Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSof NO production showing the presence of innate immunity-like responses also in fungi.549. Balanced posttranslational activation of eukaryotic translation initiation factor 5A is required for pathogenesis in Fusarium graminearum. Ana LiliaMartinez-Rocha 1 , Mayada Woriedh 1,2 , Jan Chemnitz 3 , Peter Willingmann 1 , Joachim Hauber 3 , Wilhelm Schäfer 1 . 1) Molecular Phytopathology, University ofHamburg, Hamburg, Hamburg, Germany; 2) Cell Biology and Plant Biochemistry, University of Regensburg, Germany; 3) 3Heinrich-Pette-Institute forExperimental Virology and Immunology, Martinistrasse 52, D- 20251 Hamburg, Germany.Activation of the eukaryotic translation initiation factor 5A (EIF5A) requires a posttranslational modification, changing a lysine into the unique amino acidhypusine. This activation is a two steps reaction mediated by deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). First DHS cleavageand transfers the 4-aminobutyl moiety from the spermidine to an specific lysine in EIF5A to form the deoxyhypusine intermediate, during the second stepDOHH hydroxylate the 4-amonibutyl moiety to create the active form of eIF5A containing hypusine. The activated protein transports mRNAs from thenucleus to the ribosomes, where initiates protein biosynthesis. This system is conserved from Archea to humans and is involved in diseases as diverse asHIV infection, malaria, cancer, and diabetes. Until now, inhibition or silencing of DHS or DOHH has been tested to modify hypusination. For the first time,we evaluate its importance by over-expressing the enzymes that control hypusination of EIF5A. Over-expression of DOHH (oexDOHH) prevents virulence ofFusarium graminearum to wheat and maize. In contrast, over-expression of DHS (oexDHS) leads to an increase of virulence to wheat. Simultaneous overexpressionof both enzymes results in virulence comparable to the wild type strain. GFP assisted histology revealed that oexDOHH mutant is unable toform infection structures on wheat flower leaves and barely grows in point inoculated wheat spikelets. OexDHS results in an increase of infectionstructures and accordingly in an increase of virulence. We determined general hypusine formation by incorporating radiolabeled spermidine in EIF5Aduring in culture growth. Wild type and oexDHS showed similar hypusination intensity, whereas oexDOHH showed an increased incorporation. Thedifferential hypusination state of ElF5A was determined by 2D gels and western blot due to the difference in isoelectric point of the three states; inactive(lysine), intermediate (deoxyhypusine) or active (hypusine). Preliminary results show the wild type strain with all three hypusination states, oexDHS anddouble over-expressing mutants with an increased inactive and intermediate forms, and oexDOHH mutant lacked the inactive and intermediate form, onlythe activated form was detectable. We conclude that a balanced hypusination is required for proper function of EIF5A.550. The Con7 transcription factor, essential for pathogenicity, regulates the expression of genes involved in glycolysis and virulence in Fusariumoxysporum. Carmen Ruiz-Roldán 1,2 , Yolanda Pareja-Jaime 1,2 , M. Isabel G. Roncero 1,2 . 1) Department of Genetics, University of Cordoba, Spain; 2) Campusde Excelencia Agroalimentario (ceiA3).Transcription factors (TF) regulating the different stages of infection like adhesion to the host surface, differentiation of infection structures andpenetration represent potential molecular targets for fungicides with specific modes of action. Our studies on Fusarium oxysporum have demonstrated theessential role of morphogenetic regulation in pathogenesis, including processes such as cell-wall biogenesis, cell division and differentiation of infectionstructures. We identified the Con7 TF whose inactivation produces non-pathogenic mutants with altered morphogenesis, including abnormal polar growthand hyphal branching. To identify genes dependent on Con7 protein profiles of wild type and Dcon7 mutant were compared by 2D electrophoresis.Expression of 126 proteins varied quantitatively between both strains with statistical significance. Among the 80 proteins identified by MALDI-TOF/TOF-MS15, 9, and 4 were associated with secondary metabolism, glycolysis/gluconeogenesis (Gly/Glu) and pentose and glucuronate interconversions,respectively. Additionally, 6 proteins were known virulence factors, including citochrome P450 