The Pseudomonas syringae pv. tomato HrpW protein has domains similar to harpins and pectate lyases and can elicit the plant hypersensitive response and bind to pectate

Abstract

The host-specific plant pathogen Pseudomonas syringae elicits the hypersensitive response (HR) in nonhost plants and secretes the HrpZ harpin in culture via the Hrp (type III) secretion system. Previous genetic evidence suggested the existence of another harpin gene in the P. syringae genome. hrpW was found in a region adjacent to the hrp cluster in P. syringae pv. tomato DC3000. hrpW encodes a 42. 9-kDa protein with domains resembling harpins and pectate lyases (Pels), respectively. HrpW has key properties of harpins. It is heat stable and glycine rich, lacks cysteine, is secreted by the Hrp system, and is able to elicit the HR when infiltrated into tobacco leaf tissue. The harpin domain (amino acids 1 to 186) has six glycine-rich repeats of a repeated sequence found in HrpZ, and a purified HrpW harpin domain fragment possessed HR elicitor activity. In contrast, the HrpW Pel domain (amino acids 187 to 425) is similar to Pels from Nectria haematococca, Erwinia carotovora, Erwinia chrysanthemi, and Bacillus subtilis, and a purified Pel domain fragment did not elicit the HR. Neither this fragment nor the full-length HrpW showed Pel activity in A230 assays under a variety of reaction conditions, but the Pel fragment bound to calcium pectate, a major constituent of the plant cell wall. The DNA sequence of the P. syringae pv. syringae B728a hrpW was also determined. The Pel domains of the two predicted HrpW proteins were 85% identical, whereas the harpin domains were only 53% identical. Sequences hybridizing at high stringency with the P. syringae pv. tomato hrpW were found in other P. syringae pathovars, Pseudomonas viridiflava, Ralstonia (Pseudomonas) solanacearum, and Xanthomonas campestris. DeltahrpZ::nptII or hrpW::OmegaSpr P. syringae pv. tomato mutants were little reduced in HR elicitation activity in tobacco, whereas this activity was significantly reduced in a hrpZ hrpW double mutant. These features of hrpW and its product suggest that P. syringae produces multiple harpins and that the target of these proteins is in the plant cell wall.

FIG. 1

Physical map of the P. syringae pv. tomato DC3000 hrpW region and structural features of HrpW. (A) The physical map of the DC3000 genome adjacent to the hrp gene cluster is shown with open arrowheads denoting putative ς⁵⁴ promoters and with filled arrowheads denoting putative HrpL-dependent promoters that control previously defined transcriptional units (38). ORFs in polycistronic operons are marked by divisions in the open arrows denoting the operons. hrpR and hrpS encode regulatory proteins and are located at the right end of the hrp cluster. The region carried in pCPP2373 is indicated above the physical map, and inverted filled triangles mark the location of the ΩSp^r insertions in pCPP2374 and pCPP2375. (B) The diagram of HrpW indicates the harpin-like and Pel-like domains, with numbers on the top corresponding to DC3000 and on the bottom to B728a. The DC3000 hrpW PCR subclone-generated His₆-tagged harpin domain fragment encompasses amino acids 1 to 186; the His₆-tagged Pel domain has amino acids 187 to 425. (C) The sequences of the region in the middle of the HrpW proteins from P. syringae pv. tomato DC3000 (HrpW_Pto) and P. syringae pv. syringae B728a (HrpW_Psy), which contains glycine-rich repeats (denoted by the bar in panel B), are shown in the boxes. Dashes were inserted where necessary to permit alignment.

FIG. 2

DNA gel blot analysis of the hybridization of hrpW under high-stringency conditions to total DNA from other bacterial plant pathogens. DNA from the indicated pathogens was isolated, digested with EcoRI, resolved on a 0.5% agarose gel, transferred to an Immobilon-N membrane, and hybridized with a ³²P-labeled hrpW subclone at 62°C. Abbreviations: Pto, P. syringae pv. tomato; Psy, P. syringae pv. syringae; Pgy, P. syringae pv. glycinea; Ppp, P. syringae pv. papulans; Ppi, P. syringae pv. pisi; Pph, P. syringae pv. phaseolicola; Pta, P. syringae pv. tabaci; Pvf, P. viridiflava; Rso, Ralstonia solanacearum; Xam, Xanthomonas campestris pv. amoraciae; Xvs, X. campestris pv. vesicatoria; Ea, Erwinia amylovora; Ecc, E. carotovora subsp. carotovora; Ech, E. chrysanthemi.

FIG. 3

SDS-PAGE and immunoblot analysis of preparations containing the P. syringae pv. tomato HrpW and its harpin domain and Pel domain fragments. (A) His₆-tagged full-length HrpW and the two domain fragments were partially purified by Ni-NTA chromatography, separated by SDS-PAGE, and stained with Coomassie blue R250. The arrowhead indicates the full-length HrpW, which is produced in very small amounts. Lanes: 1, Pel domain fragment; 2, harpin domain fragment; 3, HrpW. (B) The same HrpW derivatives were also visualized on immunoblots with anti-HrpW antibodies used in conjunction with the Western light chemiluminescence assay. Lanes: 4, Pel domain fragment; 5, harpin domain fragment; 6, HrpW.

FIG. 4

Elicitation in tobacco leaves of active tissue death indicative of the HR by cell-free preparations containing the P. syringae pv. tomato HrpW and its N-terminal fragment. The protein preparations analyzed in Fig. 3 were infiltrated into tobacco leaves, in some cases with 1.0 mM lanthanum chloride. Leaves were photographed 48 h later. (A) P. syringae pv. syringae 61 HrpZ (0.12 μg/ml); (B) HrpW; (C) harpin domain fragment of HrpW (0.22 μg/ml); (D) HrpZ plus lanthanum chloride; (E) HrpW plus lanthanum chloride; (F) Pel domain fragment of HrpW (1.40 μg/ml).

FIG. 5

Binding of the HrpW Pel domain to calcium pectate beads. Purified HrpW Pel domain or marker proteins were mixed with calcium pectate or calcium alginate beads in 50 mM MES (pH 5.6), 500 mM NaCl and washed several times with the same buffer. In panels A through D, equal volumes from the original protein-bead mix (Total), the supernatant from the original mix (Unbound), the last buffer wash (Wash), and the washed beads (Bound) were analyzed by SDS-PAGE to determine if proteins bound to calcium pectate or calcium alginate beads. (A) HrpW Pel domain with calcium pectate beads; (B) HrpW Pel domain with calcium alginate beads; (C) mixed low-molecular-weight, low-pI, and high-pI marker proteins (Pharmacia, Uppsala, Sweden) with calcium pectate beads; (D) HrpZ with calcium pectate beads. In panels E and F, equal volumes from the original protein mix (Total), the supernatant, and the pellet were similarly analyzed to determine protein precipitation. (E) HrpW Pel domain with CaCl₂; (F) HrpW Pel domain with soluble pectate.