Identification of harpins in Pseudomonas syringae pv. tomato DC3000, which are functionally similar to HrpK1 in promoting translocation of type III secretion system effectors.

Harpins are a subset of type III secretion system (T3SS) substrates found in all phytopathogenic bacteria that utilize a T3SS. Pseudomonas syringae pv. tomato DC3000 was previously reported to produce two harpins, HrpZ1 and HrpW1. DC3000 was shown here to deploy two additional proteins, HopAK1 and HopP1, which have the harpin-like properties of lacking cysteine, eliciting the hypersensitive response (HR) when partially purified and infiltrated into tobacco leaves, and possessing a two-domain structure similar to that of the HrpW1 class of harpins. Unlike the single-domain harpin HrpZ1, the two-domain harpins have C-terminal enzyme-like domains: pectate lyase for HopAK1 and lytic transglycosylase for HopP1. Genetic techniques to recycle antibiotic markers were applied to DC3000 to generate a quadruple harpin gene polymutant. The polymutant was moderately reduced in the elicitation of the HR and translocation of the T3SS effector AvrPto1 fused to a Cya translocation reporter, but the mutant was unaffected in the secretion of AvrPto1-Cya. The DC3000 hrpK1 gene encodes a putative translocator in the HrpF/NopX family and was deleted in combination with the four harpin genes. The hrpK1 quadruple harpin gene polymutant was strongly reduced in HR elicitation, virulence, and translocation of AvrPto1-Cya into plant cells but not in the secretion of representative T3SS substrates in culture. HrpK1, HrpZ1, HrpW1, and HopAK1, but not HopP1, were independently capable of restoring some HR elicitation to the hrpK1 quadruple harpin gene polymutant, which suggests that a consortium of semiredundant translocators from three protein classes cooperate to form the P. syringae T3SS translocon.

Identification of Pseudomonas syringae pv. syringae 61 type III secretion system Hrp proteins that can travel the type III pathway and contribute to the translocation of effector proteins into plant cells.

Pseudomonas syringae translocates effector proteins into plant cells via an Hrp1 type III secretion system (T3SS). T3SS components HrpB, HrpD, HrpF, and HrpP were shown to be pathway substrates and to contribute to elicitation of the plant hypersensitive response and to translocation and secretion of the model effector AvrPto1.

The complete genome sequence of the Arabidopsis and tomato pathogen Pseudomonas syringae pv. tomato DC3000.

We report the complete genome sequence of the model bacterial pathogen Pseudomonas syringae pathovar tomato DC3000 (DC3000), which is pathogenic on tomato and Arabidopsis thaliana. The DC3000 genome (6.5 megabases) contains a circular chromosome and two plasmids, which collectively encode 5,763 ORFs. We identified 298 established and putative virulence genes, including several clusters of genes encoding 31 confirmed and 19 predicted type III secretion system effector proteins. Many of the virulence genes were members of paralogous families and also were proximal to mobile elements, which collectively comprise 7% of the DC3000 genome. The bacterium possesses a large repertoire of transporters for the acquisition of nutrients, particularly sugars, as well as genes implicated in attachment to plant surfaces. Over 12% of the genes are dedicated to regulation, which may reflect the need for rapid adaptation to the diverse environments encountered during epiphytic growth and pathogenesis. Comparative analyses confirmed a high degree of similarity with two sequenced pseudomonads, Pseudomonas putida and Pseudomonas aeruginosa, yet revealed 1,159 genes unique to DC3000, of which 811 lack a known function.

A pseudomonas syringae pv. tomato DC3000 Hrp (Type III secretion) deletion mutant expressing the Hrp system of bean pathogen P. syringae pv. syringae 61 retains normal host specificity for tomato.

The plant pathogenic species Pseudomonas syringae is divided into numerous pathovars based on host specificity. For example, P. syringae pv. tomato DC3000 is pathogenic on tomato and Arabidopsis, whereas P. syringae pv. syringae 61 is pathogenic on bean. The ability of P. syringae strains to elicit the hypersensitive response (HR) in non-hosts or be pathogenic (or parasitic) in hosts is dependent on the Hrp (type III secretion) system and effector proteins this system is thought to inject into plant cells. To test the role of the Hrp system in determining host range, the hrp/hrc gene cluster (hrpK through hrpR) was deleted from DC3000 and complemented in trans with the orthologous cluster from strain 61. Mutant CUCPB5114 expressing the bean pathogen Hrp system on plasmid pCPP2071 retained the ability of wild-type DC3000 to elicit the HR in bean, to grow and cause bacterial speck in tomato, and to elicit a cultivar-specific (gene-for-gene) HR in tomato plants carrying the Pto resistance gene. However, the symptoms produced in compatible tomato plants involved markedly reduced chlorosis, and CUCPB5114(pCPP2071) did not grow or produce symptoms in Arabidopsis Col-0 although it was weakly virulent in NahG Arabidopsis. A hypersensitive-like collapse was produced by CUCPB5114(pCPP2071) in Arabidopsis Col-0 at 1 x 10(7) CFU/ml, but only if the bacteria also expressed AvrB, which is recognized by the RPM1 resistance gene in Col-0 and confers incompatibility. These observations support the concept that the P. syringae effector proteins, rather than secretion system components, are the primary determinants of host range at both the species and cultivar levels of host specificity.