Global Regulator PhoP is Necessary for Motility, Biofilm Formation, Exoenzyme Production, and Virulence of Xanthomonas citri Subsp. citri on Citrus Plants

Citrus canker caused by Xanthomonas citri subsp. citri is one of the most important bacterial diseases of citrus, impacting both plant growth and fruit quality. Identifying and elucidating the roles of genes associated with pathogenesis has aided our understanding of the molecular basis of citrus-bacteria interactions. However, the complex virulence mechanisms of X. citri subsp. citri are still not well understood. In this study, we characterized the role of PhoP in X. citri subsp. citri using a phoP deletion mutant, ΔphoP. Compared with wild-type strain XHG3, ΔphoP showed reduced motility, biofilm formation, as well as decreased production of cellulase, amylase, and polygalacturonase. In addition, the virulence of ΔphoP on citrus leaves was significantly decreased. To further understand the virulence mechanisms of X. citri subsp. citri, high-throughput RNA sequencing technology (RNA-Seq) was used to compare the transcriptomes of the wild-type and mutant strains. Analysis revealed 1017 differentially-expressed genes (DEGs), of which 614 were up-regulated and 403 were down-regulated in ΔphoP. Gene ontology functional enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses suggested that the DEGs were enriched in flagellar assembly, two-component systems, histidine metabolism, bacterial chemotaxis, ABC transporters, and bacterial secretion systems. Our results showed that PhoP activates the expression of a large set of virulence genes, including 22 type III secretion system genes and 15 type III secretion system effector genes, as well as several genes involved in chemotaxis, and flagellar and histidine biosynthesis. Two-step reverse-transcription polymerase chain reaction analysis targeting 17 genes was used to validate the RNA-seq data, and confirmed that the expression of all 17 genes, except for that of virB1, decreased significantly. Our results suggest that PhoP interacts with a global signaling network to co-ordinate the expression of multiple virulence factors involved in modification and adaption to the host environment during infection.


Introduction
Citrus crop canker, caused by Xanthomonas citri subsp. citri and generally associated with a characteristic embossment of necrotic lesions on infected leaves, stems, and fruit [1,2], is considered we examined the role of PhoP with respect to virulence in citrus to provide new information about the virulence of this important plant pathogen.

Bacterial Strains, Culture Media, and Growth Conditions
The bacterial strains and plasmids used in this study are listed in Table 1. Wild type strain XHG3 was isolated from citrus cultivar of Xinhugang in Guangdong province of China. This strain has a high virulence on citrus plants and is preserved in Guangdong Province Key Laboratory of Microbial Signals and Disease Control. Xanthomonas citri subsp. citri was routinely grown at 28 • C in YEB medium (10 g/L tryptone, 5 g/L yeast extract, 5g/L NaCl, 0.5 g/L MgSO 4 ·7H 2 O, 5g/L sucrose, pH7.0) or NA (5 g/L tryptone, 10 g/L sucrose, 1 g/L yeast extract, 3 g/L beef extract, 15 g/L agar, pH7.0). Transformants bearing the first crossover for the gene knockout were cultured on NA without 10% sucrose medium. Transformants for the second crossover were plated on NA medium. E. coli was grown at 37 • C in LB medium (10 g/L Bacto tryptone, 5 g/L yeast extract, and 10 g/L NaCl, pH7.0). NYGA medium (5 g/L tryptone, 3 g/L yeast extract, 20 mL/L glycerol, 15 g/L agar, pH7.0) was used for enzyme assays. Antibiotics were added at the following final concentrations when required: streptomycin sulphate (Sm), 50 µg·mL −1 ; kanamycin (Km), 50 µg·mL −1 ; Ampicillin (Amp), 100 µg·mL −1 . The optical density of the cultures at 600 nm (OD 600 ) was measured by using a UNIC 7200 spectrophotometer (UNIC Apparatus Co). All antibiotics were purchased from Sigma-Aldrich.  Table S1. Susceptible cultivars of Citrus reticulata, commonly known as Xinhuigan and Orah were maintained in a greenhouse.

