XerR, a negative regulator of XccR in Xanthomonas campestris pv. campestris, relieves its repressor function in planta

We previously reported that XccR, a LuxR-type regulator of Xanthomonas campestris pv. campestris (Xcc), activates the downstream proline iminopeptidase virulence gene (pip) in response to certain host plant factor(s). In this report, we further show that the expression of the xccR gene was repressed in the culture medium by an NtrC-type response regulator, which we named XerR (XccR expression-related, repressor), and that this repression was relieved when the bacteria were grown in planta. Such a regulatory mechanism is reinforced by the observations that XerR directly bound to the xccR promoter in vitro, and that mutations at the phosphorylation-related residues of XerR resulted in the loss of its repressor function. Furthermore, the expression level of xccR increased even in XerR-overexpressing Xcc cells when they were vacuum infiltrated into cabbage plants. We also preliminarily characterized the host factor(s) involved in the above mentioned interactions between Xcc and the host plant, showing that a plant material(s) with molecular weight(s) less than 1 kDa abolished the binding of XerR to the xccR promoter, while the same material enhanced the binding of XccR to the luxXc box in the pip promoter. Taken together, our results implicate XerR in a new layer of the regulatory mechanism controlling the expression of the virulence-related xccR/pip locus and provide clues to the identification of plant signal molecules that interact with XerR and XccR to enhance the virulence of Xcc.     

Phytopathogenic bacteria utilize host glucose as a signal to stimulate virulence through LuxR homologues

Chemical signal-mediated biological communication is common within bacteria and between bacteria and their hosts. Many plant-associated bacteria respond to unknown plant compounds to regulate bacterial gene expression. However, the nature of the plant compounds that mediate such interkingdom communication and the underlying mechanisms remain poorly characterized. Xanthomonas campestris pv. campestris (Xcc) causes black rot disease on brassica vegetables. Xcc contains an orphan LuxR regulator (XccR) which senses a plant signal that was validated to be glucose by HPLC-MS. The glucose concentration increases in apoplast fluid after Xcc infection, which is caused by the enhanced activity of plant sugar transporters translocating sugar and cell-wall invertases releasing glucose from sucrose. XccR recruits glucose, but not fructose, sucrose, glucose 6-phosphate, and UDP-glucose, to activate pip expression. Deletion of the bacterial glucose transporter gene sglT impaired pathogen virulence and pip expression. Structural prediction showed that the N-terminal domain of XccR forms an alternative pocket neighbouring the AHL-binding pocket for glucose docking. Substitution of three residues affecting structural stability abolished the ability of XccR to bind to the luxXc box in the pip promoter. Several other XccR homologues from plant-associated bacteria can also form stable complexes with glucose, indicating that glucose may function as a common signal molecule for pathogen–plant interactions. The conservation of a glucose/XccR/pip-like system in plant-associated bacteria suggests that some phytopathogens have evolved the ability to utilize host compounds as virulence signals, indicating that LuxRs mediate an interkingdom signalling circuit.     

The conserved Xanthomonas campestris pv. vesicatoria effector protein XopX is a virulence factor and suppresses host defense in Nicotiana benthamiana

