A gamma-lactamase from cereal infecting Fusarium spp. catalyses the first step in the degradation of the benzoxazolinone class of phytoalexins

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

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

Fusarium head blight and crown rot, caused by the fungal plant pathogen Fusarium graminearum, impose a major threat to global wheat production. During the infection, plants are contaminated with mycotoxins such as deoxynivalenol (DON), which can be toxic for humans and animals. In addition, DON is a major virulence factor during wheat infection. However, it is not fully understood how DON production is regulated in F. graminearum. In order to identify regulators of DON production, a high-throughput mutant screen using Fluorescence Activated Cell Sorting (FACS) of a mutagenised TRI5-GFP reporter strain was established and a mutant over-producing DON under repressive conditions identified. A gain-of-function mutation in the F. graminearum adenylyl cyclase (FAC1), which is a known positive regulator of DON production, was identified as the cause of this phenotype through genome sequencing and segregation analysis. Our results show that the high-throughput mutant screening procedure developed here can be applied for identification of fungal proteins involved in diverse processes

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

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.pseudograminearumFDB2 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

Evolutionary origins of a bioactive peptide buried within preproalbumin

The de novo evolution of proteins is now considered a frequented route for biological innovation, but the genetic and biochemical processes that lead to each newly created protein are often poorly documented. The common sunflower (Helianthus annuus) contains the unusual gene PawS1 (Preproalbumin with SFTI-1) that encodes a precursor for seed storage albumin; however, in a region usually discarded during albumin maturation, its sequence is matured into SFTI-1, a protease-inhibiting cyclic peptide with a motif homologous to unrelated inhibitors from legumes, cereals, and frogs. To understand how PawS1 acquired this additional peptide with novel biochemical functionality, we cloned PawS1 genes and showed that this dual destiny is over 18 million years old. This new family of mostly backbone-cyclic peptides is structurally diverse, but the protease-inhibitory motif was restricted to peptides from sunflower and close relatives from its subtribe. We describe a widely distributed, potential evolutionary intermediate PawS-Like1 (PawL1), which is matured into storage albumin, but makes no stable peptide despite possessing residues essential for processing and cyclization from within PawS1. Using sequences we cloned, we retrodict the likely stepwise creation of PawS1's additional destiny within a simple albumin precursor. We propose that relaxed selection enabled SFTI-1 to evolve its inhibitor function by converging upon a successful sequence and structure