Program Book - 27th Fungal Genetics Conference

FULL POSTER SESSION ABSTRACTS38. Metabolomics of growth and type B trichothecenes production in Fusarium graminearum. L. Legoahec 1 , V. Atanasova-Penichon 1 , N. Ponts 1 , C.Deborde 2,3 , M. Maucourt 3,4 , S. Bernillon 2,3 , A. Moing 2,3 , F. Richard-Forget 1 . 1) 1INRA, UR1264 MycSA, 71 avenue Edouard Bourlaux, BP81, F-33140 Villenaved’Ornon, France; 2) INRA, UMR1332 Fruit Biology and Pathology, 71 avenue Edouard Bourlaux, BP81, F-33140 Villenave d’Ornon, France; 3) MetabolomeFacility of Bordeaux Functional Genomics Center, IBVM Centre INRA de Bordeaux, F-33140 Villenave d'Ornon, France; 4) Univ. Bordeaux, UMR1332 FruitBiology and Pathology, Centre INRA de Bordeaux, F-33140 Villenave d'Ornon, France.The plant fungal pathogen Fusarium graminearum can produce type B trichothecenes, a family of sesquiterpene molecules with toxic properties uponhuman or animal ingestion. Deoxynivalenol, or DON, and its acetylated forms belong to this family of secondary metabolites and are frequentcontaminants of cereals worldwide. The biosynthesis of trichothecenes initiates with the condensation of two molecules of farnesyl pyrophosphate, at theend of the mevalonate pathway in Fusarium, and is under the control of various factors such as the redox parameters of the environment or the carbonsource. For example, supplementing liquid submerged cultures of F. graminearum with caffeic acid, a phenolic acid with known antioxidant properties,reduces the accumulation of DON and its acetylated forms in the medium. Such a result, however, gives a partial glimpse of the effect of phenolic acids,from the trichothecene production point of view only. The present study analyzes F. graminearum metabolome in conditions when DON and its acetylatedforms are produced. Liquid chromatography coupled with mass spectrometry and proton nuclear magnetic resonance were used to characterize themetabolites produced by the fungus, secreted in the culture medium or not, over the course of 14 days. Fifty-two polar and semi-polar metabolites wereidentified in the culture medium, i.e., the exo-metabolites, and/or in the mycelium, i.e., the endo-metabolites, comprising amino acids and derivatives,sugars, polyketides, and terpenes including trichothecenes and DON precursors. Sample composition varied over time in terms of primary metabolites aswell as secondary metabolites. Data analysis further revealed correlations, positive or negative, between metabolic pathways. In the presence of caffeicacid, metabolomic profiles were modified, counting those resulting from primary metabolism even though fungal biomass production was not affected bythe treatment. Several metabolites affected by the treatment were identified for both the exo- and endo-metabolome, in particular DON and itsprecursors. For the first time, these results expose a unique outlook of a hidden aspect of Fusarium’s response to antioxidant treatment.39. Diversity of telomeric sequences and telomerase RNA structures within Ascomycetes. Xiaodong Qi, Yang Li, Dustin P. Rand, Julian J-L Chen.Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ, 85287-1604.Telomeres are specialized DNA-protein complexes that cap chromosome ends. Telomeric DNA is composed of repetitive short sequences synthesized bytelomerase, an RNA-containing DNA polymerase. The integral telomerase RNA (TER) contains telomerase provides a short template for telomeric DNAsynthesis and two highly conserved structural elements essential for enzymatic function. Fungal telomerase from budding and fission yeasts has beenstudied extensively. We have recently developed Neurospora crassa as a new fungal model organism for telomere and telomerase studies, and haveidentified TER structural domains highly conserved in vertebrate and Pezizomycotina, but not in budding yeasts (Qi et al. 2012). N. crassa telomeraseprocessively synthesizes the (TTAGGG)n telomere repeats, an attribute conserved in vertebrate but not yeast telomerases. In contrast, both budding andfission yeast telomerases synthesize irregular telomere repeats non-processively. Two structural elements of TER, the template-pseudoknot and the threewayjunction (TWJ) domain, are conserved in vertebrates, Pezizomycotina as well as Taphrinomycotina. Both of these elements are necessary fortelomerase activity in vitro for Pezizomycotina and Taphrinomycotina telomerases, while the TWJ is dispensable for budding yeast telomerase.Furthermore, spliceosome-mediated TER 3’-end processing is conserved in Pezizomycotina and Taphrinomycotina, but not in budding yeasts. Incomparison, the budding yeast (e.g. S. cerevisiae) TER employs a nuclease-mediated mechanism for the 3’ end processing. Our results indicate thatPezizomycotina telomerase preserved ancestral features that budding and fission yeast species lost during evolution and supports N. crassa as an excellentmodel for the study of telomere and telomerase. (Reference: Qi, X., Y. Li, S. Honda, S. Hoffmann, M. Marz. A. Mosig, J.D. Podlevsky, P.F. Stadler, E. Selkerand J.J.