Nicotiana benthamiana leaves display a visible plant cell death response when infiltrated with a high titer inoculum of the non-host pathogen, Xanthomonas campestris pv. vesicatoria (Xcv). This visual phenotype was used to identify overlapping cosmid clones from a genomic cosmid library constructed from the Xcv strain, GM98-38. Individual cosmid clones from the Xcv library were conjugated into X. campestris pv. campestris (Xcc) and exconjugants were scored for an altered visual high titer inoculation response in N. benthamiana. The molecular characterization of the cosmid clones revealed that they contained a novel gene, xopX, that encodes a 74-kDa type III secretion system (TTSS) effector protein. Agrobacterium-mediated transient expression of XopX in N. benthamiana did not elicit the plant cell death response although detectable XopX protein was produced. Interestingly, the plant cell death response occurred when the xopX Agrobacterium-mediated transient expression construct was co-inoculated with strains of either XcvDeltaxopX or Xcc, both lacking xopX. The co-inoculation complementation of the plant cell death response also depends on whether the Xanthomonas strains contain an active TTSS. Transgenic 35S-xopX-expressing N. benthamiana plants also have the visible plant cell death response when inoculated with the non-xopX-expressing strains XcvDeltaxopX and Xcc. Unexpectedly, transgenic 35S-xopX N. benthamiana plants displayed enhanced susceptibility to bacterial growth of Xcc as well as other non-xopX-expressing Xanthomonas and Pseudomonas strains. This result is also consistent with the increase in bacterial growth on wild type N. benthamiana plants observed for Xcc when XopX is expressed in trans. Furthermore, XopX contributes to the virulence of Xcv on host pepper (Capsicum annuum) and tomato (Lycopersicum esculentum) plants. We propose that the XopX bacterial effector protein targets basic innate immunity in plants, resulting in enhanced plant disease susceptibility.     

Type III secretion-dependent host defence elicitation and type III secretion-independent growth within leaves by Xanthomonas campestris pv. campestris

In many plant-bacterial interactions, loss of the type III secretion system (T3SS) severely reduces bacterial growth, symptom causation and suppression of defences in host plants. In the present study of Xanthomonas campestris pv. campestris (Xcc), Xcc strain B305 grew better than strain B186 in Arabidopsis thaliana after hydathode inoculation, and B305 strains mutated to the loss of T3SS (ΔhrcC and/or ΔhrpE; also ΔhrcCΔflgBC) grew similarly to wild-type B305 in Arabidopsis leaves. Unlike Xcc strain B186, wild-type B305 was relatively inefficient in secreting the exogenous T3S effector AvrBsT, but ΔhrcC and/or ΔhrpE attenuated the disease symptoms caused by Xcc B305, showing that the partially compromised T3SS of this strain still promotes necrotic leaf symptoms. In contrast with the T3SS-dependent defence suppression that has been observed for some other plant pathogenic bacteria, the Xcc B186 and B305 wild-type strains (which are virulent on Arabidopsis) caused greater elicitation of host PR-1 and PR-5 expression and callose deposition in comparison with their respective T3SS mutants. A defence-suppressing/virulence-enhancing activity of the Xcc T3SS effector suite was detectable when co-inoculation with wild-type Xcc B186 increased the growth of ΔhrcC Xcc, but this activity did not prevent the above defence elicitation. Experiments using T3SS mutants and Arabidopsis fls2 mutants suggested that FLS2 does not play a prominent role in restriction of the examined Xcc strains. However, ectopic overexpression of the Pseudomonas syringae effector AvrPto promoted in planta growth of wild-type and ΔhrcC Xcc. In summary, the T3SS components or effector suite from virulent Xcc strains elicit some host defence responses, but suppress other defences and stimulate more severe disease symptoms, AvrPto-disruptable elements other than FLS2 apparently contribute to the host restriction of Xcc, and in some virulent Xcc strains the T3SS is not absolutely required for wild-type levels of bacterial growth within the plant.     

hxc2, an Arabidopsis mutant with an altered hypersensitive response to Xanthomonas campestris pv. campestris

A chemical mutagenized population of Arabidopsis Col-0-gl plants was screened for an altered hypersensitive response (HR) after spray inoculation with an HR-inducing isolate of Xanthomonas campestris pv. campestris (strain 147). Three classes of mutant were identified: those exhibiting an HR- phenotype or partial loss of HR; hyper-responsive mutants showing necrotic lesions rapidly leading to the collapse of leaves; and susceptible mutants. One mutant belonging to the susceptible class, hxc-2, was extensively characterized. The compatible phenotype observed several days after initiation of the interaction was confirmed by measurement of in planta bacterial growth and use of bacterial strains constitutively expressing the GUS reporter gene. In the same way, accumulation of autofluorescent compounds, salicylic acid production and defence gene expression in the mutant were found to be similar to that displayed by the susceptible ecotype. Inoculation of hxc-2 with different avirulent bacteria suggests that the mutation is specific for the interaction with the Xcc 147 strain, although the mutation has been shown to affect a single dominant locus, different from the resistance locus defined by genetic analysis of resistance to Xcc 147. Genetic mapping of the mutation indicated that it is located on chromosome III, defining a previously unknown resistance function in response to X. c. campestris.     

