Degradation of the benzoxazolinone class of phytoalexins is important for virulence of Fusarium pseudograminearum towards wheat

Abstract

Wheat, maize, rye and certain other agriculturally important species in the Poaceae family produce the benzoxazolinone class of phytoalexins on pest and pathogen attack. Benzoxazolinones can inhibit the growth of pathogens. However, certain fungi can actively detoxify these compounds. Despite this, a clear link between the ability to detoxify benzoxazolinones and pathogen virulence has not been shown. Here, through comparative genome analysis of several Fusarium species, we have identified a conserved genomic region around the FDB2 gene encoding an N-malonyltransferase enzyme known to be involved in benzoxazolinone degradation in the maize pathogen Fusarium verticillioides. Expression analyses demonstrated that a cluster of nine genes was responsive to exogenous benzoxazolinone in the important wheat pathogen Fusarium pseudograminearum. The analysis of independent F. pseudograminearum FDB2 knockouts and complementation of the knockout with FDB2 homologues from F. graminearum and F. verticillioides confirmed that the N-malonyltransferase enzyme encoded by this gene is central to the detoxification of benzoxazolinones, and that Fdb2 contributes quantitatively to virulence towards wheat in head blight inoculation assays. This contrasts with previous observations in F. verticillioides, where no effect of FDB2 mutations on pathogen virulence towards maize was observed. Overall, our results demonstrate that the detoxification of benzoxazolinones is a strategy adopted by wheat-infecting F. pseudograminearum to overcome host-derived chemical defences.

High-throughput FACS-based mutant screen identifies a gain-of-function allele of the Fusarium graminearum adenylyl cyclase causing deoxynivalenol over-production

“…Extension was performed for 2 minutes. Each primer used for fragment amplification contained at least 15 bp of sequence (30 bp of total overlap) at the 5' end to allow yeast mediated homologous recombination of the fragments into a HindIII/XbaI linearized pYES2 with yeast transformation as described elsewhere (Kettle et al, 2015). Transformation of the isolate CS3005 was conducted as previously described (Desmond et al, 2008).…”

Degradation of the benzoxazolinone class of phytoalexins by Fusarium pseudograminearum, a pathogenic fungus of wheat

“…checking both if the target gene has been deleted without introduction of any background mutation), genome sequencing to further analyse the Δfdb1 strains was performed as previously described (Kettle et al, 2015a). CLCbio Genomics Workbench version 7.0 was used for mapping.…”

“…To delete F. graminearum FDB1 the construct was synthesised as previously described (Kettle et al, 2015a Table 2.1) PCR assay using Red Extract-N-Amp kit (Sigma-Aldrich) was used to identify transformants lacking FGSG_00079 (the F. graminearum CS3005 strain annotation locus tag is FG05_00079 (Gardiner et al, 2014)). …”

“…However, the specific genetic identity of Fdb1, proposed to be a hydrolytic enzyme that performs transformation of benzoxazolinones, remains to be identified (Glenn et al, 2003). Genomic comparisons of the FDB gene cluster in F. pseudograminearum to that in F. verticillioides (Glenn et al, 2002) identified a single lactamase-encoding gene that was previously proposed (but not demonstrated) to encode the Fdb1 function (Kettle et al, 2015a).…”

“…We have previously shown that detoxification of aminophenols by a N-acyl transferase, Fdb2, in the F. pseudograminearum FDB gene cluster is important for virulence on wheat (Kettle et al, 2015a). However, the specific genetic identity of Fdb1, proposed to be a hydrolytic enzyme that performs transformation of benzoxazolinones, remains to be identified (Glenn et al, 2003).…”