Separable roles of the Pseudomonas syringae pv. phaseolicola accessory protein HrpZ1 in ion-conducting pore formation and activation of plant immunity

The HrpZ1 gene product from phytopathogenic Pseudomonas syringae is secreted in a type-III secretion system-dependent manner during plant infection. The ability of HrpZ1 to form ion-conducting pores is proposed to contribute to bacterial effector delivery into host cells, or may facilitate the nutrition of bacteria in the apoplast. Furthermore, HrpZ1 is reminiscent of a pathogen-associated molecular pattern (PAMP) that triggers immunity-associated responses in a variety of plants. Here, we provide evidence that the ion pore formation and immune activation activities of HrpZ1 have different structure requirements. All HrpZ1 orthologous proteins tested possess pore formation activities, but some of these proteins fail to trigger plant defense-associated responses. In addition, a C-terminal fragment of HrpZ1 retains the ability to activate plant immunity, whereas ion pore formation requires intact HrpZ1. Random insertion mutagenesis of HrpZ1 further revealed the C terminus to be important for the PAMP activity of the protein. HrpZ1 binds to plant membranes with high affinity and specificity, suggesting that the activation of plant immunity-associated responses by HrpZ1 is receptor-mediated. Our data are consistent with dual roles of HrpZ1 as a virulence factor affecting host membrane integrity, and as a microbial pattern governing the activation of plant immunity during infection.     

Diversifying selection drives the evolution of the type III secretion system pilus of Pseudomonas syringae

The plant pathogenic bacterium Pseudomonas syringae uses a type III secretion system to inject virulence proteins directly into the cytoplasm of its hosts. The P. syringae type III secretion apparatus is encoded, in part, by the HrpZ operon, which carries the hrpA gene encoding the pilin subunit of the pilus, various components of the structural apparatus, and the HrpZ harpin protein that is believed to produce pores in the host cell membrane. The pilus of the type III system comes into direct contact with the host cell and is, therefore, a likely target of the host's pathogen surveillance systems. We sequenced and analyzed 22 HrpZ operons from P. syringae strains spanning the diversity of the species. Selection analyses, including K(a)/K(s) tests and Tajima's D, revealed strong diversifying selection acting on the hrpA gene. This form of selection enables pathogens to maintain genetic diversity within their populations and is often driven by selection imposed by host defense systems. The HrpZ operon also revealed a single significant recombination event that dramatically changed the evolutionary relationships among P. syringae strains from 2 quite distinct phylogroups. This recombination event appears to have introduced genetic diversity into a clade of strains that may now be undergoing positive selection. The identification of diversifying selection acting on the Hrp pilus across the whole population sample and positive selection within one P. syringae lineage supports a trench warfare coevolutionary model between P. syringae and its plant hosts.     

Harpin of Pseudomonas syringae pv. phaseolicola harbors a protein binding site

Harpin HrpZ of plant-pathogenic bacterium Pseudomonas syringae elicits a hypersensitive response (HR) in some nonhost plants, but its function in the pathogenesis process is still obscure. HrpZ-interacting proteins were identified by screening a phage-display library of random peptides. HrpZ of the bean pathogen P. syringae pv. phaseolicola (HrpZPph) shows affinity to peptides with a consensus amino acid motif W(L)ARWLL(G/L). To localize the peptide-binding site, the hrpZPph gene was mutagenized with randomly placed 15-bp insertions, and the mutant proteins were screened for the peptide-binding ability. Mutations that inhibited peptide-binding localized to the central region of hrpZPph, which is separate from the previously determined HR-inducing region. Antiserum raised against one of the hrpZPph-binding peptides recognized small proteins in bean, tomato, parsley, and Arabidopsis thaliana but none in tobacco. On native protein blots, hrpZPph bound to a bean protein with similar pI as the protein recognized by the peptide antiserum. The result suggests a protein-protein interaction between the harpin and a host plant protein, possibly involved in the bacterial pathogenesis.     