The bifunctional effector AvrXccC of Xanthomonas campestris pv. campestris requires plasma membrane-anchoring for host recognition

Bacterial pathogens use type III secretion systems (TTSS) to deliver effector proteins into eukaryotic cells for pathogenesis. In bacterial–plant interactions, one effector may function as an avirulence factor to betray the pathogen to the plant surveillance system and induce the hypersensitive response (HR) in the resistant host carrying a corresponding resistance ( R ) gene. However, the same effector can also sustain the growth of the pathogen by acting as a virulence factor to modulate plant physiology in the susceptible host lacking the corresponding R gene. Here, we identified and characterized a bifunctional TTSS effector AvrXccC belonging to the AvrB effector family in Xanthomonas campestris pv. campestris 8004. This effector is required for full bacterial virulence in the susceptible host cabbage ( Brassica oleracea ) and avirulence in the resistant host mustard ( Brassica napiformis L.H. Baily). Expressing avrXccC in mustard-virulent strain Xcc HRI 3849A converts its virulence to avirulence. The effector AvrXccC is anchored to the plant plasma membrane, and the N-terminal myristoylation site (amino acids 2–7: GLcaSK) is essential for its localization. In addition, the avirulence function of AvrXccC for host recognition depends on its plasma membrane localization. Promoter activity assays showed that the expression of avrXccC is hrpG/hrpX -dependent. Moreover, the secretion of AvrXccC displayed hrp -dependency and the core sequence for AvrXccC translocation was defined to the N-terminal 40 amino acids.     

Co-regulation of Xanthomonas campestris virulence by quorum sensing and a novel two-component regulatory system RavS/RavR

Xanthomonas campestris pv. campestris ( Xcc ) is known to regulate virulence through a quorum-sensing mechanism. Detection of the quorum-sensing signal DSF by sensor RpfC leads to activation of the response regulator RpfG, which influences virulence by degrading cyclic-di-GMP and by subsequent increasing expression of the global regulator Clp. In this study, we show that mutation of a response regulator RavR containing the GGDEF–EAL domains decreases Xcc virulence factor production . The functionality of RavR is dependent on its EAL domain-associated cyclic-di-GMP phosphodiesterase activity. Deletion of a multidomain sensor gene ravS , which shares the same operon with ravR , results in similar phenotype changes as the ravR mutant. In addition, the sensor mutant phenotypes can be rescued by in trans expression of the response regulator, supporting the notion that RavS and RavR constitute a two-component regulatory system. Significantly, mutation of either the PAS domain or key residues of RavS implicated in sensing low-oxygen tension abrogates the sensor activity in virulence regulation. Moreover, similar to the DSF signalling system, RavS/RavR regulates virulence gene expression through the global regulator Clp. These results outline a co-regulation mechanism that allows Xcc to integrate population density and environmental cues to modulate virulence factor production and adaptation.     

Cyclic di-GMP signalling in the virulence and environmental adaptation of Xanthomonas campestris

Cyclic di-GMP is a second messenger with a role in regulation of a range of cellular functions in diverse bacteria including the virulence of pathogens. Cellular levels of cyclic di-GMP are controlled through synthesis, catalysed by the GGDEF protein domain, and degradation by EAL or HD-GYP domains. Here we report a comprehensive study of cyclic di-GMP signalling in bacterial disease in which we examine the contribution of all proteins with GGDEF, EAL or HD-GYP domains to virulence and virulence factor production in the phytopathogen Xanthomonas campestris pathovar campestris (Xcc). Genes with significant roles in virulence to plants included those encoding proteins whose probable function is in cyclic-di-GMP synthesis as well as others (including the HD-GYP domain regulator RpfG) implicated in cyclic di-GMP degradation. Furthermore, RpfG controlled expression of a subset of these genes. A partially overlapping set of elements controlled the production of virulence factors in vitro. Other GGDEF-EAL domain proteins had no effect on virulence factor synthesis but did influence motility. These findings indicate the existence of a regulatory network that may allow Xcc to integrate information from diverse environmental inputs to modulate virulence factor synthesis as well as of cyclic di-GMP signalling systems dedicated to other specific tasks.     

