Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTS108. The role of hydrophobins in sexual development of Botrytis cinerea. Razak Bin Terhem 1 , Matthias Hahn 2 , Jan van Kan 1 . 1) Laboratory ofPhytopathology, Wageningen University, Wageningen, The Netherlands; 2) Department of Biology, University of Kaiserslautern, Kaiserslautern, Germany.Hydrophobins are small secreted proteins that play a role in a broad range of developmental processes in filamentous fungi, e.g. in the formation ofaerial structures. Hydrophobins allow fungi to escape their aqueous environment and confer hydrophobicity to fungal surfaces. In Botrytis cinerea(teleomorph Botryotinia fuckeliana), one class I and two class II hydrophobin genes have been identified, as well as a number of hydrophobin-like genes.Previous studies showed that hydrophobins are not required for conferring surface hydrophobicity to conidia and aerial hyphae. We investigated the roleof hydrophobins in sclerotium and apothecium development. RNA seq analysis of gene expression during different stages of apothecium developmentrevealed high expression of the Bhp1 (class I hydrophobin) gene and of the Bhl1 (hydrophobin-like) gene in certain stages, whereas Bhp2 and Bhp3 (class IIhydrophobin) genes were always expressed at very low levels. We characterized different hydrophobin mutants: four single gene knockouts, three doubleknockouts as well as a triple knockout. Sclerotia of DBhp1/DBhp3 (double knock out) and DBhp1/DBhp2/DBhp3 (triple knock out) mutants showed easilywettable phenotypes. These results indicate that hydrophobins Bhp1 and Bhp3 are important for normal development of sclerotia of B. cinerea. Foranalyzing apothecial development, a reciprocal crossing scheme was set up. Morphological aberrations were observed in crosses with some hydrophobinmutants. When the DBhp1/DBhp2 (double knock out) and DBhp1/DBhp2/DBhp3 (triple knockout) mutants bearing a MAT1-1 mating type were used asmaternal parents (sclerotia), and fertilized with microconidia of a wild type MAT1-2 strain, the resulting apothecia were swollen, dark brown in color andhad a blotted surface. Instead of growing upwards, the apothecia in some cases fell down. This aberrant apothecial development was not observed in thereciprocal cross, when the same mutants were used as paternal parent (microconidia). These results indicate that the presence of hydrophobins Bhp1 andBhp2 in maternal tissue is important for normal development of apothecia of B. cinerea.109. The pescadillo homolog, controlled by Tor, coordinates proliferation and growth and response in Candida albicans yeast. Tahmeena Chowdhury 1 ,Niketa Jani 1 , Folkert J. Van Werven 2 , Robert J. Bastidas 3 , Joseph Heitman 3 , Julia R. Köhler 1 . 1) Division of Infectious Diseases, Boston Children'sHospital/Harvard Medical School, Boston, MA; 2) Institute for Integrative Cancer Research, MIT, Cambridge, MA; 3) Dept. of Genetics and MolecularBiology, Duke University, NC.Candida albicans has evolved as a colonizer and opportunistic pathogen of mammals. Among fungi infecting humans, it is unique in the frequency withwhich it switches between growth as budding yeast and growth as pseudohyphal and hyphal filaments. In vitro and presumably in vivo, filamentsconstitutively produce yeast from their sub-apical compartments. This developmental step is required for dispersal of planktonic yeast from biofilms. TheC. albicans pescadillo homolog PES1 is required for this lateral yeast growth. In eukaryotes, pescadillo homologs are involved in cell cycle progression andribosome biogenesis, processes that respond to nutrient availability. This work investigated the potential role of C. albicans PES1 in the Tor signalingpathway, which is a major nutrient signaling cascade. Results show that Tor signaling controls Pes1 expression and localization. C. albicans yeast but nothyphae require Pes1 for proliferation, and for proliferation arrest upon Tor1 inhibition with rapamycin. Pes1 inactivation via a temperature-sensitive alleleleads to defective exit of starved cells from the cell cycle. Pes1 inactivation also leads to rapid loss of phosphorylation of ribosomal protein S6, a marker oftranslational activity, as does Tor1 inhibition and genetic perturbation of Tor1 activation. These data support a role for Pes1 downstream of Tor1 incoordinating cell cycle progression with protein synthesis. As all cells must coordinate proliferation and growth, investigating why the requirement forPes1 in this role is yeast-specific will inform our understanding of morphogenesis and Tor signaling in C. albicans.110. Uncovering the mechanisms of thermal adaptation in Candida albicans. Michelle Leach 1,2 , Susan Budge 2 , Louise Walker 2 , Carol Munro 2 , AlistairBrown 2 , Leah Cowen 1 . 