The hrpK Operon of Pseudomonas syringae pv. tomato DC3000 Encodes Two Proteins Secreted by the Type III (Hrp) Protein Secretion System: HopB1 and HrpK, a Putative Type III Translocator

Abstract

Pseudomonas syringae is a gram-negative bacterial plant pathogen that is dependent on a type III protein secretion system (TTSS) and the effector proteins it translocates into plant cells for pathogenicity. The P. syringae TTSS is encoded by hrp-hrc genes that reside in a central region of a pathogenicity island (Pai). Flanking one side of this Pai is the exchangeable effector locus (EEL). We characterized the transcriptional expression of the open reading frames (ORFs) within the EEL of P. syringae pv. tomato DC3000. One of these ORFs, PSPTO1406 (hopB1) is expressed in the same transcriptional unit as hrpK. Both HopB1 and HrpK were secreted in culture and translocated into plant cells via the TTSS. However, the translocation of HrpK required its C-terminal half. HrpK shares low similarity with a putative translocator, HrpF, from Xanthomonas campestris pv. vesicatoria. DC3000 mutants lacking HrpK were significantly reduced in disease symptoms and multiplication in planta, whereas DC3000 hopB1 mutants produced phenotypes similar to the wild type. Additionally, hrpK mutants were reduced in their ability to elicit the hypersensitive response (HR), a programmed cell death associated with plant defense. The reduced HR phenotype exhibited by hrpK mutants was complemented by hrpK expressed in bacteria but not by HrpK transgenically expressed in tobacco, suggesting that HrpK does not function inside plant cells. Further experiments identified a C-terminal transmembrane domain within HrpK that is required for HrpK translocation. Taken together, HopB1 is a type III effector and HrpK plays an important role in the TTSS and is a putative type III translocator.

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

“…Because our complementation experiments with the ⌬hrpK1 quadruple harpin gene polymutant suggest that HrpK1 and three of the harpins have overlapping functions, it is important to assess the evidence that HrpK1 is a translocator. An overriding problem in this regard is that P. syringae hrpK1 mutants of various pathovars have been reported to have only variably reduced or conditional phenotypes (6,7,16,45,54). A recent report by Petnicki-Ocwieja et al (54) showed that P. syringae pv.…”

“…6A), which is consistent with previous reports of the moderate HR phenotype of a hrpK1 P. syringae pv. tomato DC3000 mutant (54). The ⌬hrpK1 ⌬hrpZ1 double mutant showed a stronger reduction in HR elicitation ability, and the ⌬hrpK1 ⌬hrpZ1 ⍀hrpW1 ⌬hopAK1 ⌬hopP1 polymutant had the strongest phenotype, failing to elicit the HR even at the high concentration of 3 ϫ 10 8 CFU ml Ϫ1 (Fig.…”

“…Furthermore, HrpF has been shown to form pores in planar lipid bilayers (10). The P. syringae HrpK1 protein is also a member of the HrpF/NopX family with a predicted TM domain (47,54). HrpK1 is encoded in the hrp-hrc gene cluster, which suggests a central role in the T3SS.…”

“…HrpK1 is encoded in the hrp-hrc gene cluster, which suggests a central role in the T3SS. However, hrpK1 mutants have relatively weak and variable T3SS phenotypes, which brings into question the role of HrpK1 as a translocator (47,54).…”

Protein Export According to Schedule: Architecture, Assembly, and Regulation of Type III Secretion Systems from Plant- and Animal-Pathogenic Bacteria

“…In contrast to Xanthomonas campestris pv. vesicatoria, several predicted translocon proteins from other plant-pathogenic bacteria are not essential for pathogenicity, suggesting that additional proteins such as harpins are involved in effector protein translocation (47,300,364,438). Harpins are small, heat-stable T3S substrates from plant-pathogenic bacteria that are rich in glycine and can elicit plant defense responses when infiltrating the plant apoplast at high concentrations.…”