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。     

Cloning of a locus involved in pathogenicity and production of extracellular polysaccharide and protease in Xanthomonas campestris pv. campestris

Abstract A Tn5-induced mutant of Xanthomonas campestris pv campestris deficient in protease and extracellular polysaccharide production, and non-pathogenic to turnip seedlings, was isolated. Recombinant clones which restored all three characteristics concomitantly were obtained from a wild-type genomic library. One of the plasmid clones, pIJ3090, showed no hybridization to previously isolated clones involved In protease production. The mutant was not complemented by other known clones involved in pathogenicity, in xanthan gum production or in protease production.     

Requirement of a mip-like gene for virulence in the phytopathogenic bacterium Xanthomonas campestris pv. campestris

Macrophage infectivity potentiators (Mips) are FKBP domain-containing proteins reported as virulence factors in several human pathogens, such as members of genera Legionella, Salmonella and Chlamydia. The putative peptidylprolyl cis-trans isomerase (PPIase) encoded by XC2699 of the plant bacterial pathogen Xanthomonas campestris pv. campestris 8004 exhibits a 49% similarity at the amino-acid level to the Mip protein of Legionella pneumophila. This mip-like gene, XC2699, was overexpressed in Escherichia coli and the purified (His)6-tagged Mip-like protein encoded by XC2699 exhibited a PPIase activity specifically inhibited by FK-506. A mutation in the mip-like gene XC2699 led to significant reductions in virulence and replication capacity in the host plant Chinese radish (Raphanus sativus L. var. radiculus Pers.). Furthermore, the production of exopolysaccharide and the activity of extracellular proteases, virulence factors of X. campestris pv. campestris, were significantly decreased in the mip-like mutant. These results reveal that the mip-like gene is involved in the pathogenesis of X. campestris pv. campestris through an effect on the production of these virulence factors.     

The zinc uptake regulator Zur is essential for the full virulence of Xanthomonas campestris pv. campestris

Zur is a regulator of the high-affinity zinc uptake system in many bacteria. In Xanthomonas campestris pv. campestris 8004, a putative protein encoded by the open reading frame designated as XC1430 shows 42% amino acid similarity with the Zur of Escherichia coli. An XC1430-disrupted mutant 1430nk was constructed by homologous suicide plasmid integration. 1430nk failed to grow in rich medium supplemented with Zn2+ at a concentration of 400 microM and in nonrich medium supplemented with Zn2+ at a concentration of 110 microM, whereas the wild-type strain grew well in the same conditions. In rich medium with 400 microM Zn2+, 1430nk accumulated significantly more Zn2+ than the wild-type strain. 1430nk showed a reduction in virulence on the host plant Chinese radish (Raphanus sativus L. var. radiculus Pers.) and produced less extracellular polysaccharide (EPS) than did the wild-type strain in the absence of added zinc. These results revealed that XC1430 is a functional member of the Zur regulator family that controls zinc homeostasis, EPS production, and virulence in X. campestris pv. campestris.     

The Host Plant Metabolite Glucose Is the Precursor of Diffusible Signal Factor (DSF) Family Signals in Xanthomonas campestris

Plant pathogen Xanthomonas campestris pv. campestris produces cis-11-methyl-2-dodecenoic acid (diffusible signal factor [DSF]) as a cell-cell communication signal to regulate biofilm dispersal and virulence factor production. Previous studies have demonstrated that DSF biosynthesis is dependent on the presence of RpfF, an enoyl-coenzyme A (CoA) hydratase, but the DSF synthetic mechanism and the influence of the host plant on DSF biosynthesis are still not clear. We show here that exogenous addition of host plant juice or ethanol extract to the growth medium of X. campestris pv. campestris could significantly boost DSF family signal production. It was subsequently revealed that X. campestris pv. campestris produces not only DSF but also BDSF (cis-2-dodecenoic acid) and another novel DSF family signal, which was designated DSF-II. BDSF was originally identified in Burkholderia cenocepacia to be involved in regulation of motility, biofilm formation, and virulence in B. cenocepacia. Functional analysis suggested that DSF-II plays a role equal to that of DSF in regulation of biofilm dispersion and virulence factor production in X. campestris pv. campestris. Furthermore, chromatographic separation led to identification of glucose as a specific molecule stimulating DSF family signal biosynthesis in X. campestris pv. campestris. ¹³C-labeling experiments demonstrated that glucose acts as a substrate to provide a carbon element for DSF biosynthesis. The results of this study indicate that X. campestris pv. campestris could utilize a common metabolite of the host plant to enhance DSF family signal synthesis and therefore promote virulence.     

Two Xanthomonas extracellular polygalacturonases, PghAxc and PghBxc, are regulated by type III secretion regulators HrpX and HrpG and are required for virulence

Xanthomonas campestris pv. campestris, the causal agent of black rot disease, produces a suite of extracellular cell-wall degrading enzymes (CWDE) that are involved in bacterial virulence. Polygalacturonase (PG) is an important CWDE and functions to degrade the pectic layers of plant cell walls. Although previous studies have documented the virulence functions of PG in Erwinia and Ralstonia species, the regulation of PG genes still needs to be elucidated. In this study, we identified two novel PG genes (pghAxc and pghBxc) encoding functional PG from X. campestris pv. campestris 8004. The expressions of these two PG genes are regulated by the type III secretion regulators HrpX and HrpG and the global regulator Clp. These PG genes could be efficiently induced in planta and were required for the full virulence of X. campestris pv. campestris to Arabidopsis. In addition, these PG were confirmed to be secreted via the type II secretion system in an Xps-dependent manner.     

