HexR Transcription Factor Contributes to Pseudomonas cannabina pv. alisalensis Virulence by Coordinating Type Three Secretion System Genes

Pseudomonas cannabina pv. alisalensis (Pcal) causes bacterial blight on cabbage. We previously conducted a screening for reduced virulence using Tn5 transposon mutants and identified one of the transcriptional factors, HexR, as a potential Pcal virulence factor. However, the role of HexR in plant pathogenic Pseudomonas virulence has not been investigated well. Here, we demonstrated that the Pcal hexR mutant showed reduced disease symptoms and bacterial populations on cabbage, indicating that HexR contributes to Pcal virulence. We used RNA-seq analysis to characterize the genes regulated by HexR. We found that several type three secretion system (T3SS)-related genes had lower expression of the Pcal hexR mutant. Five genes were related to T3SS machinery, two genes were related to type three helper proteins, and three genes encoded type three effectors (T3Es). We also confirmed that T3SS-related genes, including hrpL, avrPto, hopM1, and avrE1, were also down-regulated in the Pcal hexR mutant both in culture and in vivo by using RT-qPCR. T3SS functions to suppress plant defense in host plants and induce hypersensitive response (HR) cell death in non-host plants. Therefore, we investigated the expression profiles of cabbage defense-related genes, including PR1 and PR5, and found that the expressions of these genes were greater in the Pcal hexR mutant. We also demonstrated that the hexR mutant did not induce HR cell death in non-host plants, indicating that HexR contributes in causing HR in nonhost plants. Together, these results indicate that the mutation in hexR leads to a reduction in the T3SS-related gene expression and thus an impairment in plant defense suppression, reducing Pcal virulence.


Introduction
In natural habitats, plants are constantly surrounded by an enormous number of microorganisms, including potential pathogens. Therefore, plants have developed monitoring systems that recognize invading pathogens and regulate immune responses to defend themselves against pathogens. The first line of defense is pathogen-associated molecular patterns (PAMPs)-triggered immunity (PTI). PTI is initiated after recognition of PAMPs, including flagellin and elongation factor Tu (EF-Tu), using plasma membrane-localized pattern recognition receptors (PRRs) such as FLAGELLIN-SENSING2 (FLS2) and EF-Tu

Bacterial Strains, Plasmids, and Growth Conditions
The bacterial strains and plasmids used in this study are described in Table S1. Pseudomonas cannabina pv. alisalensis strain KB211 (Pcal KB211) was used as the pathogenic strain. Pcal wild-type (WT) was grown on King's B (KB; [26]) medium at 28 • C. The hexR mutant was grown on KB containing kanamycin (Km) (10 µg/mL). The hexR mutant complemented with pDSKG-hexR was grown on KB containing Km (10 µg/mL) and gentamicin (Gen) (25 µg/mL) (Table S1). Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) was used as the pathogenic strain on Arabidopsis thaliana. Pst wild-type (WT) and ∆hexR was grown on KB medium at 28 • C. Before Pcal and Pst inoculation, bacteria were suspended in sterile distilled H 2 O, and the bacterial cell densities at 600 nm (OD 600 ) were measured using a Biowave CO8000 Cell Density Meter (Funakoshi, Tokyo, Japan).

Complementation of the hexR Mutant
The hexR-complemented strain was constructed as described in Ishiga et al. (2018) [27]. Briefly, the pDSKG vector [28] replaced the kanamycin cassette to gentamycin. The hexR was transferred into the pDSKG vector to generate pDSKG-hexR. The pDSKG-hexR construct was introduced into the hexR mutant by electroporation to generate the complemented strain.

Generation of the Pseudomonas syringae pv. tomato ∆hexR Mutant
The mutant was generated as described previously (Ishiga et al. 2018) [27]. Briefly, the genetic regions containing hexR and surrounding regions were amplified using PCR primer sets (Table S2) and inserted into the pGEM-T Easy vector (Promega, Madison, WI, USA). The inverse PCR was carried out, using a primer set (Table S2), to delete the hexR open reading frame. After digesting with BamHI and DpnI, the resultant DNA was self-ligated with T4 DNA ligase (Ligation-Convenience kit, Nippon Gene, Tokyo). The hexR-deleted DNA constructs were introduced into pK18mobsacB [29] and then transformed into E. coli S17-1. The deletion mutant was obtained by conjugation and homologous recombination according to the method previously reported [30].