Molecular evolution of virulence in natural field strains of Xanthomonas campestris pv. vesicatoria

The avrBs2 avirulence gene of the bacterial plant pathogen Xanthomonas campestris pv. vesicatoria triggers disease resistance in pepper plants containing the Bs2 resistance gene and contributes to bacterial virulence on susceptible host plants. We studied the effects of the pepper Bs2 gene on the evolution of avrBs2 by characterizing the molecular basis for virulence of 20 X. campestris pv. vesicatoria field strains that were isolated from disease spots on previously resistant Bs2 pepper plants. All field strains tested were complemented by a wild-type copy of avrBs2 in their ability to trigger disease resistance on Bs2 plants. DNA sequencing revealed four mutant alleles of avrBs2, two of which consisted of insertions or deletions of 5 nucleotides in a repetitive region of avrBs2. The other two avrBs2 alleles were characterized by point mutations with resulting single amino acid changes (R403P or A410D). We generated isogenic X. campestris pv. vesicatoria strains by chromosomal avrBs2 gene exchange to study the effects of these mutations on the dual functions of avrBs2 in enhancing bacterial virulence and inducing plant resistance by in planta bacterial growth experiments. The deletion of 5 nucleotides led to loss of avrBs2-induced resistance on Bs2 pepper plants and abolition of avrBs2-mediated enhancement of fitness on susceptible plants. Significantly, the point mutations led to minimal reduction in virulence function of avrBs2 on susceptible pepper plants, with either minimal (R403P allele) or an intermediate level of (A410D allele) triggering of resistance on Bs2 plants. Consistent with the divergent selection pressures on avrBs2 exerted by the Bs2 resistance gene, our results show that avrBs2 is evolving to decrease detection by the Bs2 gene while at the same time maintaining its virulence function.     

Light signaling mediated by PAS domain-containing proteins in Xanthomonas campestris pv. campestris

Per-ARNT-Sim (PAS) domains are important signalling modules that possibly monitor changes in various stimuli such as light. For the majority of PAS domains that have been identified by sequence similarity, the biological function of the signalling pathways has not yet been experimentally investigated.Thirty-three PAS proteins were discovered in Xanthomonas campestris pv. campestris(Xcc) by genome/proteome analysis. Thirteen PAS proteins were identified as contributing to light signalling and Xcc growth, motility or virulence using molecular genetics and bioinformatics methods. The PAS domains played important roles in light signalling to regulate the growth, motility and virulence of Xcc. They might be regulated by not only light quality (wavelength)but also quantity (intensity) as potential light-signalling components. Evaluating the light wavelength, three light-signalling types of PAS proteins in Xcc were shown to be involved in blue light signalling, tricolour (blue, red and far red)signalling or red/far-red signalling. This showed that Xcc had evolved a complicated light-signalling system to adapt to a complex environment.     

DsbB is required for the pathogenesis process of Xanthomonas campestris pv. campestris

The DsbA/DsbB oxidation pathway is one of the two pathways that catalyze disulfide bond formation of proteins in the periplasm of gram-negative bacteria. It has been demonstrated that DsbA is essential for multiple virulence factors of several animal bacterial pathogens. In this article, we present genetic evidence to show that the open reading frame XC_3314 encodes a DsbB protein that is involved in disulfide bond formation in periplasm of Xanthomonas campestris pv. campestris, the causative agent of crucifer black rot disease. The dsbB mutant of X. campestris pv. campestris exhibited attenuation in virulence, hypersensitive response, cell motility, and bacterial growth in planta. Furthermore, mutation in the dsbB gene resulted in ineffective type II and type III secretion systems as well as flagellar assembly. These findings reveal that DsbB is required for the pathogenesis process of X. campestris pv. campestris.     

