The transcription regulator ChpA affects the global transcriptome including quorum sensing‐dependent genes in Ralstonia pseudosolanacearum strain OE1‐1

Abstract The gram‐negative plant‐pathogenic β‐proteobacterium Ralstonia pseudosolanacearum strain OE1‐1 produces methyl 3‐hydroxymyristate as a quorum sensing (QS) signal through methyltransferase PhcB and senses the chemical via the sensor histidine kinase PhcS. This leads to activation of the LysR family transcription regulator PhcA, which regulates the genes (QS‐dependent genes) responsible for QS‐dependent phenotypes, including virulence. The transcription regulator ChpA, which possesses a response regulator receiver domain and also a hybrid sensor histidine kinase/response regulator phosphore‐acceptor domain but lacks a DNA‐binding domain, is reportedly involved in QS‐dependent biofilm formation and virulence of R. pseudosolanacearum strain GMI1000. To explore the function of ChpA in QS of OE1‐1, we generated a chpA‐deletion mutant (ΔchpA) and revealed that the chpA deletion leads to significantly altered QS‐dependent phenotypes. Furthermore, ΔchpA exhibited a loss in its infectivity in xylem vessels of tomato plant roots, losing virulence on tomato plants, similar to the phcA‐deletion mutant (ΔphcA). Transcriptome analysis showed that the transcript levels of phcB, phcQ, phcR, and phcA in ΔchpA were comparable to those in OE1‐1. However, the transcript levels of 89.9% and 88.9% of positively and negatively QS‐dependent genes, respectively, were significantly altered in ΔchpA compared with OE1‐1. Furthermore, the transcript levels of these genes in ΔchpA were positively correlated with those in ΔphcA. Together, our results suggest that ChpA is involved in the regulation of these QS‐dependent genes, thereby contributing to the behaviour in host plant roots and virulence of OE1‐1.


| INTRODUC TI ON
Bacteria monitor quorum sensing (QS) signals to track changes in abundance and to activate QS for the synchronous control of the expression of genes beneficial for vigorous replication, adaptation to environmental conditions, and virulence (Ham, 2013;Rutherford & Bassler, 2012).The soilborne gram-negative β-proteobacterium Ralstonia solanacearum species complex (RSSC; Remenant et al., 2010) infects more than 250 plant species in over 50 families, causing a devastating bacterial wilt disease that damages crop production worldwide (Mansfield et al., 2012).A phylotype I strain of RSSC, R. pseudosolanacearum OE1-1 (Kanda et al., 2003), first attaches to the surface of the epidermis in meristematic zones and elongation zones in roots of 4-day-old tomato seedlings without secondary roots and then colonizes intercellular spaces between the epidermis and cortex, activating QS (Inoue et al., 2023).This leads to degradation of the cell wall of cortical cells adjacent to the epidermis by plant cell wall-degrading enzymes such as cellobiohydrolase (Liu et al., 2005), endoglucanase (Roberts et al., 1988), and pectin methylesterase (Tans-Kersten et al., 1998), which are produced in the active state of QS and secreted through type II secretion machinery (Tsujimoto et al., 2008), and infection of the cell wall-denatured cortical cells (Inoue et al., 2023).The infection of cell wall-denatured cortical cells by OE1-1 leads to further infection of OE1-1 in xylem vessels.Therefore, QS is required for OE1-1 virulence.
OE1-1 produces methyl 3-hydroxymyristate (3-OH MAME) as the QS signal (Kai et al., 2015;Ujita et al., 2019).The QS signal is synthesized by the methyltransferase PhcB and is sensed through the sensor histidine kinase PhcS.The 3-OH MAME sensing through PhcS leads to the phosphorylation of the regulators PhcQ and PhcR; PhcQ and PhcR strongly and partially contribute, respectively, to the regulation of QS-dependent genes by the LysR family transcription regulator PhcA, which has a conserved structure with an N-terminal DNA-binding helix-turn-helix motif and a C-terminal co-inducer-binding domain (Maddocks & Oyston, 2008;Takemura et al., 2021).The sensor histidine kinase PhcK is required for full expression of phcA, independent of 3-OH MAME sensing (Senuma et al., 2020).In the active state of QS, PhcA regulates QS-dependent genes responsible for QS-dependent phenotypes including virulence and induces the production of the virulencerelated aryl-furanone secondary metabolites, ralfuranones (Kai et al., 2014;Kai et al., 2016), and the major exopolysaccharide EPS I (Garg et al., 2000;Schell, 2000).These secondary metabolites are associated with the feedback loop of QS-dependent gene regulation (Hayashi, Senuma, et al., 2019;Mori et al., 2018).In the QS active state, expression of lecM, encoding the lectin LecM, is induced, which affects the activation of QS through regulating the stability of extracellularly secreted 3-OH MAME (Hayashi, Kai, et al., 2019).Furthermore, QS-inducible β-1,4-cellobiohydrolase is involved not only in plant cell wall degradation (Liu et al., 2005;Tsujimoto et al., 2008), but also in the full expression of phcA, thereby contributing to the QS feedback loop and virulence of OE1-1 (Senuma et al., 2023).
The deduced amino acid sequence of ChpA based on its nucleotide sequence confirms the presence of not only a response regulator receiver domain but also a hybrid sensor histidine kinase/ response regulator phosphor-acceptor receiver domain.ChpA is conserved among RSSC strains.ChpA is an intracellular soluble protein that lacks transmembrane helices and a DNA-binding domain (Corral et al., 2020;Shen et al., 2020).ChpA is involved in the QS-dependent regulation of biofilm formation (Corral et al., 2020), chemotaxis (Corral et al., 2020), exopolysaccharide production (Shen et al., 2020), cellulase activity (Shen et al., 2020), and swarming activity (Corral et al., 2020), but not the swimming activity (Corral et al., 2020) of GMI1000.Furthermore, deletion of chpA in GMI1000 also leads to significantly reduced virulence (Corral et al., 2020;Shen et al., 2020).However, how ChpA regulates these QS-dependent phenotypes of RSSC including virulence remains unclear.
To elucidate the virulence mechanisms of OE1-1, it is important to comprehensively analyse its QS signalling pathway.Here, to elucidate the role of ChpA in QS-dependent phenotypes including their virulence on tomato plants, we analysed the transcriptomes of a chpA-deletion mutant (ΔchpA, Table 1) from OE1-1 and a transformant of ΔchpA with native chpA (chpA-comp, Table 1) using reverse transcription-quantitative real-time PCR (RT-qPCR) and RNAsequencing (RNA-seq).
It has been reported that the phcA mutant exhibits faster in vitro growth than the parent strain (Khokhani et al., 2017;Peyraud et al., 2016), suggesting that the in vitro growth rate of RSSC is negatively associated with QS activation.Compared with OE1-1 and chpA-comp, ΔchpA exhibited faster in vitro growth, like ΔphcA (p < 0.05, t test; Figure 1d).

