The bacterial strains and plasmids used in this study are detailed in Supplementary Table S1 . Ralstonia solanacearum strains were cultured in CTG medium (casein acid hydrolysate, 1 g/L; tryptone, 10 g/L; and glucose, 5 g/L) at 28°C. The CTG agar plate was supplemented with 0.005% 2,3,5-triphenyltetrazolium chloride (final concentration). The upstream and downstream flanking regions of the shk gene and the spectinomycin resistance gene sequence were amplified by PCR with the primer pairs shk-1F/shk-2R, shk-3F/shk-4R, and pHSE401spcF/pHSE401spcR, respectively, cloned into the pK18mobsacB vector using the Gibson Assembly^® Master Mix (NEB, Ipswich, MA, USA). The resultant plasmid was electroporated into R. solanacearum-competent cells, creating the Δshk mutant via sacB counterselection (Chen et al., 2018; Yan et al., 2022b). Transformants were selected on 50-mg/L spectinomycin-containing CTG plates. The Δshk mutant was verified by PCR and DNA sequencing. DNA fragment of native shk was amplified by PCR using the primer pairs shk-5F/shk-6R with R. solanacearum GMI1000 genomic DNA as the template. The resultant plasmid was electroporated into the Δshk-competent cell to generate an shk-complemented Δshk(shk) strain. Similarly, a DNA fragment of the shk gene, fused with a 6×His tag sequence, was amplified by PCR using the primer pairs shkHis-7F/shkHis-8R and subsequently cloned into the pK18mobsacB vector. The recombinant plasmid was transformed into the Δshk mutant, yielding the shk-His strain. A DNA fragment of the shk gene fused with the Flag sequence was amplified by PCR using the primer pairs shk-FlagUPF/shk-FlagUPR and shk-FlagDownF/shk-FlagDownR, and then cloned into the pK18mobsacB vector, yielding the pK18mobsacB-shk-Flag vector. Furthermore, DNA fragments of the RSc0291 and RSc0040 genes fused with a 6×His sequence were amplified by PCR with the primer pairs RSc0291UPF/RSc0291UPR/RSc0291-CDSF-HIS/RSc0291-CDSR-HIS/RSc0291DownF/RSc0292DownR and RSc0040UPF/RSc0040UPR/RSc0040-CDSF-His/RSc0040-CDSR-His/RSc0040DownF/RSc0040DownR, cloned into the pK18mobsacB-shk-Flag vector, thereby yielding pK18mobsacB-shk-Flag-RSc0291-6×His and pK18mobsacB-shk-Flag-RSc0040-6×His vector, respectively. The resultant plasmids were transformed into the Δshk mutant, ultimately yielding shk-Flag/RSc0291-6×His and shk-Flag/RSc0040-6×His strains.

Four-week-old tomato plants (Moneymaker) were inoculated with Δshk, Δshk(shk), and wild-type GMI1000 strains (10⁹ CFU/mL) by soil soaking with 20 mL of bacterial suspension as previously described by Takemura et al. (2023). The inoculated plants were then maintained in an incubator at 28°C with a 14-h light and 10-h dark cycle. Each treatment consisted of 20 plants, and the experiment was repeated three times. The disease symptoms were monitored and recorded daily according to the following disease index scale: 0, no wilting; 1, 1%–25% wilting; 2, 26%–50% wilting; 3, 51%–75% wilting; 4, 76%–99% wilting; and 5, dead.

Tomato plants were sectioned into 2 cm pieces, placed in a 2-mL tube containing 1 mL of sterile water at 4°C, and vigorously shaken to release the bacteria. Total RNA was extracted from plant-deprived R. solanacearum or 1 mL cultures of R. solanacearum (OD₆₀₀ = 1) using the Eastep™ Super Total RNA Extraction Kit (Promega, Shanghai, China). Complementary DNA (cDNA) synthesis was performed with the HiScript III RT SuperMix for quantitative PCR (Vazyme, Nanjing, China). RT-qPCR analyses were carried out with the ChamQ Universal SYBR qPCR master mix (Vazyme, Nanjing, China). The transcript level of gdhA was used as a reference control for RT-qPCR analysis. The relative expression values of the target genes were calculated using the threshold cycle (2^−ΔΔCT) method, as described by Yan et al. (2022a). The primers used in RT-qPCR analysis are detailed in Supplementary Table S2 . RT-qPCR experiments were performed in triplicate.

