Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTSand the double deletion mutant Dctf1Dctf2. In vitro qRT-PCR expression analyses of twelve structural lipase genes in the regulatory mutants Dctf1, Dctf2and Dctf1Dctf2, in comparison with the wild type strain, demonstrate the existence of a complex lipase regulation network in F. oxysporum. The reductionof total lipase activity (10-30%), besides the severe altered pathogenic behaviour on tomato plants shown by the single Dctf1, Dctf2, and the doubleDctf1Dctf2 mutants suggest an important role of the lipolytic system of this fungus in pathogenicity.553. Role of glycogen metabolism in the pathotypic behavior of Fusarium oxysporum f.sp. lycopersici on tomato plants. C. Corral Ramos 1,2 , C. RuizRoldan 1,2 , M. I. González Roncero 1,2 . 1) Departamento de Genética, Universidad de Córdoba; 2) Campus de Excelencia Agroalimentario (ceiA3), E-14071Córdoba, Spain.Glycogen can play different roles in biological systems, such as storage carbohydrate for energy and/or carbon or control of glucose metabolism. In mosteukaryotes, glycogen is built on a self-glucosylating initiator protein core, glycogenin (gnn), which acts as a primer for glycogen synthase (gls). Thebranches are created by a specific enzyme (gbe) which transfers a block of 6-7 residues from the end of a linear chain of glucose units to an internalglucose residue by an a-1,6 linkage. De-branching, which is essential for the degradation of glycogen, is catalyzed by a distinct enzyme (gdb) which acts onbranches containing only four residues, transferring three of them to the end of a linear chain and then hydrolyzing the final residue. The level of glycogenfound in a particular situation results from the balance between glycogen synthase and glycogen de-branching activities, resulting in the synthesis anddegradation of this compound, respectively. Additionally, both activities are regulated by the action of a glycogen phosphorilase (gph). In order to studythe role of glycogen metabolism in Fusarium oxysporum pathotypic behavior we generated single deletion mutants of the genes Dgnn, Dgls, Dgbe, Dgdband Dgph, and the double mutant DgphDgdb, by direct targeted replacement. Quantification of glycogen reserves during in vitro growth indicated anincrease along the time period (72 h) up 700mg glucose equivalents per mg protein in a pH independent manner. As expected, no detectable glycogen wasaccumulated in any of three single structural deletion mutants Dgnn5, Dgls10 or Dgbe17. By contrast, glycogen levels were 10% higher in the single Dgdb2and Dgph8, and the double Dgph8Dgdb2 mutants in comparison to wild type. Similar hyphal agglutination patterns were observed in the three singlemutants Dgnn5, Dgls10 and Dgbe17 compared to the wild type, whereas those strains affected in glycogen catabolism, Dgdb2, Dgph8, and the doublemutant Dgph8Dgdb2, showed a dramatic reduction in hyphal agglutination. This phenotype did not exactly correlate with conidial anastomosis tube (CAT)formation since all the mutants, except Dgls10, showed a 40-50% reduction in hyphal fusions. We are currently performing tomato plant infection assayswhat will help us to gain insight into the role of glycogen metabolism in the virulence of F. oxysporum.554. Identification of chemoattractant compounds from tomato root exudate that trigger chemotropism in Fusarium oxysporum. El Ghalid Mennat,David Turra, Antonio Di Pietro. Departamento de Genética, Universidad de Córdoba, 14071 Córdoba, Spain.Fusarium oxysporum is a soilborne pathogen that causes vascular wilt disease on a wide range of plant species, including tomato (Solanum lycopersicum).The host signals that trigger fungal infection are currently unknown. A chemotropic response of F. oxysporum towards tomato root exudate was observedusing a plate assay that measures directed growth of fungal germ tubes towards chemoattractants. To purifiy the chemoattractant coumpound(s) fromtomato root exudate, we applied a series of purification methods including extraction with organic and inorganic solvents, fractionation by size exclusionand ion exchange chromatography. The compound(s) showing chemoattractant activity were found in the hydophilic fraction, had a molecular weightbetween 30 and 50 kDa and were sensitive to