Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSfusion. We hypothesize that starvation might be a driving force for horizontal chromosome transfer in order to increase the chance of survival.Reference:[1] Ma, L.-J. et al; Nature 464, 367-373 (2010) [2] Manners, JM & He,C; Mycol Progress 10:383-388 (2011) [3] Ishikawa, FH et al; PLoS ONE7(2): e31175. doi:10.1371/journal.pone.0031175 (2012).149. Requirements for horizontal chromosome transfer in the plant pathogenic fungus Fusarium oxysporum. Ido Vlaardingerbroek, Martijn Rep. FNWI,University of Amsterdam, Amsterdam, Netherlands.Strains within the Fusarium oxysporum species complex are clonal and diverse. A number of them are pathogenic to plants but rarely can they infectmore than one host. Host specificity is determined by the presence of a set of secreted effector genes. These genes typically reside on Lineage Specific (LS)chromosomes that can be transferred between strains, even if they are vegetatively incompatible. These extra chromosomes typically carry nohousekeeping genes and have many more transposable elements then the non-LS or core chromosomes. If a strain receives one of these chromosomes itcan acquire the ability to infect a new host, compatible with the effector genes the chromosome harbours. Our main interests at this moment are (1)determining which chromosomes are amenable for transfer, and (2) which cellular processes are involved in transfer. To determine which chromosomescan be transferred, we created a bank of random insertional mutants carrying an antibiotic resistance marker. These have been tested for chromosometransfer. A few of these showed consistent transfer of the chromosome tagged with the marker. By screening a large number of transformants we shouldcover the entire genome. In addition to the screen we tagged an LS chromosome that we know can be transferred, as well as the smallest of the corechromosomes, with GFP. In this way we can directly compare transfer capability of these chromosomes. We hypothesize that the LS chromosomes’ uniquemake-up is required for transfer. We will also test these strains for stability of the tagged chromosome by screening spores for the loss of GFP expressionusing FACS. In this way we can test whether transferrable (LS) chromosomes differ in stability from core chromosomes under varying conditions. Toidentify cellular processes involved in chromosome transfer, we are making deletion mutants for genes required for cellular processes we suspect might beinvolved in chromosome transfer. These will be tested for transfer efficiency compared to the wild-type strains. We are currently investigating hyphalfusion, heterochromatin formation and programmed cell death. By combining the results from these two research lines we should be able to discoverwhich chromosomes can be transferred as well as the chromosomal features and processes involved.150. Characterization of the endocytotic proteins Yel1-Arf3-Gts1 in Ashbya gossypii and the role of Gts1 in endocytosis, actin localization andfilamentous growth. Therése Oskarsson, Klaus Lengeler, Jürgen Wendland. Carlsberg Laboratory, Copenhagen, Denmark.Endocytic vesicle formation and regulation thereof is performed by a complex protein machinery, coordinating every detail of the endocytic process frominitiation and pit formation to vesicle scission and uncoating.We have used the filamentous fungi Ashbya gossypii to study three proteins that are involved in uncoating of vesicles in clathrin-mediated endocytosis.We deleted the corresponding genes encoding the GTP-binding protein Arf3 and its regulators; the Guanine nucleotide Exchange Factor Yel1 and theArfGAP protein Gts1, using PCR-based gene targeting methods. We then characterized these mutant strains under various conditions.While no deletion-specific phenotypes could be observed in the Darf3 and Dyel1, the Dgts1 strain shows several severe mutant phenotypes. Deletion ofGTS1 results in a strong growth defect and renders mycelia with severe endocytotic deficiencies indicated by distinctly reduced endocytic rates, and largeimmobile vacuoles. Other phenotypic observations in A. gossypii Dgts1 strains indicate that Gts1 may have additional functions other than regulating theactivity of Arf3. We have observed effects of Gts1 on temperature stress resistance, actin