-L. Chen (2012) The common ancestral core of vertebrate and fungal telomerase RNAs. Nucleic Acids Research 40: doi:10.1093/nar/gks980.).40. Characterizing a putative three-step formaldehyde oxidation pathway in Neurospora crassa. Ethan Addicott 1 , Kolea Zimmerman 2 , Anne Pringle 2 . 1)Faculty of Arts and Sciences, Harvard College, Harvard University, Cambridge, MA; 2) Organismic and Evolutionary Biology, Harvard University, Cambridge,MA.Using bioinformatic analyses, we identified 13 Neurospora genes that code for putative secreted-proteins. One of these proteins, NCU01056 - a proposedS-(hydroxymethyl)glutathione synthase, is implicated in a highly conserved formaldehyde oxidation pathway involving two other genes, NCU06652 - anNAD and GSH dependent formaldehyde dehydrogenase and NCU0173- an S-formylglutathione hydrolase. Knockout strains for the three genes in thispathway were obtained from the FGSC and confirmed by PCR. We conducted standard phenotypic assays on the three knock-outs and WT controls,including growth morphology, growth rate, and mating ability. Additionally, growth in the presence of methanol, the compound just upstream offormaldehyde in the pathway, was tested by biomass and flow cytometry. Two key observations were made: (1) NCU06652 knockouts showed significantgrowth defects compared to the WT (2) Knockouts for NCU01056 (hypothesized to be upstream of the critical enzyme) showed increased pigmentation onSC media (3) NCU6652 knockouts germinated significantly slower than other strains in the presence of methanol compared to a control treatment. Thedata suggest NCU06652 is involved in the critical oxidation step of the pathway and that the absence of NCU01056 may induce stress, which points to itsrole in the formation of a formaldehyde-glutathione complex, immediately upstream of NCU06652. The fact that NCU01056 codes a secreted protein maysuggest that N. crassa may detoxify formaldehyde extracellularly or in membrane-bound vesicles. Further exploration will involve determining a doseresponsecurve for formaldehyde, confirming the localization of the proteins, and investigating the GSH balance in each of the strains.41. Nitrate assimilation in Neurospora crassa. Oleg Agafonov, Tina Marie Monge Are, Peter Ruoff. Centre for Organelle Research, University of Stavanger,Stavanger, Norway.Nitrogen is one of the essential components for a variety of cellular elements. Regulation of nitrogen assimilation can be critical for the evolutionaryadvantage of an organism and it has been extensively studied in filamentous fungi Neurospora crassa. Nitrate is an important source of inorganic nitrogenfor N. crassa, but it is not utilized unless favored nitrogen sources such as ammonium, glutamine or glutamate are absent in the environment. It wasshown that nitrate is transported into the cell by high affinity transporter, NIT10, where it is stepwise reduced, first, by nitrate reductase, NIT3 to nitrite,and then by nitrite reductase, NIT6 to ammonia, which is then converted to organic nitrogen in a form of glutamate, making it available for furtherutilization by the cell.Although biochemical pathways of nitrate assimilation have been extensively studied, there is a certain disagreement in literature about therequirement of functional nitrate reductase activity for nitrate uptake. In the paper by Schloemer and Garrett, 1974, it was shown that nitrate transport isnot dependent upon nitrate reduction. However, later Unkles et. al., 2004, concluded that functional nitrate reductase is required for the nitrateaccumulation in Neurospora crassa.The goal of this work was to investigate nitrate assimilation and involvement of nitrate reductase in this process in N. crassa. 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