| Deletion of chpA significantly affected the expression levels of QS-dependent genes
The expression of three genes-epsB, a component of the eps operon that is required for EPS I biosynthesis (Huang & Schell, 1995); ralA, encoding ralfuranone synthase (Kai et al., 2014); and lecM (Mori et al., 2016)-is dependent on QS.The expression of the flagellar motility-related gene fliC, encoding flagellin, is suppressed during QS (Tans-Kersten et al., 2001).Using RT-qPCR, we analysed the transcript levels of these genes in the R. pseudosolanacearum strains grown in quarter-strength M63 medium until OD 600 = 0.3.

| Deletion of chpA did not influence the regulation of QS-related genes
The QS-dependent phenotypes differed significantly between ΔchpA and OE1-1.To explore the reason for these differences, we conducted RT-qPCR analyses to determine the transcript levels of QS-related genes, phcB, phcQ, phcR, phcK, and phcA, in R. pseudosolanacearum strains grown in quarter-strength M63 medium until OD 600 = 0.3.The transcript levels of these QS-related genes did not differ significantly between ΔchpA and OE1-1 (Figure 2a).Furthermore, the chpA transcript levels were not significantly different among OE1-1, the phcBdeletion mutant (ΔphcB; Table 1; Kai et al., 2015), the phcQ-deletion mutant (ΔphcQ;  2b).

| Deletion of chpA resulted in a loss of virulence
To investigate the effects of chpA on the virulence of OE1-1, we inoculated 8-week-old tomato plants with R. pseudosolanacearum strains by the root-dip method.The plants inoculated with OE1-1 exhibited wilt symptoms at 5 days after inoculation (DAI) and died by 9 DAI (Figure 3a).Like ΔphcA, ΔchpA was not virulent on tomato plants.
To investigate the behaviour of ΔchpA in roots of 4-day-old tomato seedlings using an in vitro pathosystem (Inoue et al., 2023), we observed toluidine blue-dyed sections of tomato roots co-incubated with ΔchpA under an optical microscope.After 24 h of co-incubation (HOI), we observed detached epidermis in meristematic zones and elongation zones of tomato roots co-incubated with ΔchpA, similar to those co-incubated with OE1-1 or with ΔphcA (Figure 3b).After 48 HOI, cell walls of cortical cells adjacent to the epidermis of tomato roots co-incubated with OE1-1 but not ΔchpA or ΔphcA were degraded.Furthermore, no ΔchpA or ΔphcA cells were present in cortical cells (Figure 3c).After 120 HOI, no ΔphcA cells were observed in xylem vessels.However, after 120 HOI a smaller number of ΔchpA cells was observed in xylem vessels compared to the number of OE1-1 cells (Figure 3b).

