The Pseudomonas syringae pv. syringae 61 hrpH product, an envelope protein required for elicitation of the hypersensitive response in plants.

Pseudomonas syringae pv. syringae 61 contains a 25-kb cluster of hrp genes that are required for elicitation of the hypersensitive response (HR) in tobacco. TnphoA mutagenesis of cosmid pHIR11, which contains the hrp cluster, revealed two genes encoding exported or inner-membrane-spanning proteins (H.-C. Huang, S. W. Hutcheson, and A. Collmer, Mol. Plant-Microbe Interact. 4:469-476, 1991). The gene in complementation group X, designated hrpH, was subcloned on a 3.1-kb SalI fragment into pCPP30, a broad-host-range, mobilizable vector. The subclone restored the ability of hrpH mutant P. syringae pv. syringae 61-2089 to elicit the HR in tobacco. DNA sequence analysis of the 3.1-kb SalI fragment revealed a single open reading frame encoding an 81,956-Da preprotein with a typical amino-terminal signal peptide and no likely inner-membrane-spanning hydrophobic regions. hrpH was expressed in the presence of [35S]methionine by using the T7 RNA polymerase-promoter system and vector pT7-3 in Escherichia coli and was shown to encode a protein with an apparent molecular weight of 83,000 on sodium dodecyl sulfate-polyacrylamide gels. The HrpH protein in E. coli was located in the membrane fraction and was absent from the periplasm and cytoplasm. The HrpH protein possessed similarity with several outer membrane proteins that are known to be involved in protein or phage secretion, including the Klebsiella oxytoca PulD protein, the Yersinia enterocolitica YscC protein, and the pIV protein of filamentous coliphages. All of these proteins possess a possible secretion motif, GG(X)12VP(L/F)LXXIPXIGXL(F/L), near the carboxyl terminus, and they lack a carboxyl-terminal phenylalanine, in contrast to other outer membrane proteins with no known secretion function. These results suggest that the P. syringae pv. syringae HrpH protein is involved in the secretion of a proteinaceous HR elicitor.     

Molecular cloning of a Pseudomonas syringae pv. syringae gene cluster that enables Pseudomonas fluorescens to elicit the hypersensitive response in tobacco plants.

A cosmid clone isolated from a genomic library of Pseudomonas syringae pv. syringae 61 restored to all Tn5 mutants of this strain studied the ability to elicit the hypersensitive response (HR) in tobacco. Cosmid pHIR11 also enabled Escherichia coli TB1 to elicit an HR-like reaction when high levels of inoculum (10(9) cells per ml) were infiltrated into tobacco leaves. The cosmid, which contains a 31-kilobase DNA insert, was mobilized by triparental matings into Pseudomonas fluorescens 55 (a nonpathogen that normally causes no plant reactions), P. syringae pv. syringae 226 (a tomato pathogen that causes the HR in tobacco), and P. syringae pv. tabaci (a tobacco pathogen that causes the HR in tomato). The plant reaction phenotypes of all of the transconjugants were altered. P. fluorescens(pHIR11) caused the HR in tobacco and tomato leaves and stimulated an apparent proton influx in suspension-cultured tobacco cells that was indistinguishable from the proton influx caused by incompatible pathogenic pseudomonads. P. syringae pv. tabaci(pHIR11) and P. syringae pv. syringae 226(pHIR11) elicited the HR rather than disease symptoms on their respective hosts and were no longer pathogenic. pHIR11 was mutagenized with TnphoA (Tn5 IS50L::phoA). One randomly chosen mutant, pHIR11-18, no longer conferred the HR phenotype to P. fluorescens. The mutation was marker-exchanged into the genomes of P. syringae pv. syringae strains 61 and 226. The TnphoA insertions in the two pseudomonads abolished their ability to elicit any plant reactions in all plants tested. The results indicate that a relatively small portion of the P. syringae genome is sufficient for the elicitation of plant reactions.     

