Jia-Xun Feng

Biofilm dispersal in Xanthomonas campestris is controlled by cell–cell signaling and is required for full virulence to plants

The rpf gene cluster of Xanthomonas campestris pathovar campestris (Xcc) is required for the pathogenesis of this bacterium to plants. Several rpf genes are involved in the coordinate positive regulation of the production of virulence factors mediated by the small diffusible molecule DSF (for diffusible signal factor). RpfF directs the synthesis of DSF, and a two-component sensory transduction system comprising RpfC and RpfG has been implicated in the perception of the DSF signal and signal transduction. In L medium, rpfF, rpfG, rpfC, and rpfGHC mutants grew as matrix-enclosed aggregates, whereas the wild type grew in a dispersed planktonic fashion. Synthesis of the extracellular polysaccharide xanthan was required for aggregate formation. Addition of DSF triggered dispersion of the aggregates formed by the rpfF strain, but not those of rpf strains defective in DSF signal transduction. An extracellular enzyme from Xcc whose synthesis was positively controlled by the DSF/rpf system could disperse the aggregates produced by all rpf strains. The enzyme was identified as the single endo-β-1,4-mannanase encoded by the Xcc genome. This enzyme had no detectable activity against soluble xanthan. The endo-β-1,4-mannanase was required for the full virulence of Xcc to plants. On the basis of this model system, we propose that one role of the β-mannanase during disease is to promote transitions from an aggregated or biofilm lifestyle to a planktonic lifestyle in response to the DSF signal.

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.

Colonization of Rice Leaf Blades by an African Strain of Xanthomonas oryzae pv. oryzae Depends on a New TAL Effector That Induces the Rice Nodulin-3 Os11N3 Gene

African strains of Xanthomonas oryzae pv. oryzae contain fewer TAL effectors than Asian strains, and their contribution to pathogenicity is unknown. Systematic mutagenesis of tal genes was used to decipher the contribution of each of the eight TAL effector paralogs to pathogenicity of African X. oryzae pv. oryzae BAI3. A strain mutated in talC was severely affected in the production of disease symptoms. Analysis of growth in planta upon leaf-clip inoculation showed that mutant bacteria multiplied only at the site of inoculation at the apex of the leaf, suggesting a requirement for talC during colonization of vascular tissues. Such tissue-specific effect of a tal mutant is a novel phenotype, which has not yet been characterized in other xanthomonads. Microarray experiments comparing the host response of rice leaves challenged with BAI3(R) vs. BAI3(R)ΔtalC were performed to identify genes targeted by TalC. A total of 120 upregulated and 21 downregulated genes were identified, among them Os11N3, which is a member of the MtN3/saliva family. Based on semiquantitative reverse transcription-polymerase chain reaction and β-glucuronidase reporter assays, we show that Os11N3 is directly upregulated by TalC and identify a TalC DNA target box within the Os11N3 upstream sequence.

Identification of Cellulase Genes from the Metagenomes of Compost Soils and Functional Characterization of One Novel Endoglucanase

Metagenomics, a new research field developed over the past decade, aims to identify potential enzymes from nonculturable microbes. In this study, genes encoding three glycoside hydrolase family (GHF) 9 endoglucanases and one GHF 5 endoglucanase were cloned and identified from the metagenome of the compost soils. The shared identities between the predicted amino acid sequences of these genes and their closest homologues in the database were less than 70%. One GHF 9 endoglucanase, Umcel9B, was further characterized. The recombinant protein, Umcel9B, showed activity against carboxymethyl cellulose, indicating that Umcel9B is an endoactive enzyme. Enzymatic activity occurs optimally at a pH of 7.0 and a temperature of 25°C.