monooxygenase, O-methyltransferase, peptidyl-prolyl cistransisomerase D, peroxidase/catalase, phospholipase C and superoxide dismutase. Expression of the responsible genes was confirmed by qRT-PCR. Toverify the role of Con7 in Gly/Glu pathways, the intracellular ATP levels and alcohol dehydrogenase (ADH) activity in Dcon7 were compared with wt. Wedetected 2.4 fold increased ATP and 27.5% reduced ADH activity in Dcon7. Additionally, Dcon7 showed a dramatic growth reduction in the presence ofglucose, glycin or polygalacturonic acid, indicating nutrient assimilation defects. No significant differences were detected in intracellular glucoseaccumulated by both strains, while extracellular glucose levels in Dcon7 were significantly higher, suggesting defective sugar transport. RT-PCR analyses inthe wt revealed the existence of four con7 transcripts that differ in size and abundance, indicating alternative intron splicing. To determine thefunctionality of the different deduced proteins, Dcon7 was complemented with cDNA fragments from each version of the mature Con7 protein.Phenotypic and pathotypic characterization of the transformants should reveal their role in the different phenotypes observed in the Dcon7.551. Fusarium oxysporum produces volatile organic compounds that enhance the growth and stress resistance of Arabidopsis thaliana. Vasileios Bitas 1 ,Michael Axtell 2 , James Tumlinson 3 , Seogchan Kang 1 . 1) Department Plant Pathology and Environmental Microbiology, Pennsylvania State Univ, UniversityPark, PA; 2) Department of Biology, Pennsylvania State Univ, University Park, PA; 3) Department of Entomology, The Pennsylvania State University,University Park, PA.Production of volatile organic compounds (VOCs) as signal molecules is a widespread and efficient mode of communication utilized by all organisms.Microbial VOCs promoting plant growth and stress resistance present an environmentally and economically attractive alternative to fertilizers andpesticides. Fungi are some of the most predominant and yet, under-investigated organisms that employ VOCs in order to regulate and affect surroundingenvironments including neighboring organisms. Certain isolates of F. oxysporum, a well-known soilborne fungus, produce VOCs that promote plantgrowth, alter morphological and physiological properties, and enhance biotic and abiotic stress resistance in the model plant Arabidopis thaliana. In orderto investigate the mode of action by which those volatiles function, we have employed genetic and molecular resources and tools including A. thalianamutants that are defective hormonal signaling pathways and gene expression analysis. We are also trying to identify those compounds that affect A.thaliana through the use of GC-MS. Identification of the fungal VOCs triggering these plant responses and elucidation of the physiological and molecularalterations occurring in plants will help us shed light into the mechanism underpinning complex VOC-mediated interactions.552. Lipolytic system of the tomato pathogen Fusarium oxysporum f.sp. lycopersici. G. A. Bravo Ruiz 1,2 , C. Ruiz Roldán 1,2 , M. I. González Roncero 1,2 . 1)Departamento de Genética, Universidad de Córdoba; 2) Campus de Excelencia Agroalimentario (ceiA3), E-14071 Cordoba, Spain.The lipolytic profile of Fusarium oxysporum f.sp lycopersici has been determined on the basis of in silico analyses search and validated by biochemicalenzyme activity determination of Wheat Germ Oil (WGO) induced cultures. Twenty five identified structural secreted lipases show the conservedpentapeptide -Gly-X-Ser-X-Gly- characteristic of fungal lipases and the signal sequence for extracellular secretion. On the other hand, two transcriptionalregulatory predicted lipase genes have been identified showing nuclear localization signals (NLS) and the Zn2Cys6 zinc finger DNA binding motifs. Thetranscription profile of twelve structural genes, during tomato plant colonization in the wild type strain, shows variable expression levels (100 fold-range)being lip1, lip3, and lip22 the highest induced (20% relative to the actin gene). The maximal level of expression is observed in roots at 21-96 hours postinoculation.Targeted replacement of four structural (lip1, lip2, lip3 and lip5), and two regulatory ctf1 (orthologue to Fusarium solani ctf1 and to Aspergillusnidulans farA) and ctf2 (orthologue to F. solani ctf2 and to A. nidulans farB), lipase predicted genes originated the corresponding single deletion mutants256