Bacterial In Vitro Growth Measurements
Pcal WT, the Pcal hexR mutant, and the Pcal hexR mutant complemented with pDSKG-hexR were grown at 28 • C on KB medium. The bacterial suspensions were standardized to an OD 600 of 0.01 with KB, and bacterial growth was measured at OD 600 for 24 h.

Plant Materials
Cabbage (Brassica oleracea var. capitata) cv. Kinkei 201 was used for Pcal virulence assays. All plants were grown from seed at 23-25 • C with a light intensity of 200 µEm −2 s −1 and a 16 h light/8 h dark photoperiod.
A. thaliana (Col-0) was used for Pst virulence assays. A. thaliana seeds were germinated and grown on 1/2 strength Murashige and Skoog (MS) medium (0.3% phytagel) with Gamborg vitamins (Sigma-Aldrich, St. Louis, MO, USA). Seedlings were incubated in a growth chamber at 24 • C with a light intensity of 200 µEm −2 s −1 and a 12 h light/12 h dark photoperiod.
Nicotiana tabacum var. Xanthi was used for hypersensitive reaction (HR) cell assays. The plants were grown from seed at 23-25 • C with a light intensity of 200 µEm −2 s −1 and a 16 h light/8 h dark photoperiod.

Bacterial Populations Measurements
To assess bacterial growth in plants, the internal bacterial populations in the plant were evaluated. Dip-and flood-inoculated leaves were surface-sterilized with 10% H 2 O 2 for 3 min. After washing with sterile distilled water, the leaves were homogenized in sterile distilled water, and diluted samples were plated onto solid KB agar medium. The bacterial populations at 0 dpi were estimated using leaves harvested 1 h post-inoculation (hpi) without surface sterilization. For syringe inoculation, to assess bacterial growth in cabbage, leaf discs were harvested using a cork borer. The plant samples were homogenized in sterile distilled water, and diluted samples were plated onto solid KB agar medium. The bacterial colony-forming units (CFUs) were counted and normalized as CFU per gram or CFU per cm 2 , using the total leaf weight or leaf square centimeters. The bacterial populations were evaluated in at least three independent experiments.

RNA Purification
For expression profiles of Pcal WT and hexR mutant genes in culture, bacteria were grown in KB broth for 24 h, then adjusted to an OD 600 of 0.3 with fresh KB broth and grown for 3 h further, and then incubated in MG medium for 30 min. Total RNA was extracted using Reliaprep (Promega) according to the manufacturer's protocol.
To analyze Pcal and cabbage gene expression profiles during infection, we dip-inoculated cabbage plants with Pcal at 1 × 10 8 CFU/mL, and at 4 and 48 h the total RNAs including plant and bacterial RNAs were extracted from infected leaves and purified using RNAiso Plus (Takara Bio, Kusatsu, Japan).

