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Keywords:

  • Xanthomonas oryzae pv. oryzae;
  • hrp;
  • type III secretion system;
  • histone-like nucleoid-structuring protein

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Authors' contribution
  7. References
  8. Supporting Information

hrp genes encode components of a type III secretion (T3S) system and play crucial roles in the pathogenicity of the rice pathogen Xanthomonas oryzae pv. oryzae (Xoo). A histone-like nucleoid-structuring (H-NS) protein binds DNA and acts as a global transcriptional repressor. Here, we investigated the involvement of an h-ns-like gene, named xrvB, in the expression of hrp genes in Xoo. Under the hrp-inducing culture condition, the expression of a key hrp regulator HrpG increased in the XrvB mutant, followed by activation of the downstream gene expression. Also, in planta, the secretion of a T3S protein (XopR) was activated by the mutation in xrvB. Gel retardation assay indicated that XrvB has DNA-binding activity, but without a preference for the promoter region of hrpG. The results suggest that XrvB negatively regulates hrp gene expression and that an unknown factor(s) mediates the regulation of hrpG expression by XrvB.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Authors' contribution
  7. References
  8. Supporting Information

Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of bacterial leaf blight of rice (Swings et al., 1990; Niño-Liu et al., 2006). Like other Gram-negative phytopathogenic bacteria in the genera Erwinia, Pseudomonas, Ralstonia and Xanthomonas, Xoo possesses hypersensitive response and pathogenicity (hrp) genes, which play critical roles in conferring pathogenicity on host plants and triggering a hypersensitive response in nonhost plants (Alfano & Collmer, 1997). The hrp genes are involved in the construction of a type III secretion (T3S) apparatus, through which bacterial virulence-associated proteins (effectors) are directly delivered into plant cells (Büttner & Bonas, 2002).

The expression of hrp genes is tightly regulated and is induced in planta, but suppressed in complex media. Appropriate hrp-inducing media have been established for several bacteria; the media are generally nutrient poor and likely to mimic plant conditions (Schulte & Bonas, 1992; Xiao et al., 1992; Wengelnik et al., 1996a; Brito et al., 1999; Tsuge et al., 2002).

In xanthomonads, hrpG and hrpX, which are located apart from the clustered hrp genes, are known to be key regulatory genes for hrp gene expression (Wengelnik & Bonas, 1996; Wengelnik et al., 1996b, 1999). The HrpG protein belongs to an OmpR family of two-component regulatory systems, and phosphorylated HrpG is predicted to regulate the expression of hrpX. Another hrp-regulatory protein, HrpX, belongs to the AraC regulator family and activates the transcription of other hrp genes and genes that encode effectors secreted via a T3S apparatus.

In addition to HrpG and HrpX, several hrp regulatory proteins have been identified, for example Trh, a member of the GntR transcriptional regulator family, and PhoP, a response regulator of a two-component regulatory system, both of which are involved in the expression of HrpG (Tsuge et al., 2006; Lee et al., 2008; Zhang et al., 2008; Huang et al., 2009). Recently, a histone-like nucleoid-structuring (H-NS) protein, named XrvA, was shown to be associated with the positive regulation of hrpG expression in Xoo (Feng et al., 2009). An H-NS protein is a small DNA-binding protein, which is widely conserved in Gram-negative bacteria (Tendeng & Bertin, 2003; Dorman 2004; Fang & Rimsky, 2008). The protein is an important global regulator and, usually as a repressor of transcription, regulates a wide range of genes including virulence-related genes and environment-responsive genes.

