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SESSION 4: GENETICS OF HOSTS – PLANT RESISTANCE TO FUSARIUM, VARIETY DEVELOPMENT P100 - S-Methyl-DON: Chemical synthesis and toxicity of a novel DON metabolite T. Weigl-Pollack 2 , G. Wiesenberger 1 , P. Fruhmann 2 , H. Mikula 2 , C. Hametner 2 , B. Kluger 1 , R. Schuhmacher 1 , R. Krska 1 , J. Fröhlich 2 , G. Adam 1 1 University of Natural Resources and Life Sciences, Vienna (BOKU), Konrad Lorenz Str, 20 - 24, A 3430 Tulln Austria; 2 University of Technology, Institute of Applied Synthetic Chemistry, A-1060 Vienna, Austria E-mail: gerlinde.wiesenberger@boku.ac.at Previously performed microarray studies of deoxynivalenol (DON) treated barley revealed a strong up-regulation of cysteine biosynthesis genes, providing the first evidence for glutathione-mediated detoxification of DON (Gardiner et al., 2010). S-Methyl-DON (SMD) is a plant metabolite of DON first described to occur in Fusarium infected barley and wheat by Kushalappa et al. in 2010. Our hypothesis is that SMD is derived from a DON-cysteine adduct, which is again generated by processing of a DON-glutathione adduct. The in planta occurrence of a DONglutathione conjugate, and of DON-S-Cys-Gly and DON-S-Cys after DON treatment has recently been demonstrated (Kluger et al., 2012). Presumably SMD is generated by activity of a cysteine-S-conjugate beta-lyase and subsequent methylation of DON-SH. The aim of this study was to synthesize SMD for structure determination, as reference substance for quantification, and for toxicity testing. SMD was obtained by reaction of DON with iodomethane and thiourea in the presence of sodium carbonate and wet polyethylene glycol. 2D-NMR was used to elucidate the structure of the purified main product, which revealed the desired addition of a methylthio group to the double bond of DON (C10), but also formation of an intramolecular hemiketal (C15-OH reacting with the C8-keto group). The SMD concentration required for 50% growth inhibition of a sensitive yeast bioindicator strain was about 9-fold higher than for DON. Tests for the inhibition of protein synthesis of wheat ribosomes (using a wheat germ in vitro translation system) showed that SMD is at least 12-fold less toxic than DON. This strongly indicates that addition of the much larger cysteine and glutathione substituents should also lead to detoxification due to steric hindrance preventing interaction of the toxin-conjugate with the ribosomal target. Keywords: glutathione, conjugate, processing, detoxification 193
SESSION 4: GENETICS OF HOSTS – PLANT RESISTANCE TO FUSARIUM, VARIETY DEVELOPMENT P101 - Functional characterization of a lipid transfer protein associated with Qfhs.ifa-5A G. Siegwart, J. J. Peter, W. Schweiger, H. Buerstmayr BOKU - University of Natural Resources and Life Sciences, A-1180 Vienna - Department for Biotechnology in Plant production, A-3430 Tulln E-mail: gerald.siegwart@boku.ac.at Fusarium head blight (FHB) is a substantial disease for wheat and other small grain cereals. Resistance is generated by many QTL, all providing a distinct part to the mutually produced quantitative resistance. Beneath these many QTL, two of them are outstanding as they provide together about 40% of the total resistance – these QTL are designated Fhb1 and Qfhs.ifa-5A. (Buerstmayr 2002, Buerstmayr 2003). It is not known which or how many genes are responsible for the effect of these QTL, but potential candidates could be used as target for virusinduced gene silencing (VIGS) in order to transiently silence the gene and hence experience differences in spread of the disease. A candidate gene that was shown strongly associated with the major type I resistance QTL Qfhs.ifa-5A in Affymetrix microarray as well as in RNA-seq encodes a Lipid transfer protein (LTP). LTPs are small and abundant proteins involved in membrane biosynthesis, lipid trafficking, cutin/suberin formation as well as in stress response and defense against bacterial or fungal pathogens. (Kirubakaran, 2008)The candidate emerged due to its high upregulation (150x), when comparing mock-inoculated near-isogenic lines (NIL) that only differed in Qfhs.ifa-5A, suggesting constitutive expression associated with the QTL. We mapped the LTP close to the centromere on chromosome 5AL, proving that it only seems to be associated with Qfhs.ifa-5A, but not the causal protein itself, as this is located on 5AS. Nevertheless there is a strong connection between the LTP and the QTL, so we subjected it to our VIGSpipeline. We transiently silenced the gene in a NIL only harboring Qfhs.ifa-5A, which should facilitate comparison between silenced- and control plants. We observed a faster spread of disease symptoms and earlier wilting, compared to controls. Results of two independent biological replications using two different silencing-constructs both times as well as qPCR validation will be presented. Keywords: LTP, Virus induced gene silencing, Qfhs.ifa-5A, wheat 194
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Bordeaux EFS12 12 th European Fusar
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TABLE OF CONTENTS Welcome from the
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4 SPONSORSHIP The 12 th European Fu
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8 13:00 - 14:00 Lunch 14:00 - 15:00
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10 Session 4: Genetics of Hosts - P
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12 Thursday, May 16 th Session 6: F
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Evidence for birth-and-death evolut
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Future challenges of Fusarium and m
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P35 - Screening deoxynivalenol in o
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P76 - Fusarium verticillioides and
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P113 - Agronomic practices and risk
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ORAL PRESENTATIONS 25
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KEYNOTE LECTURE SESSION 1: FUSARIUM
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SESSION 1: FUSARIUM - GENETICS, GEN
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KEYNOTE LECTURE SESSION 2: SECONDAR
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SESSION 2: SECONDARY METABOLITES -
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SESSION 3: PATHOGENESIS - EPIDEMIOL
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KEYNOTE LECTURE 1 SESSION 4: GENETI
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SESSION 4: GENETICS OF HOSTS - PLAN
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KEYNOTE LECTURE 2 SESSION 4: GENETI
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KEYNOTE LECTURE SESSION 5: DISEASE
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SESSION 5: DISEASE CONTROL AND FORE
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SESSION 6: FUTURE CHALLENGE FOR EUR
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SESSION 6: FUTURE CHALLENGE FOR EUR
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POSTER PRESENTATIONS 91
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Page 241 and 242: de Hoog G. S., 89, 114, 236 de Kler
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BOKU-University of Natural Resource
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touatisihem03@yahoo.fr He Xinyao In
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steve.oliver@bioc.cam.ac. uk Ortega
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Subramaniam Gopal Agriculture and A
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