| RNA-seq transcriptome analysis of R. pseudosolanacearum strains
To comprehensively analyse the effects of chpA deletion on the regulation of QS-dependent genes, we performed RNA-seq transcriptome analyses of R. pseudosolanacearum strains grown in quarter-strength M63 medium until OD 600 = 0.3.Mapping of RNAseq reads of OE1-1 to the GMI1000 genome (Salanoubat et al., 2002) resulted in the identification of 4311 protein-coding transcripts (Table S1).The following thresholds were applied to identify genes with significant changes in transcript levels: q < 0.05 and log 2 (fold change) ≥ |2|.To identify positively and negatively QS-dependent genes, we compared their transcript levels in OE1-1 with those in ΔphcB and ΔphcA.In both ΔphcB and ΔphcA, 315 and 180 genes commonly exhibited significant down-regulation and up-regulation, respectively, and were thus inferred to be positively and negatively QS-dependent genes, respectively (Table S2).
The transcriptome analyses revealed that 549 genes (positively ChpA-dependent genes) exhibited significant down-regulation in ΔchpA compared with their transcript levels in OE1-1, while 333 genes (negatively ChpA-dependent genes) were significantly up-regulated in ΔchpA (Table S3).Among the positively ChpAdependent genes, 283 were positively QS-dependent genes (Figure 4a, Table S3).Among the negatively ChpA-dependent genes, 160 were negatively QS-dependent genes (Figure 4b, Table S3).The expression in a similar manner to PhcA (Figure 5a).On the other hand, a PCA plot for all genes or negatively QS-dependent genes suggested that the effect of ChpA on the transcriptome is different from that of PhcA, suggesting the effect of ChpA on transcriptional control is distinct for other gene sets (Figures 5b and S2).Hierarchical clustering with a heatmap representing the global gene expression pattern in OE1-1, ∆phcA, and ∆chpA showed that the effect of ChpA on the gene expression pattern is close to that of PhcA, though they are different for some gene sets (Figure 5c).The Gene Ontology (GO) terms enriched in cluster I include terms related to QS-related genes like "quorum sensing (GO:0009372)" and "lipopolysaccharide biosynthesis process (GO:0009103)", with a similar expression pattern between ∆phcA and ∆chpA (Figure 5c,d).Also, the cluster V genes, which are enriched in negatively QS-dependent genes, showed decreased expression in ∆phcA and ∆chpA, including terms like "chemotaxis (GO:0006935)" and "bacterial-type flagellum-dependent cell motility (GO:0071973)".Interestingly, some gene sets belonging to cluster II, III, or IV showed either ∆phcA-or ∆chpA-specific gene expression (Figure 5c).Cluster II, with genes up-regulated only in ∆phcA, showed significant enrichment of the terms "siderophore (GO:0019290)" and "acid-amino acid ligase activity (GO:0016881)", whereas cluster III showed ∆chpA-specific down-regulation including the enriched terms "2-dehydropantoate 2-reductase activity (GO:0008677)" and "efflux transmembrane transporter activity (GO:0015562)" (Figure 5c,d).
These results suggest that ChpA has an effect on transcriptional control in a similar manner to PhcA on QS-dependent genes, though it also has PhcA-independent effects on the R. pseudosolanacearum transcriptome and vice versa for PhcA.iron from the extracellular environment.PhcA negatively controls siderophore-mediated iron acquisition (Takemura et al., 2021).