The genome database for a Japanese strain of Xoo, MAFF311018, predicts three H-NS-like proteins with the conserved C-terminal domain: proteins XOO0736, XOO2588 (corresponds to XrvA) and XOO3168, which are all conserved in two other sequenced Xoo strains (Lee et al., 2005; Ochiai et al., 2005; Salzberg et al., 2008) (Supporting Information, Fig. S1). Although XOO2588 and XOO3168 have homology with each other (positives=68%), XOO0736 has only partial homology with others. Here, we show experimental data suggesting that, unlike XrvA, XOO0736, named XrvB, is involved in the negative regulation of hrp gene expression in Xoo.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Authors' contribution
  7. References
  8. Supporting Information

Bacterial strains and plasmids

The bacterial strains and plasmids used in this study are listed in Table S1. Escherichia coli DH5αMCR was generally cultured at 37 °C in Luria–Bertani (LB) medium. Xoo strains were usually grown at 28 °C in nutrient broth–yeast extract (NBY) medium (Vidaver, 1967) or in the hrp-inducing medium, XOM2 (Tsuge et al., 2002). All media were supplemented with the following antibiotics: ampicillin, 50 μg mL−1 for E. coli; kanamycin, 25 μg mL−1 for Xoo and 50 μg mL−1 for E. coli; and spectinomycin, 25 μg mL−1 for Xoo and 50 μg mL−1 for E. coli.

Generation of an XrvB-deficient mutant

We constructed a plasmid harboring a c. 3.8-kb xrvB-containing PvuII fragment of the genomic DNA, and then transposon EZ∷TN<KAN-2>(Epicentre) was randomly inserted into the plasmid according to the manufacturer's instructions. Using a plasmid with a transposon at +292 (+1 represents A of the start codon) of xrvB, marker-exchange mutagenesis was conducted on Xoo MAFF311018 (Tsuge et al., 2001). A mutant, named MAFF/XrvB∷Km, was confirmed by Southern blot analysis (data not shown).

Assay of β-glucuronidase (GUS) activity

GUS activity was assayed as described previously (Jefferson et al., 1987; Tsuge et al., 2002). One unit of GUS activity was defined as nanomoles of p-nitrophenol released per hour. Simultaneously, the concentration of bacterial proteins per assay was examined. Bacterial cells in 1 mL of culture were pelleted and resuspended with 100 μL of B-PER Bacterial Protein Extraction Reagent (Pierce) to extract bacterial proteins. Protein concentrations were measured using a Protein Assay kit (Bio-Rad) and bovine serum albumin as a reference. GUS activity of each sample was calculated as U μg−1 bacterial proteins.

Semi-quantified reverse transcription and PCR (semi-qRT-PCR) analysis

Total RNA was extracted from bacteria incubated for 16 h using an RNeasy Mini Kit (Qiagen). Two hundred nanograms of each RNA sample was used for the synthesis of cDNA using a reverse-transcriptase ReverTra-Ace (Toyobo), followed by PCR with a DNA polymerase BlendTaq (Toyobo). Amplified fragments were visualized by staining with ethidium bromide after agarose gel electrophoresis. As a control, 16S rRNA gene was used. The gene-specific primer sets used in this study are listed in Table S2.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis

Xoo strains incubated in XOM2 for 24 h were diluted with the medium to A600 nm=0.3 (c. 108 CFU mL−1). Bacterial cells in 300 μL of diluted culture were pelleted and resuspended with 150 μL Laemmli buffer (Laemmli, 1970), then used for SDS-PAGE, followed by Western blot analysis using rabbit anti-Hpa1 (Tsuge et al., 2006) as the primary antibody and alkaline phosphatase-conjugated anti-rabbit IgG as the secondary antibody (Bio-Rad).

Adenylate cyclase (Cya) reporter assay

The Bordetella pertussis calmodulin-dependent adenylate cyclase (Cya) reporter assay was conducted as described previously (Sory & Cornelis, 1994; Furutani et al., 2009). Bacterial strains with a plasmid harboring an effector gene (xopR) and cya fusion gene (Furutani et al., 2009) were suspended in distilled water (A600 nm=0.3), and then infiltrated into Nicotiana benthamiana leaves using a needleless syringe. After 3- and 6-h incubations, the translocation of the fusion protein into plant cells was examined by measuring cAMP accumulation using the cAMP Biotrak enzyme-immunoassay system (GE Healthcare).

