Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTS455. The Sclerotinia sclerotiorum mating type locus (MAT) contains a 3.6-kb region that is inverted in every generation. Patrik Inderbitzin 1 , PeriasamyChitrampalam 2 , Karunakaran Maruthachalam 1 , Bo-Ming Wu 3 , Krishna Subbarao 1 . 1) Department of Plant Pathology, University of California-Davis, Davis,CA, USA; 2) Department of Plant Sciences, University of Arizona, Tucson, AZ, USA; 3) Department of Plant Pathology, China Agricultural University, 2 WestYuanmingyuan Rd., Haidian District, Beijing, China.Sclerotinia sclerotiorum is a filamentous ascomycete in the Sclerotiniaceae (Pezizomycotina) and a necrotrophic pathogen of more than 400 hostsworldwide, including many important agricultural crops. In California, the biggest lettuce producer in the United States, S. sclerotiorum is a causal agent oflettuce drop that reduces overall annual lettuce yield by 15%. Little is known about the details of sexual reproduction in S. sclerotiorum, but the structureof the S. sclerotiorum mating type locus MAT, the master regulator of sexual reproduction in ascomycetes, has previously been reported. As in otherhomothallic (self-fertile) ascomycetes, S. sclerotiorum MAT contains both idiomorphs (divergent alleles) fused end-to-end at a single locus. Using 283isolates from lettuce in California and from other states and hosts, we investigated the diversity of S. sclerotiorum MAT, and identified a novel version ofMAT that differed by a 3.6-kb inversion and was designated Inv+, as opposed to the previously known S. sclerotiorum MAT that lacked the inversion andwas Inv-. The inversion affected three of the four MAT genes: MAT1-2-1 and MAT1-2-4 were inverted and MAT1-1-1 was truncated at the 3’-end.Expression of MAT genes differed between Inv+ and Inv- isolates. In Inv+ isolates, only one of the three MAT1-2-1 transcript variants of Inv- isolates wasdetected, and the alpha1 domain of Inv+ MAT1-1-1 transcripts was truncated. Both Inv- and Inv+ isolates were self-fertile, and the inversion segregated ina 1:1 ratio regardless of whether the parent was Inv- or Inv+. This suggested the involvement of a highly regulated process in maintaining equalproportions of Inv- and Inv+, likely associated with the sexual state. The MAT inversion region, defined as the 3.6-kb MAT inversion in Inv+ isolates and thehomologous region of Inv- isolates, was flanked by a 250-bp inverted repeat on either side. The 250-bp inverted repeat was a partial MAT1-1-1 thatthrough mediation of loop formation and crossing over, may be involved in the inversion process. Inv+ isolates were widespread, and in California andNebraska constituted half of the isolates examined. We speculate that a similar inversion region may be involved in mating type switching in thefilamentous ascomycetes Chromocrea spinulosa, Sclerotinia trifoliorum and in certain Ceratocystis species.456. Repression of the phosphor-transmitter gene ypdA resulting in growth defect in Aspergillus fumigatus. Daisuke Hagiwara 1 , Hiroki Takahashi 1 ,Mayumi Nakayama 2 , Keietsu Abe 2 , Tohru Gonoi 1 , Susumu Kawamoto 1 . 1) Medical Mycology Research Center, Chiba university, Chiba, Japan; 2) NICHe,Tohoku university, Sendai, Japan.Two-component system (TCS) is a conserved signal transduction system implicated in cellular responses to a variety of environmental stimuli in fungi.Aspergillus fumigatus has 13 histidine kinases, single HPt (histidine-containing phosphor transmitter), and 3 response regulators, which together constitutea TCS signaling. According to studies of HPt in several fungi, ypdA encoding a single HPt of A. fumigatus has been thought to be an essential gene. In thisstudy, we tested if absence of YpdA leads to cell lethality in A. fumigatus, and investigated what the molecular mechanisms underlying the lethality is. Toaddress these questions, we constructed a conditional ypdA-expressing strain by replacing a native promoter of ypdA with thiA promoter (PthiA). PthiA isstrictly repressed in the presence of thiamine, while moderately expressed in the absence of thiamine. The conditional PthiA-ypdA strain showed severegrowth defect on a plate containing thiamine, while the strain grew normally on a plate without thiamine as the wild-type stain. We, then, investigated theexpression profiles of catA, dprA, and dprB genes, which are regulated under the control of SakA MAPK cascade, since the SakA MAPK cascade