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The GO terms enriched in cluster II showed ∆phcA-but not ∆chpA-specific gene expression (Figure 5c), and cluster II, with genes up-regulated only in ∆phcA, showed significant enrichment of siderophore-related terms (Figure 5d).To determine the effect Compared with OE1-1, ΔphcA showed even higher siderophoremediated iron acquisition activity (p < 0.05, t test; Figure 6).In contrast, ΔchpA had significantly lower siderophore-mediated iron acquisition activity than OE1-1.

| ChpA contributes to the regulation of genes involved in twitching motility governed by type IV pili in a QS-independent manner
Twitching movement is impaired in the chpA mutant generated from GMI1000 (Corral et al., 2020).To analyse the influence of chpA deletion on the transcript levels of genes involved in twitching motility governed by type IV pili, we carried out hierarchical clustering of ΔphcA and ΔchpA as well as OE1-1, based on their relative transcript levels normalized against those of genes involved in twitching motility governed by type IV pili (Table S4).The expression patterns of pilN, pilB, pilO, pilT, pilA, pilD, pilZ, pilK, and pilY1 differed between ΔchpA and OE1-1 (Figure 7).Furthermore, the expression patterns of pilD, pilZ, and pilW differed between ΔchpA and ΔphcA.
2.9 | Swimming motility of the ΔchpA mutant is enhanced compared with that of OE1-1 when incubated on modified CPG medium with 0.3% agar When incubated on modified CPG medium containing 0.3% wt/ vol agar, the ΔchpA strain generated from GMI1000 exhibits similar swimming motility to that of the parent strain GMI1000 (Corral et al., 2020).In this study, however, the ΔchpA strain generated from OE1-1 exhibited significantly enhanced swimming motility compared with that of OE1-1 when incubated on quarter-strength M63 medium containing 0.25% wt/vol agar.We then analysed the swimming motility of ΔchpA on modified CPG medium containing 0.3% wt/vol agar.Compared with the swimming motility of OE1-1, the swimming motility of the fliC-deletion mutant (ΔfliC) was significantly reduced, while those of the ΔchpA and ΔphcA mutants were significantly enhanced (p < 0.05, t test; Figure 8a).
We carried out hierarchical clustering of ΔphcA and ΔchpA as well as OE1-1 cultured on quarter-strength M63 based on their relative transcript levels normalized against those of swimming motility-related genes (Table S5).The expression patterns of chemotaxis-related genes were similar in ΔchpA and ΔphcA, but those of flagella biogenesis-related genes differed between ΔchpA and ΔphcA (Figure 8b).

| DISCUSS ION
Recently, we proposed a scenario to explain the QS signalling cascade of OE1-1 (Takemura et al., 2021).In this scenario, the sensor histidine kinase PhcK is required for full expression of The RSSC strains bear type IV pili, which are surface structures associated with a hypothetical pilus-mediated chemotaxis pathway encoded by pil-chp genes (Kang et al., 2002;Siri et al., 2014).The regulator ChpA controls twitching motility in strain GMI1000, suggesting that ChpA is a regulator of type IV pilus assembly (Corral et al., 2020).The dendrogram of swarming-related pil genes based on the transcriptome data showed significantly reduced transcript levels of pilA in ΔchpA and ΔphcA.In contrast, the expression patterns of pilD, pilZ, and pilW differed between ΔchpA and ΔphcA.
Among the positively and negatively ChpA-dependent genes, 60.7% and 56.5% were positively and negatively QS-dependent genes, respectively (Table S3).Interestingly, ChpA was found to be involved in the positive regulation of the negatively PhcA-regulated gene ripAB, encoding a type III effector.Thus, ChpA may be involved not only in the regulation of QS-dependent genes, but also in the regulation of genes including pilD, pilZ, and pilW by the alternative transcription regulator(s).
Swimming motility is repressed when QS is active.The swimming motility of ΔchpA and ΔphcA on quarter-strength M63 medium containing 0.25% wt/vol agar was significantly enhanced compared with that of OE1-1.However, the dendrogram of swimming motilityrelated genes based on the transcriptome data showed that the transcript levels of some flagella biogenesis-related genes such as fliE, flgA, fliH, fliI, and fliK differed between ΔphcA and ΔchpA, but were similar in ΔphcA and OE1-1.It is thus thought that these genes may be regulated by alternative transcription regulator(s) rather than PhcA and that ChpA may participate in this regulation.When incubated on modified CPG medium containing 0.3% wt/vol agar, the ΔchpA mutant derived from strain GMI1000 exhibits similar swimming motility to that of the parent strain GMI1000 (Corral et al., 2020).However, in our swimming assay using modified CPG medium containing 0.3% wt/vol agar, the swimming motility of ΔchpA was higher than that of OE1-1.Furthermore, chpA deletion in strain OE1-1 led to a loss in virulence.However, chpA deletion in strain GMI1000 led to moderately reduced virulence (Corral et al., 2020) or a loss in virulence (Shen et al., 2020).QS is conserved in all RSSC strains and is required for RSSC virulence (Castillo & Agathos, 2019;Hikichi et al., 2017).It is thus thought that the gene regulation by the alternative transcription F I G U R E 6 Siderophore-mediated iron acquisition activity of Ralstonia pseudosolanacearum strain OE1-1 and the phcA-deletion (ΔphcA) and chpA-deletion (ΔchpA) mutants grown in PY medium.
Three replicate experiments were conducted using independent samples, with eight technical replicates per assay.Means were analysed for significant differences between R. pseudosolanacearum strains by analysis of variance followed by Tukey-Kramer's honestly significant difference test.Statistically significant differences are indicated by different lowercase letters (p < 0.05).All R. pseudosolanacearum strains were routinely grown in quarterstrength M63 medium at 30°C.Escherichia coli strains were grown in lysogeny broth medium (Hanahan, 1983) at 37°C.Gentamycin (50 μg/mL) was used in selective media.