RNA-Seq Analysis
After total RNAs were isolated from Pcal WT and the hexR mutant, rRNAs were almost depleted from the total RNAs by using the RiboMinus Transcriptome Isolation Kit for bacteria (Thermo Fisher Scientific Inc., Waltham, MA, USA). The RNA-Seq library was constructed from sample RNAs with the Ion Total RNA-Seq Kit v2 (Thermo Fisher Scientific Inc., Waltham, MA, USA) and Ion Xpress RNA-Seq barcode (Thermo Fisher Scientific Inc., Waltham, MA, USA). Subsequently, using the Ion 540 Chef Kit on an Ion Chef system (Thermo Fisher Scientific Inc., Waltham, MA, USA), RNA-Seq templates were prepared. Sequencing of the amplicon libraries was performed using an Ion 540 Chip with the Ion GeneStudio S5 system (Thermo Fisher Scientific Inc., Waltham, MA, USA). Sequence data were assembled and analyzed with the CLC Genomics Workbench (Qiagen, Valencia, CA, USA). The P. syringae pv. maculicola ES4326 genome sequence (GeneBank accession number: CP047260) was used as the reference for RNA-Seq mapping and assembly of sequence reads. The gene expression value was calculated from the ratio of the number of mapped reads to "reads per kilobase million (RPKM)" in Pcal WT and the hexR mutant. Since the mutant strain used in this study might affect the expression profiles of the downstream genes, the analysis was conducted by using RPKM, which is more suitable for characterizing each strain compared to "transcripts per million (TPM)". The subsequent mathematical analyses were all performed by the R program as follows: expression ratio of the hexR mutant against Pcal WT was calculated using the GLM method implemented in the edgeR package according to McCarthy et al. (2021) [32]. At this time statistical data were obtained by one-way ANOVA. The adjusted p-value and FDR values shown in this study are cited values of these statistical results. Additionally, as is well known, when the sample parameter is small or the number of iterations is small, the p-value tends to be large [33]. Moreover, because expression comparison is performed by a large number of genes between Pcal WT and the hexR mutant in this study, the p-value may become large. Thus, the second type of error in statistical analysis (Type II error), which produces false negative results, is likely to occur. Therefore, to avoid a Type II error as much as possible, genes were filtered at 0.1 of permissive FDR value, and the effect size (eta-squared) was calculated and attached to the statistical data of each gene expression. The eta-squared was calculated by the R program using the etaSquared method implemented in the Isr package.

RT-qPCR
The DNase-treated RNA was reverse-transcribed using the ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan). The cDNA (1:10) was then used for RT-qPCR using the primers shown in Table S2 with THUNDERBIRD SYBR qPCR Mix (TOYOBO) on a Thermal Cycler Dice Real Time System (Takara Bio). Pcal KB211 outer membrane porin F (oprF) and recombinase A (recA) were used to normalize gene expression. Cabbage UBIQ-UITIN EXTENSION PROTEIN 1 (BoUBQ1) was used as an internal control to normalize gene expression.

Hypertrophy-Inducing Activity Assay on Potato Tuber Tissue
Hypertrophy-inducing activity assay was conducted as described in Nguyen et al. (2021) [28]. Briefly, potato tuber discs were inoculated using toothpicks by placing the tip in Pcal WT and the hexR mutant on a KB medium plate, and then placing the toothpick on the potato tuber disc. The discs were then placed at 23 • C in an incubator (darkness) for 5 days. Photographs were taken at 5 dpi.

Hypersensitive Response Assays
For HR assays, 2-month-old Nicotiana tabacum var. Xanthi plants were used. The leaves were syringe-infiltrated with Pcal WT and the hexR mutant with an OD 600 of 0.1 (5 × 10 7 CFU/mL). HR cell death symptoms were photographed at 24 hpi.

