RESUMEN
Dickeya dadantii 3937 is a gram-negative phytopathogenic bacterium that expresses genes encoding a type III secretion system (T3SS) in a bistable pattern when cultured in a homogeneous minimal media. In this work, we further characterized the bistable gene expression of T3SS at the single-cell level. We demonstrated that bistable expression of the HrpL-regulon genes, such as hrpA and hrpN, is controlled by the same regulatory mechanism. We also showed that the expression level of the T3SS master regulatory gene hrpL plays an important role in the development of the bistable expression of hrpA. A high expression level of hrpL is required but unable to guarantee the high-state expression of hrpA in a cell. In addition, bistable expression patterns of T3SS genes in other gram-negative pathogens of the Enterobacteriaceae and Pseudomonadaceae families were also described in this study. This suggests that the T3SS bistability might be a conserved population behavior in several gram-negative bacterial pathogens.
Asunto(s)
Proteínas Bacterianas/genética , Sistemas de Secreción Bacterianos/genética , Enterobacteriaceae/genética , Regulación Bacteriana de la Expresión Génica/genética , Genes Reporteros , Bacterias Gramnegativas/genética , Modelos Genéticos , Mutación , Plásmidos , Regiones Promotoras Genéticas/genética , ARN Bacteriano/genética , Factores de Virulencia/genéticaRESUMEN
Xanthomonas oryzae pv. oryzicola, the causal agent of bacterial leaf streak in the model plant rice, possesses a hypersensitive response and pathogenicity (hrp), hrp-conserved (hrc), hrp-associated (hpa) cluster (hrp-hrc-hpa) that encodes a type III secretion system (T3SS) through which T3SS effectors are injected into host cells to cause disease or trigger plant defenses. Mutations in this cluster usually abolish the bacterial ability to cause hypersensitive response in nonhost tobacco and pathogenicity in host rice. In Xanthomonas spp., these genes are generally assumed to be regulated by the key master regulators HrpG and HrpX. However, we present evidence that, apart from HrpG and HrpX, HrpD6 is also involved in regulating the expression of hrp genes. Interestingly, the expression of hpa2, hpa1, hpaB, hrcC, and hrcT is positively controlled by HrpD6. Transcriptional expression assays demonstrated that the expression of the hrcC, hrpD5, hrpE, and hpa3 genes was not completely abolished by hrpG and hrpX mutations. As observed in analysis of their corresponding mutants, HrpG and HrpX exhibit contrasting gene regulation, particularly for hpa2 and hrcT. Other two-component system regulators (Zur, LrpX, ColR/S, and Trh) did not completely inhibit the expression of hrcC, hrpD5, hrpE, and hpa3. Immunoblotting assays showed that the secretion of HrpF, which is an HpaB-independent translocator, is not affected by the mutation in hrpD6. However, the mutation in hrpD6 affects the secretion of an HpaB-dependent TAL effector, AvrXa27. These novel findings suggest that, apart from HrpG and HrpX, HrpD6 plays important roles not only in the regulation of hrp genes but also in the secretion of TAL effectors.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/genética , Enfermedades de las Plantas/microbiología , Xanthomonas/genética , Proteínas Bacterianas/genética , Células Cultivadas , ADN Bacteriano/genética , Genes Bacterianos/genética , Genes Reguladores/genética , Genes Reporteros , Prueba de Complementación Genética , Familia de Multigenes/genética , Operón/genética , Oryza/microbiología , Regiones Promotoras Genéticas/genética , ARN Bacteriano/genética , Plantones/microbiología , Eliminación de Secuencia , Transcripción Genética , Virulencia , Xanthomonas/metabolismo , Xanthomonas/patogenicidadRESUMEN
Xanthomonas oryzae pv. oryzicola, the causative agent of bacterial leaf streak, injects a plethora of effectors through the type III secretion system (T3SS) into rice cells to cause disease. The T3SS, encoded by the hrp genes, is essential for the pathogen to elicit the hypersensitive response (HR) in nonhost tobacco and for pathogenicity in host rice. Whether or not a putative lytic transglycosylase, Hpa2, interacts with a translocon protein, HrpF, to facilitate bacterial pathogenicity remains unknown. Here we demonstrated that both the hpa2 and hrpF genes are required for the pathogenicity of X. oryzae pv. oryzicola strain RS105 in rice but not for HR induction in tobacco. The expression of hpa2 was positively regulated by HrpG and HrpD6 but not by HrpX. In vivo secretion and subcellular localization analyses confirmed that Hpa2 secretion is dependent on HpaB (a T3SS exit protein) and that Hpa2 binds to the host cell membrane. Protein-protein assays demonstrated that Hpa2 interacts with HrpF. In planta translocation of AvrXa10 indicated that the mutation in hpa2 and hrpF inhibits the injection of the HpaB-dependent transcriptional activator-like (TAL) effector into rice. These findings suggest that Hpa2 and HrpF form a complex to translocate T3S effectors into plant cells for pathogenesis in host rice.