Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSmultiple penetration events underneath infection cushions by scanning electron microscopy. Colonization of the underlying plant tissue was studied bybright field microscopy and transmission electron microscopy of LR-White serial sections. To understand the molecular basis of initial colonization of theleaf surface followed by infection cushion development, a laser capture microdissection (LCM) approach was established to isolate specifically runnerhyphae and infection cushions. Several hundred runner hyphae and infection cushions grown on wheat glumes were collected using the PALM system(Zeiss) avoiding contamination with plant tissue. Total mRNA of runner hyphae and infection cushions were isolated and amplified. The cDNA library ofeach developmental stage was used for next generation sequencing with Illumina HiSeq 2000. Quantitative expression analysis show marked differences ingene expression patterns between runner hyphae and infection cushions. Different functional pathways specific for each infection stage were identified.Thereby new insights in the initial infection process of FHB disease are gained. To our knowledge, we provide the first transcriptome data of runnerhyphae and infection cushions from a fungal plant pathogen obtained under in planta conditions. In summary, the power of combined microscopic andmolecular approaches to analyze cell type-specific gene expression during fungal-plant-interactions is demonstrated.141. Biochemical and biophysical analysis of the CarO rhodopsin of Fusarium fujikuroi. Jorge García-Martínez 1 , Marta Castrillo 1 , Javier Avalos 1 , UlrichTerpitz 2 . 1) Departamento de Genética, Universidad de Sevilla, Sevilla, Spain; 2) Lehrstuhl für Biotechnologie und Biophysik, Julius-Maximilians-UniversitätWürzburg, Biozentrum / Am Hubland, Würzburg, Germany.Light controls many substantial processes in filamentous fungi, such as reproduction and pathogenicity. Fungi naturally possess light sensors, which reactto a broad range of wavelengths with absorption maxima in the blue, green or red regions of the spectrum. Rhodopsins are green light-absorbingmembrane-integrated photoreceptors consisting of seven transmembrane helices forming an interior pocket for the chromophore, either all-trans or 11-cis retinal, covalently bound to the protein via a protonated Schiff-base. Type I rhodopsins, predicted to bind all-trans retinal, are widespread inascomycota and basidiomycota. Upon light-activation, type I rhodopsins act as proton pumps or sensory proteins; however, detailed knowledge of theirphysiological function and biological role in fungi is still missing. The gibberellin-producing fungus Fusarium fujikuroi contains two rhodopsin encodinggenes, carO and opsA, whose mutations produce no external phenotypic alterations. The carO gene is linked and co-regulated with genes coding forenzymes for retinal-synthesis, whose expression is strongly induced by light. To gain information on CarO biological role, we have combined biophysicalmethods to analyse the localisation and function of this rhodopsin in F. fujikuroi mycelia. We established a strain expressing CarO fused to a yellowfluorescent protein (YFP) under control of the carO promoter. This strain was investigated with confocal laser scanning microscopy (cLSM) and superresolutionfluorescence imaging (dSTORM) to reveal the subcellular localisation of CarO. Protein-localisation was compared with data recorded from a S.cerevisiae DSY5 strain overexpressing CarO-YFP. Additionally, the carO-YFP gene fusion was expressed in neuroblastoma cells, where it exhibited anefficient ion pump-activity, as demonstrated by Patch-clamp techniques. The results suggest a light dependent ion-pumping role in the fungus,nonessential under standard laboratory conditions.142. Roles of membrane and organellar calcium channels and transporters in controlling pulsatile [Ca 2+ ] c signatures. Hye-Seon Kim 1 , Jung-Eun Kim 2 , KirkCzymmek 1 , Robert Cirino 1 , Randall Duncan 1 , Hokyoung Son 3 , Yin-Won Lee 3 , Seogchan Kang 2 . 