Importance of HexR in Pseudomonas Virulence
We first measured the Pseudomonas cannabina pv. alisalensis (Pcal) WT, hexR mutant, and hexR-complemented strain growth in vitro. The Pcal hexR mutant tended to be defective in growth in comparison with that of Pcal WT and the complemented strain at 24 h and 48 h after incubation in KB media ( Figure S1). To investigate the importance of HexR in Pcal virulence, we challenged cabbage with Pcal WT, the hexR mutant, and the hexRcomplemented strain by dip inoculation. Cabbage leaves inoculated with Pcal WT showed severe chlorosis, but the Pcal hexR mutant showed no symptoms ( Figure 1A), indicating that the Pcal hexR mutant had lost its pathogenicity. Moreover, bacterial populations of the Pcal hexR mutant were significantly reduced compared to Pcal WT and the complemented strain ( Figure 1B). To further investigate whether HexR is important for virulence in the intercellular space (apoplast), we also conducted syringe inoculation. When infiltrated directly into the cabbage apoplast, the Pcal hexR mutant also showed no symptoms and reduced bacterial populations compared to Pcal WT and the complemented strain ( Figure 1C,D). The disease symptoms and bacterial populations of the complemented strains were greater than that of the Pcal hexR mutant, but less than that of Pcal WT, indicating that the complemented strains partially restored their virulence ( Figure 1A-D). Together, these results indicate that HexR is required for Pcal virulence in cabbage. gae pv. tomato (Pst) DC3000 WT and the hexR mutant (ΔhexR) on A. thaliana. Disease symptoms caused by Pst WT showed severe chlorosis ( Figure S2A). Although disease symptoms by Pst ΔhexR showed less chlorosis compared to Pst WT, the Pst ΔhexR also still caused disease symptoms ( Figure S2A). Bacterial populations did not show any significant differences between Pst WT and ΔhexR ( Figure S2B). These results suggest that HexR does not contribute to Pst virulence in A. thaliana. and bacterial populations (B) in cabbage dip-inoculated with WT, the hexR mutant, and the hexR mutant complemented with pDSKG-hexR. Disease symptoms (C) and bacterial populations (D) in cabbage syringe-inoculated with WT, the hexR mutant, and the hexR mutant complemented with pDSKG-hexR. Cabbage was dip-inoculated with 5 × 10 7 CFU/mL of inoculum containing 0.025% SilwetL-77 and syringe-inoculated with 5 × 10 4 CFU/mL of inoculum, respectively. Bacterial populations in the plant leaves were evaluated at 0, 3, and 5 dpi. The leaves were photographed 5 days after dip inoculation, and 3 days after syringe inoculation. Vertical bars indicate the standard error for at least three independent experiments. ns indicates not significant. Statistical analysis was performed using one-way ANOVA with Tukey's HSD test. Scale bar shows 1 cm.

Gene Expression Profiles of Pseudomonas cannabina pv. alisalensis WT and the hexR Mutant
To compare gene expression profiles, Pcal WT and the hexR mutant were incubated in culture, and the complete transcriptome was determined for each strain using RNA-seq analysis. This analysis identified 21 HexR-dependent genes for which expression changed by twofold or more and had FDR values of less than 1% (Table 1). Several genes that encode genes related to the type three secretion system (T3SS), coronatine (COR), ABC transporter, and auxin biosynthesis, had less expression in the Pcal hexR mutant compared to Cabbage was dip-inoculated with 5 × 10 7 CFU/mL of inoculum containing 0.025% SilwetL-77 and syringe-inoculated with 5 × 10 4 CFU/mL of inoculum, respectively. Bacterial populations in the plant leaves were evaluated at 0, 3, and 5 dpi. The leaves were photographed 5 days after dip inoculation, and 3 days after syringe inoculation. Vertical bars indicate the standard error for at least three independent experiments. ns indicates not significant. Statistical analysis was performed using one-way ANOVA with Tukey's HSD test. Scale bar shows 1 cm.
We also investigated the importance of HexR in the virulence of another plant pathogenic Pseudomonas. Therefore, we conducted inoculation assays with Pseudononas syringae pv. tomato (Pst) DC3000 WT and the hexR mutant (∆hexR) on A. thaliana. Disease symptoms caused by Pst WT showed severe chlorosis ( Figure S2A). Although disease symptoms by Pst ∆hexR showed less chlorosis compared to Pst WT, the Pst ∆hexR also still caused disease symptoms ( Figure S2A). Bacterial populations did not show any significant differences between Pst WT and ∆hexR ( Figure S2B). These results suggest that HexR does not contribute to Pst virulence in A. thaliana.