isdownstream of A. fumigatus TCS. In a liquid culture, expressions of catA, dprA, and dprB were gradually increased after addition of thiamine, suggestingthat inactivation of ypdA expression leads to the activation of SakA MAPK cascade. To get more insight into a response to ypdA-repression, thetranscriptome profiles were obtained by RNA-seq. Expression levels of each gene at 3h, 6h, and 9h after addition of thiamine were compared to that at 0h.More than 2-fold or less than 1/2-fold of expression changes were regarded as up- or down-regulated, respectively. Through statistical analysis oncategory of gene ontology, the groups concerning ribosome biogenesis or RNA metabolism were found to be significantly down-regulated after theinactivation of ypdA. Based on these results, we will discuss the cellular responses to YpdA deprivation and try to find out the molecular mechanismsattributed to the lethality.457. Unravelling the GTPase polarity complex in Claviceps purpurea. Andrea Herrmann 1 , Janine Schürmann 1 , Britta Tillmann 2 , Michael Bölker 2 , PaulTudzynski 1 . 1) IBBP, WWU Muenster, Schlossplatz 8, 48143 Muenster, Germany; 2) Philipps-Universität, Karl-von-Frisch-Strasse 8, 35032 Marburg,Germany.Claviceps purpurea is a plant pathogen infamous for its production of toxic alkaloids on infected host plants like barley. Consumption of infected grainsleads to severe symptoms up to the death of the patient. Infection patterns are complex and the topic of intensive research. One interesting aspect is thestrict polarity of the hyphal growth during the first infection stage which seems to be crucial for the non-recognition of C. purpurea as a pathogen by thehost. To address the question of the importance of polarity the structure and dynamics of the polarity complex are the focus of this work. The guaninenucleotide exchange factors (GEFs) Cdc24 and Dock180 belong to different families, Cdc24 being a member of the Dbl GEF family and Dock180 a CZH GEF.Cdc24-GFP localises cytosolically and to hyphal tips whereas Dock180-GFP is present in small vesicles in the hypha, though concentrated at the tip region,too. Cdc24 DHPH domains are able to activate the small GTPases Rac and Cdc42 of C. purpurea and U. maydis In vitro, whereas the catalytic domain ofDock180 only activates Rac in both organisms. Despite the proven activation Cdc24 does not interact with any GTPase in yeast two hybrid assays. Dock180shows a weak interaction with Rac and the two p21-activated kinases (PAKs) Ste20 and Cla4. Thus, both GEFs do not share many characteristics apart fromtheir GEF activity. The PAKs Ste20 and Cla4 and the scaffold protein Bem1 are involved in the polarity complex, too. Ste20 localises to hyphal tips andinteracts with Cdc42 in a loading status dependent manner, whereas Cla4 is the main partner of Rac. Other interactions of Ste20 with Dock180 and Cla4could also be shown. Bem1 is present in the cytosol - concentrated at the hyphal tip - and links most of the proteins of the polarity complex as interactionswith Cdc24, Cla4, Ste20 and Dock180 have been detected. Taken together we postulate at least two different polarity complexes, the Rac complex and theCdc42 complex. Both are gathered by Bem1, but Cla4 is the main partner of Rac, whereas Ste20 plays a similar role for Cdc42. Dock180 is mainly linked toRac, Cdc24 can be active in both complexes. We are interested in the spatial and temporal formation and regulation of these complexes and its influenceon polarity and virulence which will be the subject of further studies.458. Atypical Gb and RACK homolog Gib2 is a signal transducing adaptor protein affecting growth and virulence of Cryptococcus neoformans. YanliWang 1 , Gui Shen 1 , Jinjun Gong 1 , Amy Whittington 2 , Ping Wang 1,2,3 . 1) Res Inst for Children, Children's Hospital, New Orleans, LA USA; 2) Dept Microbiology,Immunology and Parasitology, LSUHSC, New Orleans, LA USA; 3) Dept Pediatrics, LSUHSC, New Orleans, LA USA.Virulence in Cryptococcus neoformans is a multifaceted trait underpinned by complex signaling pathways. The atypical G-protein b subunit Gib2 displaysversatility in interactions with signaling molecules such as Ga Gpa1 that governs cAMP signaling and intersectin Cin1 that regulates intracellular trafficking.This and the conserved seven-bladed b-propeller motif are highly suggestive that Gib2 functions as an adaptor protein. We here show that Gib2 binds to27th Fungal Genetics Conference | 233