Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTS212. The C 2H 2 transcription factor HgrA promotes hyphal growth in the dimorphic pathogen Penicillium marneffei. Hayley E. Bugeja, Michael J. Hynes,Alex Andrianopoulos. 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.213. Involvement of a specific ubiquitin ligase in the assembly of the dynein motor. Ryan Elsenpeter, Robert Schnittker, Michael Plamann. Sch BiologicalSci, Univ Missouri, Kansas City, Kansas City, MO.Cytoplasmic dynein is a large, microtubule-associated motor complex that facilitates minus-end-directed transport of various cargoes. The dynein heavychain (DHC) is >4000 residues in length, with the last two-thirds of the heavy chain forming the motor head. Six domains within the dynein motor exhibitvarying degrees of homology to the AAA+ superfamily of ATPases. These domains form a ring-like structure from which a microtubule-binding domainprotrudes. Using a genetic assay, we have isolated over 30 DHC mutants of Neurospora that produce full-length proteins that are defective in function. Toexplore the mechanism by which mutations in the C-terminal region of the DHC affect function, we have identified both intragenic and extragenicsuppressors. Interestingly, analysis of the extragenic suppressors revealed that loss of function for a putative E3 ubiquitin ligase restored dynein function ina select set of C-terminal DHC mutants. Our results suggest that these C-terminal DHC mutations block assembly of the dynein motor and loss of activity ofa specific E3 ubiquitin ligase restores dynein assembly.214. Identification and characterization of new alleles required for microtubule-based transport of nuclei, endosomes, and peroxisomes. K. Tan, A. J.Roberts, M. Chonofsky, M. J. Egan, S. L. Reck-Peterson. Dept Cell Biology, Harvard Medical School, Boston, MA.Eukaryotic cells use the microtubule-based molecular motors dynein and kinesin to transport a wide variety of cargos. Cytoplasmic dynein is responsiblefor minus-end-directed microtubule transport (from the cell periphery towards the nucleus), while kinesins-1, -2 and -3 move cytoplasmic cargo in theopposite direction. While much is known about how these motors work in vitro, many questions regarding the mechanism and regulation of microtubulebasedcargo transport in cells remain. To identify novel alleles and genes required for microtubule-based transport, we have performed a genetic screen inthe filamentous fungus, Aspergillus nidulans. We fluorescently-labeled three different organelle populations that are known to be cargo of dynein andkinesin in Aspergillus: nuclei, endosomes, and peroxisomes. After mutagenesis we used a fluorescence microscopy-based screen to identify mutants withdefects in the distribution or motility of these organelles. Here, we report the identification and characterization of new alleles of kinesin, dynein and thedynein regulatory factors, Lis1 and Arp1 (a component of the dynactin complex). In vivo analysis of two new dynein alleles revealed that mutations in twoof dynein’s nucleotide binding sites (termed AAA1 and AAA3), led to the accumulation of endosomes and peroxisomes at the hyphal tip, with more subtledefects on nuclear distribution compared to dynein null alleles. In vitro studies of the AAA3 motor mutation showed dramatic reduction in velocity andprolonged binding to the microtubules in single molecule motility assays.215. Pheromone-induced G2 cell cycle arrest in Ustilago maydis requires inhibitory phosphorylation of Cdk1. Sónia M. Castanheira, José Perez-Martín.Centro Nacional de Biotecnología. CSIC. Darwin 3, Campus de Cantoblanco, 28049 Madrid, Spain.Ustilago maydis is a dimorphic basidiomycete that infects maize. In this fungus virulence and sexual development are intricately interconnected.Induction of pathogenicity program requires that two haploid compatible cells fuse and form an infective filament after pheromone signaling. Thepheromone signal is transmitted by a well-known MAPK cascade. Interestingly, Saccharomyces cerevisiae and Ustilago maydis use a similar MAPK cascadeto respond to sexual pheromone and in both cases a morphogenetic response is provided (shmoo and conjugative hypha, respectively). However, while S.cerevisiae arrests its cell cycle in G1 in response to pheromone, U. maydis does this by arresting at G2. The mechanisms and physiological reasons involvedin the distinct cell cycle response to pheromone in U. maydis are largely unknown. In this communication we will introduce our attempts to characterizethe molecular mechanisms behind pheromone-induced cell cycle arrest in U. maydis .Our results have indicated that inhibitory phosphorylation of Cdk1 ispart of the mechanism of the pheromone-induced G2 cell cycle arrest. This inhibitory phosphorylation depends on the essential kinase Wee1. We analyzedthe transcriptional pattern of cell cycle related genes in response to overactivation of pheromone pathway (using a constitutively activated allele of fuz7,the MAPKK of the cascade) and found that two main G2/M regulators -Hsl1, a kinase involved in downregulation of Wee1 and Clb2, the mitotic cyclinweredownregulated at transcriptional level. Using chimeric promoter fusions we found that transcriptional downregulation was not as important forpheromone-induced cell cycle arrest as expected and we are analyzing other possible regulatory options such as stability or subcellular localization ofthese regulators.216. Microtubule-dependent mRNA transport and mitochondrial protein import in Ustilago maydis. T. Langner 1 , T. Pohlmann 1 , C. Haar 1 , J. Koepke 2 , V.Goehre 1 , M. Feldbruegge 1 . 1) Institute for Microbiology, Heinrich-Heine University, Duesseldorf, Northrhine-Westfalia, Germany; 2) MARA, Philipps-University, Marburg, Hesse, Germany.Transport, subcellular localization, and local translation of mRNAs constitute a very important mechanism to ensure correct targeting of proteins todistinct subcellular domains. Although mRNA transport is well studied in various organisms, its function in regulating specific cellular processes likemitochondrial protein import is still ambiguous. We use the corn pathogen Ustilago maydis as a model system to study microtubule-dependent mRNAtransport during formation of infectious filaments. The key RNA-binding protein Rrm4 is an integral part of this long-distance transport machinery.Combining proteomics, in vivo UV cross-linking, and biochemical approaches, we uncovered that Rrm4 plays a crucial role in active transport of mRNAsencoding mitochondrial proteins. In Rrm4 loss-of-function mutants, mitochondrial proteins are altered in expression and localization, which correlateswith impaired production of reactive oxygen species (ROS). We propose that microtubule-dependent mRNA transport and local translation are crucial forcorrect import of mitochondrial proteins. This work is funded by iGRAD-plant graduate school (German research council, DFG/ GRK1525).27th Fungal Genetics Conference | 173