Genome scale analysis of diffusible signal factor regulon in Xanthomonas campestris pv. campestris: identification of novel cell-cell communication-dependent genes and functions

The bacterial pathogen Xanthomonas campestris pv. campestris (Xcc) recruits a diffusible signal factor (DSF), which has recently been structurally characterized as cis-11-methyl-2-dodecenoic acid, as a cell-cell communication signal to synchronize virulence gene expression and biofilm dispersal. In this study, we showed that despite the existance of phenotype variations in different Xcc isolates, the DSF-mediated functions were in general conserved. To investigate the genomic profiles of DSF regulation, we designed and conducted oligomicroarray analysis by comparison of the gene expression patterns of wild-type strain XC1 and its DSF-deficient mutant XC1dF, as well as those of XC1dF in the presence or absence of DSF signals. The analyses led to identification of 165 genes, whose expression was significantly influenced by DSF signals. These genes encode proteins and enzymes belonging to at least 12 functional groups. In addition to those previously known DSF-dependent activities such as production of extracellular enzymes and extracellular polysaccharides, microarray analyses also revealed new functions mediated by DSF, such as flagellum synthesis, resistance to toxins and oxidative stress, and aerobic respiration. Phenotype analyses confirmed that DSF signalling contributed to resistance to toxin acriflavin and hydrogen peroxide, and to the survival of bacterial cells at different temperatures. We conclude that DSF cell-cell signalling is not only essential for co-ordinating the expression of virulence genes but also plays a vital role in keeping up the general competence of the pathogen in ecosystems.     

MarR家族转录因子HpaR和XC0449协同调控野油菜黄单胞菌致病性

MarR家族转录因子广泛存在于细菌及古生菌中,并灵活、精细地调控多种毒力、抗胁迫及抗生素相关的生理生化途径。在野油菜黄单胞菌中,MarR家族转录因子HpaR (XC2827)的失活会显著降低细菌对于寄主甘蓝的致病力,同时会导致胞外蛋白酶的过量表达。本研究进一步发现,Xcc 8004基因组一共编码9个MarR家族转录因子。表达并纯化其中的HpaR (XC2827)和XC0449,体外微量热泳动(MST)实验及Pull-down实验证明二者可以在体外特异性结合。同时,表型检测发现XC0449突变会导致细菌致病力显著下降。通过体外凝胶迁移阻滞试验(EMSA)、体内qRT-PCR和GUS检测证明,XC0449和HpaR均作为转录激活子协同调控下游致病相关基因XC0705的表达,最终调控细菌毒力及胞外酶合成。     

The Blight-Associated Epitope and DNA Fragment from Xanthomonas campestris pv. campestris are not Required for Blight

Abstract: It has been reported that all tested naturally occurring strains of Xanthomonas campestris pv. campestris that are known to be capable of inducing blight symptoms in cabbage react with MAb A11 and hybridize with a 5.4-kb DNA fragment (in plasmid pJC41), cloned from the Xanthomonas campestris species type strain, Xcc528^T, whereas all tested naturally occurring strains that do not cause blight react with MAb X21 and do not hybridize to pJC41. The roles of the 5.4-kb DNA in pJC41 and the epitope recognized by MAb A11 in the pathogenicity of X. c. campestris strains that cause blight were examined by mutational analyses. A 4.0-kb deletion of the pJC41 region on the Xcc528^T chromosome was created by marker exchange, but the derivatives were evidently not affected in their ability to elicit blight symptoms. Nitrosoguanidine was used to mutagenize two blight strains, Xcc528^T and CAM19, and mutants were selected that were not reactive to MAb A11. The MAb A11-negative mutant of Xcc528^T was reactive to MAb X21, but was evidently not affected in the ability to elicit blight symptoms. MAb A11-negative mutants of CAM19, however, were not reactive to MAb X21, and showed reduction or loss of virulence, which suggested the requirement for at least one of the two antigens (to MAbs A11 or X21) for pathogenicity. A genomic library of CAM19 was made and screened for genes responsible for the production of the A11 antigen. Cosmid clones were identified that restored MAb A11 reactivity to the mutants. None of these cosmids restored the virulence of the mutant strains that had lost virulence. Therefore, neither the blight-associated 5.4-kb DNA fragment nor the MAb A11 antibody marker were required for blight symptom elicitation or pathogenicity.     