The role of PilZ domain proteins in the virulence of Xanthomonas campestris pv. campestris

Cyclic di-GMP [(bis-(3-5)-cyclic di-guanosine monophosphate)] is an almost ubiquitous second messenger in bacteria that is implicated in the regulation of a range of functions that include developmental transitions, aggregative behaviour, adhesion, biofilm formation and virulence. Comparatively little is known about the mechanism(s) by which cyclic di-GMP exerts these various regulatory effects. PilZ has been identified as a cyclic di-GMP binding protein domain; proteins with this domain are involved in regulation of specific cellular processes, including the virulence of animal pathogens. Here we have examined the role of PilZ domain proteins in virulence and the regulation of virulence factor synthesis in Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot of crucifers. The Xcc genome encodes four proteins (XC0965, XC2249, XC2317 and XC3221) that have a PilZ domain. Mutation of XC0965, XC2249 and XC3221 led to a significant reduction of virulence in Chinese radish. Mutation of XC2249 and XC3221 led to a reduction in motility whereas mutation of XC2249 and XC0965 affected extracellular enzyme production. All mutant strains were unaffected in biofilm formation in vitro. The reduction of virulence following mutation of XC3221 could not be wholly attributed to an effect on motility as mutation of pilA, which abolishes motility, has a lesser effect on virulence.

AvrACXcc8004, a Type III Effector with a Leucine-Rich Repeat Domain from Xanthomonas campestris Pathovar campestris Confers Avirulence in Vascular Tissues of Arabidopsis thaliana Ecotype Col-0

Xanthomonas campestris pathovar campestris causes black rot, a vascular disease on cruciferous plants, including Arabidopsis thaliana. The gene XC1553 from X. campestris pv. campestris strain 8004 encodes a protein containing leucine-rich repeats (LRRs) and appears to be restricted to strains of X. campestris pv. campestris. LRRs are found in a number of type III-secreted effectors in plant and animal pathogens. These prompted us to investigate the role of the XC1553 gene in the interaction between X. campestris pv. campestris and A. thaliana. Translocation assays using the hypersensitive-reaction-inducing domain of X. campestris pv. campestris AvrBs1 as a reporter revealed that XC1553 is a type III effector. Infiltration of Arabidopsis leaf mesophyll with bacterial suspensions showed no differences between the wild-type strain and an XC1553 gene mutant; both strains induced disease symptoms on Kashmir and Col-0 ecotypes. However, a clear difference was observed when bacteria were introduced into the vascular system by piercing the central vein of leaves. In this case, the wild-type strain 8004 caused disease on the Kashmir ecotype, but not on ecotype Col-0; the XC1553 gene mutant became virulent on the Col-0 ecotype and still induced disease on the Kashmir ecotype. Altogether, these data show that the XC1553 gene, which was renamed avrAC(Xcc8004), functions as an avirulence gene whose product seems to be recognized in vascular tissues.

hpaR, a Putative marR Family Transcriptional Regulator, Is Positively Controlled by HrpG and HrpX and Involved in the Pathogenesis, Hypersensitive Response, and Extracellular Protease Production of Xanthomonas campestris Pathovar campestris

The MarR family of transcriptional regulators of bacteria are involved in the regulation of many cellular processes, including pathogenesis. In this work, we have demonstrated genetically that hpaR (hpa, hrp associated), which encodes a putative MarR family regulator, is involved in the hypersensitive response (HR), pathogenicity, and extracellular protease production of the phytopathogenic bacterium Xanthomonas campestris pathovar campestris. A mutation in hpaR resulted in complete loss of virulence in the host plant cabbage, a delayed and weakened HR in the nonhost plant pepper ECW-10R, and an increase in extracellular protease production. Detection of the beta-glucuronidase activity of a plasmid-driven hpaR promoter-gusA reporter revealed that the expression of hpaR is positively controlled by HrpG and HrpX and is suppressed in rich medium while being strongly induced in minimal and hrp-inducing media and inside the host. These findings indicate that hpaR belongs to the hrpG and hrpX regulon and that HrpX regulates the extracellular protease production via hpaR in X. campestris pv. campestris.