Gene Knockout and Complementation of phoP Gene
A phoP deletion mutant was generated from wild-type X. citri subsp. citri strain XHG3 using homologous recombination [28]. All primers used in the generation of the mutants are shown in Supplementary Table S1. The left and right homologous arms of phoP were amplified by PCR using genomic DNA from XHG3 as a template and primer pairs phoP-U-F/phoP-U-R and phoP-D-F/phoP-D-R, respectively. The two amplified fragments, which flank phoP, were fused together by PCR using primers phoP-U-F/phoP-D-R. Following digestion with SpeI and BamHI, the fusion fragment was purified and ligated into suicide vector pKNG101 to obtain recombinant plasmid pKNG-∆phoP. pKNG-∆phoP was then transformed into wild-type strain XHG3 by electroporation, and transformants were selected for on NAN agar medium supplemented with Sm (50 µg/mL). Sucrose-sensitive clones were grown in the absence of antibiotics, and double recombination events were selected for on NAS agar plates supplemented with 10% sucrose. The resulting ∆phoP mutant was confirmed by PCR and sequencing.
For complementation of the ∆phoP mutant, a 684-bp fragment containing the entire gene of phoP was amplified from XHG3 genomic DNA using primers phoP-F/phoP-R. The resulting amplicon was digested with BamHI and HindIII and then cloned into the corresponding sites of plasmid vector PBBR1MCS4 so that expression of the target gene was under the control of the lac promoter present in the vector. The ligated mixture was transfected into E. coli DH5α competent cells, and transformants were selected on LB agar supplemented with 100 µg·mL −1 of Amp. Following PCR-based verification using primers MCS4-F/R, the recombinant plasmid was transformed into ∆phoP by electroporation to produce the complemented strain, R-phoP.

Bacterial Growth Curve
To examine the growth of bacterial strains, cells from overnight cultures of the tested strains were harvested and were inoculated into fresh YEB broth. The strains were then cultured at 30 • C with shaking at 200 rpm. Every 2 h, 2-mL volumes of each culture were collected to measure the optical density at 600 nm (OD 600 ). Three replicates were performed for each strain, and bacterial growth curves were plotted based on the average values.

Motility and Biofilm-Formation Assay
Motility assays were performed as described by [29] with modification. Briefly, bacterial strains were cultured in NB medium overnight to an OD 600 = 0.5. Aliquots (2 µL) of the bacterial suspensions were then inoculated onto the surface of agar plates (10 g tryptone, 5 g NaCl, and 3.0 g agar per liter) and incubated for 72 h at 28 • C, Motility was then assessed by measuring the diameter of the growth circle around the inoculation site. Each assay was repeated three times, and strains were examined in triplicate.
The ability of the strains to form a biofilm was examined as described by [6] with modification. Briefly, a 10-µL aliquot of bacterial cell culture (OD 600 = 0.5) was inoculated into tubes containing SONG medium (20 g/L of tryptone, 5 g/L of yeast extract, 5 g/L of NaCl, 2.4 g/L of MgSO 4 ·7H 2 O, 0.186 g/L of KCl, 50 ml/L of 40% glycerin) and incubated for 72 h at 28 • C. The culture medium was then discarded, and attached bacterial cells were gently washed three times with distilled water, incubated for 20 min at 60 • C, and then stained with 1.5 mL of 0.1% (w/v) crystal violet for 45 min. The unbound crystal violet was discarded, and the wells were washed with water prior to interpretation.

Determination of Extracellular Enzymes Activity
Bacterial protease, amylase and cellulase activities were measured using protease, polygalacturonase (PG) and cellulase test plates, respectively, as described by [30]. Relative levels of exoenzyme production were assessed by radial diffusion assays. Bacterial strains were grown in YEB at 28 • C overnight, and the supernatants were collected by centrifugation. Thirty microliters aliquots of the filter-sterilized supernatant were added to the wells of the exoenzyme test plates for further cultivation at 28 • C for 24 h. Each assay was repeated three times, with each strain examined in triplicate. Enzyme activity was estimated from the diameter of the zones surrounding the culture supernatants of each strain.

Pathogenicity Assay
Pathogenicity assays were conducted in a quarantine greenhouse facility. Bacterial strains were cultured in YEB for 24 h at 28 • C. The cells were then pelleted by centrifugation and resuspended in sterile water to an OD 600 = 0.01. The bacterial suspensions were then inoculated onto the tender leaves of cultivars of Xinhuigan and Orah using needleless syringes [31]. Sterile water was used as a control. The inoculated plants were maintained in the greenhouse at 30 • C, and symptoms were observed for three days following inoculation.