1) Department of Biological Sciences, University of Delaware,Newark, DE 19711; 2) Department of Plant Pathology & Environmental Microbiology, The Pennsylvania State University, University Park, PA 16802; 3)Department of Agricultural Biotechnology and Center for Fungal Pathogenesis, Seoul National University, Seoul 151-921.Calcium ions translate diverse environmental stimuli into many different physiological and developmental functions in fungi via an evolutionaryconserved cell-signaling pathway. Using the expression of Yellow Cameleon YC3.60, a fluorescent protein-based, ratiometric Ca 2+ sensor in Magnaportheoryzae, Fusarium oxsyporum, and F. graminearum, we reported that cytoplasmic tip high Ca 2+ signatures exhibited distinct species-specific and agedependentpulsatile patterns (FGB 49:589). We successfully expressed a new circularly permuted Ca 2+ sensor, GCaMP5, in F. graminearum and F.oxysporum and GCaMP3 in Neurospora crassa. The improved sensitivity, photostability, and fast kinetics of GCaMP5 enabled us to image smaller Ca 2+changes in hyphae tips with high-speed imaging that showed that the tip high Ca 2+ gradient has multiple origins. Disruption of F. graminearum genesencoding plasma membrane Ca 2+ channels (Mid1, Cch1, and Fig1), vacuole/ER Ca 2+ pumps (Pmc, Pmr), calcineurin transcription factor (Crz1), and vacuoleH + /Ca 2+ exchanger (Vcx1 and Vcx2) significantly altered the amplitude, interval, and origin of Ca 2+ pulses and also affected growth. Additional phenotypesassociated with these mutants are currently being characterized. The combination of molecular genetics, genomics, live cell imaging, and correlativemicroscopy will help us study the mechanism underpinning fungal Ca 2+ signaling at multiple scales ranging from the function and mode-of-action ofindividual genes to nano-scale dynamics of individual proteins and subcellular machineries.143. Characterization of positive regulator for asexual and sexual reproduction in the cereal head blight pathogen Gibberella zeae. Jungkwan Lee 1 ,Boknam Jung 1 , Hokyoung Son 2 , Yin-Won Lee 2 . 1) Department of Applied Biology, Dong-A University, Busan 604-714, Republic of Korea; 2) Department ofAgricultural Biotechnology and Center for Fungal Pathogenesis, Seoul National University, Seoul 151-921, Republic of Korea.Gibberella zeae is an important plant pathogen that causes cereal head blight and produces mycotoxins that are harmful to animals and humans.Ascospores and conidia contribute to the primary inoculums and propagation for disease epidemics. In this study, we identified one putative C2H2 zincfinger transcription factor (prd1) that is required for both conidiation and sexual reproduction, as screening transcription factor mutant collection wepreviously generated. prd1 deletion mutants impaired conidial production and lost both self-fertility and female fertility, but retain male fertility. Theoverexpression of the gene increased the amount of conidial production and resulted in earlier maturation of fruiting body formation than the wild-typestrain. The vegetative growth of deletion and overexpression mutants was increased and decreased on nutrient-rich mediua, respectively, but was notdifferent from the wild-type strain on nutrient-poor media. This study was the first report for transcription factor which positively regulates bothconidiation and sexual reproduction, and the characterization of genes regulated by this gene will be further studied.144. Functional analysis of Elongator complex protein 3 in Gibberella zeae. Y. J. Lee 1 , H. Son 1 , J.-C. Kim 2 , G. J. Choi 2 , Y.-W. Lee 1 . 1) Department ofAgricultural Biotechnology and Center for Fungal Pathogenesis, Seoul National University, Seoul 151-921, Republic of Korea; 2) Eco-friendly New MaterialsResearch Group, Research Center for Biobased Chemistry, Division of Convergence Chemistry, Korea Research Institute of Chemical Technology, Daejeon305-343, Republic of Korea.Gibberella zeae (anamorph: Fusarium graminearum) is a causal agent of Fusarium head blight (FHB) which causes huge economic losses in cereal cropssuch as wheat and barley. In addition to yield reduction, mycotoxin contamination of grain presents a threat to human safety. We examined one ofRestriction-Enzyme-Mediated Integration (REMI) mutants Z43R9282 showing defects in virulence and sexual development and identified a gene encodingElongator complex protein 3 (ELP3). ELP3 is a catalytic subunit of Elongator complex and contains histone acetyltransferase (HAT) domain. The biologicalfunction of ELP3 gene was studied by targeted deletion in G. zeae. Deletion of ELP3 resulted in retarded growth and delay of sexual developmentcompared to the wild-type strain. Most of the ascospores had two cells in the ELP3 deletion mutants, while wild-type ascospores usually had four cells. The156