1. 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 one of the most serious crop diseases worldwide [
3]. Citrus yields and quality are greatly impaired by citrus canker because of defoliation, blemished fruit, and in serious cases, premature fruit drop [
4].
X. citri subsp.
citri invasion of the citrus host occurs directly through natural openings, including stomata, or wounds, with the bacteria then acquiring nutrients from host cells and proliferating in the apoplast [
4].
X. citri subsp.
citri is an important model
Xanthomonas pathogen and is used in studies investigating plant-microbe interactions and virulence mechanisms. The pathogen has evolved several strategies, including a type III secretion system (T3SS), extracellular enzymes, and polysaccharides, to adapt to and successfully establish an
in planta niche, conquering plant defenses and creating a favorable environment for growth [
5,
6]. Previous studies have characterized the major pathogenicity and virulence genes responsible for secretion systems such as the T3SS components and effector molecules, as well as bacterial adhesins, extracellular enzymes, toxins, surface structural elements, and
rpf (regulation of pathogenicity factors)-encoded cell-cell signaling proteins [
7,
8,
9,
10,
11,
12]. PthA, an effector of the T3SS, is a critical factor for citrus canker symptoms [
13], while GalU, a UTP-glucose-1-phosphate uridylyltransferase, contributes to the growth of
X. citri subsp.
citri in intercellular spaces and is involved in the synthesis of extracellular polysaccharide (EPS) and lipopolysaccharide (LPS), as well as in biofilm formation [
14]. Genome sequencing of
X. citri subsp.
citri has greatly increased our understanding of
X. citri subsp.
citri-citrus plant interaction [
7,
15].
Bacterial two-component regulatory systems (TCS) regulate a wide gamut of biological processes in response to fluctuating environmental stimuli. However, only a few
X. citri subsp.
citri TCS have been investigated for their contributions to virulence. For example, the HrpG/HrpX TCS of
X. citri subsp.
citri interacts with a global signaling network to co-ordinate the expression of multiple virulence factors required for the modification of and adaption to the host environment during infection [
8]. The ColR/ColS system is also critical for
X. citri subsp.
citri virulence, contributing to growth
in planta, biofilm formation, LPS production, catalase activity, and resistance to environmental stress [
6]. PhoQ/PhoP, which modulates adaptive responses to changes in levels of divalent cations, including magnesium, in the environment [
16,
17], is an evolutionarily active system involved in bacterial adaptation to various ecological niches [
18]. In animal-pathogenic bacterium
Salmonella enterica subsp.
enterica serovar Typhimurium, antimicrobial peptides serve as direct signals for the activation of PhoQ [
19], while regulatory protein PhoP controls susceptibility to the host inflammatory response in
Shigella flexneri [
20]. PhoP also controls virulence in
Yersinia pestis, the etiological agent of plague, with a
Y. pestis phoP mutant showing decreased survival within macrophages and increased sensitivity to low pH, oxidative killing, and high osmolarity [
21].
In plant-pathogenic bacteria, the PhoQ/PhoP system has been implicated in the regulation of several virulence determinants in
Dickeya dadantii (formerly known as
Erwinia chrysanthemi), a pectinolytic enterobacterium causing soft rot in several plant species [
22], and is required for
hrpG expression, the stress defense response, cation transportation, and virulence in
Xanthomonas oryzae pv.
oryzae AvrXa21 [
23,
24]. The expression of the set of genes activated by PhoP varies among different bacterial species [
25,
26]. Comparison of the downstream genes regulated by PhoP in
S. Typhimurium and in
Escherichia coli showed that gene expression was very different between the two species [
27]. In
X. campestris pv.
campestris, PhoP controls the transcription of many essential, structural genes by directly binding to their
cis-regulatory elements. However, it does not control the same essential genes in
Pseudomonas aeruginosa, as no PhoP binding sites exist in the promoter regions of the genes [
18]. A comparative analysis of the genomes of
P. aeruginosa,
X. campestris pv.
campestris and
X. oryzae pv.
oryzae found that only five genes with expression putatively regulated by PhoP were shared among the three species. Although the PhoP amino acid sequences from
X. campestris pv.
campestris and
X. oryzae pv.
oryzae were identical, approximately 70% of the PhoP-binding sequences differed between the two bacteria, indicating that the effects and regulatory mechanisms of PhoQ/PhoP may differ amongst pathogenic bacteria, even those belonging to the same genus.