| General DNA manipulations
Isolation of genomic DNA, plasmid DNA manipulations, PCR, and Southern blot analyses were performed using standard techniques (Sambrook et al., 1989).OE1-1 was transformed by electroporation (Kanda et al., 2003).Double-stranded DNA sequencing templates were prepared with the GenElute Plasmid Miniprep Kit (Sigma).
Sequences were determined using an Automated DNA Sequencer (ABI Prism 3100-Avant Genetic Analyzer, Applied Biosystems).DNA sequence data were analysed using DNASIS-Mac software (Hitachi Software Engineering).

| Generation of a chpA-deletion mutant transformed with native chpA
The 7496-bp DNA fragment was amplified by PCR from OE1-1 using the primers 0672-1-FW and 0672-2-RV and then digested with BamHI and HindIII to yield a 7.5-kb fragment.The 7.5-kb fragment was ligated into the BamHI and HindIII sites of the pUC18-mini-Tn7T-Gm vector (Choi et al., 2005) to produce pUC18-mini-Tn7T-Gm-chpA.This plasmid was electroporated into ΔchpA competent cells with a Tn7 transposase expression vector pTNS2 (Choi et al., 2005).Finally, a gentamycin-resistant transformant, chpAcomp (Table 1), was selected.

| Transcriptome analysis based on RNA-seq
Total RNA was extracted from R. pseudosolanacearum strains grown in quarter-strength M63 until OD 600 = 0.3 with a High Pure RNA Isolation kit (Roche Diagnostics).Ribosomal RNA was eliminated from the extracted total RNA using a Ribo-Zero rRNA Removal kit (Gram-negative bacteria; Illumina) as previously described (Hayashi, Kai, et al., 2019).Then, oriented, paired-end RNA-seq (2 × 100 bp) was performed on an Illumina HiSeq 2500 system and DNBSEQ-G400.

| Differential gene expression analysis
Statistical analysis of the RNA-seq data was performed using R cran (R Core Team, 2022).Genes with zero counts in at least one OE1-1 sample in the raw count data set were excluded.The RNA-seq read counts of the remaining genes were normalized using the function calcNormFactors (trimmed mean of M-values normalization) using Three replicate experiments were conducted using independent samples, with eight technical replicates per assay.Means were analysed for significant differences between R. pseudosolanacearum strains by analysis of variance followed by Tukey-Kramer's HSD test (p < 0.05).
The infection process of R. pseudosolanacearum strains in tomato roots was observed using the tomato-R.solanacearum model system (Inoue et al., 2023).In brief, seeds of tomato (S. lycopersicum 'Ohgata-Fukuju') plants were sterilized with 70% ethanol solution for 20 s and with 1% sodium hypochlorite solution for 20 min.The seeds were sown on 1% agar in Petri dishes at 30°C under dark and axenic conditions.Four days after sowing, seedlings with approximately 20-mm long roots were placed on agar plates, and 200 μL of OE1-1 suspension at 10 8 cfu/mL was added to the centre of the plate, 10 mm away from the tomato roots.The tomato seedlings were then incubated at 30°C under dark and axenic conditions.
Root pieces were excised 4 mm from the tip, fixed in half-strength Karnovsky's fixative overnight at 4°C, and then post-fixed in 1% osmium tetroxide for 1 h at room temperature (Karnovsky, 1965).The specimens were dehydrated in an ethanol series (three times at 30%, 50%, 70%, 80%, 90%, and 100%) for 10 min each at 25°C and then embedded in Quetol-812 resin (Nisshin EM).Serial semithin sections (800 nm thick) were cut with a diamond knife, mounted on glass slides, stained with toluidine blue, and observed under a BX53 optical microscope equipped with a DP74 camera (Olympus).
To assay the influence of the chpA deletion on the infectivity of R. pseudosolanacearum strains in cortical cells, we counted the number of cortical cells with a nucleus and infected with R. pseudosolanacearum adjacent to the epidermis in the elongation zone using microscopic imaging data of serial semithin sections of tomato roots after 48 HOI with R. pseudosolanacearum.Means were analysed for significant differences between R. pseudosolanacearum strains by analysis of variance followed by Tukey-Kramer's HSD test (p < 0.05).

