Identification of Harpins in Pseudomonas syringae pv. tomato DC3000, Which Are Functionally Similar to HrpK1 in Promoting Translocation of Type III Secretion System Effectors

Abstract

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.The type III secretion system (T3SS) is an important virulence determinant in gram-negative bacterial pathogens of animals and plants (17). In Pseudomonas syringae pv. tomato DC3000, the T3SS, which is encoded by the hrp-hrc (hypersensitive response [HR] and pathogenicity-hr conserved) gene cluster, is required for elicitation of the defense-associated HR in nonhost plants like tobacco and for pathogenicity in host plants like tomato. Both HR elicitation and pathogenicity require the translocation into plant cells of effectors, which are also known as Avr (avirulence) proteins or Hops (Hrp outer proteins) (44).The T3SS of pathogens must cross three biological membranes, the inner and outer bacterial membranes and the host plasma membrane. Breaching the host plasma membrane is expected to involve a T3SS pore-forming translocon complex (17). In addition, the T3SS of phytopathogens such as P. syringae, Erwinia amylovora, Ralstonia solanacearum, Pantoea spp., and Xanthomonas spp. must cross the plant cell wall, which can be several hundred nanometers in width and is constructed primarily of a meshwork of polysaccharides, with pectic polymers being particularly important in controlling pore size and integrity (59,66). Because of this additional barrier, the Hrp pilus of phytopathogens is much longer than the T3SS extracellular appendages of animal p...

Genetic disassembly and combinatorial reassembly identify a minimal functional repertoire of type III effectors in Pseudomonas syringae

“…S2 A and C). DC3000D28E carrying pCPP5702, a plasmid expressing avrPto-cya from its native promoter (15), translocated the reporter as well as DC3000(pCPP5702) (with two levels of inoculum used to ensure that the assay was not saturated) (15) (Fig. 2B).…”

“…DC3000D28E seems to elicit plant defenses that are T3SS-dependent and additional to basal PTI. In this regard, it is noteworthy that DC3000D28E has the WT complement of T3SS helper proteins (except HrpW1), which fall into the overlapping functional classes of harpins, translocators, and lytic transglycosylases, and several of these proteins can elicit plant defenses (15). T3Es in the minimal functional repertoire restore virulence to DC3000D28E in the following approximate hierarchy.…”

“…Primers for plasmid and mutant construction are given in Table S2. Culture conditions, plant virulence assays, and Cya reporter translocation assays have been previously described (13,15).…”

Rhizobium etli HrpW is a pectin-degrading enzyme and differs from phytopathogenic homologues in enzymically crucial tryptophan and glycine residues

“…syringae, as it was reported that virulence of hrpW mutants in these bacteria was at wild-type level (Charkowski et al, 1998;Kim & Beer, 1998). Only recently, Kvitko et al (2007) succeeded in shedding light on the biological role of HrpW. They did so by constructing a polymutant that is severely affected in its capacity to direct effector proteins through the host cell membrane, and by studying complementation with individual proteins supposedly involved in this activity.…”

Expression of hpa1 Gene Encoding a Bacterial Harpin Protein in Xanthomonas oryzae pv. oryzae Enhances Disease Resistance to Both Fungal and Bacterial Pathogens in Rice and Arabidopsis

“…They are secreted from bacteria through type III secretion system and are known to be located at intercellular space of plant tissues (Alfano and Collmer, 1997). Harpin proteins have been identified from diverse Gram-negative plantpathogenic bacteria such as Erwinia amylovora (Kim and Beer, 1998;Wei et al, 1992), Pseudomonas syringae (Charkowski et al, 1998;He et al, 1993;Kvitko et al, 2007), Ralstonia solanacearum (Arlat et al, 1994), and Xanthomonas species (Kim et al, 2003;Noel et al, 2002;Zhu et al, 2000). For example, Hpa1 is the harpin protein that is produced and secreted from X. oryzae pv.…”