Three amino acid residues are required for the recognition of Ralstonia solanacearum RipTPS in Nicotiana tabacum

Abstract

Ralstonia solanacearum causes devastating diseases in a wide range of economically important crops. It secretes a large number of virulence factors, also known as effectors, to promote its infection, and some of them are recognized when the host plant contains corresponding resistance genes. In this study we showed that a type III effector RipTPS from the avirulent R. solanacearum strain GMI1000 (RipTPSG) specifically induced cell death in Nicotiana tabacum, but not in Nicotiana benthamiana, whereas the RipTPS homolog in the virulent strain CQPS-1 (RipTPSC) induced cell death in neither N. tabacum nor N. benthamiana. These results indicated that RipTPSG is recognized in N. tabacum. Expression of RipTPSG induced upregulation of hypersensitive response (HR) -related genes in N. tabacum. The virulence of CQPS-1 was reduced when RipTPSG was genetically introduced into CQPS-1, further confirming that RipTPSG functions as an avirulence determinant. Protein sequence alignment indicated that there are only three amino acid polymorphisms between RipTPSG and RipTPSC. Site-directed mutagenesis analyses confirmed that the three amino acid residues are jointly required for the recognition of RipTPSG in N. tabacum. Expression of either RipTPSG or RipTPSC suppressed flg22-triggered reactive oxygen species (ROS) burst in N. benthamiana, suggesting that RipTPS contributes to pathogen virulence. Mutating the conserved residues in RipTPS’s trehalose-phosphate synthase (TPS) domain did not block its HR induction and defense suppression activity, indicating that the TPS activity is not required for RipTPS’s avirulence and virulence function.

Comparative genomics and host range analysis of four Ralstonia pseudosolanacearum strains isolated from sunflower reveals genomic and phenotypic differences

“…The effectors (T3Es) secreted by T3SS play a critical role in bacterial virulence and determination of host range specificity [ 42 ]. RipP1, RipAA (formerly AvrA) and RipTPS triggered HR cell death and induced resistance to bacterial wilt in Nicotiana tabacum [ 43 – 45 ]. RipG7, the essential determinant of R. solanacearum strains for virulence on the legume plant Medicago truncatula [ 46 ].…”

A robust high‐throughput functional screening assay for plant pathogen effectors using the TMV‐GFP vector

“…Total RNA was extracted from N. benthamiana leaves sampled at 48 hpi using the RNAsimple Total RNA Extraction Kit (TIANGEN). The quantitative expression of GFP , the HR marker gene NbHsr203J (Takahashi et al., 2004), and the defense‐related gene NbPR1 (Delaney et al., 1994) was measured by qRT‐PCR, as previously described (An et al., 2022). The primers used for the amplification of these genes and the internal control are listed in Table S1.…”

Ubiquitin E3 ligase activity of Ralstonia solanacearum effector RipAW is not essential for induction of plant defense in Nicotiana benthamiana

“…Total RNA was extracted using the RNAsimple Total RNA Extraction Kit (TIANGEN), and quantitative expression of the defense-related genes NbHIN1 and NbPR1a was determined by qRT-PCR with SYBR Green PCR Master Mix as previously described ( An et al, 2022 ). NbEF1α was selected as an internal control.…”