Relatedness of Pseudomonas syringae pv. tomato, Pseudomonas syringae pv. maculicola and Pseudomonas syringae pv. antirrhini

The relationships among strains of Pseudomonas syringae pv. tomato, Ps. syr. antirrhini, Ps. syr. maculicola, Ps. syr. apii and a strain isolated from squash were examined by restriction fragment length polymorphism (RFLP) patterns, nutritional characteristics, host of origin and host ranges. All strains tested except for Ps. syr. maculicola 4326 isolated from radish ( Raphanus sativus L.) constitute a closely related group. No polymorphism was seen among strains probed with the 5.7 and 2.3 kb Eco RI fragments which lie adjacent to the hrp cluster of Ps. syr. tomato and the 8.6 kb Eco RI insert of pBG2, a plasmid carrying the β-glucosidase gene(s). All strains tested had overlapping host ranges. In contrast to this, comparison of strains by RFLP patterns of sequences homologous to the 4.5 kb Hind III fragment of pRut2 and nutritional properties distinguished four groups. Group 1, consisting of strains of pathovars maculicola, tomato and apii , had similar RFLP patterns and used homoserine but not sorbitol as carbon sources. Group 2, consisting of strains of pathovars maculicola and tomato , differed from Group 1 in RFLP patterns and did not use either homoserine or sorbitol. Group 3 was similar to Group 2 in RFLP patterns but utilized homoserine and sorbitol. This group included strains of the pathovars tomato and antirrhini , and a strain isolated from squash. Group 4, a single strain of Ps. syr. maculicola isolated from radish, had unique RFLP patterns and resembled Group 3 nutritionally. The evolutionary relationships of these strains are discussed.     

Conversion of compatible plant-pathogen interactions into incompatible interactions by expression of the Pseudomonas syringae pv. syringae 61 hrmA gene in transgenic tobacco plants

The hrmA gene from Pseudomonas syringae pv. syringae has previously been shown to confer avirulence on the virulent bacterium P. syringae pv. tabaci in all examined tobacco cultivars. We expressed this gene in tobacco plants under the control of the tobacco Delta0. 3 TobRB7 promoter, which is induced upon nematode infection in tobacco roots (Opperman et al. 1994, Science, 263, 221-223). A basal level of hrmA expression in leaves of transgenic plants activated the expression of pathogenesis-related genes, and the transgenic plants exhibited high levels of resistance to multiple pathogens: tobacco vein mottling virus, tobacco etch virus, black shank fungus Phytophthora parasitica, and wild fire bacterium Pseudomonas syringae pv. tabaci. However, the hrmA transgenic plants were not significantly more resistant to root-knot nematodes. Our results suggest a potential use of controlled low-level expression of bacterial avr genes, such as hrmA, in plants to generate broad-spectrum resistance to bacterial, fungal and viral pathogens.     

Degeneration of hrpZ gene in Pseudomonas syringae pv. tabaci to evade tobacco defence: an arms race between tobacco and its bacterial pathogen

The HrpZ harpin of Pseudomonas syringae is known to induce a hypersensitive response (HR) in some plants. In P. syringae pv. tabaci (Pta), the harpin gene hrpZ has been spontaneously disrupted by an internal deletion in its open reading frame and a frame shift. The loss of the ability of the recombinant harpin polypeptide of Pta to induce HR despite the high sensitivity of tobacco plants to harpin led us to investigate the meaning of the disrupted hrpZ gene in the virulence of Pta 6605. The hrpZ gene from P. syringae pv. pisi was introduced into wild-type (WT) Pta. The hrpZ-complemented Pta secreted harpin into the culture medium, but failed to cause disease symptoms by both infiltration and spray inoculation. Inoculation with the hrpZ-complemented Pta induced defence responses in tobacco plants, whereas the defence responses of tobacco plants were not prominent on inoculation with WT Pta. These results indicate that an ancestor of Pta might have disrupted hrpZ by an internal deletion to evade plant defences and confer the ability to cause disease in tobacco plants.     

Inhibition by Agrobacterium tumefaciens and Pseudomonas savastanoi of development of the hypersensitive response elicited by Pseudomonas syringae pv. phaseolicola.