Identification of genes in Xanthomonas campestris pv. vesicatoria induced during its interaction with tomato

Xanthomonas campestris pv. vesicatoria is the causal agent of bacterial spot disease of tomato and pepper. The disease process is interactive and very intricate and involves a plethora of genes in the pathogen and in the host. In the pathogen, different genes are activated in response to the changing environment to enable it to survive, adapt, evade host defenses, propagate, and damage the host. To understand the disease process, it is imperative to broaden our understanding of the gene machinery that participates in it, and the most reliable way is to identify these genes in vivo. Here, we have adapted a recombinase-based in vivo expression technology (RIVET) to study the genes activated in X. campestris pv. vesicatoria during its interaction with one of its hosts, tomato. This is the first study that demonstrates the feasibility of this approach for identifying in vivo induced genes in a plant pathogen. RIVET revealed 61 unique X. campestris pv. vesicatoria genes or operons that delineate a picture of the different processes involved in the pathogen-host interaction. To further explore the role of some of these genes, we generated knockout mutants for 13 genes and characterized their ability to grow in planta and to cause disease symptoms. This analysis revealed several genes that may be important for the interaction of the pathogen with its host, including a citH homologue gene, encoding a citrate transporter, which was shown to be required for wild-type levels of virulence.     

Mutagenesis of all eight avr genes in Xanthomonas campestris pv. campestris had no detected effect on pathogenicity, but one avr gene affected race specificity

Suppression subtractive hybridization (SSH) was used to identify genes present in the systemic crucifer black rot pathogen Xanthomonas campestris pv. campestris 528T but missing from the nonsystemic crucifer leaf spot pathogen, X. campestris pv. armoraciae 417. Among the DNA fragments unique to 528T was Xcc2109, one of eight putative avr genes identified in the published 528T genome (NC_003902). Individual and sequential deletion, insertion mutations, or both of all eight 528T avr gene loci were made, but no change in pathogenicity was observed with any combination of avr mutations, including a strain with all eight avr genes deleted. However, insertion or deletion mutants affecting the Xcc2109 locus lost avirulence (i.e., became virulent) on Florida Mustard, an X. campestris pv. campestris race-determining, differential host. The Xcc2109 open reading frame as annotated was cloned and found to be nonfunctional. A longer gene, encompassing Xcc2109 and here designated avrXccFM, was cloned and found to complement the Xcc2109 mutants and to confer avirulence to two additional wild-type X. campestris pv. campestris strains, thereby changing their races. Resistance in Florida Mustard to 528T strains carrying avrXccFM occurred without a typical hypersensitive response (HR) on leaves, although a vascular HR was observed in seedlings.     

Differential accumulation of Xanthomonas campestris pv. campestris proteins during the interaction with the host plant: Contributions of an in vivo system

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot, a highly destructive disease that affects all brassicas. This work aimed to study the interaction Xcc–Brassica oleracea using an in vivo system in an attempt to identify proteins involved in pathogenicity. We used label‐free shotgun 2D‐nanoUPLC/MS^E to analyze Xcc proteins in three conditions: in the interaction with susceptible (REK) and resistant (REU) plants and in culture medium (control condition). A model of Xcc–susceptible host interaction is proposed and shows that Xcc increases the abundance of several crucial proteins for infection and cell protection. In this study, we also confirmed the differential expression by qPCR analysis of selected genes. This is the first report showing a large‐scale identification of proteins in an in vivo host plant condition. Considering that most studies involving phytopathogens are in vitro (growth in culture medium or in plant extract), this work contributes with relevant information related to the plant–pathogen interaction in planta.