High-throughput RNA Sequencing (RNA-seq) and Data Analysis
Wild-type strain XHG3 and the ∆phoP mutant were cultured in YEB medium at 28 • C to OD 600 = 1.0, with two biological repeats performed for each strain. Total bacterial RNA was extracted using the SV Total RNA Isolation System (Promega), with eluted RNA then DNase-treated using a Turbo DNA-free Kit (Promega) as per the manufacturer's instructions. The quality and quantity of the total RNA was assessed using agarose gel electrophoresis, spectrophotometry, and an Agilent 2100 Bioanalyzer. Transcriptome library construction and sequencing were performed by Novogene (Beijing, China). Clean read data were obtained following sequencing and data filtering, and resulting reads were mapped to the X. citri subsp. citri strain 306 reference genome. Gene expression, as indicated by the expected number of fragments per kilobase of transcript sequence per million base pairs sequenced (FPKM), was calculated using HTSeq, with difference in expression between strains ∆phoP and XHG3 determined using DESeq.
Differentially-expressed genes (DEGs) were designated based on q-values < 0.005 and a minimum absolute |log 2 (Fold Change)| > 1, and were exported as a tabular file (Supplementary Table S2). DEGs were then classified and enriched based on gene ontology (GO) database (http://www.geneontology.org/ and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses.

Quantitative Real-time PCR (qRT-PCR) Analysis
To verify the RNA-seq results and identify the downstream genes regulated by PhoP, the expression of several genes was examined using qRT-PCR analysis. RNA samples were reverse-transcribed using a PrimeScript RT Master Mix (TaKaRa). Gene-specific primers were designed using DNASTAR software (DNASTAR) to generate 100-250 bp products from the X. citri subsp. citri genome (Supplementary Table S1). qRT-PCR analysis was performed using three biological replicates on a Bio-Rad CFX96 Real-Time PCR System using TaKaRa SYBR Premix Ex Taq II (Tli RNaseH Plus) as per the manufacturer's instructions. The 16S rRNA gene was used as an endogenous control to normalize gene expression data. Target genes included hrpG and hrpX (T3SS regulators), avrBs2 and avrXacE1 (avirulence protein), and egl0028 (cellulase). The relative fold change in gene expression was calculated using the formula 2 -∆∆CT [32]. Fold change values were log 2 transformed to allow comparison with values generated from RNA-seq analysis.

