Functional characterization of two putative DAHP synthases of AroG1 and AroG2 and their links with type III secretion system in ralstonia solanacearum

Abstract

Type three secretion system (T3SS) is essential for Ralstonia solanacearum to cause disease in host plants and we previously screened AroG1 as a candidate with impact on the T3SS expression. Here, we focused on two putative DAHP synthases of AroG1 and AroG2, which control the first step of the shikimate pathway, a common route for biosynthesis of aromatic amino acids (AAA), to characterize their functional roles and possible links with virulence in R. solanacearum. Deletion of aroG1/2 or aroG1, but not aroG2, significantly impaired the T3SS expression both in vitro and in planta, and the impact of AroG1 on T3SS was mediated with a well-characterized PrhA signaling cascade. Virulence of the aroG1/2 or aroG1 mutants was completely diminished or significantly impaired in tomato and tobacco plants, but not the aroG2 mutants. The aroG1/2 mutants failed to grow in limited medium, but grew slowly in planta. This significantly impaired growth was also observed in the aroG1 mutants both in planta and limited medium, but not in aroG2 mutants. Complementary aroG1 significantly restored the impaired or diminished bacterial growth, T3SS expression and virulence. Supplementary AAA or shikimic acid, an important intermediate of the shikimate pathway, significantly restored diminished growth in limited medium. The promoter activity assay showed that expression of aroG1 and aroG2 was greatly increased to 10-20-folder higher levels with deletion of the other. All these results demonstrated that both AroG1 and AroG2 are involved in the shikimate pathway and cooperatively essential for AAA biosynthesis in R. solanacearum. The AroG1 plays a major role on bacterial growth, T3SS expression and pathogenicity, while the AroG2 is capable to partially carry out the function of AroG1 in the absence of AroG1.