Tomato roots or stems were harvested at 5 days after inoculation (DAI), weighed, cut into small pieces, and then placed in tubes containing 1 mL of sterile water (de Pedro-Jové et al., 2021). After shaking for 20 min, the suspensions were serially diluted and spread on CPG plates. The plates were incubated at 28°C for 48 h. Subsequently, the numbers of R. solanacearum cells were counted. Each treatment consisted of 10 plants. The experiments were repeated in triplicate.

EPS quantification was conducted as described previously (Song et al., 2022). In brief, R. solanacearum was cultured in a sucrose-peptone liquid medium (peptone, 5 g/L; sucrose, 20 g/L; KH₂PO₄, 0.5 g/L; MgSO₄, 0.5 g/L; pH 7.2). A 100-mL aliquot of the culture (OD₆₀₀ = 3.0) was harvested and subjected to centrifugation at 12,000 rpm for 20 min. The collected supernatants were mixed with 4 volumes of ethanol and incubated at 4°C overnight. The precipitated EPSs were isolated via centrifugation, dried at 55°C overnight, and weighed. The independent experiments were repeated in triplicate.

Biofilm formation was performed in polystyrene tubes as described previously (Song et al., 2022). In brief, the bacterial suspensions were adjusted to an OD₆₀₀ of 0.2 with sterile water. The bacteria were cultured 1:20 in CTG medium in 96-well polystyrene plates, statically incubated at 28°C for 48 h, and finally stained with 0.1% crystal violet. The stained samples were repeatedly rinsed with sterile water and dissolved in 95% ethanol. Biofilm formation was quantified by measuring absorbance at 570 nm (A ₅₇₀). The independent experiments were repeated in triplicate.

Swarming motility was assessed on 0.3% semisolid agar plates described previously (Song et al., 2022). In brief, bacterial suspensions were adjusted to an OD₆₀₀ of 0.2 with sterile water. Aliquots (1 µL) of the suspensions were added at the center of the tryptone-solidified medium (tryptone 10 g/L and agar 3 g/L). The plates were incubated at 28°C for 48 h before the diameter of the swarming halos was measured. The independent experiments were repeated in triplicate.

Cellulase activity was determined as described previously (Song et al., 2022). In brief, bacterial suspensions were adjusted to an OD₆₀₀ of 0.2 with sterile water. Aliquots (1 µL) of the suspensions were added to carboxymethylcellulose sodium (CMS) solid medium (CMS 1 g/L, Na₃PO₄ 3.8 g/L, agar 8 g/L, pH 7.0). The plates were incubated at 28°C for 48 h. The plates were stained with 0.5% Congo red for 30 min. The plates were rinsed three times with 1 M of NaCl. The diameter of the transparent circles was observed and measured. The independent experiments were repeated in triplicate.

Ralstonia solanacearum shk-His cells were harvested from CTG medium (OD₆₀₀ = 1) and 4-week-old tomato plants. The bacterial cells were centrifuged and resuspended in a lysis buffer (50 mM of NaH₂PO₄, 300 mM of NaCl, pH 8.0, and 1 mM of phenylmethylsulfonyl fluoride). The suspension was sonicated, centrifuged, and sequentially added with Ni-NTA magnetic agarose beads. The beads were then washed with a wash buffer (50 mM of NaH₂PO₄, 300 mM of NaCl, 10 mM of imidazole, pH 8.0). The Shk-6×His complex was eluted with an elution buffer (50 mM of NaH₂PO₄, 300 mM of NaCl, 200 mM of imidazole, pH 8.0) at 4°C. Finally, the protein complex was analyzed by the LC-MS/MS system (Thermo Scientific, Shanghai, China).

For the modified pull-down assay in vivo, the fusion gene constructs were transformed into Δshk cells via electroporation as described by Wise and Binns (2016). The transformed cells were cultured and lysed in 10 mL of protein extraction buffer (150 mM of NaCl, 1% Triton X-100, 50 mM of Tris, pH 7.5 and 1× protease inhibitor cocktail) and sonicated on ice. The samples were centrifuged at 12,000 rpm for 15 min at 4°C. The supernatant was added with 50 µL of Ni-NTA magnetic agarose beads and incubated on a shaker at 4°C overnight. After incubation, the beads were washed three times with a wash buffer (150 mM of NaCl, 50 mM of Tris, pH 7.5). The input and bead-bound proteins were mixed with 6× SDS loading buffer and boiled for 5 min. The proteins were separated on 12% SDS-PAGE gels and detected by Western blot analysis with anti-Flag or anti-His antibodies.