Assay for bacterial growth in medium

Bacterial strains grown on NBY medium were washed twice and resuspended in distilled water to a concentration of A600 nm=1.0. Samples (1 mL) of the bacterial suspension were added to 25 mL synthetic medium XOM2 containing 0.18% glucose (Originally, we used xylose to induce hrp gene expression, but here, we used glucose for more active growth.) and incubated (120 r.p.m., 28 °C). The bacterial population of cultures (A600 nm) was measured every 12 h after inoculation.

Expression and purification of XrvB

A coding region of XrvB, amplified by PCR (Table S2 for primers) and digested with NdeI and EcoRI, was cloned in the expression vector pET28b(+) (Merck), followed by transformation into E. coli BL21(DE3). The transformant was incubated in LB medium for 3 h, and then isopropyl-β-d-1-thiogalactopyranoside. was added for a final concentration of 1 mM, followed by incubation for 1 h. The bacteria were pelleted and suspended with the binding buffer (0.5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9), and then sonicated. After centrifugation at 10 000 g for 10 min at 4 °C, the insoluble fraction was solubilized in the binding buffer with 6 M urea during an overnight incubation on ice. The His(6)-tagged XrvB protein, which was included in the soluble fraction, was purified using a His-Bind Resin column (Merk).

Gel retardation experiments

The target plasmid, which is a pBluescript II SK+ derivative with the putative promoter region of hrpG (−686 to +56) amplified by PCR (Table S2 for primers), was digested with SspI, PvuII and BamHI, and incubated with the purified His(6)-tagged XrvB for 15 min at 37 °C in the reaction buffer described by Soutourina et al. (1999). After the reaction, the samples were loaded onto a 1% TBE-agarose gel, followed by staining with ethidium bromide.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Authors' contribution
  7. References
  8. Supporting Information

Involvement of XrvB in the expression of hrp regulatory genes hrpG and hrpX

First, we examined the expression of xrvB under culture conditions. Semi-qRT-PCR analysis using total RNA extracted from bacteria after a 16-h incubation in the hrp-inducing (XOM2) or the hrp-noninducing medium (NBY) as templates revealed that xrvB is expressed under both conditions (data not shown).

To investigate the involvement of XrvB in the expression of hrp regulatory gene hrpG, we transformed MAFF/XrvB∷Km with a plasmid that harbored the GUS gene preceded by the hrpG promoter (pHMHrpG∷GUS) (Tsuge et al., 2006). The transformant was incubated in XOM2 for 16 h, and then GUS activity was measured. GUS activity was approximately two times higher in the mutant strain than in the parental strain (Table 1), indicating higher hrpG expression in MAFF/XrvB∷Km. The expression level of a phosphoglucose isomerase gene (pgi) in the mutant, which is independent of the hrp-regulatory system (Tsuge et al., 2004, 2006) and was used as a control, was similar to that in the wild type. Semi-qRT-PCR using bacterial total RNA extracted after a 16-h incubation in XOM2 revealed that more hrpG transcript was produced in MAFF/XrvB∷Km with the empty vector pHM1 than in the wild-type derivative and that the hrpG transcript was reduced by the introduction of the complementary plasmid pHMXrvB harboring a PCR-amplified 550-bp fragment containing xrvB and the preceding putative promoter region (−93 to −1) (Fig. 1). The results suggest that, unlike another H-NS protein, XrvA, XrvB is involved in the negative regulation of hrpG expression.

Table 1.   Expression of hrpGgus and hrpXgus in the wild type (WT) and the XrvB mutant (XrvB∷Km) of Xanthomonas oryzae pv. oryzae
Fusion genehrp-inducinghrp-noninducing
WTXrvB∷KmWTXrvB∷Km
  1. The wild type and the XrvB mutant were transformed with pHMHrpG∷GUS or pHMHrpX∷GUS and transformants were incubated in the hrp-inducing (XOM2) or the hrp-noninducing (NBY) medium for 16 h. Numbers represent mean GUS activity (units) per mg of bacterial inner proteins with standard deviation (n=5). One unit of GUS activity is defined as nanomoles of p-nitrophenol released per hour. We used a phosphoglucose isomerase gene (pgi), which is independent of an hrp-regulatory system, as a control (Tsuge et al., 2004, 2006).

