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.     

Chemotaxis by Pseudomonas syringae pv. tomato

Optimal laboratory conditions for studying chemotaxis by Pseudomonas syringae pv. tomato were determined by using the Adler capillary tube assay. Although they are not an absolute requirement for chemotaxis, the presence of 0.1 mM EDTA and 1 mM MgCl₂ in the chemotaxis buffer (10 mM potassium phosphate [pH 7.2]) significantly enhanced the response to attractant. The addition of mannitol as an energy source had little effect. The optimal temperature for chemotaxis was 23°C, which is 5°C below the optimal growth temperature for this pathogen. The best response occurred when the bacteria were exposed to attractant for 60 min at a concentration of approximately 5 × 10⁶ CFU/ml. P. syringae pv. tomato was strongly attracted to citric and malic acids, which are the predominant organic acids in tomato fruit. With the exception of asparagine, the major amino acids of tomatoes were weak to moderate attractants. Glucose and fructose, which account for approximately 47% of tomato dry matter, also elicited poor responses. In assays with tomato intercellular fluid and leaf surface water, the bacterial speck pathogen could not chemotactically distinguish between a resistant and a susceptible cultivar of tomato.     

Expression of a RING-HC protein from rice improves resistance to Pseudomonas syringae pv. tomato DC3000 in transgenic Arabidopsis thaliana

A cDNA clone (OsRHC1) was obtained, which encodes a novel RING zinc finger protein sharing similar structural features (multiple transmembrane domains at the N-half; a unique RING zinc finger consensus Cys-X(2)-Cys-X(11)-Cys-X-His-X(3)-Cys-X(2)-Cys-X(6)-Cys-X(2)-Cys at the C terminus) to a group of closely related annotated proteins from both monocots and dicots. OsRHC1 was found to be localized on plasma membrane of rice cells and induced by wounding in rice lines containing Xa loci. Ecotopic expression of the OsRHC1 cDNA from rice (a monocot) in transgenic Arabidopsis thaliana (a dicot) enhanced the defence response toward Pseudomonas syringae pv. tomato DC3000, suggesting that OsRHC1 may confer broad-spectrum disease resistance. The protective effects were neutralized in the presence of MG132 or in an npr1-3 mutation background, indicating that the function of OsRHC1 is dependent on the ubiquitin-mediated protein degradation via the 26S proteasome and the presence of the key defence response regulator NPR1.     

Molecular cloning of copper resistance genes from Pseudomonas syringae pv. tomato.

A cosmid library of copper-resistant (Cur) Pseudomonas syringae pv. tomato PT23 plasmid DNA was constructed and mobilized into the copper-sensitive recipient P. syringae pv. syringae PS61. One resultant cosmid clone, pCOP1 (46 kilobases), conferred copper resistance. The PT23 Cur gene(s) was located on pCOP1 by subcloning PstI restriction endonuclease fragments of pCOP1 in the broad-host-range vector pRK404. A subclone containing a 4.4-kilobase PstI fragment conferred Cur on PS61. The Cur gene(s) was further located by insertional inactivation with Tn5. A subcloned fragment internal to the Cur determinant on pCOP2 was probed to plasmid and chromosomal DNA of four copper-resistant and three copper-sensitive strains of P. syringae pv. tomato. The probe hybridized to plasmids in resistant strains, but showed no detectable homology to copper-sensitive strains.     

Genetics of resistance to bacterial speck of tomato caused by Pseudomonas syringae pv. tomato

Two tomato cultivars, Ontario 7710 and Rehovot 13, and their F₁, F₂, F₃ and backcross progenies were screened for resistance to bacterial speck (Pseudomonas syringae pv. tomato) of tomato. The results support the hypothesis that the resistance factors contained in the two parents are non-allelic and controlled by two different genes.     

Identification of Pseudomonas syringae pv. tomato genes induced during infection of Arabidopsis thaliana

Phytopathogenic bacteria possess a large number of genes that allow them to grow and cause disease on plants. Many of these genes should be induced when the bacteria come in contact with plant tissue. We used a modified in vivo expression technology (IVET) approach to identify genes from the plant pathogen Pseudomonas syringae pv. tomato that are induced upon infection of Arabidopsis thaliana and isolated over 500 in planta-expressed (ipx) promoter fusions. Sequence analysis of 79 fusions revealed several known and potential virulence genes, including hrp/hrc, avr and coronatine biosynthetic genes. In addition, we identified metabolic genes presumably important for adaptation to growth in plant tissue, as well as several genes with unknown function that may encode novel virulence factors. Many ipx fusions, including several corresponding to novel genes, are dependent on HrpL, an alternative RNA polymerase sigma factor that regulates the expression of virulence genes. Expression analysis indicated that several ipx fusions are strongly induced upon inoculation into plant tissue. Disruption of one ipx gene, conserved effector locus (CEL) orf1, encoding a putative lytic murein transglycosylase, resulted in decreased virulence of P. syringae. Our results demonstrate that this screen can be used successfully to isolate genes that are induced in planta, including many novel genes potentially involved in pathogenesis.     

