Ralstonia solanacearum elicitor RipX Induces Defense Reaction by Suppressing the Mitochondrial atpA Gene in Host Plant

Abstract

RipX of Ralstonia solanacearum is translocated into host cells by a type III secretion system and acts as a harpin-like protein to induce a hypersensitive response in tobacco plants. The molecular events in association with RipX-induced signaling transduction have not been fully elucidated. This work reports that transient expression of RipX induced a yellowing phenotype in Nicotiana benthamiana, coupled with activation of the defense reaction. Using yeast two-hybrid and split-luciferase complementation assays, mitochondrial ATP synthase F1 subunit α (ATPA) was identified as an interaction partner of RipX from N. benthamiana. Although a certain proportion was found in mitochondria, the YFP-ATPA fusion was able to localize to the cell membrane, cytoplasm, and nucleus. RFP-RipX fusion was found from the cell membrane and cytoplasm. Moreover, ATPA interacted with RipX at both the cell membrane and cytoplasm in vivo. Silencing of the atpA gene had no effect on the appearance of yellowing phenotype induced by RipX. However, the silenced plants improved the resistance to R. solanacearum. Moreover, qRT-PCR and promoter GUS fusion experiments revealed that the transcript levels of atpA were evidently reduced in response to expression of RipX. These data demonstrated that RipX exerts a suppressive effect on the transcription of atpA gene, to induce defense reaction in N. benthamiana.

Keywords: Ralstonia solanacearum; RipX; atpA gene; gene expression; susceptibility.

Figure 1

Defense reaction induced by transient expression of ripX1 and ripX3 in Nicotiana benthamiana. (A) Schematic diagram of expressed RipX1 and RipX3. (B) Yellowing phenotypes induced by RipX1 and RipX3. The infiltration areas were indicated by circles. All the experiments were repeated three times. (C) Activation of the defense genes hsr203J, hin1, and PR1a. The transcript level of each gene in plants transformed with empty vector pHB was set to 1, and levels in other plant samples were calculated relative to that. Data represent mean and standard deviation of three technical replicates. The asterisks denote statistical significance (** p < 0.01).

Figure 2

HR reactions induced by RipX1 and RipX3 proteins. (A) Macroscopic HR reaction induced by GST-RipX1 or GST-RipX3. A GST tag was used as a negative control. (B) Activation of the defense genes hsr203J, hin1, and PR1a examined by qRT-PCR analyses. The expression level for each gene in plants transformed with empty vector pHB was set to 1, and the level in other samples was calculated relative to that. Error bars indicate standard deviations of three individual replicates, and asterisks denote statistical significance (** p < 0.01). (C) Identification of the minimum concentration of the RipX protein essential for HR induction. All experiments were repeated three times.

Figure 3

Identification of the ATPA protein that interacted with RipX1. (A) Y2H assays showing the interaction between ATPA and RipX1. The yeast strains were grown on SD/-Ade/-Leu/-Trp/-His/ plates supplied with X-α-galactosidase. The interaction between VemR and RpoN2 was used as a positive control. Y2H assays were repeated three times. (B) Split-luciferase assays of the interaction of ATPA with RipX1. This experiment was repeated three times, with similar results. Relative light units are shown below. (C) Evolutionary relationships of 12 ATPAs from representative plant species.

Figure 4

Subcellular localization of ATPA and its interaction with RipX1 in N. benthamiana cells. (A) Subcellular localization of RFP-RipX1. (B) Subcellular localization of YFP-ATPA. The arrows indicate the localization of YFP-ATPA in mitochondria. (C) Colocalization of RFP-RipX1 and YFP-ATPA in N. benthamiana cells. (D) BiFC analysis showing the spatial interaction of ATPA and RipX1 at the cell membrane. (E) Protein expression levels in the infiltrated plant leaves were collected for immunoblotting assays with anti-GFP and anti-Myc antibody. Ponceau S staining indicates the equal loading of the proteins. All experiments were repeated three times. The scale bar represents 50 μm.

Figure 5

Involvement of the atpA gene in bacterial wilt disease. (A) Detection of the transcript level of atpA gene in gene-silenced plants by qRT-PCR. The transcript level in wild type was set to 1, and the level in other samples was calculated relative to that. Error bars indicate standard deviations of three individual replicates, and asterisks denote statistical significance (** p < 0.01). (B) Examination of the HR and yellowing phenotypes in atpA-silenced plants induced by RipX. (C) Disease development in atpA-silenced N. benthamiana plants. Each point represents the mean disease index of 24 plants combined from four separate experiments. Asterisks indicate the significant difference by ANOVA (p < 0.05).

Figure 6

RipX1 suppressed atpA transcription. (A) Reduction in atpA transcription in N. benthamiana transiently expressing RipX1. The plants transformed with the pHB empty vector were used as controls. (B) Examination of atpA promoter activity. For qRT-PCR analyses, the transcript level in uninoculated plants was set to 1, and the level in other samples was calculated relative to that. Error bars indicate standard deviations of three individual replicates, and asterisks denote statistical significance (** p < 0.01).