Gene Expression Profiles of Pseudomonas cannabina pv. alisalensis WT and the hexR Mutant
To compare gene expression profiles, Pcal WT and the hexR mutant were incubated in culture, and the complete transcriptome was determined for each strain using RNA-seq analysis. This analysis identified 21 HexR-dependent genes for which expression changed by twofold or more and had FDR values of less than 1% (Table 1). Several genes that encode genes related to the type three secretion system (T3SS), coronatine (COR), ABC transporter, and auxin biosynthesis, had less expression in the Pcal hexR mutant compared to Pcal WT ( Table 1). The expression of several T3SS-related genes was suppressed in the Pcal hexR mutant compared to Pcal WT (Table 1). Five genes were related to T3SS machinery, two genes were related to type three helper proteins, and three genes encoded type three effectors (T3Es) ( Table 1). Moreover, hrpL, which encodes an alternate RNA polymerase sigma factor required for the expression of T3SS genes, was suppressed in the Pcal hexR mutant (Table 1). Additionally, corR, which encodes a response regulator transcriptional factor required for the expression of COR biosynthesis genes, was also suppressed in the Pcal hexR mutant compared to Pcal WT (Table 1). Therefore, we focused on genes related to the T3SS and COR. We next confirmed the gene expression profiles in culture by using RT-qPCR. The T3SS-related genes, including hrpL, avrPto, hopM1, and avrE1, were all down-regulated in the Pcal hexR mutant (Figure 2A-D). corR gene expression was also significantly reduced, but that of cmaA and cfl was not down-regulated ( Figure 2E-G). We also investigated COR production by using a hypertrophy-inducing activity test on potato tuber tissues [34]. While Pcal ∆cmaA, which is a deletion mutant for a COR biosynthesis gene, showed no hypertrophy response, the Pcal WT and hexR mutant-inoculated potato tuber tissues showed hypertrophy response ( Figure S3), indicating that the Pcal hexR mutant was not impaired in COR production. ture and the Pcal hexR mutant did not abrogate COR production, we investigated the gene expression profiles of T3SS-related genes, including hrpL, avrPto, hopM1, and avrE1, during infection. The expression of hrpL and hopM1 were not significantly different between Pcal WT and the hexR mutant at 4 hpi, but were down-regulated in the Pcal hexR mutant at 48 hpi ( Figure 3A,C). Furthermore, the expressions of avrPto and avrE1 were up-regulated in the Pcal hexR mutant at 4 hpi ( Figure 3B,D). These results indicate that HexR regulates the expression of T3SS-related genes. , hopM1 (C), and avrE1 (D)) and COR-biosynthesis-related genes (including corR (E), cfl (F), and cmaA (G)) were investigated. Total RNA was extracted for use in real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) with gene-specific primer sets (Table S2). Expression was normalized using oprE and recA. Vertical bars indicate the standard error for at least six biological replicates. Asterisks indicate a significant difference from WT in a t-test (** p < 0.01).  , hopM1 (C), and avrE1 (D)) and COR-biosynthesis-related genes (including corR (E), cfl (F), and cmaA (G)) were investigated. Total RNA was extracted for use in real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) with gene-specific primer sets (Table S2). Expression was normalized using oprE and recA. Vertical bars indicate the standard error for at least six biological replicates. Asterisks indicate a significant difference from WT in a t-test (** p < 0.01).
Since the genes related to COR biosynthesis, cmaA and cfl, were not regulated in culture and the Pcal hexR mutant did not abrogate COR production, we investigated the gene expression profiles of T3SS-related genes, including hrpL, avrPto, hopM1, and avrE1, during infection. The expression of hrpL and hopM1 were not significantly different between Pcal WT and the hexR mutant at 4 hpi, but were down-regulated in the Pcal hexR mutant at 48 hpi ( Figure 3A,C). Furthermore, the expressions of avrPto and avrE1 were up-regulated in the Pcal hexR mutant at 4 hpi ( Figure 3B,D). These results indicate that HexR regulates the expression of T3SS-related genes. showed hypertrophy response ( Figure S3), indicating that the Pcal hexR mutant was not impaired in COR production.
Since the genes related to COR biosynthesis, cmaA and cfl, were not regulated in culture and the Pcal hexR mutant did not abrogate COR production, we investigated the gene expression profiles of T3SS-related genes, including hrpL, avrPto, hopM1, and avrE1, during infection. The expression of hrpL and hopM1 were not significantly different between Pcal WT and the hexR mutant at 4 hpi, but were down-regulated in the Pcal hexR mutant at 48 hpi ( Figure 3A,C). Furthermore, the expressions of avrPto and avrE1 were up-regulated in the Pcal hexR mutant at 4 hpi ( Figure 3B,D). These results indicate that HexR regulates the expression of T3SS-related genes.  and avrE1 (D)) and COR-biosynthesis-related genes (including corR (E), cfl (F), and cmaA (G)) were investigated. Total RNA was extracted for use in real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) with gene-specific primer sets (Table S2). Expression was normalized using oprE and recA. Vertical bars indicate the standard error for at least six biological replicates. Asterisks indicate a significant difference from WT in a t-test (** p < 0.01).   (Table S2). Expression was normalized using oprE and recA. Vertical bars indicate the standard error for at least six biological replicates. Asterisks indicate a significant difference from WT in a t-test (* p < 0.05, ** p < 0.01).