The Hrp pilus of Pseudomonas syringae elongates from its tip and acts as a conduit for translocation of the effector protein HrpZ

The type III secretion system (TTSS) is an essential requirement for the virulence of many Gram-negative bacteria infecting plants, animals and man. Pathogens use the TTSS to deliver effector proteins from the bacterial cytoplasm to the eukaryotic host cell, where the effectors subvert host defences. Plant pathogens have to translocate their effector proteins through the plant cell wall barrier. The best candidates for directing effector protein traffic are bacterial appendages attached to the membrane-bound components of the TTSS. We have investigated the protein secretion route in relation to the TTSS appendage, termed the Hrp pilus, of the plant pathogen Pseudomonas syringae pv. tomato. By pulse expression of proteins combined with immunoelectron microscopy, we show that the Hrp pilus elongates by the addition of HrpA pilin subunits at the distal end, and that the effector protein HrpZ is secreted only from the pilus tip. Our results indicate that both HrpA and HrpZ travel through the Hrp pilus, which functions as a conduit for the long-distance translocation of effector proteins.     

Analysis of the role of the Pseudomonas syringae pv. syringae HrpZ harpin in elicitation of the hypersensitive response in tobacco using functionally non-polar hrpZ deletion mutations, truncated HrpZ fragments, and hrmA mutations

Pseudomonas syringae pv. syringae , like many plant pathogenic bacteria, secretes a 'harpin' protein that can elicit the hypersensitive response (HR), a defensive cellular suicide, in non-host plants. The harpin-encoding hrpZ gene is located in an operon that also encodes Hrp secretion pathway components and is part of the functional cluster of hrp genes carried on cosmid pHIR11 that enables saprophytic bacteria like Escherichia coli and Pseudomonas fluorescens to elicit the HR in tobacco leaves. We have constructed functionally non-polar hrpZ deletion mutations, revealing that HrpZ is necessary for saprophytic bacteria carrying pHIR11 to elicit a typical HR, whereas it only enhances the elicitation activity of P. s. syringae . Partial deletion mutations revealed that the N-terminal 153 amino acids of HrpZ can enable E. coli MC4100-(pHIR11) to elicit a strong HR. hrpZ subclone products comprising the N-terminal 109 amino acids and C-terminal 216 amino acids, respectively, of the 341 amino acid protein were isolated and found to elicit the HR. P. fluorescens (pHIR11 hrmA  ::Tn phoA ) mutants do not elicit the HR, but cell fractionation and immunoblot analysis revealed that they produce and secrete wild-type levels of HrpZ. Therefore, elicitor activity resides in multiple regions of HrpZ, P. syringae produces elicitor(s) in addition to HrpZ, and HrpZ is essential but not sufficient for HR elicitation by saprophytic bacteria carrying pHIR11.     

The Pseudomonas syringae HrpJ protein controls the secretion of type III translocator proteins and has a virulence role inside plant cells

The bacterial plant pathogen Pseudomonas syringae injects effector proteins into plant cells via a type III secretion system (T3SS), which is required for pathogenesis. The protein HrpJ is secreted by P. syringae and is required for a fully functional T3SS. A hrpJ mutant is non-pathogenic and cannot inject effectors into plant cells or secrete the harpin HrpZ1. Here we show that the hrpJ mutant also cannot secrete the harpins HrpW1 and HopAK1 or the translocator HrpK1, suggesting that these proteins are required in the translocation (injection) of effectors into plant cells. Complementation of the hrpJ mutant with secretion incompetent HrpJ derivatives restores the secretion of HrpZ1 and HrpW1 and the ability to elicit a hypersensitive response, a measure of translocation. However, growth in planta and disease symptom production is only partially restored, suggesting that secreted HrpJ may have a direct role in virulence. Transgenic Arabidopsis plants expressing HrpJ-HA complemented the virulence phenotype of the hrpJ mutant expressing a secretion incompetent HrpJ derivative and were reduced in their immune responses. Collectively, these data indicate that HrpJ has a dual role in P. syringae: inside bacterial cells HrpJ controls the secretion of translocator proteins and inside plant cells it suppresses plant immunity.     