Injection into tobacco leaves of biotype 1 Agrobacterium tumefaciens or of Pseudomonas savastanoi inhibited the development of a visible hypersensitive response to the subsequent injection at the same site of Pseudomonas syringae pv. phaseolicola. This interference with the hypersensitive response was not seen with injection of bacterial growth medium or Escherichia coli cells. Live A. tumefaciens cells were required for the inhibitory effect. Various mutants and strains of A. tumefaciens were examined to determine the genes involved. Known chromosomal mutations generally had no effect on the ability of A. tumefaciens to inhibit the hypersensitive response, except for chvB mutants which showed a reduced (but still significant) inhibition of the hypersensitive response. Ti plasmid genes appeared to be required for the inhibition of the hypersensitive response. The bacteria did not need to be virulent in order to inhibit the hypersensitive response. Deletion of the vir region from pTi had no effect on the inhibition. However, the T region of the Ti plasmid was required for inhibition. Studies of transposon mutants suggested that the tms but not tmr or ocs genes were required. These genes were not acting after transfer to plant cells since they were effective in strains lacking vir genes and thus unable to transfer DNA to plant cells. The results suggest that the expression of the tms genes in the bacteria may inhibit the development of the hypersensitive response by the plant. An examination of the genes required in P. savastanoi for the inhibition of the hypersensitive response suggested that bacterial production of auxin was also required for the inhibition of the hypersensitive response by these bacteria.     

Identification and expression of the Pseudomonas syringae pv. aptata hrpZPsa gene which encodes an harpin elicitor

A sequence homologous to an internal fragment 0.75 kb BstXI of the Pseudomonas syringae pv. syringae hrpZ gene was identified in Pseudomonas syringae pv. aptata NCPPB 2664, the causal agent of bacterial blight in sugar beet, lettuce and other plants, and in E. coli DH10B (pCCP1069) containing the P. syringae pv. aptata hrp gene cluster. PCR with oligonucleotides, based on the hrpZ_Pss gene and used as primers with the total genomic DNA of P. syringae pv. aptata, amplified a 1 kb fragment that hybridized with the probe in highly stringent conditions. The amplicon was cloned into the pGEM-T® plasmid vector, amplified in E. coli DH5 and sequenced. The sequence showed 95%, 83% and 61% identity with those of hrpZ_Pss, hrpZ_Psg and hrpZ_Pst genes encoding the harpins of the P. syringae pv. syringae, glycinea and tomato, respectively. The amplicon was cloned into the pMAL® expression system. The expressed protein, fused with maltose-binding protein, was cleaved with a specific protease factor Xa, and purified using affinity chromatography. On the basis of the amino acid sequence and its ability to induce HR in tobacco leaves, it was identified as a P. syringae pv. aptata harpin.     

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.     

Influence of Pseudomonas syringae culture conditions on initiation of the hypersensitive response of culture tobacco cells.

The inhibitor sensitivity and timing of the ionic response of suspension-cultured tobacco cells were used as a bioassay for the Pseudomonas syringae signal that elicits the hypersensitive response in resistant plants. The ionic response of tobacco cell suspensions inoculated with P. syringae pv. syringae 61 and P. syringae pv. pisi grown in rich media was inhibited by rifampin, tetracycline, and streptomycin during a 2- to 2.5-h induction stage. Coculturing the bacteria with tobacco cells for 3 h or more before inoculating fresh tobacco cells specifically abolished the sensitivity of the ionic response to these inhibitors and reduced the response time of the tobacco cells from 3 to 1 h. The apparent activation of the bacteria during coculture was not dependent on the plant cells and could be achieved by incubating the bacteria in a nitrogen-deficient medium containing a metabolizable carbon source. Addition of proteose peptone and Casamino Acids to this medium suppressed activation of the bacteria. The results suggest that the hypersensitive response-eliciting signal forms late in the induction stage, perhaps as a result of the derepression of some of the P. syringae genes functional in elicitation of the hypersensitive response. The nature of the activated state remains elusive but is consistent with the accumulation of protein(s) whose activity indirectly elicits the ionic response.     

