Regulators of G-protein signalling in Fusarium verticillioides mediate differential host-pathogen responses on nonviable versus viable maize kernels.

GBB1, a heterotrimeric G-protein β-subunit gene, was shown to be a key regulator of fumonisin B(1) (FB(1) ) biosynthesis in the maize pathogen Fusarium verticillioides. In this study, we performed functional analyses of genes that encode putative RGS (regulators of G-protein signalling) proteins and PhLPs (phosducin-like proteins) in F. verticillioides. These proteins are known to regulate heterotrimeric G-protein activity by altering the intrinsic guanosine triphosphatase (GTPase) activity, which, in turn, influences the signalling mechanisms that control fungal growth, virulence and secondary metabolism. Our aim was to isolate and characterize gene(s) that are under the transcriptional control of GBB1, and to test the hypothesis that these genes are directly associated with FB(1) regulation and fungal development in F. verticillioides on maize kernels. We first identified eight genes (two PhLPs and six RGSs) in the F. verticillioides genome, and a subsequent transcriptional expression study revealed that three RGS genes were up-regulated in the gbb1 deletion (Δgbb1) mutant and one RGS gene was up-regulated in the wild-type. To characterize their function, we generated knockout mutants using a homologous recombination strategy. When grown on autoclaved nonviable kernels, two mutants (ΔflbA2 and ΔrgsB) produced significantly higher levels of FB(1) compared with the wild-type progenitor, suggesting that the two mutated genes are negative regulators of FB(1) biosynthesis. ΔflbA2 also showed a severe curly conidia germination pattern, which was contradictory to that observed in the Δgbb1 strain. Strikingly, when these mutants were grown on live maize kernels, we observed contrasting FB(1) and conidiation phenotypes in fungal mutants, which strongly suggests that these G-protein regulators have an impact on how F. verticillioides responds to host/environmental factors. Our data also provide evidence that fungal G-protein signalling is important for modulating the ethylene biosynthetic pathway in maize kernels.