For protein pull-down assay in vitro, GST, Shk-ID-6×His (intracellular domain of Shk), and GST-RSc0040 fusion proteins expressed in Escherichia coli (DE3) were purified by affinity chromatography as described by Liu et al. (2017). Fifty micrograms of GST, GST-RSc0040, was incubated with 50 µg of Shk-6×His in PBS buffer and subsequently added with 50 µL of glutathione agarose beads at 4°C under agitation for 2 h. The beads were collected by centrifugation and washed five times with pre-cooled PBS buffer. After washing, input and bead-bound proteins were detected by Western blot analysis using appropriate antibodies (Abcam, Shanghai, China).

Statistical analyses were conducted with Prism 9 software (GraphPad, Boston, MA, USA). Results were presented as the mean ± standard deviation (SD). Statistical significance was determined with a two-tailed t-test for comparison between two treatments. P-value was used to denote the statistical significance of the differences: * indicates P < 0.05, and ** indicates P < 0.01.

We screened for virulent genes by analyzing dynamic transcriptional changes in R. solanacearum UY031 under different conditions and identified a virulent HK sensor RSUY_RS17350 (de Pedro-Jové et al., 2021). We found that RSUY_RS17350 mRNA expression in R. solanacearum UY031 cells within the early xylem and late xylem exhibited 2.6- and 2.8-fold upregulation compared to that in a rich medium. The RSUY_RS17350 was homologous to the HK sensor Shk in R. solanacearum GMI1000. To investigate the effects of Shk on R. solanacearum pathogenicity, 4-week-old tomato plants were inoculated with Δshk, Δshk(shk), and wild-type GMI1000 strains using the soil soak inoculation method. The tomato plants inoculated with R. solanacearum GMI1000 and complemented strain Δshk(shk) exhibited apparent wilt symptoms at 5 DAI ( Figure 1A ). In contrast, tomato plants infected by the Δshk mutant showed a significant reduction in wilt symptoms ( Figure 1A ). A disease index was employed to monitor the progression of bacterial wilt. As illustrated in Figure 1C , the disease indices of tomato plants inoculated with GMI1000 and Δshk(shk) strains were 0.511 and 0.365 at 13 DAI. In contrast to both the GMI1000 and Δshk(shk) strains, the Δshk mutant exhibited a significant decrease in disease index, which was 0.218 at 13 DAI ( Figure 1C ). To further investigate the potential contribution of Shk to the colonization of R. solanacearum in planta, we quantified the bacterial colony-forming units (CFUs) in the stems and roots of tomato plants. The cell numbers of the Δshk(shk) and GMI1000 strains in tomato stems were 7.06 × 10⁷ CFU/g and 1.77 × 10⁸ CFU/g at 5 DAI, respectively ( Figure 1B ), whereas the cell numbers of the Δshk mutant in tomato stems were 2.31 × 10⁷ CFU/g at 5 DAI, which were significantly lower than those of the Δshk(shk) and GMI1000 strains ( Figure 1B ). The CFUs of the Δshk(shk) and GMI1000 strains in tomato roots were significantly increased in comparison to those of the Δshk mutant at 5 DAI ( Figure 1D ). These findings suggested that Shk is crucial for the pathogenicity of R. solanacearum.

To assess whether Shk regulates the expression of virulence factors in R. solanacearum, we investigated its key biological functions, including swarming motility, cellulase activity, biofilm formation, and EPS production. The Δshk mutant exhibited a significant increase in swarming motility compared to the wild-type GMI1000 strain ( Figure 2A ). Complementation of shk fully restored the phenotypic trait of the Δshk mutant to wild-type GMI1000 levels ( Figure 2A ). The deletion of shk led to a marked decrease in cellulase activity ( Figure 2B ), EPS production ( Figure 2C ), and biofilm formation ( Figure 2D ) compared to the wild-type GMI1000 strain. In addition, these indices were restored to normal levels in the complemented strain Δshk(shk). These findings revealed the important roles of Shk in regulating R. solanacearum virulence.

