Pseudomonas shiyinii ST4 inhibits sexual mating of Sporisorium scitamineum through the GacS/GacA two-component system and the mib1 gene cluster

ABSTRACT Sugarcane smut is one of the most devastating fungal diseases affecting sugarcane plantations. The virulence of the causative agent, Sporisorium scitamineum , depends on a process called sexual mating of compatible sporidia MAT-1 and MAT-2 to form infectious dikaryotic hyphae. We previously isolated Pseudomonas shiyinii strain ST4, capable of inhibiting the fungal sexual mating. However, its biocontrol mechanism remains unclear. In this study, Tn5 mutagenesis of strain ST4 led to the identification of a number of genes potentially associated with the biosynthesis of mating inhibitors. Among them, a two-component system GacS/GacA was found responsible for the inhibitory activity against fungal mating. Transcriptomic and genetic analyses unveiled that GacS/GacA positively regulates a novel gene cluster designated as mib1 (mating inhibitor biosynthesis 1), consisting of seven genes. Compared with the parental strain ST4, the mib1 deletion mutants were much compromised in the anti-mating activity. Two LysR-family transcriptional regulators, located adjacent to the mib1 gene cluster, were also shown to play key roles in regulating mib1 transcription and biosynthesis of mating inhibitor(s). In summary, the results from this study uncovered a novel gene cluster, the two-component system GacS/GacA, and two transcriptional factors associated with the regulation and biosynthesis of inhibitory compound(s) against S. scitamineum sexual mating. The findings depict the genetic regulatory network that controls the biocontrol potency of Pseudomonas shiyinii ST4 and present useful clues for identification of its anti-mating inhibitor(s). IMPORTANCE Sporisorium scitamineum is the major sugarcane pathogen that causes tremendous economic losses internationally. The sporidia sexual mating is vital for the fungus to form dikaryotic hyphae capable of infecting sugarcanes, thus is a highly attractive and promising target for screening of biocontrol agents and green chemical mating inhibitors against this devastating fungal pathogen. We previously reported that Pseudomonas shiyinii ST4 can inhibit the sexual mating of S. scitamineum without affecting its haploid sporidia growth. By using transposon mutagenesis in combination with genetic and bioinformatics analyses, this study aimed to identify the genetic components of strain ST4 associated with its biocontrol potency. The significance of this study is in (i) identifying a novel gene cluster mib1 involved in production of mating inhibitor(s) by strain ST4, (ii) demonstrating that the GacS/GacA-RsmY-CsrA regulatory cascade plays an essential role in regulation of the mib1 cluster and the bacterial anti-mating activity, and (iii) unveiling two novel transcription factors, that is, MibR1 and MibR2, which act downstream of the Gac regulatory cascade in modulation of the mib1 gene expression. The findings represent a critical step in depicting the biocontrol mechanisms of strain ST4 and provide valuable clues for further investigations.