Influence of Pseudomonas syringae culture conditions on initiation of the hypersensitive response of culture tobacco cells

The inhibitor sensitivity and timing of the ionic response of suspension-cultured tobacco cells were used as a bioassay for the Pseudomonas syringae signal that elicits the hypersensitive response in resistant plants. The ionic response of tobacco cell suspensions inoculated with P. syringae pv. syringae 61 and P. syringae pv. pisi grown in rich media was inhibited by rifampin, tetracycline, and streptomycin during a 2- to 2.5-h induction stage. Coculturing the bacteria with tobacco cells for 3 h or more before inoculating fresh tobacco cells specifically abolished the sensitivity of the ionic response to these inhibitors and reduced the response time of the tobacco cells from 3 to 1 h. The apparent activation of the bacteria during coculture was not dependent on the plant cells and could be achieved by incubating the bacteria in a nitrogen-deficient medium containing a metabolizable carbon source. Addition of proteose peptone and Casamino Acids to this medium suppressed activation of the bacteria. The results suggest that the hypersensitive response-eliciting signal forms late in the induction stage, perhaps as a result of the derepression of some of the P. syringae genes functional in elicitation of the hypersensitive response. The nature of the activated state remains elusive but is consistent with the accumulation of protein(s) whose activity indirectly elicits the ionic response.     

Functional mapping of harpin HrpZ of Pseudomonas syringae reveals the sites responsible for protein oligomerization, lipid interactions and plant defence induction

Harpin HrpZ is one of the most abundant proteins secreted through the pathogenesis-associated type III secretion system of the plant pathogen Pseudomonas syringae. HrpZ shows membrane-binding and pore-forming activities in vitro, suggesting that it could be targeted to the host cell plasma membrane. We studied the native molecular forms of HrpZ and found that it forms dimers and higher order oligomers. Lipid binding by HrpZ was tested with 15 different membrane lipids, with HrpZ interacting only with phosphatidic acid. Pore formation by HrpZ in artificial lipid vesicles was found to be dependent on the presence of phosphatidic acid. In addition, HrpZ was able to form pores in vesicles prepared from Arabidopsis thaliana plasma membrane, providing evidence for the suggested target of HrpZ in the host. To map the functions associated with HrpZ, we constructed a comprehensive series of deletions in the hrpZ gene derived from P. syringae pv. phaseolicola, and studied the mutant proteins. We found that oligomerization is mainly mediated by a region near the C-terminus of the protein, and that the same region is also essential for membrane pore formation. Phosphatidic acid binding seems to be mediated by two regions separate in the primary structure. Tobacco, a nonhost plant, recognizes, as a defence elicitor, a 24-amino-acid HrpZ fragment which resides in the region indispensable for the oligomerization and pore formation functions of HrpZ.     

Analysis of the syrP gene, which regulates syringomycin synthesis by Pseudomonas syringae pv. syringae.

Syringomycin is a lipodepsinonapeptide phytotoxin synthesized by Pseudomonas syringae pv. syringae on multienzymatic peptide synthetases. Sequence analysis of the interval between the syrB and syrD genes of P. syringae pv. syringae strain B301D revealed a 1,059-bp open reading frame (ORF), designated syrP. The predicted product of this ORF was a 39.6-kDa protein consisting of 353 amino acid residues. Searches of protein sequence databases demonstrated that SyrP was most similar to histidine kinases such as the CheA regulatory protein of Escherichia coli. The predicted SyrP sequence was aligned with the N terminus of CheA, a region corresponding to the phosphotransfer and acceptor domains of CheA. The SyrP region that aligns with the phosphotransfer domain of CheA contained a His at position 101 which is flanked by a weak consensus sequence of the unorthodox sensory kinase subfamily of two-component regulatory systems. Strain B301D-31, obtained by site-directed insertional mutagenesis of the syrP gene, exhibited an unusual pleiotropic phenotype including a failure to produce syringomycin in liquid media in contrast to production of elevated levels of the toxin on agar media. The syrP mutant was relieved of the suppression of toxin production that accompanies inorganic phosphate concentrations of > 1 mM on agar media. Nevertheless, the syrP mutant was substantially less virulent than the wild-type strain in pathogenicity assays in cherry fruits. These results suggest that the syrP gene encodes a regulatory protein that participates in a phosphorylation cascade controlling syringomycin production and virulence in P. syringae pv. syringae.     