Cellular locations of Pseudomonas syringae pv. syringae HrcC and HrcJ proteins, required for harpin secretion via the type III pathway

The complete hrp-hrc-hrmA cluster of Pseudomonas syringae pv. syringae 61 encodes 28 polypeptides. A saprophytic bacterium carrying this cluster is capable of secreting HrpZ-a harpin encoded by hrpZ-in an hrp-dependent manner, which suggests that this cluster contains sufficient components to assemble functional type III secretion machinery. Sequence data show that HrcJ and HrcC are putative outer membrane proteins, and nonpolar mutagenesis demonstrates they are all required for HrpZ secretion. In this study, we investigated the cellular localization of the HrcC and HrcJ proteins by Triton solubilization, sucrose-gradient isopycnic centrifugation, and immunogold labeling of the bacterial cell surface. Our results indicate that HrcC is indeed an outer membrane protein and that HrcJ is located between both membranes. Their membrane localization suggests that they might be involved in the formation of a supramolecular structure for protein secretion.     

Amyloidogenesis of type III-dependent harpins from plant pathogenic bacteria

Harpins are heat-stable, glycine-rich type III-secreted proteins produced by plant pathogenic bacteria, which cause a hypersensitive response (HR) when infiltrated into the intercellular space of tobacco leaves; however, the biochemical mechanisms by which harpins cause plant cell death remain unclear. In this study, we determined the biochemical characteristics of HpaG, the first harpin identified from a Xanthomonas species, under plant apoplast-like conditions using electron microscopy and circular dichroism spectroscopy. We found that His(6)-HpaG formed biologically active spherical oligomers, protofibrils, and beta-sheet-rich fibrils, whereas the null HR mutant His(6)-HpaG(L50P) did not. Biochemical analysis and HR assay of various forms of HpaG demonstrated that the transition from an alpha-helix to beta-sheet-rich fibrils is important for the biological activity of protein. The fibrillar form of His(6)-HpaG is an amyloid protein based on positive staining with Congo red to produce green birefringence under polarized light, increased protease resistance, and beta-sheet fibril structure. Other harpins, such as HrpN from Erwinia amylovora and HrpZ from Pseudomonas syringae pv. syringae, also formed curvilinear protofibrils or fibrils under plant apoplast-like conditions, suggesting that amyloidogenesis is a common feature of harpins. Missense and deletion mutagenesis of HpaG indicated that the rate of HpaG fibril formation is modulated by a motif present in the C terminus. The plant cytotoxicity of HpaG is unique among the amyloid-forming proteins that occur in several microorganisms. Structural and morphological analogies between HpaG and disease-related amyloidogenic proteins, such as Abeta protein, suggest possible common biochemical characteristics in the induction of plant and animal cell death.     

Synergistic pore formation by type III toxin translocators of Pseudomonas aeruginosa

Type III secretion/translocation systems are essential actors in the pathogenicity of Gram-negative bacteria. The injection of bacterial toxins across the host cell plasma membranes is presumably accomplished by a proteinaceous structure, the translocon. In vitro, Pseudomonas aeruginosa translocators PopB and PopD form ringlike structures observed by electron microscopy. We demonstrate here that PopB and PopD are functionally active and sufficient to form pores in lipid vesicles. Furthermore, the two translocators act in synergy to promote membrane permeabilization. The size-based selectivity observed for the passage of solutes indicates that the membrane permeabilization is due to the formation of size-defined pores. Our results provide also new insights into the mechanism of translocon pore formation that may occur during the passage of toxins from the bacterium into the cell. While proteins bind to lipid vesicles equally at any pH, the kinetics of membrane permeabilization accelerate progressively with decreasing pH values. Electrostatic interactions and the presence of anionic lipids were found to be crucial for pore formation whereas cholesterol did not appear to play a significant role in functional translocon formation.     

