Identification and Functional Characterisation of Two Oat UDP-Glucosyltransferases Involved in Deoxynivalenol Detoxification

Abstract

Oat is susceptible to several Fusarium species that cause contamination with different trichothecene mycotoxins. The molecular mechanisms behind Fusarium resistance in oat have yet to be elucidated. In the present work, we identified and characterised two oat UDP-glucosyltransferases orthologous to barley HvUGT13248. Overexpression of the latter in wheat had been shown previously to increase resistance to deoxynivalenol (DON) and nivalenol (NIV) and to decrease disease the severity of both Fusarium head blight and Fusarium crown rot. Both oat genes are highly inducible by the application of DON and during infection with Fusarium graminearum. Heterologous expression of these genes in a toxin-sensitive strain of Saccharomyces cerevisiae conferred high levels of resistance to DON, NIV and HT-2 toxins, but not C4-acetylated trichothecenes (T-2, diacetoxyscirpenol). Recombinant enzymes AsUGT1 and AsUGT2 expressed in Escherichia coli rapidly lost activity upon purification, but the treatment of whole cells with the toxin clearly demonstrated the ability to convert DON into DON-3-O-glucoside. The two UGTs could therefore play an important role in counteracting the Fusarium virulence factor DON in oat.

Keywords: UDP-glucosyltransferase; deoxynivalenol; glycosylation; oats.

Figure 1

Phylogenetic analysis of putative oat trichothecene-detoxifying UGT proteins together with barley orthologs present in orthogroup OG0000783. HORVU.MOREX.r3.5HG0464880.1 corresponding to barley HvUGT13248 is marked in red. The two oat proteins, AVESA.00010b.r2.6AG1068650.1 (AsUGT1) and AVESA.00010b.r2.6AG1068570.1 (AsUGT2), chosen for the current study are marked in blue. The clade formed by the proteins phylogenetically closest to HvUGT13248 is marked with a dotted line.

Figure 2

Relative expression of AsUGT1 and AsUGT2 genes in oat spikelets after inoculation with either DON (A,C) or F. graminearum (B,D).

Figure 3

Spottings of yeast transformants expressing oat glucosyltransferases AsUGT1 and AsUGT2 on plates with indicated concentrations of five different mycotoxins. Strains carrying barley HvUGT13248, yeast acetyltransferase ScAYT1 and the empty vector were used as controls. Toxin-containing plates are based on YPD. Control plates without toxin are SC-leu, where only transformed yeast cells can grow, and the rich medium YPD, which allows for growth of strains without a plasmid. Two independent transformants of each construct were spotted in two different dilutions.

Figure 4

(A) SDS-PAGE analysis of crude extracts of independent transformants of E. coli BL21(DE3) cells expressing either AsUGT1 (lines 1–3) or AsUGT2 (lines 4–6) as fusion proteins with the N-terminal His-tag and maltose binding protein (MalE). Transformants carrying a empty vector with only His-tag and MalE were used as a control (lines 7–8). (B) Concentrations of DON and DON-3G at 0 h and after 16 h incubation with E. coli transformants containing the expression vector with the AsUGT1 gene (1–3), AsUGT2 gene (4–6) or the empty vector (7–8).