Relationship of Pseudomonas syringae pv. tabaci races to the rhizosphere of Wisconsin-grown tobacco

Isolates of Pseudomonas syringae pv. tabaci, including 21 strains of the wildfire pathogen and 2 strains of the angular leafspot pathogen, were isolated from 143 rhizosphere and soil samples collected from 11 tobacco fields in Wisconsin. These pathogens were isolated by inoculating rhizosphere and soil washings into tobacco leaves and isolating the bacteria from wildfire or angular leafspot lesions that developed on the leaves. The wildfire isolates were from the rhizospheres of tobacco and Panicum capillare and from soil. While the majority of these were from wildfire-diseased fields, one isolate was from a field without disease symptoms; both angular leafspot isolates were from fields without angular leafspot symptoms. The majority of wildfire isolates were race 1, but three were race 0, and one was a new race. In three fields multiple races of wildfire were found. Both angular leafspot isolates were race 1. Two wildfire and one angular leafspot isolates were from fields where the cultivars were resistant to the races isolated.     

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.     

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.     

Characterization of Pyoverdinpss, the Fluorescent Siderophore Produced by Pseudomonas syringae pv. syringae

Pseudomonas syringae pv. syringae B301D produces a yellow-green, fluorescent siderophore, pyoverdin_pss, in large quantities under iron-limited growth conditions. Maximum yields of pyoverdin_pss of approximately 50 μg/ml occurred after 24 h of incubation in a deferrated synthetic medium. Increasing increments of Fe(III) coordinately repressed siderophore production until repression was complete at concentrations of ≥ 10 μM. Pyoverdin_pss was isolated, chemically characterized, and found to resemble previously characterized pyoverdins in spectral traits (absorbance maxima of 365 and 410 nm for pyoverdin_pss and its ferric chelate, respectively), size (1,175 molecular weight), and amino acid composition. Nevertheless, pyoverdin_pss was structurally unique since amino acid analysis of reductive hydrolysates yielded β-hydroxyaspartic acid, serine, threonine, and lysine in a 2:2:2:1 ratio. Pyoverdin_pss exhibited a relatively high affinity constant for Fe(III), with values of 10²⁵ at pH 7.0 and 10³² at pH 10.0. Iron uptake assays with [⁵⁵Fe]pyoverdin_pss demonstrated rapid active uptake of ⁵⁵Fe(III) by P. syringae pv. syringae B301D, while no uptake was observed for a mutant strain unable to acquire Fe(III) from ferric pyoverdin_pss. The chemical and biological properties of pyoverdin_pss are discussed in relation to virulence and iron uptake during plant pathogenesis.     

Molecular and Physiological Characterization of Pseudomonas syringae pv. tomato and Pseudomonas syringae pv. maculicola Strains That Produce the Phytotoxin Coronatine

The chlorosis-inducing phytotoxin coronatine is produced by several Pseudomonas syringae pathovars, including glycinea, morsprunorum, atropurpurea, and the closely related tomato and maculicola. To date, all coronatine-producing pv. glycinea, morsprunorum, and atropurpurea strains that have been examined carry the gene cluster that controls toxin production on a large plasmid. In the present study the genomic location of the coronatine gene cluster was determined for coronatine-producing strains of the pv. tomato-maculicola group by subjecting their genomic DNA to pulsed-field electrophoresis and Southern blot analysis with a hybridization probe from the coronatine gene cluster. The cluster was chromosomally borne in 10 of the 22 strains screened. These 10 strains infected both crucifers and tomatoes but could not use sorbitol as a sole source of carbon. The remaining 12 coronatine-producing strains had plasmid-borne toxin gene clusters and used sorbitol as a carbon source. Only one of these strains was pathogenic on both crucifers and tomatoes; the remainder infected just tomatoes. Restriction fragment length polymorphism analysis of the pv. tomato-maculicola coronatine gene clusters was performed with probes from P. syringae pv. tomato DC3000, a tomato and crucifer pathogen. Although the coronatine cluster appeared, in general, to be highly conserved across the pv. tomato-maculicola group, there were significant differences between plasmid-borne and chromosomally borne genes. The extensively studied coronatine cluster of pv. glycinea 4180 closely resembled the plasmid-borne clusters of the pv. tomato-maculicola group.