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Biofilm formation (a), production of the major exopolysaccharide EPS I (b), swimming motility (c), and the in vitro growth rate (d) of Ralstonia pseudosolanacearum strain OE1-1, the phcA-deletion (ΔphcA) and chpA-deletion (ΔchpA) mutants, and ΔchpA transformed with native chpA (chpA-comp).(a) Biofilm formation of R. pseudosolanacearum incubated in quarter-strength M63 medium was quantified by measuring absorbance at 550 nm (A 550 ) and normalized to the number of cells (optical density at 600 nm, OD 600 ).Three replicate experiments were conducted using independent samples, with seven technical replicates per experiment.(b) R. pseudosolanacearum strains were incubated on quarter-strength M63 medium solidified with 0.25% wt/vol agar.EPS I content of the supernatant was quantified by ELISA.Three replicate experiments were conducted using independent samples, with seven technical replicates per experiment.(c) R. pseudosolanacearum strains were grown on quarter-strength M63 medium solidified with 0.25% wt/vol agar.Three replicate experiments were conducted using independent samples, with five technical replicates per experiment.(d) Overnight cultures of R. pseudosolanacearum strains were diluted to OD 600 = 0.01 with quarter-strength M63 and grown with shaking.The in vitro growth rate of bacterial strains was quantified based on OD 600 of the bacterial culture.Three replicate experiments were conducted using independent samples, with three technical replicates per experiment.Bars indicate standard error.Means were analysed for significant differences between R. pseudosolanacearum strains by analysis of variance followed by Tukey-Kramer's honestly significant difference test.Statistically significant differences are indicated by different lowercase letters (p < 0.05).
transcript levels of ChpA-dependent genes among QS-dependent genes were strongly and positively correlated between ΔchpA and ΔphcA (y = 0.9903x − 0.1491, r 2 = 0.9605; where y is log[fold change in ΔphcA] and x is log[fold change in ΔchpA]; Figure 4c).These results indicate that ChpA is strongly involved in the regulation of 283 genes (89.9%) and 160 genes (88.9%) among positively and negatively QS-dependent genes, respectively.2.6 | ChpA affected the genome-wide transcriptome for some gene sets in a PhcA-independent mannerTo determine the global effect of ChpA on the R. pseudosolanacearum transcriptome, we further performed principal component analysis (PCA) on a certain gene set with the RNA-seq data of ΔchpA as well as ΔphcA.The resultant loadings for positively QS-dependent genes indicated that ChpA contributes to positively PhcA-regulated gene ChpA is involved in the positive control of siderophore-mediated iron acquisition in a QS-independent manner RSSC strains produce micacocidin(Kreutzer et al., 2011) and staphyloferrin B(Bhatt & Denny, 2004) as siderophores to acquire F I G U R E 2 Gene transcript levels in wild-type and mutant strains.(a) Transcript levels of chpA in Ralstonia pseudosolanacearum strain OE1-1 and the phcB-deletion (ΔphcB), phcQ-deletion (ΔphcQ), phcR-deletion (ΔphcR), phcKdeletion (ΔphcK), and phcA-deletion (ΔphcA) mutants.(b) Transcript levels of quorum sensing (QS)-related genes phcB, phcQ, phcR, phcK, and phcA, and QS-dependent genes epsB, ralA, lecM, and fliC, in OE1-1 and the chpA-deletion mutant (ΔchpA).Gene transcript levels were normalized to that of rpoD.The experiments were performed with three biological replicates and two technical replicates.Bars indicate standard errors.Asterisks indicate a significant difference from OE1-1 (p < 0.05, t test).

