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.

The benzoxazolinone class of phytoalexins are released by wheat, maize and rye upon pathogen attack. Benzoxazolinones have antimicrobial activity, but certain fungi have evolved mechanisms to actively detoxify these compounds, which may contribute to the virulence of these pathogens. In many Fusarium spp. a cluster of genes is thought to be involved in the detoxification of benzoxazolinones. However, only one enzyme, Fusarium Detoxification of Benzoxazolinone 2 (Fdb2), encoded in the cluster has been unequi vocally assigned a role in this process. The FDB2 gene encodes a N-malonyltransferase and was identified in the maize pathogen F. verticillioides. In this thesis, through comparative genome analysis of several Fusarium species, a conserved genomic region has been identified around FDB2. Expression analyses demonstrated that a cluster of nine genes were responsive to exogenous benzoxazolinone in the important wheat pathogen Fusarium pseudograminearum.

The benzoxazolinone class of phytoalexins are released by wheat, maize, rye and other agriculturally important species in the Poaceae family upon pathogen attack. Benzoxazolinones show antimicrobial effects on plant pathogens, but certain fungi have evolved mechanisms to actively detoxify these compounds which may contribute to the virulence of the pathogens. In many Fusarium spp. a cluster of genes is thought to be involved in the detoxification of benzoxazolinones. However, only one enzyme encoded in the cluster has been unequivocally assigned a role in this process. The first step in the detoxification of benzoxazolinones in Fusarium spp. involves the hydrolysis of a cyclic ester bond. This reaction is encoded by the FDB1 locus in F. verticillioides but the underlying gene is yet to be cloned. We previously proposed that FDB1 encodes a γ-lactamase, and here direct evidence for this is presented. Expression analyses in the important wheat pathogen F. pseudograminearum demonstrated that amongst the three predicted γ-lactamase genes only the one designated as FDB1, part of the proposed benzoxazolinone cluster in F. pseudograminearum, was strongly responsive to exogenous benzoxazolinone application. Analysis of independent F. pseudograminearum and F. graminearum FDB1 gene deletion mutants, as well as biochemical assays, demonstrated that the γ-lactamase enzyme, encoded by FDB1, catalyses the first step in detoxification of benzoxazolinones. Overall, our results support the notion that Fusarium pathogens that cause crown rot and head blight on wheat have adopted strategies to overcome host-derived chemical defences.