Gene Expression Profiles of Plant Defense-Related Genes Inoculated with Pseudomonas cannabina pv. alisalensis WT and the hexR Mutant
Since T3SS functions to suppress plant defense, we hypothesized that the downregulation of T3SS-related genes in the Pcal hexR mutant would lead to impairment in plant defense suppression. Thus, we next investigated plant defense-related gene expression, including PR1 and PR5. The expressions of both genes were significantly greater in the Pcal hexR mutant compared to Pcal WT ( Figure 4A,B). These results indicate that HexR contributes to suppressing plant defense during infection.
Microorganisms 2023, 11, x FOR PEER REVIEW 9 of 14 merase chain reaction (RT-qPCR) with gene-specific primer sets (Table S2). Expression was normalized using oprE and recA. Vertical bars indicate the standard error for at least six biological replicates. Asterisks indicate a significant difference from WT in a t-test (* p < 0.05, ** p < 0.01).

Gene Expression Profiles of Plant Defense-Related Genes Inoculated with Pseudomonas cannabina pv. alisalensis WT and the hexR Mutant
Since T3SS functions to suppress plant defense, we hypothesized that the down-regulation of T3SS-related genes in the Pcal hexR mutant would lead to impairment in plant defense suppression. Thus, we next investigated plant defense-related gene expression, including PR1 and PR5. The expressions of both genes were significantly greater in the Pcal hexR mutant compared to Pcal WT ( Figure 4A,B). These results indicate that HexR contributes to suppressing plant defense during infection.  (Table S2). Expression was normalized using BoUBQ. Vertical bars indicate the standard error for at least six biological replicates. Asterisks indicate a significant difference from WT in a t-test (** p < 0.01).

Hypersensitive Response in Nonhost Plants Inoculated with Pseudomonas cannabina pv. alisalensis WT and the hexR Mutant
Hypersensitive response (HR) cell death is induced by hrp-associated proteins [35]. Therefore, to further investigate the impact of the Pcal hexR mutant on the hrp system, HR assays were performed on non-host tobacco (Nicotiana tabacum). HR cell death was induced in tobacco plants by Pcal WT (Figure 5). Conversely, the Pcal hexR mutant did not induce HR cell death ( Figure 5).  (Table S2). Expression was normalized using BoUBQ. Vertical bars indicate the standard error for at least six biological replicates. Asterisks indicate a significant difference from WT in a t-test (** p < 0.01).

Hypersensitive Response in Nonhost Plants Inoculated with Pseudomonas cannabina pv. alisalensis WT and the hexR Mutant
Hypersensitive response (HR) cell death is induced by hrp-associated proteins [35]. Therefore, to further investigate the impact of the Pcal hexR mutant on the hrp system, HR assays were performed on non-host tobacco (Nicotiana tabacum). HR cell death was induced in tobacco plants by Pcal WT (Figure 5). Conversely, the Pcal hexR mutant did not induce HR cell death ( Figure 5).