hrpGgus123.6 ± 26.8226.4 ± 28.926.6 ± 3.966.0 ± 8.5
hrpXgus160.8 ± 10.9316.9 ± 91.73.7 ± 1.910.3 ± 1.2
pgigus322.4 ± 31.1350.2 ± 42.7345.3 ± 22.1312.1 ± 35.4
image

Figure 1.  Semi-quantified RT-PCR analysis of hrpG expression in Xanthomonas oryzae pv. oryzae strains: wild type with an empty vector (pHM1); WT, MAFF/XrvB∷Km with pHM1; -XrvB, MAFF/XrvB∷Km with pHMXrvB; -XrvB (+XrvB), wild type with pHMXrvB; WT (+XrvB). See Table S2 for primers. The PCR product obtained from wild-type genomic DNA was used as a size marker, and 16S rRNA gene was used as control. RNA samples used for the semi-qRT-PCR are shown at the bottom. We confirmed the absence of DNA contamination in RNA samples by conducting PCR with the RNA samples as a template (without the cDNA synthesis procedure).

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We also investigated the expression of another hrp-regulatory gene, hrpX, which is regulated by HrpG and regulates other hrp genes and T3S protein genes (Furutani et al., 2006, 2009; Wengelnik & Bonas, 1996), in MAFF/XrvB∷Km. When MAFF/XrvB∷Km with pHMHrpX∷GUS, harboring the GUS gene controlled by the hrpX promoter (Tsuge et al., 2006), was incubated in XOM2, GUS activity was higher than that for the wild-type derivative, indicating that the expression of hrpX also increases from the lack of XrvB (Table 1).

We found that, also in the hrp-noninducing medium NBY, the loss of XrvB resulted in increased HrpG expression, although the level of GUS activity after incubation in NBY was lower than that in XOM2 (Table 1). Not only XrvB but also another factor(s) seems to be involved in the inactivation of hrpG expression in NBY. When MAFF/XrvB∷Km (pHMHrpX∷GUS) was incubated in NBY, GUS activity remained much lower than the level in XOM2. As reported previously (Wengelnik et al., 1996b, 1999), phosphorylation of HrpG is required for the expression of HrpX. It is likely that XrvB is not involved in the phosphorylation process and that the high levels of HrpG remain nonphosphorylated and inactive for hrp gene expression during NBY incubation.

High accumulation of HrpG- and HrpX-regulated gene product Hpa1 in the XrvB mutant

To confirm the negative regulation of hrp gene expression by XrvB, we analyzed the accumulation of HrpG- and HrpX-regulated gene product Hpa1 in bacterial cells by Western blot analysis using anti-Hpa1 antibody (Fig. 2). After proteins were transferred to a membrane, we stained the upper part of the membrane, where proteins with a molecular weight >20 kDa were located (the molecular weight of Hpa1 is c. 18 kDa), with Coomassie brilliant blue and confirmed that similar amounts of proteins were loaded in each lane. Western blot analysis using the lower part of the membrane revealed that the lack of XrvB resulted in more accumulation of Hpa1 in bacterial cells than that of the wild type. Interestingly, the introduction of the complementary plasmid pHMXrvB into the mutant, as well as into the wild type, caused less Hpa1 accumulation than even in the wild type with the empty vector, likely because multiple copies of xrvB suppress the expression of hrp genes. The results strongly support that XrvB is involved in the negative regulation of hrp gene expression.

image

Figure 2.  Western blot analysis of the accumulation of Hpa1 in bacterial cells of Xanthomonas oryzae pv. oryzae strains: wild type with an empty vector (pHM1); WT, MAFF/XrvB∷Km with pHM1; -XrvB, MAFF/XrvB∷Km with pHMXrvB; -XrvB (+XrvB), wild type with pHMXrvB; WT (+XrvB) and, as a negative control, 74Hpa1∷Lux/Tet; -Hpa1. Bacteria incubated in XOM2 for 24 h were used for SDS-PAGE. After proteins were transferred to the membrane, the upper part (without Hpa1) was stained with Coomassie brilliant blue and the lower part (with Hpa1) was used for Western blot analysis using anti-Hpa1 antibody. Similar results were obtained in two independent experiments.

