Chemical Targeting and Manipulation of Type III Secretion in the Phytopathogen Xanthomonas campestris for Control of Disease.

Xanthomonas campestris pv. campestris is the causative agent of black rot disease in crucifer plants. This Gram-negative bacterium utilizes the type III secretion system (T3SS), encoded by the hrp gene cluster, to aid in its resistance to host defenses and the ability to cause disease. The T3SS injects a set of proteins known as effectors into host cells that come into contact with the bacterium. The T3SS is essential for the virulence and hypersensitive response (HR) of X. campestris pv. campestris, making it a potential target for disease control strategies. Using a unique and straightforward high-throughput screening method, we examined a large collection of diverse small molecules for their potential to modulate the T3SS without affecting the growth of X. campestris pv. campestris. Screening of 13,129 different compounds identified 10 small molecules that had a significant inhibitory influence on T3SS. Moreover, reverse transcription-quantitative PCR (qRT-PCR) assays demonstrated that all 10 compounds repress the expression of the hrp genes. Interestingly, the effect of these small molecules on hrp genes may be through the HpaS and ColS sensor kinase proteins that are key to the regulation of the T3SS in planta Five of the compounds were also capable of inhibiting X. campestris pv. campestris virulence in a Chinese radish leaf-clipping assay. Furthermore, seven of the small molecules significantly weakened the HR in nonhost pepper plants challenged with X. campestris pv. campestris. Taken together, these small molecules may provide potential tool compounds for the further development of antivirulence agents that could be used in disease control of the plant pathogen X. campestris pv. campestris.IMPORTANCE The bacterium Xanthomonas campestris pv. campestris is known to cause black rot disease in many socioeconomically important vegetable crops worldwide. The management and control of black rot disease have been tackled with chemical and host resistance methods with variable success. This has motivated the development of alternative methods for preventing this disease. Here, we identify a set of novel small molecules capable of inhibiting X. campestris pv. campestris virulence, which may represent leading compounds for the further development of antivirulence agents that could be used in the control of black rot disease.

RpfC regulates the expression of the key regulator hrpX of the hrp/T3SS system in Xanthomonas campestris pv. campestris.

BACKGROUND: The Gram-negative phytopathogenic bacterium Xanthomonas campestris pv. campestris recruits the hrp/T3SS system to inject pathogenicity effector proteins into host cells and uses the rpf/DSF cell-cell signaling system to regulate the expression of virulence factors such as extracellular enzymes and polysaccharide. Whether these two systems have any connection is unknown. METHODS: Positive regulator candidates affecting hrpX expression were identified by sacB strategy. The transcriptional expression was determined by qRT-PCR and GUS activity analysis. Transcriptome analysis was performed by RNA deep-sequencing. The hypersensitive response (HR) was determined in the nonhost plant pepper ECW-10R and electrolyte leakage assay. RESULTS: Mutation of the gene encoding the sensor RpfC of the rpf/DSF system significantly reduced the expression of hrpX, the key regulator of the hrp/T3SS system, all of the genes in the hrp cluster and most reported type III effector genes. Mutation of rpfG did not affect the expression of hrpX. The rpfC mutant showed a delayed and weakened HR induction. CONCLUSIONS: RpfC positively regulates the expression of hrpX independent of RpfG, showing a complex regulatory network linking the rpf/DSF and hrp/T3SS systems.

The C-terminal domain of the type III secretion chaperone HpaB contributes to dissociation of chaperone-effector complex in Xanthomonas campestris pv. campestris.

Many animal and plant pathogenic bacteria employ a type three secretion system (T3SS) to deliver type three effector proteins (T3Es) into host cells. Efficient secretion of many T3Es in the plant pathogen Xanthomonas campestris pv. campestris (Xcc) relies on the global chaperone HpaB. However, how the domain of HpaB itself affects effector translocation/secretion is poorly understood. Here, we used genetic and biochemical approaches to identify a novel domain at the C-terminal end of HpaB (amino acid residues 137-160) that contributes to virulence and hypersensitive response (HR). Both in vitro secretion assay and in planta translocation assay showed that the secretion and translocation of T3E proteins depend on the C-terminal region of HpaB. Deletion of the C-terminal region of HpaB did not affect binding to T3Es, self-association or interaction with T3SS components. However, the deletion of C-terminal region sharply reduced the mounts of free T3Es liberated from the complex of HpaB with the T3Es, a reaction catalyzed in an ATP-dependent manner by the T3SS-associated ATPase HrcN. Our findings demonstrate the C-terminal domain of HpaB contributes to disassembly of chaperone-effector complex and reveal a potential molecular mechanism underpinning the involvement of HpaB in secretion of T3Es in Xcc.

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.

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.

The type III secretion effector XopXccN of Xanthomonas campestris pv. campestris is required for full virulence.

XopN was originally identified from Xanthomonas campestris pv. vesicatoria as an effector translocated into plant cells via the type III secretion system (T3SS), and is required for pathogenicity. We report here that the xopN homologue in the X. campestris pv. campestris genome, named xopXccN, also encodes a T3SS effector and is required for full virulence. We further demonstrate that expression of xopXccN is positively regulated by the key hrp (hypersensitive response and pathogenicity) regulators HrpG and HrpX.