Discussion
One of the transcriptional factors, HexR, was identified as a potential Pseudomonas cannabina pv. alisalensis (Pcal) virulence factor [20]. However, the role of HexR in plant

Discussion
One of the transcriptional factors, HexR, was identified as a potential Pseudomonas cannabina pv. alisalensis (Pcal) virulence factor [20]. However, the role of HexR in plant pathogenic Pseudomonas virulence has not been investigated well. To investigate HexR roles in Pcal virulence, we firstly conducted inoculation assay and demonstrated that the Pcal hexR mutant showed reduced disease symptoms and bacterial populations on cabbage, indicating that HexR contributes to Pcal virulence. We also conducted RNA-seq analysis and revealed that several type three secretion system (T3SS)-related genes had less expression in the Pcal hexR mutant. Plant defense-related genes showed greater expression in cabbage inoculated with the Pcal hexR mutant compared to WT, suggesting that HexR contributes to suppressing plant defense. Together, these results suggest that the downregulation of T3SS-related genes in the Pcal hexR mutant leads to impairment in plant defense suppression, resulting in reduced Pcal virulence.
The disease symptoms and bacterial populations on cabbage inoculated with the Pcal hexR mutant were significantly reduced compared to that of Pcal WT after dip and syringe inoculation ( Figure 1). If HexR does not contribute to Pcal virulence after its entry into the plant, the hexR mutant would not show any significant differences compared to WT after syringe inoculation, which is an inoculation method for directly injecting bacterial suspensions into plants. Thus, these results indicate that HexR contributes to Pcal virulence even after Pcal entry into plants. Interestingly, the Pseudomonas syringae pv. tomato (Pst) ∆hexR caused similar disease symptoms to Pst WT in A. thaliana, and bacterial populations were not significantly different between Pst WT and ∆hexR ( Figure S2). Similarly, in P. savastanoi pv. glycinea (Psg), the total populations and percentages of internalized bacteria were not significantly different between Psg WT and the hexR mutant [25]. Therefore, it is tempting to speculate that HexR contributes to virulence differently among plant pathogenic Pseudomonas species.
The Pcal hexR mutant complemented with pDSKG-hexR restored its virulence partially ( Figure 1). Plasmid complementation often cannot fully restore virulence. The Pcal ∆cmaA showed fewer disease symptoms and bacterial populations compared to WT, indicating that Coronatine (COR) contributes to Pcal virulence [36]. However, the Pcal ∆cmaA complemented with pDSKG-cmaA did not restore its virulence (Sakata et al., unpublished data). Similarly, RND transporter contributes to diseases [22], and mutants complemented with pDSKG-PMA4326_12408 also did not restore virulence (Sakata et al., unpublished data). Besides Pcal, P. amygdali pv. tabaci (Pta) ∆cheA1 showed reduced swimming motility and virulence in host tobacco plants, but its complementation did not recover its swimming motility and virulence [37]. Conversely, the Pcal trpA (tryptophan synthase alpha chain)complemented strain fully restored its virulence [21]. One possibility is that the plasmid vector is effective only in the case of rescued housekeeping genes, such as trpA. Conversely, constitutive gene expression, which encodes the virulence-specific genes (e.g., hexR, cmaA, PMA4326_12408, and cheA1) might result in a disadvantage for bacterial growth. Further efforts to create complemented strains in different ways, such as by using a low copy plasmid or introducing it chromosomally, are needed. Although further quantitative analysis for spatiotemporal promoter activity is needed, these facts suggest that the regulation of virulence-related genes is important for successful infection.
Our RNA-seq analysis revealed that several T3SS-related genes had less expression in the Pcal hexR mutant compared to WT (Table 1). Moreover, the expression of hrpL and corR was less in the Pcal hexR mutant than that in WT (Table 1). We also demonstrated that hrpL and corR expression were down-regulated in the Pcal hexR mutant in culture ( Figure 2). Moreover, type three effectors (T3Es), including avrPto, hopM1, and avrE1, had less expression in the Pcal hexR mutant (Figure 2). However, cmaA and cfl expression were not down-regulated in the Pcal hexR mutant (Figure 2). Additionally, both the Pcal WT and hexR mutant induced hypertrophy response in potato tubers ( Figure S3), indicating that the Pcal hexR mutant was not impaired in COR production. These results suggest that HexR regulates T3Es, but does not regulate COR biosynthesis. HrpL regulates COR production by modulating corR expression [16]. Therefore, HexR might regulate hrpL, resulting in the down-regulation of corR gene expression.
Our results clearly demonstrated that T3SS gene expression was significantly downregulated in the Pcal hexR mutant ( Figure 3) and that plant defense-related gene expression was not suppressed in the Pcal hexR mutant (Figure 4). T3SS mutants typically lose their ability to grow parasitically or be pathogenic in host plants [38]. Moreover, Xie et al. (2023) [39] demonstrated that a nonpathogenic isolate of P. syringae pv. actinidiae biovar 3 (Psa3) was defective in hrpL transcription and lost the ability to induce T3SS-dependent phenotypes. These studies suggest that the T3SS regulated by HrpL is essential for the determination of pathogenicity. Together, these results suggest that the down-regulation of the T3SS in the Pcal hexR mutant results in the impairment of plant defense suppression. Moreover, the Pcal hexR mutant did not induce hypersensitive response (HR) cell death in nonhost plants ( Figure 5). Furthermore, the Pcal hexR mutant lost its pathogenicity on cabbage ( Figure 1). These results indicate that the secretion of hrp-associated T3Es was suppressed when hexR was mutated in Pcal. Conversely, in Psg, HexR did not influence its ability to cause a HR in nonhost plants [25]. Additionally, the bacterial populations of the Psg hexR mutant were not significantly different compared to Psg WT [25]. These results suggest that the loss of pathogenicity in the Pcal hexR mutant was mainly caused by the impairment of T3SS regulation. Kim et al. (2008) [23] demonstrated that HexR might be a dual-sensing regulator of zwf-1 (glucose-6-phosphate dehydrogenase) induction that is able to respond to both 2-keto-3 deoxy-6-phosphogluconate (KDPF) and oxidative stress in P. putida. Moreover, HexR might repress the expression of extra-cellular levansucrase in Psg [25]. These results demonstrated that HexR controls genes involved in glucose metabolites. However, any genes related to carbon metabolism and sucrose utilization were not identified from our RNA-seq analysis. Since the Pcal hexR mutant tended to be defective in growth compared to WT and the complemented strain ( Figure S1), there is a possibility that a growth deficit might contribute to a reduction in virulence.
Our RNA-seq analysis also demonstrated that HexR regulates ABC transporter and auxin metabolism as well as the T3SS (Table 1). Yan et al. (2020) [40] demonstrated that an amino acid ABC transporter pathway (AatJ-AauS-AauR) in Pst directly regulates T3SSencoding genes in response to host aspartate and glutamate signals. Furthermore, the plant hormone auxin (indole-3-acetic acid [IAA]) modulates virulence gene expression, including the T3SS and COR in Pst [41]. Further investigation into virulence factor regulation will help to understand how plant pathogenic Pseudomonas species coordinate the expression of multiple virulence factors under changing environmental conditions.

