulator of transcription at 37 o Cand 22 o C. Gene Ontology enrichment demonstrated SREB was involved with diverse processes including iron ion binding, amino acid transport, metabolic98