Pseudomonas syringae HrpJ is a type III secreted protein that is required for plant pathogenesis, injection of effectors, and secretion of the HrpZ1 Harpin

The bacterial plant pathogen Pseudomonas syringae requires a type III protein secretion system (TTSS) to cause disease. The P. syringae TTSS is encoded by the hrp-hrc gene cluster. One of the genes within this cluster, hrpJ, encodes a protein with weak similarity to YopN, a type III secreted protein from the animal pathogenic Yersinia species. Here, we show that HrpJ is secreted in culture and translocated into plant cells by the P. syringae pv. tomato DC3000 TTSS. A DC3000 hrpJ mutant, UNL140, was greatly reduced in its ability to cause disease symptoms and multiply in Arabidopsis thaliana. UNL140 exhibited a reduced ability to elicit a hypersensitive response (HR) in nonhost tobacco plants. UNL140 was unable to elicit an AvrRpt2- or AvrB1-dependent HR in A. thaliana but maintained its ability to secrete AvrB1 in culture via the TTSS. Additionally, UNL140 was defective in its ability to translocate the effectors AvrPto1, HopB1, and AvrPtoB. Type III secretion assays showed that UNL140 secreted HrpA1 and AvrPto1 but was unable to secrete HrpZ1, a protein that is normally secreted in culture in relatively large amounts, into culture supernatants. Taken together, our data indicate that HrpJ is a type III secreted protein that is important for pathogenicity and the translocation of effectors into plant cells. Based on the failure of UNL140 to secrete HrpZ1, HrpJ may play a role in controlling type III secretion, and in its absence, specific accessory proteins, like HrpZ1, may not be extracellularly localized, resulting in disabled translocation of effectors into plant cells.     

The Influence of Natural Lipid Asymmetry upon the Conformation of a Membrane-inserted Protein (Perfringolysin O)

Eukaryotic membrane proteins generally reside in membrane bilayers that have lipid asymmetry. However, in vitro studies of the impact of lipids upon membrane proteins are generally carried out in model membrane vesicles that lack lipid asymmetry. Our recently developed method to prepare lipid vesicles with asymmetry similar to that in plasma membranes and with controlled amounts of cholesterol was used to investigate the influence of lipid composition and lipid asymmetry upon the conformational behavior of the pore-forming, cholesterol-dependent cytolysin perfringolysin O (PFO). PFO conformational behavior in asymmetric vesicles was found to be distinct both from that in symmetric vesicles with the same lipid composition as the asymmetric vesicles and from that in vesicles containing either only the inner leaflet lipids from the asymmetric vesicles or only the outer leaflet lipids from the asymmetric vesicles. The presence of phosphatidylcholine in the outer leaflet increased the cholesterol concentration required to induce PFO binding, whereas phosphatidylethanolamine and phosphatidylserine in the inner leaflet of asymmetric vesicles stabilized the formation of a novel deeply inserted conformation that does not form pores, even though it contains transmembrane segments. This conformation may represent an important intermediate stage in PFO pore formation. These studies show that lipid asymmetry can strongly influence the behavior of membrane-inserted proteins.     

Expression of the Pseudomonas syringae avirulence protein AvrB in plant cells alleviates its dependence on the hypersensitive response and pathogenicity (Hrp) secretion system in eliciting genotype-specific hypersensitive cell death.

The nonpathogenic bacteria Pseudomonas fluorescens and Escherichia coli can elicit a genotype-specific hypersensitive response (HR) in plants if they express both the HR and pathogenesis (Hrp) protein secretion system and the HrpZ harpin from P. syringae pv syringae 61 and a P. syringae avirulence (avr) gene whose presence is recognized by a corresponding disease resistance gene in the plant. We have found that the recognition event appears to require transfer of the Avr protein into the plant cell. Elicitation of a genotype-specific HR was observed with avrB+ P. fluorescens in soybean and Arabidopsis plants carrying resistance genes RPG1 and RPM1, respectively, and with avrPto+ E. coll in tomato plants carrying resistance gene PTO, but only if the Hrp secretion system, HrpZ, and the appropriate Avr proteins were produced in the same bacterial cell. The failure of avrB hyperexpression and exogenous AvrB or HrpZ to alleviate these requirements in soybean and Arabidopsis suggests that the site of AvrB action is not in the bacterial cell or plant apoplast. An Arabidopsis rps3 (rpm1) glabrous1 mutant was transformed with constructs expressing avrB and was crossed with an Arabidopsis ecotype Columbia (RPM1 GLABROUS1) plant. F1 seedlings (identified by their kanamycin-resistant, pubescent phenotype) exhibited extensive necrosis on cotyledon leaves 10 days postgermination. Ecotype Columbia and rps3-1 leaves biolistically cobombarded with plasmids expressing the beta-glucuronidase (GUS) gene and avrB failed to produce GUS activity (indicative of cell death) only when RPM1 and avrB were present in the leaf. Thus, both stable and transient expression of avrB in Arabidopsis resulted in RPM1-dependent necrosis, and the only demonstrable site of action for AvrB was inside plant cells.     