Mutation of phoP does not affect the bacterial growth.
To examine the function of PhoP in X. citri subsp. citri, we successfully constructed phoP knockout mutant ∆phoP, along with its complementation strain R-phoP (Supplementary Figure S1). To determine whether mutation of phoP affects the growth and proliferation of X. citri subsp. citri, the growth of the wild-type, mutant, and complementation strains was compared in YEB medium. Compared with the wild-type, ∆phoP had a slower growth rate between 4 and 16 h post-inoculation, but reached a cell density similar to that of the wild-type by 20 h post-inoculation (Figure 1). At 24 h post-inoculation, there were no obvious differences in the growth of the three strains. Mutation of phoP reduces motility and biofilm formation in X. citri subsp. citri. Motility assays demonstrated that following incubation at 28 • C for 72 h, the ∆phoP mutant showed considerably reduced motility compared with wild-type strain XHG3 (Figure 2A), with a 70.6% reduction in colony diameter compared with that of strain XHG3 ( Figure 2B). The motility of complemented mutant strain R-phoP was comparable with that of the wild-type, suggesting that PhoP positively regulates bacterial motility.
Biofilms are bacterial communities that are attached to a surface so as to provide protection from deleterious conditions [33,34]. ∆phoP showed a significant decrease in biofilm formation in glass tubes compared with wide-type strain XHG3 and the complementation strain R-phoP ( Figure 3), indicating that PhoP regulates biofilm formation in X. citri subsp. citri.   Mutation of phoP reduces the production of polygalacturonase, amylase and cellulase in X. citri subsp. citri. In many plant pathogenic bacteria, extracellular enzymes, including protease, polygalacturonase (PG), cellulase and amylase, are important virulence factors. To investigate the role of PhoP in X. citri subsp. citri extracellular enzyme production, we compared the levels of the different exoenzymes in ∆phoP and wild type strain XHG3. The results showed a 100% reduction in PG activity ( Figure 4B,E), a 37.16% reduction in amylase production ( Figure 4C,E), and a 19.05% reduction in cellulase activity ( Figure 4D,E), in the ∆phoP mutant compared with wild-type strain XHG3. The activities of each of the enzymes were restored to wild-type levels in complementation mutant strain R-phoP. Interestingly, there was no significant difference in protease production between ∆phoP and XHG3 ( Figure 4A,E). These results confirm that PhoP is involved in the production of polygalacturonase, amylase and cellulase, in X. citri subsp. citri, but does not appear to regulate protease production.
PhoP is required for the virulence of X. citri subsp. citri on citrus plants. The tested bacterial suspensions were inoculated onto the young leaves of two citrus cultivars (Xinhuigang and Orah), which were then maintained in the greenhouse at 30 • C. At 3 days post-inoculation, the leaves inoculated with wild-type strain XHG3 showed obvious canker symptoms, while only very mild symptoms were observed on leaves inoculated with ∆phoP ( Figure 5). Canker symptoms similar to those induced by the wild-type strain were observed on leaves inoculated with the complemented mutant strain, suggesting that PhoP is required for the virulence of X. citri subsp. citri. Analysis of RNA-seq data. To clarify differences in gene expression between XHG3 and ∆phoP, transcriptome sequencing was performed. Total RNA integrity was detected by using an Agilent Technologies 2100 Bioanalyzer, and the degradation and contamination of RNA were analyzed by agarose gel electrophoresis (Supplementary Figure S2). RNA purity was determined by Nanodrop spectrophotomete (OD 260/280 ratio). All RNA evaluations meet the quality requirements for sequencing (Supplementary Figure S2). Sequencing reads were mapped against the reference genome. The XHG3 sample resulted in a total of 19,945,236 reads, with a total of 24,370,669 reads obtained for ∆phoP sample, indicating good data output quality (Supplementary Table S2).
In this study, a false discovery rate of 0.05, a (|log 2 (FoldChange)| > 1, and a q value < 0.005 were used as the cut-off for differential gene expression. There was a total of 1017 DGEs between ∆phoP and XHG3, among which 614 were up-regulated and 403 were down-regulated (Supplementary Table S3). Using a stringent P-value of > 0.005, 287 genes showing differential expression between XHG3 and ∆phoP were identified and then classified and enriched based on the GO database (http://www.geneontology.org/). The DEGs were significantly enriched in 20 GO terms (corrected_P-value < 0.1) involved in cell motility, localization of cell, flagellar motility, cellular components, bacterial-type flagellum and motor activity ( Figure 6).
KEGG database (http://www.genome.jp/kegg/) analysis assigned pathways for 2733 genes, of which 481 showed significant differences in expression. These genes were significantly enriched (P < 0.05) in six signaling pathways, including flagellar assembly, two-component system, histidine metabolism, bacterial chemotaxis, ABC transporters, and bacterial secretion system (Supplementary Table S4).
PhoP regulates histidine metabolism. Bacterial histidine metabolism involves 10 histidine biosynthesis genes coding for nine enzymes that catalyze 10 enzymatic reactions. Our result showed that eight of these histidine biosynthetic genes, hisABCDFGHI (XAC1828 to XAC1835), were dramatically down-regulated in the ∆phoP mutant, The proteins encoded by these genes include 1-(5-phosphoribosyl)-5-[(5-phosphoribosylamino) methylideneamino] imidazole-4-carboxamide isomerase, histidine biosynthesis bifunctional protein, histidinol-phosphate aminotransferase, histidinol dehydrogenase, imidazole glycerol phosphate synthase subunit, imidazole glycerol phosphate synthase subunit and ATP phosphoribosyl transferase. In addition, the expression of both hutH and hutU, which are involved in the histidine utilization pathway, was also significantly decreased (Table 6). PhoP is involved in general metabolism and transport. Many genes involved in general metabolism were regulated by PhoP, including PstS, PstC, PstA and PstB, all of which are involved in phosphate and amino acid transport. In addition, the expression of XacPNP (Xac 2654), encoding a plant natriuretic peptide (PNP)-like protein that is related to virulence in X. citri subsp. citri [35], was reduced in ∆phoP. In higher plants, PNPs elicit a number of responses that contribute to the regulation of homeostasis and growth [36], and help to induce the opening of stomata [37]. XacPNP shares significant sequence similarity and identical domain structure with other PNPs, and may also cause plant physiological responses such as stomatal opening [35].