Conclusions
In summary, our results suggest that HexR contributes to Pseudomonas cannabina pv. alisalensis (Pcal) virulence by coordinating type three secretion system (T3SS) genes. The results presented here also suggest that the mutation in hexR leads to a reduction in T3SS-related gene expression, and thus impaired plant defense suppression, reducing Pcal virulence. To our knowledge, this is the first report to demonstrate the importance of HexR in plant pathogenic Pseudomonas. Our results also provide the possibility that the role of HexR in plant pathogenic Pseudomonas species is different. Therefore, further investigation of other extracytoplasmic function (ECF) sigma factors that regulate virulence factors during infection in Pcal and other Pseudomonas species will be needed to understand the further role of HexR in virulence and other regulatory mechanisms in plant-bacterial pathogen interactions.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/microorganisms11041025/s1, Figure S1: Bacterial populations in vitro, Figure S2: Disease phenotypes and bacterial populations of Pseudomonas syringae pv. tomato DC3000 WT and the ∆hexR mutant in Arabidopsis thaliana after flood inoculation, Figure S3: Coronatine detection of Pseudomonas cannabina pv. alisalnesis KB211 WT, hexR mutant, and ∆cmaA, Table S1: Bacterial strains and plasmids used in this study, Table S2: Primer sets used in this study.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request.