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Virulence of Ralstonia pseudosolanacearum strains on tomato plants (a), microscopic observation of roots of 4-day-old tomato seedlings co-incubated with R. pseudosolanacearum strains (b), and percentages of cell wall-degraded cortex cells infected with R. pseudosolanacearum strains in the meristematic zone in roots of 4-day-old tomato seedlings after 48 h of co-incubation (HOI) with R. pseudosolanacearum strains (c).(a) Eight-week-old tomato plants were inoculated with R. pseudosolanacearum strain OE1-1, the phcAdeletion (ΔphcA) and chpA-deletion (ΔchpA) mutants, and ΔchpA transformed with native chpA (chpA-comp).Plants were rated 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; 5, dead.For each bacterial strain, three independent groups were tested, with 12 technical replicates per group.Bars indicate standard error.Means were analysed for significant differences between R. pseudosolanacearum strains by analysis of variance followed by Tukey-Kramer's honestly significant difference test.Statistically significant differences are indicated by different lowercase letters (p < 0.05).(b) Mock and 24, 48, and 120 HOI with R. pseudosolanacearum strains OE1-1, ΔphcA, and ΔchpA.Longitudinal semithin resin sections were stained with toluidine blue.Red arrows indicate bacterial cells.Bars indicate 20 μm.e, epidermal cell; c, cortical cell; en, endodermis cell; pe, pericycle cell; v, xylem vessel; mBF, mushroom-shaped biofilm.(c) Micrographs of toluidine blue-dyed serial semi-thin sections (800 nm thick) from the meristematic zone in roots were observed.The experiment was repeated five times, each with 100 technical replicates.Bars indicate standard error.Means were analysed for significant differences between R. pseudosolanacearum strains by analysis of variance followed by Tukey-Kramer's honestly significant difference test.Statistically significant differences are indicated by different lowercase letters (p < 0.05). of ChpA on siderophore-mediated iron acquisition, we measured iron acquisition activity in different R. pseudosolanacearum strains.
phcA, encoding the LysR family transcription regulator PhcA, independently of QS signal production by PhcB(Senuma et al., 2020).PhcQ contributes to the regulation of QS-dependent genes, in which PhcR is partially involved, dependent on sensing of 3-OH MAME through the sensor kinase PhcS(Takemura et al., 2021).The regulator ChpA in RSSC strains contains an REC domain and a CheA-like domain, but it lacks transmembrane helices and a DNA-binding domain.ChpA is an intracellular soluble protein that is conserved among RSSC strains(Corral et al., 2020;Shen et al., 2020).Results in this study showed that ChpA is involved in the regulation of 89.9% and 88.9% of positively and negatively QS-dependent genes, respectively, suggesting a large overlap between genes regulated by the global QS regulator PhcA and genes that are regulated by ChpA.It is thus thought F I G U R E 4 RNA-sequencing transcriptome analysis of Ralstonia pseudosolanacearum strains grown in quarter-strength M63 medium until OD 600 = 0.3.(a) Numbers of genes with transcript-level log 2 (fold change) ≤ −2 in the chpA-deletion (ΔchpA) and phcAdeletion (ΔphcA) mutants relative to their transcript levels in OE1-1 (q < 0.05).(b) Number of genes with transcript-level log 2 (fold change) ≥ 2 in ΔchpA and ΔphcA relative to their transcript levels in OE1-1 (q < 0.05).(c) Correlation of transcript levels of ChpA-dependent genes among quorum sensing (QS)-dependent genes between R. pseudosolanacearum mutants: ΔchpA versus ΔphcA.that ChpA might participate in the regulation of these genes by PhcA, similar to PhcQ and PhcR, or by alternative transcription regulator(s).The comparative transcriptome analysis showed that some gene sets belonging to cluster II, III, or IV in the hierarchical clustering analysis and the heatmap of relative transcript levels are differentially expressed between at least two genotypes in OE1-1.Furthermore, ΔphcA and ΔchpA showed either ∆phcA-or ∆chpA-specific gene expression.These results suggest that ChpA has PhcA-independent effects on the regulation of these genes.Deletion of chpA significantly reduces the bacterial population in tomato plants, leading to significantly reduced virulence (Corral et al., 2020; Shen et al., 2020), consistent with the results of our virulence assays.However, different from ΔphcA, a smaller number of ΔchpA cells infected the intercellular spaces of the cortex, leading F I G U R E 5 Global effect of ChpA on gene expression in Ralstonia pseudosolanacearum.A principal component analysis plot for the transcriptome data of positively quorum sensing (QS)-dependent genes (a) and all genes (b) in OE1-1 and phcA-deletion (ΔphcA), phcRdeletion (ΔphcR), phcQ-deletion (ΔphcQ), and chpA-deletion (ΔchpA) mutants.(c) Hierarchical clustering and heatmap analysis of relative transcript levels of genes that were differentially expressed between at least two genotypes (OE1-1, ΔphcA, and ΔchpA) based on RNAsequencing data with the samples grown in quarter-strength M63 medium until OD 600 = 0.3.Values are averages of three replicates per strain.Hierarchical clustering by the complete linkage method was applied to z-scores calculated with normalized log 2 (fold change) values of all normalized mean expression values (counts per million) using the hclust R package.The heatmap was created with the R package pheatmap.(d) GO terms enriched in each cluster shown in (c).toinfectivity to the xylem vessels as described byCorral et al. (2020) andShen et al. (2020).The colonization of intercellular spaces of the cortex and xylem vessels by OE1-1 may thus be affected by QSdependent genes but not ChpA-dependent genes.We previously reported that QS-deficient mutants lose their ability to infect cell wall-degraded cortical cells and form mushroom-shaped biofilms (mBFs) in the cortical cells(Inoue et al., 2023).The transcriptome analyses revealed significantly reduced transcript levels of plant cell wall-degrading enzyme genes in ΔchpA compared with OE1-1, similar to ΔphcA.The microscopic analyses confirmed that ΔchpA had lost its ability to degrade cortical cell walls and could not infect cell wall-denatured cortical cells and form mBFs, which ultimately significantly reduced its virulence.It is thus thought that mBF formation in cell wall-degraded cortical cells dependent on both PhcA and ChpA is required for virulence of OE1-1.

