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.