1) Department of Molecular Genetics, University of Toronto, Medical Sciences Building, 1 Kings College Circle, Toronto, Ontario,Canada, M5S 1A8; 2) Aberdeen Fungal Group, School of Medical Sciences, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen,AB25 2ZD, UK.The heat shock response is governed by one of the most highly conserved networks in eukaryotic cells. Upon sensing a sudden temperature upshift, theheat shock transcription factor (Hsf1) is rapidly phosphorylated and activated, leading to the induction of numerous genes that mediate thermaladaptation, including heat shock genes that encode molecular chaperones. We have shown that the major fungal pathogen of humans, Candida albicans,has retained a bona fide heat shock response even though it is obligatorily associated with warm-blooded animals [Molec. Micro. 74, 844]. Furthermore,this thermal adaptation is essential for the virulence of C. albicans [Fungal Gen. Biol. 48, 297]. To identify signalling pathways that contribute to long-termthermal adaptation resistance in C. albicans we performed unbiased genetic screens for protein kinase mutants that display temperature sensitivity. Thisscreen reproducibly highlighted several key signalling pathways associated with cell wall remodelling: the Hog1, Mkc1 and Cek1 pathways. None of thesepathways are essential for Hsf1 phosphorylation and activation; each pathway contributing to heat shock adaptation independently of Hsf1. Wedemonstrate that these pathways are differentially activated during heat shock, and that there is crosstalk between these pathways, with hightemperatures contributing to increased resistance to cell wall stress in the long term, and oxidative stress in the short term. Critically, this crosstalkbetween thermotolerance and other types of stress adaptation is mediated by the molecular chaperone Hsp90, whose down-regulation reduces theresistance of C. albicans to proteotoxic stresses. Hsp90 depletion also affects cell wall biogenesis by impairing activation of these signalling pathways.Furthermore, we show that Hsp90 interacts with and down-regulates Hsf1 thereby modulating short-term thermal adaptation. Therefore, Hsp90 lies at theheart of heat shock adaptation, modulating the short-term Hsf1-mediated activation of the classic heat shock response, coordinating this response withlong term thermal adaptation via Mkc1- Hog1- and Cek1-mediated cell wall remodelling.111. Characterisation of contact-dependant tip re-orientation in Candida albicans hyphae. Darren Thomson, Silvia Wehmeier, Alex Brand. AberdeenFungal Group, Aberdeen University, Aberdeen, United Kingdom.Candida albicans is a pleiomorphic fungus that lives as a commensal yeast in the human body but can become pathogenic in susceptible patient groups.Virulence is strongly linked with the production of penetrative hyphae that can adhere to and invade a wide range of substrates, including blood vessels,organ tissue, keratinised finger-nails and even soft medical plastics. Using live-cell imaging and nanofabricated surfaces, we are characterising the spatiotemporaldynamics of contact-induced hyphal tip behaviour (thigmotropism). To test whether tip re-orientation responses positively correlate with levelsof hyphal adhesion, we generated substrates with increasing adhesive force. Hyphal tip re-orientation was absent in poorly-immobilised hyphae and athreshold adhesive force was required sub-apically to generate the hyphal tip pressure required for re-orientation. Interestingly, sub-threshold adhesionresulted in sub-apical hyphal bending. Localization of fluorescent protein markers for the Spitzenkörper and the Polarisome (Mlc1-YFP and Spa2-YFP,respectively) showed that C. albicans hyphal tips grow in an asymmetric, ‘nose-down’ manner on a surface. Additionally, hyphal tips can detect surfacestiffness and show a distinct preference for nose-down growth on the softer of two substrates. Localisation of fluorescent cell-cycle reporter proteins overtime revealed that hyphal tip contact slowed the cell-cycle, suggesting that tip-contact perturbs cell-cycle mechanics. Finally, we examined the role ofcytoskeleton regulators in thigmotropism and determined the force that can be generated by the hyphal tip. Our results suggest that C. albicans hyphae148