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Hierarchical clustering of relative transcript levels of genes involved in twitching motility governed by type IV pilus genes in Ralstonia pseudosolanacearum strain OE1-1 and the phcA-deletion (ΔphcA) and chpA-deletion (ΔchpA) mutants grown in quarter-strength M63 medium until OD 600 = 0.3.Based on RNAsequencing transcriptome data for R. pseudosolanacearum strains, fragments per kilobase of open reading frame per million fragments mapped values from R. pseudosolanacearum strains OE1-1, ΔphcA, and ΔchpA were normalized prior to analysis of differentially expressed genes.Hierarchical clustering of all normalized mean expression values (counts per million) was performed using Cluster v. 3.0 software.Heatmaps were created with TreeView.regulator(s) may affect RSSC virulence independently of QS, and the regulatory effects of ChpA on gene expression may differ among RSSC strains.In response to iron limitation, genes involved in bacterial siderophore production and uptake are derepressed, leading to the production of siderophores and appropriate uptake proteins(Andrews et al., 2003;Ratledge & Dover, 2000).Compared with OE1-1, ΔphcA exhibited significantly enhanced siderophore-mediated iron acquisition activity, whereas ΔchpA exhibited significantly reduced siderophore-mediated iron acquisition activity.Though hierarchical clustering with a heatmap representing the global gene expression pattern in OE1-1, ∆phcA, and ∆chpA showed that the effect of ChpA on gene expression is close to that of PhcA, the effects are different for some gene sets.Interestingly, cluster II, with genes up-regulated F I G U R E 8 (a) Swimming motility of Ralstonia pseudosolanacearum strain OE1-1 and the phcA-deletion (ΔphcA), chpA-deletion (ΔchpA), and fliC-deletion (ΔfliC) mutants grown on modified CPG medium containing 0.3% wt/vol agar and (b) hierarchical clustering of relative transcript levels of flagellar motility-related genes in OE1-1, ΔphcA, and ΔchpA grown in quarter-strength M63 medium until OD 600 = 0.3.(a) The experiment was repeated three times, each with five technical replicates.Bars indicate standard errors.Means were analysed for significant differences between R. pseudosolanacearum strains by analysis of variance followed by Tukey-Kramer's honestly significant difference test.Statistically significant differences are indicated by different lowercase letters (p < 0.05).(b) Based on RNA-sequencing transcriptome data for R. pseudosolanacearum strains, fragments per kilobase of open reading frame per million fragments mapped values from OE1-1, ΔphcA, and ΔchpA were normalized prior to analysis of differentially expressed genes.The average value of three replicates per strain was used.Hierarchical clustering of all normalized mean expression values (counts per million) was performed using Cluster v. 3.0 software.Heatmaps were created with TreeView.in∆phcA but not in ∆chpA, showed significant enrichment of the term "siderophore (GO:0019290)".Therefore, ChpA may be involved in the regulation of siderophore-mediated iron acquisition activity, independently of QS.Results in the present study suggest that ChpA is involved in the regulation of about 90% of QS-dependent genes, thereby contributing to the behaviour in host plant roots and the virulence of strain OE1-1.ChpA is thus involved in the gene regulation required for virulence of OE1-1, though how ChpA regulates these genes remains unclear.To explore the role of ChpA in gene regulation, in vitro experiments with purified proteins are required.Such analyses will shed further light on the regulation of not only PhcA-dependent virulence-related but also alternative transcription regulator(sBacterial strains, plasmids, and growth conditions