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The loss of XrvB results in increased secretion of type III effector XopR into plant cells

We examined the activation of the T3S system in the XrvB mutant in planta using the B. pertussis calmodulin-dependent adenylate cyclase (Cya) reporter assay (Sory & Cornelis, 1994; Furutani et al., 2009). The wild type and the mutant transformed with pHMXopR∷Cya, which harbors xopR (an effector gene) and cya fusion gene (Furutani et al., 2009), were infiltrated into N. benthamiana leaves. After 3- and 6-h incubations, the translocation of the fusion protein into plant cells was examined by measuring cAMP accumulation. Higher accumulation of cAMP was observed in the leaves with MAFF/XrvB∷Km (pHMXopR∷Cya) than those with the wild-type derivative (Table 2), indicating that more XopR∷Cya fusion protein was secreted into the plant cells. These results suggest that, also in planta, the loss of XrvB activates the expression of T3S-related genes (hrp genes and effector genes), followed by active secretion.

Table 2.   Levels of cAMP in Nicotiana benthamiana leaves infiltrated with the wild-type Xanthomonas oryzae pv. oryzae or the XrvB mutant transformed with a plasmid harboring the xopRcya fusion gene
StraincAMP (pmol cm−2 leaf section)
3 hai6 hai
  1. Accumulation of cAMP was assayed 3 and 6 h after infiltration (hai) using infiltrated leaf sections (1 cm2 each). Values are the mean amounts of cAMP (pmol cm−2 leaf section) with the SD (n=3). 74HrcV∷Km is a T3S-system-deficient mutant (Furutani et al., 2004).

MAFF31101818.2 ± 4.8170.6 ± 28.1
MAFF/XrvB∷Km49.2 ± 9.0321.8 ± 39.0
74HrcV∷Km9.1 ± 1.113.4 ± 2.9

Growth of the XrvB mutant in culture

Generally, H-NS proteins are involved in regulating multiple gene expression and, as a result, are involved in regulating multiple cellular functions (Tendeng & Bertin, 2003; Dorman 2004). When MAFF/XrvB∷Km was incubated in synthetic medium XOM2 containing 0.18% glucose, bacterial growth was delayed compared with the wild type, and the delayed growth was complemented by the introduction of pHMXrvB (Fig. 3). Thus, XrvB may regulate not only hrp gene expression but also the expression of genes involved in bacterial growth.

image

Figure 3.  Growth of wild type and XrvB mutant of Xanthomonas oryzae pv. oryzae in a nutrient-poor synthetic medium (XOM2 containing 0.18% glucose) at 28°C. Concentration (A600 nm) of MAFF311018 (open circle), MAFF/XrvB∷Km (open triangle) and the complementary strain MAFF/XrvB∷Km (pHMXrvB) (closed circle) was measured every 12 h. Similar results were obtained in two independent experiments.

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DNA-binding activity of XrvB

H-NS protein binds preferentially to curved DNA, which is commonly associated with promoters, via its conserved C-terminal domain (Tendeng & Bertin, 2003; Dorman 2004). Generally, the binding of H-NS leads to repression of gene expression, while its release leads to gene activation. We found that XrvB has an amino acid sequence similar to the core motif in the C-terminal domain of H-NS (Fig. S1). We, therefore, investigated whether XrvB has DNA-binding activity and whether the protein binds to the promoter region of hrpG. The XrvB protein tagged with six histidine residues at its C-terminus was extracted and purified from E. coli transformed with pETXrvB harboring the coding region of xrvB (Fig. 4a), and then incubated with SspI-, PvuII- and BamHI-digested pBSHrpG-Pro, in which the putative promoter region of hrpG (−686 to +56) is contained. Electrophoresis, followed by staining with ethidium bromide revealed that, like other H-NS proteins reported previously (Zuber et al., 1994; Tendeng et al., 2003), XrvB bound to a 500-bp SspI–PvuII fragment containing the bla promoter with a curved structure, along with the 1900-bp fragment derived from the vector sequence (Fig. 4b). The electrophoretic mobility of the fragment was completely retarded at a protein concentration of 1.8 μM. Under the same conditions, the 740-bp BamHI fragment containing the hrpG promoter was not retarded as much as the 500- and 1900-bp fragments. When the predicted promoter region of hrpG was examined using the bend-it computer program (http://www.icgeb.org/dna/bend_it.html), the possibility that it possesses curved regions was low (data not shown). The results suggest that XrvB possesses DNA-binding activity, but that it does not bind to the hrpG promoter. It is likely that the regulation of hrpG expression by XrvB is indirect and that some unknown gene(s)/protein(s) mediate the regulation.

