Functional Characterization of Two Putative DAHP Synthases of AroG1 and AroG2 and Their Links With Type III Secretion System in Ralstonia solanacearum.

Weiqi Zhang,Jing Li,Xiaojun Shi,Kouhei Ohnishi,Yasufumi Hikichi,Yong Zhang

doi:10.3389/fmicb.2019.00183

Weiqi Zhang, Jing Li + Show 4 more

Open Access

https://doi.org/10.3389/fmicb.2019.00183

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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.

Highlights

Like in many pathogenic bacteria of animals and plants, the syringe-like type three secretion system (T3SS) is essential for Ralstonia solanacearum to cause disease in host plants, which is a causal agent of bacterial wilt disease on more than 450 plant species worldwide (Genin et al, 2005; Genin and Denny, 2012; Jiang et al, 2017)
We provided genetic evidence to demonstrate that two putative D- arabino-heptulosonate7-phosphate (DAHP) synthases of AroG1 and AroG2 are cooperatively essential for biosynthesis of aromatic amino acids (AAA) in R. solanacearum
The aroG1/2 mutants were auxotrophic in limited medium, and supplementary AAA or shikimic acid (SA) significantly restored the diminished growth of aroG1/2 mutants in limited medium, confirming that AroG1 and AroG2 are involved in the shikimate pathway and are responsible for AAA biosynthesis in R. solanacearum

Summary

Introduction

Like in many pathogenic bacteria of animals and plants, the syringe-like type three secretion system (T3SS) is essential for Ralstonia solanacearum to cause disease in host plants, which is a causal agent of bacterial wilt disease on more than 450 plant species worldwide (Genin et al, 2005; Genin and Denny, 2012; Jiang et al, 2017). A LysR type of transcriptional regulator PhcA negatively regulates hrpG expression, which is activated at high cell density and binds to promoters of prhI/R genes to repress their expression, and in tandem to repress the hrpB expression, while the PhcA positively regulates prhG expression (Genin et al, 2005; Yoshimochi et al, 2009a; Plener et al, 2010; Zhang et al, 2013). This results in dual regulation pathways of PhcA on hrpB expression, and R. solanacearum might switch from using the HrpG to PrhG for hrpB activation in a cell density-dependent manner (Zhang et al, 2013)

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