Comparative and functional genomics reveals genetic diversity and determinants of host specificity among reference strains and a large collection of Chinese isolates of the phytopathogen Xanthomonas campestris pv. campestris

BackgroundXanthomonas campestris pathovar campestris (Xcc) is the causal agent of black rot disease of crucifers worldwide. The molecular genetic diversity and host specificity of Xcc are poorly understood.ResultsWe constructed a microarray based on the complete genome sequence of Xcc strain 8004 and investigated the genetic diversity and host specificity of Xcc by array-based comparative genome hybridization analyses of 18 virulent strains. The results demonstrate that a genetic core comprising 3,405 of the 4,186 coding sequences (CDSs) spotted on the array are conserved and a flexible gene pool with 730 CDSs is absent/highly divergent (AHD). The results also revealed that 258 of the 304 proved/presumed pathogenicity genes are conserved and 46 are AHD. The conserved pathogenicity genes include mainly the genes involved in type I, II and III secretion systems, the quorum sensing system, extracellular enzymes and polysaccharide production, as well as many other proved pathogenicity genes, while the AHD CDSs contain the genes encoding type IV secretion system (T4SS) and type III-effectors. A Xcc T4SS-deletion mutant displayed the same virulence as wild type. Furthermore, three avirulence genes (avrXccC, avrXccE1 and avrBs1) were identified. avrXccC and avrXccE1 conferred avirulence on the hosts mustard cultivar Guangtou and Chinese cabbage cultivar Zhongbai-83, respectively, and avrBs1 conferred hypersensitive response on the nonhost pepper ECW10R.ConclusionAbout 80% of the Xcc CDSs, including 258 proved/presumed pathogenicity genes, is conserved in different strains. Xcc T4SS is not involved in pathogenicity. An efficient strategy to identify avr genes determining host specificity from the AHD genes was developed.

Identification of Six Type III Effector Genes with the PIP Box in Xanthomonas campestris pv. campestris and Five of Them Contribute Individually to Full Pathogenicity

Xanthomonas campestris pv. campestris is the pathogen of black rot of cruciferous plants. The pathogenicity of the pathogen depends on the type III secretion system (T3SS) that translocates directly effector proteins into plant cells, where they play important roles in the molecular interaction between the pathogen and its hosts. The T3SS of Xanthomonas spp. is encoded by a cluster of hypersensitive response and pathogenicity (hrp) genes. It has been demonstrated that the expression of hrp genes and some type III secreted (T3S)-effector genes is coactivated by the key hrp regulatory protein HrpX. The regulation by HrpX can be mediated by the binding of HrpX protein to a cis-regulatory element named the plant-inducible promoter (PIP) box present in the promoter region of HrpX-regulated genes. A genome screen revealed that X. campestris pv. campestris 8004 possesses 56 predicted genes with the PIP box. Nine of these genes have been shown to encode T3S effectors, Hrp, and Hrp-associated proteins. In this study, we employed an established T3S effector translocation assay with the hypersensitive-reaction-inducing domain of X. campestris pv. campestris AvrBs1 as a reporter to characterize the remaining 47 genes with the PIP box and showed that 6 of them, designated as XopXccE1, XopXccP, XopXccQ, XopXccR1, XopXccLR, and AvrXccB, harbor a functional translocation signal in their N-terminal regions, indicating that they are T3S effectors of X. campestris pv. campestris. We provided evidence to demonstrate that all these effectors are expressed in an HrpX-dependent manner and their translocation into plant cells relies on the translocon protein HrpF and the chaperone HpaB. Mutational analyses demonstrated that all these effectors, except AvrXccB, are individually required for full virulence and growth of X. campestris pv. campestris in the host plant Chinese radish.

Xanthomonas campestris pv. campestris Possesses a Single Gluconeogenic Pathway That Is Required for Virulence

Disruption of ppsA, a key gene in gluconeogenesis, of Xanthomonas campestris pv. campestris resulted in the failure of the pathogen to grow in medium with pyruvate or C4-dicarboxylates as the sole carbon source and a significant reduction in virulence, indicating that X. campestris pv. campestris possesses only the malic enzyme-PpsA route in gluconeogenesis, which is required for virulence.