The Avr (effector) proteins HrmA (HopPsyA) and AvrPto are secreted in culture from Pseudomonas syringae pathovars via the Hrp (type III) protein secretion system in a temperature- and pH-sensitive manner.

We present here data showing that the Avr proteins HrmA and AvrPto are secreted in culture via the native Hrp pathways from Pseudomonas syringae pathovars that produce these proteins. Moreover, their secretion is strongly affected by the temperature and pH of the culture medium. Both HrmA and AvrPto were secreted at their highest amounts when the temperature was between 18 and 22 degrees C and when the culture medium was pH 6.0. In contrast, temperature did not affect the secretion of HrpZ. pH did affect HrpZ secretion, but not as strongly as it affected the secretion of HrmA. This finding suggests that there are at least two classes of proteins that travel the P. syringae pathway: putative secretion system accessory proteins, such as HrpZ, which are readily secreted in culture; and effector proteins, such as HrmA and AvrPto, which apparently are delivered inside plant cells and are detected in lower amounts in culture supernatants under the appropriate conditions. Because HrmA was shown to be a Hrp-secreted protein, we have changed the name of hrmA to hopPsyA to reflect that it encodes a Hrp outer protein from P. syringae pv. syringae. The functional P. syringae Hrp cluster encoded by cosmid pHIR11 conferred upon P. fluorescens but not Escherichia coli the ability to secrete HopPsyA in culture. The use of these optimized conditions should facilitate the identification of additional proteins traveling the Hrp pathway and the signals that regulate this protein traffic.     

The ShcA protein is a molecular chaperone that assists in the secretion of the HopPsyA effector from the type III (Hrp) protein secretion system of Pseudomonas syringae

Pseudomonas syringae uses a type III protein secretion system encoded by the Hrp pathogenicity island (Pai) to translocate effector proteins into plant cells. One of these effector proteins is HopPsyA. A small open reading frame (ORF), named shcA, precedes the hopPsyA gene in the Hrp Pai of P. s. syringae 61. The predicted amino acid sequence of shcA shares general characteristics with chaperones used in type III protein secretion systems of animal pathogens. A functionally non-polar deletion of shcA in P. s. syringae 61 resulted in the loss of detectable HopPsyA in supernatant fractions, consistent with ShcA acting as a chaperone for HopPsyA. Cosmid pHIR11 carries a functional set of type III genes from P. s. syringae 61 and confers upon saprophytes the ability to secrete HopPsyA in culture and to elicit a HopPsyA-dependent hypersensitive response (HR) on tobacco. P. fluorescens carrying a pHIR11 derivative lacking shcA failed to secrete HopPsyA in culture, but maintained the ability to secrete another type III-secreted protein, HrpZ. This pHIR11 derivative was also greatly reduced in its ability to elicit an HR, indicating that the ability to translocate HopPsyA into plant cells was compromised. Using affinity chromatography, we showed that ShcA binds directly to HopPsyA and that the ShcA binding site must reside within the first 166 amino acids of HopPsyA. Thus, ShcA represents the first demonstrated chaperone used in a type III secretion system of a bacterial plant pathogen. We searched known P. syringae type III-related genes for neighbouring ORFs that shared the general characteristics of type III chaperones and identified five additional candidate type III chaperones. Therefore, it is likely that chaperones are as prevalent in bacterial plant pathogen type III systems as they are in their animal pathogenic counterparts.     