Discussion
PhoQ/PhoP is one of the best characterized TCS, a family of bacterial systems that sense environmental cues and effectors and trigger gene expression in response to these cues to enhance bacterial survival under stressful conditions or within host cells. The PhoQ/PhoP system has so far been characterized in S. Typhimurium, Shigella sp., D. dadantii, X. oryzae pv. oryzae, and X. campestris pv. campestris [18,20,22,23,38,39]. However, little is known about the PhoQ/PhoP proteins in the important plant pathogen X. citri subsp. citri. In this study, we investigated the potential contribution of PhoP to the virulence and fitness of X. citri subsp. citri, identified PhoP-regulated genes by RNA-seq analysis and demonstrated that PhoP is an important regulator involved in motility, biofilm formation, exoenzymes production and virulence of X. citri subsp. citri in citrus plants. In addition, we found that PhoP positively regulates the expression of genes involved in the T3SS, chemotaxis, flagella biosynthesis, and histidine metabolism in X. citri subsp. citri.
Chemotaxis is essential for the initial stages of bacterial infection, with several genes involved in chemotaxis signaling shown to be critical for entry of a pathogen into host cells [40]. Our study indicated that of the 17 bacterial chemotaxis genes, 16 of them showed decreased expression in the ∆phoP mutant, including cheW, cheA, cheY and cheZ.
In animal-pathogenic species Yersinia enterocolitica and Shewanella oneidensis, bacterial motility and biofilm formation are closely related to pathogenicity [41,42]. Flagella play a critical role in biofilm formation in Y. enterocolitica. Likewise, in plant-pathogenic bacterium, X. campestris pv. campestris, biofilm formation is controlled by cell-cell signaling and is required for full virulence in plant hosts [43], in X. citri subsp. citri, swimming motility, biofilm formation is required for canker development [28,44]. Our RNA-seq analysis showed that 25 genes involved in flagellar assembly, including fliACEFGILMNPR, flhABF, and flgBCDEFGHIJKL, had distinctly lower levels of expression in ∆phoP compared with the wild-type strain. Although primarily associated with cell movement, flagella also act as virulence determinants [44]. The expression of chemotaxis and motility genes by X. oryzae pv. oryzae is required for entry, colonization, and virulence in the host [45]. In the current study, the ∆phoP mutant showed a significant reduction in motility and biofilm formation compared with wide-type strain XHG3, further qRT-PCR analysis further confirmed the decreased expression of flagella-and chemotaxis-associated genes fliC, flhF, cheA, cheY, and rpoN in ∆phoP. Therefore, we concluded that the observed repression of motility and biofilm formation in the mutant strain results from the altered expression of genes involved in chemotaxis and flagellar biosynthesis.
X. citri subsp. citri uses several mechanisms to colonize host plants. These include the T3SS, which delivers virulence effector proteins [9,10,46] and the type II secretion system (T2SS), which is thought to be involved in canker development [11,47,48]. Mutation of xpsD, coding for a component of the T2SS, hinders the secretions of cellulase, protease, and amylase, and affects the ability of X. citri subsp. citri to colonize tissues and hydrolyze cellulose [47]. BglC3, an extracellular endoglucanase, is necessary for the full virulence of X.citri subsp. citri [12]. Extracellular enzymes have been extensively characterized in Pectobacterium and Dickeya species because of their essential functions in the development of soft rot symptoms [49]. Our results showed that the activities of extracellular enzymes such as polygalacturonase (PG), cellulase and amylase were significantly decreased in ∆phoP. In addition, the ∆phoP mutant demonstrated significantly reduced virulence compared with the wild-type strain when inoculated onto citrus leaves. Using qRT-PCR analysis, we also showed that the expression of egl0028, encoding cellulose, was reduced in ∆phoP.
The T3SS plays an important role in bacterial pathogenicity. It is encoded by a cluster of hypersensitive response and pathogenicity (hrp) genes that are critical for bacterial virulence. The T3SS translocates effector proteins into plant cells, where they either suppress the host defense system or interfere with host cellular processes [12]. The expression of hrp genes is controlled by two regulators HrpG and HrpX, which are important pathogenicity regulators in X. citri subsp. citri [8,50]. The transcriptome analysis carried out in the current study showed that PhoP positively regulates many genes involved in the T3SS, including 22 hrp genes and 15 putative and known T3SS effectors, while further qRT-PCR analysis confirmed that PhoP positively regulates the expression of hrpG, hrpX, hrcN, hrcQ, and pthA (T3SS effector). PhoQ/PhoP is also required for hrpG expression and virulence in X. oryzae pv. oryzae [23]. Accordingly, we believe that PhoP can influence the pathogenicity of X. citri subsp. citri through the expression of hrp genes and T3SS effectors.