image

Figure 4.  (a) Purification of His(6)-tagged XrvB. The recombinant protein expressed in Escherichia coli was purified using a His-Bind Resin column (Merck) and separated by SDS-PAGE. T, bacterial total protein; P, purified His-tagged XrvB; M, molecular marker. (b) Gel retardant assay using His(6)-tagged XrvB and a plasmid, in which the putative promoter region of hrpG is inserted into pBluescript II SK+. The plasmid, digested with SspI, PvuII and BamHI, was incubated for 15 min with various concentrations of the purified His(6)-tagged XrvB. Samples were then separated by agarose gel electrophoresis and stained with ethidium bromide. ▸, 740-bp fragment containing the putative promoter region of hrpG; ▹, 1900- and 500-bp vector-derived fragments that interact with XrvB. M, λ DNA digested with HindIII.

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Although many researchers have contributed to identifying various hrp regulatory genes in Xoo and other Xanthomonas spp., the entire scheme of the complicated hrp-regulatory cascade remains unclear (Tsuge et al., 2006; Lee et al., 2008; Zhang et al., 2008; Feng et al., 2009; Huang et al., 2009). Here, our study suggests that the H-NS-like, DNA-binding protein XrvB is involved in the negative regulation of hrp gene expression in Xoo by repressing the expression of a key hrp regulatory gene hrpG.

Besides the regulation of hrp gene expression, XrvB is likely to be involved in the regulation of various genes because the growth of the mutant decreased under the culture conditions. Moreover, virulence of the XrvB mutant on rice decreased compared with the wild type (data not shown). Generally, H-NS proteins are involved in bacterial nucleoid organization and in the regulation of various genes associated with adaptation to environmental challenges, such as pH, temperature, osmolarity and growth phase (Atlung & Ingmer, 1997), and mutations in h-ns are highly pleiotropic. Like other H-NS proteins, XrvB may regulate various genes, which may include pathogenicity-related genes other than hrp.

Feng et al. (2009) reported that another H-NS-like protein XrvA functions in the positive regulation of hrp gene expression in the bacterium. They showed that, besides playing a role in hrp gene expression, XrvA is also involved in the expression of rpfC, rpfF, rpfG and gumB, which play important roles in virulence and extracellular polysaccharide production (Tang et al., 1996; Chatterjee & Sonti, 2002; Jeong et al., 2008). When the expression of rpfC was examined by semi-qRT-PCR, little difference was observed between the wild type and the XrvB mutant, and there seems to be no difference in extracellular polysaccharide production between the two strains (data not shown). The target genes of the two H-NS-like proteins, XrvA and XrvB, are likely to be different, but they may function cooperatively to enable the adequate expression of Xoo hrp genes in the infection process.

The regulatory mechanisms of XrvB for hrp gene expression remain unclear. In a future study, a microarray assay comparing gene expression between the XrvB mutant and the wild type or the chromatin immunoprecipitation assay should reveal target genes that are directly regulated by XrvB, leading to the clarification of XrvB functions, including the interactions between XrvB and XrvA and/or other hrp regulatory proteins.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Authors' contribution
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Authors' contribution
  7. References
  8. Supporting Information

Fig. S1. Alignment of the conserved C-terminal region in H-NS-like proteins XOO0736, XOO2588 and XOO3168 of Xanthomonas oryzae pv. oryzae MAFF311018.

Table S1. Bacterial strains and plasmids used in this study.

Table S2. Primers used in this study.

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