Transmembrane pore formation by the carboxyl terminus of Bax protein

Bax is a cytosolic protein that responds to various apoptotic signals by binding to the outer mitochondrial membrane, resulting in membrane permeabilization, release of cytochrome c, and caspase-mediated cell death. Currently discussed mechanisms of membrane perforation include formation of hetero-oligomeric complexes of Bax with other pro-apoptotic proteins such as Bak, or membrane insertion of multiple hydrophobic helices of Bax, or formation of lipidic pores physically aided by mitochondrial membrane-inserted proteins. There is compelling evidence provided by our and other groups indicating that the C-terminal “helix 9” of Bax mediates membrane binding and pore formation, yet the mechanism of pore forming capability of Bax C-terminus remains unclear. Here we show that a 20-amino acid peptide corresponding to Bax C-terminus (VTIFVAGVLTASLTIWKKMG) and two mutants where the two lysines are replaced with glutamate or leucine have potent membrane pore forming activities in zwitterionic and anionic phospholipid membranes. Analysis of the kinetics of calcein release from lipid vesicles allows determination of rate constants of pore formation, peptide–peptide affinities within the membrane, the oligomeric state of transmembrane pores, and the importance of the lysine residues. These data provide insight into the molecular details of membrane pore formation by a Bax-derived peptide and open new opportunities for design of peptide-based cytotoxic agents.     

The preprotein conducting channel at the inner envelope membrane of plastids

The preprotein translocation at the inner envelope membrane of chloroplasts so far involves five proteins: Tic110, Tic55, Tic40, Tic22 and Tic20. The molecular function of these proteins has not yet been established. Here, we demonstrate that Tic110 constitutes a central part of the preprotein translocation pore. Dependent on the presence of intact Tic110, radiolabelled preprotein specifically interacts with isolated inner envelope vesicles as well as with purified, recombinant Tic110 reconstituted into liposomes. Circular dichroism analysis reveals that Tic110 consists mainly of beta-sheets, a structure typically found in pore proteins. In planar lipid bilayers, recombinant Tic110 forms a cation-selective high-conductance channel with a calculated inner pore opening of 1.7 nm. Purified transit peptide causes strong flickering and a voltage-dependent block of the channel. Moreover, at the inner envelope membrane, a peptide-sensitive channel is described that shows properties basically identical to the channel formed by recombinant Tic110. We conclude that Tic110 has a distinct preprotein binding site and functions as a preprotein translocation pore at the inner envelope membrane.     

The hrpZ gene of Pseudomonas syringae pv. phaseolicola enhances resistance to rhizomania disease in transgenic Nicotiana benthamiana and sugar beet

To explore possible sources of transgenic resistance to the rhizomania-causing Beet necrotic yellow vein virus (BNYVV), Nicotiana benthamiana plants were constructed to express the harpin of Pseudomonas syringae pv. phaseolicola (HrpZ(Psph)). The HrpZ protein was expressed as an N-terminal fusion to the PR1 signal peptide (SP/HrpZ) to direct harpin accumulation to the plant apoplast. Transgene integration was verified by mPCR in all primary transformants (T0), while immunoblot analysis confirmed that the protein HrpZ(Psph) was produced and the signal peptide was properly processed. Neither T0 plants nor selfed progeny (T1) showed macroscopically visible necrosis or any other macroscopic phenotypes. However, plants expressing the SP/HrpZ(Psph) showed increased vigor and grew faster in comparison with non-transgenic control plants. Transgenic resistance was assessed after challenge inoculation with BNYVV on T1 progeny by scoring of disease symptoms and by DAS-ELISA at 20 and 30 dpi. Transgenic and control lines showed significant differences in terms of the number of plants that became infected, the timing of infection and the disease symptoms displayed. Plants expressing the SP/HrpZ(Psph) developed localized leaf necrosis in the infection area and had enhanced resistance upon challenge with BNYVV. In order to evaluate the SP/HrpZ-based resistance in the sugar beet host, A. rhizogenes-mediated root transformation was exploited as a transgene expression platform. Upon BNYVV inoculation, transgenic sugar beet hairy roots showed high level of BNYVV resistance. In contrast, the aerial non-transgenic parts of the same seedlings had virus titers that were comparable to those of the seedlings that were untransformed or transformed with wild type R1000 cells. These findings indicate that the transgenically expressed SP/HrpZ protein results in enhanced rhizomania resistance both in a model plant and sugar beet, the natural host of BNYVV. Possible molecular mechanisms underlying the enhanced resistance and plant growth phenotypes observed in SP/HrpZ transgenic plants are discussed.