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.     

Differential expression of conserved protease genes in crucifer-attacking pathovars of Xanthomonas campestris.

Strains of Xanthomonas campestris pathovars armoraciae and raphani, which cause leaf spotting diseases in brassicas, produce a major extracellular protease in liquid culture which was partially purified. The protease (PRT 3) was a zinc-requiring metalloenzyme and was readily distinguishable from the two previously characterized proteases (PRT 1 and PRT 2) of X. campestris pv. campestris by the pattern of degradation of beta-casein and sensitivity to inhibitors. PRT 3 was produced at a low level in the vascular brassica pathogen X. campestris pv. campestris (five strains tested), in which PRT 1 and PRT 2 predominate. In contrast, expression of PRT 1, a serine protease, could not be detected in the six tested strains of the leaf spotting mesophyll pathogens. However, all these strains had DNA fragments which hybridized to a prtA probe and which probably carry a functional prtA (the structural gene for PRT 1). The structural gene for PRT 3 (prtC) was cloned by screening a genomic library of X. campestris pv. raphani in a protease-deficient X. campestris pv. campestris strain. Subcloning and Tn5 mutagenesis located the structural gene to 1.2 kb of DNA. DNA fragments which hybridized to the structural gene were found in all strains of the crucifer-attacking X. campestris pathovars tested as well as in a number of other pathovars. Experiments in which the pattern of protease production of the pathovars was manipulated by introduction of cloned genes into heterologous pathovars suggested that no determinative relationship exists between the pattern of protease gene expression and the (vascular or mesophyllic) mode of pathogenesis.     

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.     

Deoxyribonucleic acid relatedness of 24 xanthomonad strains representing 23 Xanthomonas campestris pathovars and Xanthomonas fragariae

Twenty Xanthomonas campestris pathotype strains, three non-pathotype strains, and one strain of X. fragariae were studied by S1 DNA:DNA hybridization tests. The results of these tests do not support the retention of X. campestris as a single species. DNA reassociation values among many of the strains were low. Three clusters of closely related strains were observed, but nine strains did not cluster. Xanthomonas campestris pv. secalis was more closely related to X. fragariae than to any other X. campestris pathovar. Mapping the host family upon a three-dimensional genomic distance matrix of the xanthomonads suggested that strains attacking the same plant family usually show some relationship, but only a distant one. Thus, pathogenicity toward members of the same host family is not a measure of the genomic relationships of xanthomonads.     

Cell wall degrading enzymes in Orobanche aegyptiaca and its host Brassica campestris

Activities of the cell-wall degrading enzymes cellulase (EC 3. 2. 1. 4). poly-galacturonase (EC 3. 2. 1. 15) and xylanase (EC 3. 2. 1. 8), and also that of protease (EC 3. 4. 21. 14) have been determined in the parasite Orobanche aegyiptiaca Pers. and in the roots of infected and control host plants of Brassica campestris L. var. rapa Hartm. Shoots, roots and root-freed tubers of Orobanche showed pronounced activities of these enzymes. When expressed in terms of specific activity, protease, cellulase and polygalacturonase activities were higher and xylanase lower in all parts of Orobanche than in the roots of infected or control host plants. Orobanche roots had higher activities of cellulase, polygalacturonase and protease than tuber or shoot. While xylanase activity was higher in the shoot than in roots or tuber. Infection by Orobanche significantly enhanced the cellulase activity of host roots. Cellulase and polygalacturonase in the Orobanche roots appear as the main enzymes involved in establishing haustorial connection with the host root. Protease might be involved in degrading proteins and lipoproteins of the cell walls and membranes of the host tissues.     

Extracellular proteases from Xanthomonas campestris pv. campestris, the black rot pathogen

Two proteases (PRT1 and PRT2) were fractionated from culture supernatants of wild-type Xanthomonas campestris pv. campestris by cation-exchange chromatography on SP-5PW. Inhibitor experiments showed that PRT 1 was a serine protease which required calcium ions for activity or stability or both and that PRT 2 was a zinc-requiring metalloprotease. PRT 1 and PRT 2 showed different patterns of degradation of beta-casein. The two proteases comprised almost all of the extracellular proteolytic activity of the wild type. A protease-deficient mutant which lacked both PRT 1 and PRT 2 showed considerable loss of virulence in pathogenicity tests when bacteria were introduced into mature turnip leaves through cut vein endings. This suggests that PRT 1 and PRT 2 have a role in black rot pathogenesis.     

Extracellular proteases from Xanthomonas campestris pv. campestris, the black rot pathogen.

Two proteases (PRT1 and PRT2) were fractionated from culture supernatants of wild-type Xanthomonas campestris pv. campestris by cation-exchange chromatography on SP-5PW. Inhibitor experiments showed that PRT 1 was a serine protease which required calcium ions for activity or stability or both and that PRT 2 was a zinc-requiring metalloprotease. PRT 1 and PRT 2 showed different patterns of degradation of beta-casein. The two proteases comprised almost all of the extracellular proteolytic activity of the wild type. A protease-deficient mutant which lacked both PRT 1 and PRT 2 showed considerable loss of virulence in pathogenicity tests when bacteria were introduced into mature turnip leaves through cut vein endings. This suggests that PRT 1 and PRT 2 have a role in black rot pathogenesis.