RNA-Seq and qRT-PCR analyses also confirmed that XacPNP (Xac2654), which encodes a PNP-like protein, was positively regulated by PhoP. PNPs contributed to the regulation of homeostasis reponses and growth. XacPNP plays an important role in the infection process by modifying host responses to create favorable conditions for X. citri subsp. citri growth [35]. Therefore, we can infer that XacPNP is involved in PhoP-mediated regulation of pathogenicity. In addition to altering plant defenses, X. citri subsp. citri must adapt its metabolism to the nutrient-poor and toxin-laden (either preformed or induced) intercellular spaces of host cells [51], which form part of the host defense responses [52,53]. Our transcriptomic analysis indicated that PhoP alters the expression of many genes involved in metabolic processes. For example, in the histidine metabolism, our results showed that eight histidine biosynthetic genes (hisABCDFGHI) were dramatically down-regulated in ∆phoP, while the expressions of both hutH and hutU, which are involved in the histidine utilization pathway, was also significantly decreased. Histidine biosynthesis requires 10 enzymatic reactions involving proteins encoded by seven bacterial genes, Imidazoleglycerol-phosphate dehydratase (IGPD) catalyzes the sixth step in the histidine biosynthesis pathway, and is the first identified enzyme exclusively dedicated to histidine biosynthesis in bacteria. Mutations in hisB and IGPD inhibit biofilm formation in Staphylococcus xylosus [54], while mutations within histidine metabolism genes hisD, FhisF, hisG and hutG lead to decreased virulence of Pseudomonas savastanoi pv. savastanoi in olive [55]. Further, a recent study showed that the histidine utilization (Hut) pathway is involved in quorum sensing and contributes to virulence in X. oryzae pv. oryzae, as X. oryzae pv. oryzae ∆hutG and ∆hutU deletion mutants showed reduced virulence [56]. Therefore, we deduce that histidine metabolism may also be necessary for the virulence of X. citri subsp. citri.
T4SS play a fundamental role in disease progression in several important animal and human pathogens [57] as well as in plant pathogens Agrobacterium tumefaciens [58] and Erwinia sp. [59]. However, our findings showed that 11 T4SS genes and 13 genes coding for T4SS-interacting proteins were up-regulated in ∆phoP. Thus, exactly how PhoP affects the expression of T4SS genes is not yet clear, and requires further investigation.
In plant-pathogenic bacteria, PhoP controls a key aspect of X. oryzae pv. oryzae AvrXA21 virulence through regulation of hrpG [23]. Among all PhoP-regulated genes, 137 genes were down-regulated while 77 genes were upregulated in the phoP knockout mutant of X. oryzae pv. oryzae PXO99A, which encode primarily hypothetical proteins or proteins associated with the cell envelope, protein fate, regulatory functions, or transport and binding [24]. In Dickeya dadantii 3937, a mutation in phoQ affects transcription of at least 40 genes. However, there was no investigation involving PhoP [22]. Our results in X.citri subsp. citri show that PhoP regulates more genes and has more extensive functions. Therefore, we may conclude that PhoP is a global regulatory factor in X. citri subsp. citri, affecting the pathogenicity and virulence of this important citrus pathogen by regulating chemotaxis and motility, biofilm formation, T3SS proteins, histidine biosynthesis, and the production of extracellular enzymes. However, further investigation of these regulatory mechanisms is needed.
Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4425/10/5/340/ s1, Figure S1: Agarose gel showing PCR detection of wild-type strain XHG3, deletion mutant ∆phoP, and complementation strain R-phoP. Figure S2: Agarose gel electrophoresis of total RNA. Figure S3: Verification of log 2 .Fold_change values for 16 virulence-associated genes by qRT-PCR and RNA-seq analyses. Table S1: Primers used in this study. Table S2: Statistics of reads map to reference genome. Funding: This work was supported by the National Natural Science Foundation of China (31371903).

Conflicts of Interest:
The authors declare no conflict of interest.