boiling and treatment with proteinase K, suggesting that they correspond to one or several secreted tomatoproteins. Polyacrylamide gel electrophoresis of the active fraction revealed multiple protein bands of the expected size, two of which displayedchemoattractant activity when eluted from the gel. Identification of the active protein(s) by LC-ESI-MS is currently ongoing. Identification of the secretedchemoattractant(s) from tomato roots will advance our understanding of the molecular events that trigger fungus-root interactions.555. TOR-mediated control of virulence functions in the trans-kingdom pathogen Fusarium oxysporum. Gesabel Y. Navarro Velasco, Antonio Di Pietro.Departamento de Genética, Universidad de Córdoba, 14071 Córdoba, Spain.Infectious growth of fungal pathogens is controlled by environmental cues, including nutrient status. The soilborne fungus Fusarium oxysporum producesvascular wilt disease in more than a hundred different crop species and can cause lethal systemic infections in immunodepressed humans. Previous workshowed that the preferred nitrogen source ammonium causes repression of infection-related processes in F. oxysporum that could be reversed byrapamycin, a specific inhibitor of the conserved protein kinase TOR. Here we generated mutations in upstream components that should result inconstitutive activation of TOR, including null mutants in tuberous sclerosis complex 2 (TSC2), a small GTPase that represses TOR activity, as well as strainsexpressing a dominant activating allele of the small GTPase Rag (ragA Q86L ), an activator of TOR. The Dtsc2 mutants and, to a minor extent, the ragA Q86Lstrains showed defects in hyphal growth and colony morphology on several amino acids, as well as decreased efficiency in cellophane penetration andvegetative hyphal fusion. These phenotypes were exacerbated in Dtsc2ragA Q86L double mutants and could be reversed by rapamycin, suggesting that theyare caused by hyperactivation of TOR. The mutants caused significantly lower mortality on tomato plants and on larvae of the animal model host Galleriamellonella. These results suggest that TOR functions as a negative regulator of fungal virulence on plant and animal hosts.556. Components of the urease complex govern virulence of Fusarium oxysporum on plant and animal hosts. Katja Schaefer, Elena Pérez-Nadales,Antonio Di Pietro. Departamento de Genética, Universidad de Córdoba, 14071 Cordoba, Spain.In the soilborne pathogen Fusarium oxysporum, a mitogen-activated protein kinase (MAPK) cascade homologous to the yeast filamentous growthpathway controls invasive growth and virulence on tomato plants. Full phosphorylation of Fmk1 requires the transmembrane protein Msb2, a member ofthe family of signalling mucins that have emerged as novel virulence factors in fungal plant pathogens. A yeast two-hybrid screen for proteins interactingwith the Msb2 cytoplasmic tail identified UreG, a component of the urease enzymatic complex. UreG belongs to a set of accessory proteins needed toactivate Apo- urease, which converts urea to yield ammonia and carbon dioxide. The F. oxysporum genome contains two structural urease genes, ure1 andure2. Mutants in ureG or ure1 showed reduced growth on urea as the sole carbon and nitrogen source. Lack of urease activity in the mutants resulted infailure to secrete ammonia and to increase the extracellular pH. The DureG mutants caused significantly reduced mortality on tomato plants and on theanimal model host Galleria melonella, while Dure1 mutants only showed reduced virulence on tomato plants. Real-time qPCR analysis of key genesinvolved in nitrogen uptake and assimilation, as well as in the urea cycle, during infectious growth of F. oxysporum in G. melonella revealed increasedtranscript levels of arginase, which converts arginine to urea. Our results suggest a role for the urease accessory protein UreG in fungal virulence on plantand animal hosts.557. Knock-out of the Fusarium oxysporum f.sp. lycopersici homologs of the DNA-methylation genes DIM2 and HP1 does not affect effector geneexpression. Charlotte van der Does, Jerom van Gemert, Karlijn Klei, Ido Vlaardingerbroek, Martijn Rep. Molecular plant pathology, SILS, University ofAmsterdam, Amsterdam, Netherlands.27th Fungal Genetics Conference | 257