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. 2001 Jan 2;98(1):289-94.
doi: 10.1073/pnas.98.1.289.

HrpZ(Psph) from the plant pathogen Pseudomonas syringae pv. phaseolicola binds to lipid bilayers and forms an ion-conducting pore in vitro

J Lee  1 , B Klusener, G Tsiamis, C Stevens, C Neyt, A P Tampakaki, N J Panopoulos, J Nöller, E W Weiler, G R Cornelis, J W Mansfield, T Nürnberger
Affiliations

HrpZ(Psph) from the plant pathogen Pseudomonas syringae pv. phaseolicola binds to lipid bilayers and forms an ion-conducting pore in vitro

J Lee et al. Proc Natl Acad Sci U S A. .

Abstract

The hrp gene clusters of plant pathogenic bacteria control pathogenicity on their host plants and ability to elicit the hypersensitive reaction in resistant plants. Some hrp gene products constitute elements of the type III secretion system, by which effector proteins are exported and delivered into plant cells. Here, we show that the hrpZ gene product from the bean halo-blight pathogen, Pseudomonas syringae pv. phaseolicola (HrpZ(Psph)), is secreted in an hrp-dependent manner in P. syringae pv. phaseolicola and exported by the type III secretion system in the mammalian pathogen Yersinia enterocolitica. HrpZ(Psph) was found to associate stably with liposomes and synthetic bilayer membranes. Under symmetric ionic conditions, addition of 2 nM of purified recombinant HrpZ(Psph) to the cis compartment of planar lipid bilayers provoked an ion current with a large unitary conductivity of 207 pS. HrpZ(Psph)-related proteins from P. syringae pv. tomato or syringae triggered ion currents similar to those stimulated by HrpZ(Psph). The HrpZ(Psph)-mediated ion-conducting pore was permeable for cations but did not mediate fluxes of Cl-. Such pore-forming activity may allow nutrient release and/or delivery of virulence factors during bacterial colonization of host plants.

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Figures

Figure 1
Figure 1
Hydrophobicity plot of HrpZPsph based on the Eisenberg algorithm (35).
Figure 2
Figure 2
Analysis of HrpZPsph secretion by Psph and by Y. enterocolitica. (A) hrp-dependent secretion of HrpZPsph. Psph race-6 wild-type (wt), race-6 hrpA− mutant (hrpA−), and a type IV pili mutant of strain HB10Y (pilM−) were grown in complex media or hrp-inducing minimal media (8). Bacteria were pelleted by centrifugation, and secreted proteins were precipitated from the culture supernatant by 5% (vol/vol) trichloroacetic acid. Proteins prepared from the supernatant (Upper) and pellet (Lower) were analyzed by SDS/PAGE and immunoblotting by using an antiserum raised against recombinant HrpZPsph (1:5,000 dilution). Numbers below individual lanes represent β-glucuronidase activity (nmol 4-methylumbelliferone released per minute per bacterium × 1010), which was determined in uidA-transformed Psph strains grown in complex and minimal growth medium (30). (B) Type III-dependent secretion of HrpZPsph. Y. enterocolitica wild-type strain KNG22703(pYV227) (wt), this strain carrying pBluescript SK(+)-hrpZ (wt∷hrpZ), and Y. enterocolitica type III secretion-deficient yscN- mutant KNG22703(pYV2276) transformed with plasmid pBluescript SK(+)-hrpZ (yscN∷hrpZ) were grown under permissive conditions. Analysis of secreted proteins was performed by using antisera raised against HrpZPsph or YopE, respectively.
Figure 3
Figure 3
HrpZPsph interacts with lipid membranes. (A) Psph race-6 wild-type (wt) and race-6 mutant hrpA− were grown in hrp-inducing minimal media (8) in the absence (−) or presence (+) of liposomes. Proteins prepared from the supernatant or proteoliposomes were separated and analyzed by SDS/PAGE immunoblotting with an anti-HrpZPsph antiserum. (B) TRANSIL beads coated with 1-hexadecanoyl-2-(cis-9-octadecenoyl)-sn-glycero-3-phosphocholine (POPC)/1-hexadecanoyl-2-(cis-9-octadecenoyl)-sn-glycero-3-phosphoethanolamine (POPE) (80:20) were incubated for 1 h with 1 μM of the proteins indicated (Total). After separation of lipid-bound (Bound) from unbound material, proteins were analyzed by SDS/PAGE and silver staining. HrpZPst, HrpZ from P. syringae pv. tomato DC3000; HrpZPss, HrpZ from P. syringae pv. syringae; AA 1–80, AA 100–200, AA 201–345, HrpZPsph fragment encompassing the N-terminal 80 amino acid (AA) residues, amino acids 100–200, or the C-terminal amino acids 201–345, respectively.
Figure 4
Figure 4
HrpZPsph and related HrpZPst and HrpZPss trigger ion-current fluctuations in planar lipid bilayers. (A) Protein from E. coli contaminants was prepared as described in Materials and Methods; purified recombinant HrpZPsph (B), HrpZPst (C), or HrpZPss (D) was added to the cis-aqueous solution of the bilayer cuvette, and the induced current traces were recorded. In B, HrpZPsph-induced traces were recorded at different membrane potentials, as indicated. Electrolyte solutions (cis/trans) contained 100 mM KCl and 10 mM Hepes, pH 7.0. Dashed lines indicate different open states. c, closed, o1-o3, open states. Note different y axes in A–D.
Figure 5
Figure 5
Electrophysiological properties of HrpZPsph-induced currents. (A) HrpZPsph-induced currents do not exhibit rectifying properties. After addition of HrpZPsph to the cis compartment of the bilayer cuvette, ion currents were recorded at a membrane potential of −100 mV and after immediately switching the membrane potential to +100 mV after 2 sec of total recording. (B) Linear current–voltage relationship of HrpZPsph-induced ion fluxes. Current–voltage relationships of HrpZPsph-induced nonselective cation currents were established in three different electrolyte solutions: 100 mM KCl and 10 mM Hepes, pH 7.0 (○); 100 mM NaCl and 10 mM Hepes, pH 7.0 (▫); and 90 mM K+-gluconate, 10 mM KCl, and 10 mM Hepes, pH 7.0 (▵). The means of three recordings are given.
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