Despite the information available on other species, the function of PhoP in X. citri subsp. citri remains unclear. Therefore, as well as analyzing PhoP-regulated gene expression in X. citri subsp. citri, we examined the role of PhoP with respect to virulence in citrus to provide new information about the virulence of this important plant pathogen.
3. Results
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 Δ
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, Δ
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 Δ
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]. Δ
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 Δ
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 Δ
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 Δ
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 Δ
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 Δ
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 Δ
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 Δ
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 Δ
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 the expression of T3SS-associated genes in X. citri subsp. citri. Transcriptome analysis revealed that the complete T3SS-encoding
hrp gene cluster, which contains 24 genes (
Xac0393 to
Xac0417), including
hrpF,
hpaB,
hrpE, hrpD6, hrpD5, hpaA, hrcS, hrcR, hrcQ, hrcV, hrcU, hrpB1, hrpB2, hrcJ, hrpB4, hrpB5, hrcN, hrpB7, hrcT, hrcC and
hpa1, was down-regulated (expect for
Xac 0393 and
Xac 0417), in Δ
phoP compared with the wild-type strain (
Table 2). Among 24 putative and known T3SS effectors in the
X. citri subsp.
citri genome [
15], 15 effector genes, including
AvrBs2, HrpW (PopW), XopAD, XopAI, XopAK, XopE1, XopE3, XopI, XopK, XopN, XopQ, XopR, XopX, XopZ, were down-regulated in Δ
phoP (
Table 3). These results suggested that PhoP positively regulates the expression of T3SS-associated genes.
PhoP regulates the expression of most T4SS genes. The genome of
X. citri subsp.
citri contains two T4SS clusters, one in the chromosome and the other on a plasmid [
7]. We observed an increase in the expression of more than 10 genes (
virB4, virB1, virB11, virB10, virB8, virD4, XAC0096, XAC1918, XAC2609, XAC2610, phlA, XAC0323, HI and
Cw1L) coding for components of the T4SS or T4SS-interacting proteins [
15] in the Δ
phoP mutant (
Table 4), indicating that PhoP negatively regulates theT4SS.
PhoP positively regulates the expression of chemotaxis and flagella genes in X. citri subsp. citri. A number of genes involved in motility and chemotaxis were regulated at a transcriptional level in the Δ
phoP mutant. Twenty-five genes involved in flagellar assembly were distinctly down-regulated in the Δ
phoP mutant compared with the wild-type, including
fliACEFGILMNPR, flhABF, and
flgBCDEFGHIJKL, all of which had log
2.Fold_change values between −1.7 and −4.30. In addition, of the 17 bacterial chemotaxis genes, 16 of them showed decreased expression in the Δ
phoP, including
cheW,
cheA,
cheY, and
cheZ (
Table 5).
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 Δ
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 Δ
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].
PhoP regulates the expression of some virulence-related genes. To verify the RNA-Seq results and confirm the downstream genes regulated by PhoP, the expression of some target genes was examined using qRT-PCR -based analysis. The target genes included
hrpG, hrpX,
hrcN and
hrcQ (T3SS regulators),
avrBs2 and
avrXacE1 (avirulence protein),
egl0028 (Cellulase),
cheA and
cheY (chemotaxis protein),
fliC (flagellin),
flhF(flagellar biosynthesis regulator),
pqqG (pyrroloquinoline quinone biosynthesis protein),
rpoN (RNA polymerase factor sigma-54),
XacPNP (plant natriuretic peptide-like protein),
virB1 (T4SS protein) and
pthA (T3SS effector). Results showed that the expression of
hrpG, hrpX,
hrcN,
hrcQ,
avrBs2,
avrXacE1,
egl0028 (Cellulase),
cheA,
cheY,
fliC,
flhF,
pqqG,
rpoN,
XacPNP and
pthA was significantly lower in the Δ
phoP mutant compared with XHG3, while the expression of
virB1 was increased (
Figure 7), which was consistent with the RNA-sequencing results (
Supplementary Figure S3).
4. 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 Δ
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 Δ
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 Δ
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 Δ
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 Δ
phoP. In addition, the Δ
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 Δ
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 Δ
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 Δ
hutG and Δ
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 Δ
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.