To unveil the regulatory role of Shk on QS-dependent genes, we conducted RT-qPCR analysis to quantify the transcript levels of phcA, epsB (EPS I polysaccharide export protein), cbhA (1,4-β-cellobiosidase), and fliC (flagellin protein) in R. solanacearum, which were released from infected tomato plants. At 2 DAI, the phcA transcript level in wild-type GMI1000 cells was approximately 5.7-fold lower than that of Δshk cells harvested from tomato stems ( Figure 3A ). The phcA expression in Δshk cells from tomato stems at 5 DAI was significantly reduced relative to wild-type GMI1000 cells ( Figure 3A ). Notably, complementation of shk in the Δshk mutant restored phcA expression to wild-type GMI100 levels. Similar expression patterns of the phcA were observed in tomato roots ( Figure 3A ). The transcript levels of epsB and cbhA in the Δshk mutant derived from the roots at both 2 DAI and 5 DAI were significantly downregulated compared to the Δshk(shk) and wild-type GMI1000 strains ( Figures 3B, C ). Likewise, epsB and cbhA were expressed at lower levels in Δshk cells harvested from tomato stems at 2 DAI and 5 DAI than those in Δshk(shk) and wild-type GMI1000 strains. Similar results of epsB and cbhA were observed in tomato roots ( Figures 3B, C ). However, the transcript levels of the fliC in the Δshk mutant derived from the roots at both 2 DAI and 5 DAI were significantly higher than those in the Δshk(shk) and wild-type GMI1000 strains ( Figure 3D ). Similar expression patterns of the fliC were detected in tomato stems ( Figure 3D ). These findings indicated that Shk positively modulates the expression of phcA, epsB, and cbhA, while it negatively regulates the expression of fliC of R. solanacearum in planta.

To determine whether the expression levels of phcA, epsB, cbhA, and fliC were regulated by Shk in CTG medium, we also used RT-qPCR to analyze the four QS-dependent genes. When the bacterial density reached an OD₆₀₀ of 1, R. solanacearum cells were harvested for RT-qPCR analysis. As illustrated in Supplementary Figures S1A, D , the transcript levels of the phcA and fliC genes did not exhibit significant differences among the Δshk, Δshk(shk), and GMI1000 strains. However, the expression of epsB and cbhA in Δshk(shk) cells was moderately upregulated compared to that of the Δshk and GMI1000 strains ( Supplementary Figures S1B, C ). These findings suggested that the RNA expression patterns of the QS-dependent genes phcA, epsB, cbhA, and fliC of R. solanacearum in planta differ from those grown in CTG medium.

The two-component system is one of the major signal transduction pathways in R. solanacearum, which consists of an HK and an RR. Extracellular signals are sensed by the HK, leading to the phosphorylation of the downstream RR and ultimately triggering the expression of virulence factors. Shk was predicted to possess seven transmembrane regions, a histidine phosphotransfer domain (DHp), and a histidine kinase-like ATPase domain (HATPase) according to the SMART (Simple Modular Architecture Research Tool) web service. To identify the Shk-interacting response regulator, we isolated the Shk-6×His complex from CTG medium and plant-derived R. solanacearum cells and further conducted LC-MS/MS analysis. As shown in Supplementary Table S3 , four response regulators—PhcR, VsrC, RSc0291, and RSc0040—were identified in the Shk complex from the medium-derived R. solanacearum cells. To confirm these findings, we proceeded with LC-MS/MS analysis of the Shk-interacting complex in R. solanacearum cells released from tomato plants. The LC-MS/MS analysis revealed that the response regulators RSc0291 and RSc0040 were also co-precipitated with Shk-6×His in plant-derived R. solanacearum cells ( Supplementary Table S3 ).

To further substantiate the interaction between Shk and these response regulators, we performed a modified pull-down assay to assess the interactions between Shk and RSc0291 or RSc0040. For this purpose, constructs of shk-Flag/RSc0291-6×His and shk-Flag/RSc0040-6×His were respectively cloned into the vector pK18mobsacB and subsequently introduced into Δshk cells via electroporation. As shown in Figure 4A , the co-precipitation of Shk-Flag with RSc0040-6×His using Ni-NTA beads was successfully detected with anti-Flag antibody in vivo. However, no interaction was observed between Shk-Flag and RSc0291-6×His ( Figure 4A ). These results indicated that the sensor Shk forms a direct association with the response regulator RSc0040 in vivo.

To evaluate whether the sensor Shk interacts with the RSc0040, pull-down assays were carried out in vitro. Shk-ID-6×His, GST-RSc0040, and GST were expressed in E. coli and purified with affinity chromatography. In order to test whether the used pull-down assay is appropriate, we analyzed the Shk-ID-6×His interaction with GST alone. The result showed that Shk-ID-6×His did not associate with GST. The results of the pull-down assays with the recombinant proteins showed that Shk-ID-6×His was pulled down by GST-RSc0040 ( Figure 4B ), indicating that the Shk was directly associated with the response regulator RSc0040 in vitro.