localization and polar- as well as filamentous growth.The importance of GTS1 for polarized hyphal growth leads us to studying the GTS1 homolog of the human fungal pathogen Candia albicans in an effortto elucidate its role for the yeast-to-hyphal transition in this dimorphic fungi.151. A Late Embryogenesis Abundant (LEA) protein in Neosartorya fischeri confers protection against desiccation. Martin Richard van Leeuwen, Timon TWyatt, Tineke M van Doorn, Jan Dijksterhuis. Applied and Industrial Mycology, CBS-KNAW Fungal Biodiversity Centre, Utrecht, Netherlands.Late Embryogenesis Abundant (LEA) proteins were first characterized in cotton and wheat and are synthesized in abundance during the late maturationstage of seed development. As the seed matures, water content decreases greatly inducing severe desiccation stress. Expression of LEA proteins is linkedto the acquisition of desiccation tolerance. Using BLAST to search for LEA like proteins in various filamentous fungal genomes (Aspergillus niger, Aspergillusflavus, Emericella nidulans, Penicillium chrysogenum, Talaromyces stipitatus and Neosartorya fischeri) resulted in orthologs in each mentioned species,indicating the wide spread appearance of LEA proteins in fungi. Ascospores produced by N. fischeri are able to survive long periods under various stressors.However, deletion of the LEA gene resulted in diminished tolerance against desiccation and high temperatures. In addition, heterologous expression ofLEA in Escherichia coli conferred increased tolerance against osmotic- and salt stress. Interestingly, LEA was able to function as protectant for enzymes thatnormally lose activity under influence of stress. Lactate dehydrogenase (LDH) was inactivated by heat stress and freeze-thaw cycles. In the presence ofLEA, LDH activity was maintained. Our results show that LEA are wide spread in filamentous fungi and function in tolerance against stressors like heat,freeze-thaw and desiccation. LEA could play an important role in stress tolerance of survival propagules like ascospores and conidia.152. Coordination of polarized secretion by the exocyst complex is critical for filamentous growth and cytokinesis in Ustilago maydis. Michaela Wehr 1 ,Kay Oliver Schink 2 , Michael Bölker 1 . 1) Philipps University, FB Biologie, AG Boelker Marburg, Hessen, Germany; 2) Department of Biochemistry, Institute forCancer Research The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway.To establish and sustain their polarity, cells have to transport proteins and membrane lipids to defined locations at the growing tip. This is achieved bydirectional transport of vesicles that fuse with the plasma membrane. Vesicle fusion and active exocytosis requires the presence of an octameric proteincomplex, the exocyst. In S. cerevisiae, two proteins of the exocyst complex, Sec3 and Exo70, were shown to serve as landmark proteins for exocytosis. Theother components of the exocyst tether secretory vesicles carrying the Rab GTPase Sec4 to the membrane. Fusion of secretory vesicles occurs viainteraction of the exocyst with SNARE proteins. To elucidate the function and regulation of the exocyst complex and its associated proteins in Ustilagomaydis, we have characterized the Rab GTPase Sec4 and the exocyst proteins Sec3, Exo70 and Sec15 by genetic, cell biological and biochemicalapproaches. We found that of the two landmark proteins, only one is important for polar growth in U. maydis. Interestingly, this gene is not essential,suggesting that in U. maydis exocytosis sites can be also marked by alternative mechanisms. Another essential player for polar growth in U. maydis is theexocyst subunit Sec15, which mediates the interaction of the exocyst with incoming secretory vesicles. Conditional mutants of sec15 are defective inhyphal tip growth and are affected in long-distance transport of secretory vesicles. In contrast to S. cerevisiae where Sec4 vesicles are transported alongthe actin cytoskeleton, long distance transport of vesicles depends in U. maydis on the microtubule cytoskeleton. Furthermore, we studied mutants ofdifferent motor proteins to get insights into the molecular mechanisms of secretory vesicle trafficking.153. Localization of Neurospora crassa Cell Fusion Proteins. Ci Fu, Stephen J. Free. Biological Sciences, University at Buffalo, Buffalo, NY.158