Cloning and heterologous expression of candidate DON-inactivating UDP-glucosyltranferases from rice and wheat in yeast.
Abstract
Fusarium graminearum and related species causing Fusarium head blight of cereals and ear rot of maize produce the trichothecene toxin and virulence factor deoxynivalenol (DON). Plants can detoxify DON to a variable extent into deoxynivalenol-3-O-glucoside (D3G). We have previously reported the DON inactivat- ing glucosyltransferase (UGT) AtUGT73C5 from Arabidopsis thaliana (Poppenberger et al, 2003). Our goal was to identify UGT genes from monocotyledonous crop plants with this enzymatic activity. The two selected rice candidate genes with the highest sequence similarity with AtUGT73C5 were expressed in a toxin sensitive yeast strain but failed to protect against DON. A full length cDNA clone corresponding to a transcript derived fragment (TDF108) from wheat, which was reported to be specifically expressed in wheat genotypes contain- ing the quantitative trait locus Qfhs.ndsu-3BS for Fusarium spreading resistance (Steiner et al, 2009) was reconstructed. Only cDNAs with a few sequence deviations from TF108 could be cloned. However, toxin sensitive yeast strains expressing this wheat UGT cDNA did not show a resistant phenotype. The main diffi- culty in generating full length cDNAs for functional validation by heterologous expression in yeast is the enormous number of the UGT superfamily members in plants, with 107 UGT genes plus some pseudogenes in Arabidopsis thaliana and about 150 putative UGT genes in grasses. We conclude that neither sequence simi- larity nor inducibility are good predictors of substrate specificity.
Keywords
deoxynivalenol; Fusarium graminearum; phase II detoxification; rice; UDP-glucosyltranferase; wheat
References
Berthiller F., Dall'asta C., Corradini R., Marchelli R., Sulyok M., Krska R., Adam G., Schuhmacher R. 2009. Occurrence of deoxynivalenol and its 3-beta-D-glucoside in wheat and maize. Food Addit. Contam. 26:507-511.
Boddu J., Cho S., Muehlbauer G.J. 2007. Transcriptome analysis of trichothecene-induced gene expression in barley. Mol. Plant-Microbe Interact. 20:1364-1375.
Desmond O.J., Manners J.M., Schenk P.M., Maclean D.J., Kazan K. 2008. Gene expression analysis of the wheat response to infection by Fusarium pseudograminearum. Physiol. Mol. Plant Path. 73:40–47.
Lemmens M., Scholz U., Berthiller F., Dall'Asta C., Koutnik A., Schuhmacher R., Adam G., Buerstmayr H., Mesterházy A., Krska R., Ruckenbauer P. 2005. The ability to detoxify the mycotoxin deoxynivalenol colocalizes with a major quantitative trait locus for Fusarium head blight resistance in wheat. Mol. Plant- Microbe Interact.18:1318-1324.
Lulin M., Yi S., Aizhong C., Zengjun Q., Liping X., Peidu C., Dajun L., Xiu-E W. 2009. Molecular cloning and characterization of an up-regulated UDP-glucosyltransferase gene induced by DON from Triticum aestivum L. cv. Wangshuibai. Mol. Biol. Rep. 37:785-795.
Mitterbauer R., Karl T., Adam G. 2002. Saccharomyces cerevisiae URH1 (encoding uridine-cytidine N- ribohydrolase): functional complementation by a nucleoside hydrolase from a protozoan parasite and by a mammalian uridine phosphorylase. Appl. Environ. Microbiol. 68:1336-1343.
Poppenberger B., Berthiller F., Lucyshyn D., Sieberer T., Schuhmacher R., Krska R., Kuchler K., Glössl J., Luschnig C., Adam G. 2003. Detoxification of the Fusarium mycotoxin deoxynivalenol by a UDP- glucosyltransferase from Arabidopsis thaliana. J. Biol. Chem. 278:47905-47914.
Schweiger W., Boddu J., Shin S., Poppenberger B., Berthiller F., Lemmens M., Muehlbauer G.J., Adam G. 2010. Validation of a candidate deoxynivalenol-inactivating UDP-glucosyltransferase from barley by heterologous expression in yeast. Mol. Plant. Microbe Interact. 23:977-986.
Steiner B., Kurz H., Lemmens M., Buerstmayr H. 2009. Differential gene expression of related wheat lines with contrasting levels of head blight resistance after Fusarium graminearum inoculation. Theor. Appl. Genet. 118:753-764.
Department of Applied Genetics and Cell Biology, University of Natural Resources and Applied Life Sciences, A-1190 Vienna, Austria. Austria
Institute of Biotechnology in Plant Production, Department of Agrobiotechnology, University of Natural Resources and Applied Life Sciences, A-3430 Tulln, Austria. Austria
Institute of Biotechnology in Plant Production, Department of Agrobiotechnology, University of Natural Resources and Applied Life Sciences, A-3430 Tulln, Austria. Austria
Institute of Chemical Engineering, Vienna University of Technology, A-1060 Vienna, Austria. Austria
Institute of Biotechnology in Plant Production, Department of Agrobiotechnology, University of Natural Resources and Applied Life Sciences, A-3430 Tulln, Austria. Austria
Center for Analytical Chemistry, Depart- ment of Agrobiotechnology, University of Natural Resources and Applied Life Sciences, A-3430 Tulln, Austria. Austria
Institute of Biotechnology in Plant Production, Department of Agrobiotechnology, University of Natural Resources and Applied Life Sciences, A-3430 Tulln, Austria. Austria
Department of Applied Genetics and Cell Biology, University of Natural Resources and Applied Life Sciences, A-1190 Vienna, Austria. Austria
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