A genetic screen reveals Arabidopsis stomatal and/or apoplastic defenses against Pseudomonas syringae pv. tomato DC3000

Bacterial infection of plants often begins with colonization of the plant surface, followed by entry into the plant through wounds and natural openings (such as stomata), multiplication in the intercellular space (apoplast) of the infected tissues, and dissemination of bacteria to other plants. Historically, most studies assess bacterial infection based on final outcomes of disease and/or pathogen growth using whole infected tissues; few studies have genetically distinguished the contribution of different host cell types in response to an infection. The phytotoxin coronatine (COR) is produced by several pathovars of Pseudomonas syringae. COR-deficient mutants of P. s. tomato (Pst) DC3000 are severely compromised in virulence, especially when inoculated onto the plant surface. We report here a genetic screen to identify Arabidopsis mutants that could rescue the virulence of COR-deficient mutant bacteria. Among the susceptible to coronatine-deficient Pst DC3000 (scord) mutants were two that were defective in stomatal closure response, two that were defective in apoplast defense, and four that were defective in both stomatal and apoplast defense. Isolation of these three classes of mutants suggests that stomatal and apoplastic defenses are integrated in plants, but are genetically separable, and that COR is important for Pst DC3000 to overcome both stomatal guard cell- and apoplastic mesophyll cell-based defenses. Of the six mutants defective in bacterium-triggered stomatal closure, three are defective in salicylic acid (SA)-induced stomatal closure, but exhibit normal stomatal closure in response to abscisic acid (ABA), and scord7 is compromised in both SA- and ABA-induced stomatal closure. We have cloned SCORD3, which is required for salicylic acid (SA) biosynthesis, and SCORD5, which encodes an ATP-binding cassette (ABC) protein, AtGCN20/AtABCF3, predicted to be involved in stress-associated protein translation control. Identification of SCORD5 begins to implicate an important role of stress-associated protein translation in stomatal guard cell signaling in response to microbe-associated molecular patterns and bacterial infection.     

Functions of pipecolic acid on induced resistance against Botrytis cinerea and Pseudomonas syringae pv. tomato DC3000 in tomato plants

Amino acid metabolic pathways are involved in the plant immune system. Pipecolic acid (Pip), a lysine-derived non-protein amino acid, acts as an important regulator of disease resistance. Here, we report the functions of Pip on tomato disease resistance. Tomato seedlings treated with 0.5 mM Pip showed increased resistance to Pst DC3000 and B. cinerea compared with the control. After pathogen infection, the expression of defence-related genes increased in plants pretreated with Pip, while reactive oxygen species (ROS) accumulation decreased. These data demonstrated that exogenous application of Pip induced resistance against Pst DC3000 and B. cinerea in tomatoes, possibly through the regulation of ROS accumulation and defence-related gene expression.     

Nucleotide sequence and organization of copper resistance genes from Pseudomonas syringae pv. tomato.

The nucleotide sequence of a 4.5-kilobase copper resistance determinant from Pseudomonas syringae pv. tomato revealed four open reading frames (ORFs) in the same orientation. Deletion and site-specific mutational analyses indicated that the first two ORFs were essential for copper resistance; the last two ORFs were required for full resistance, but low-level resistance could be conferred in their absence. Five highly conserved, direct 24-base repeats were found near the beginning of the second ORF, and a similar, but less conserved, repeated region was found in the middle of the first ORF.     

Inheritance and sources of resistance to bacterial speck of tomato caused by Pseudomonas syringae pv. tomato

Inheritance of resistance to bacterial speck of tomato was determined by analysing F₁ F₂ and backcross progenies of crosses involving a susceptible (VF-198) and a resistant cultivar (Rehovot-13). The results fit the hypothesis that resistance is controlled by a single dominant gene in interaction with minor genes. Cultivar susceptibility to Pseudomonas syringae pv. tomato was tested under greenhouse conditions under high inoculum pressure using infested tomato seeds together with infested soils and spray-inoculated wounded plants. Of 21 species, cultivars and lines, Rehovot-13, Ontario 7710 and Lycopersiconpimpinellifolium P.I. 126927 were found to be resistant to the pathogen. VF-198 and Tropic-VF were the most susceptible. Extra Marmande, Saladette, Acc.339944–3 and the wild type Lycopersicon esculentum var. cerasiforme were moderately resistant.     

Pseudomonas syringae pv. tomato hijacks the Arabidopsis abscisic acid signalling pathway to cause disease

We have found that a major target for effectors secreted by Pseudomonas syringae is the abscisic acid (ABA) signalling pathway. Microarray data identified a prominent group of effector-induced genes that were associated with ABA biosynthesis and also responses to this plant hormone. Genes upregulated by effector delivery share a 42% overlap with ABA-responsive genes and are also components of networks induced by osmotic stress and drought. Strongly induced were NCED3, encoding a key enzyme of ABA biosynthesis, and the abscisic acid insensitive 1 (ABI1) clade of genes encoding protein phosphatases type 2C (PP2Cs) involved in the regulation of ABA signalling. Modification of PP2C expression resulting in ABA insensitivity or hypersensitivity led to restriction or enhanced multiplication of bacteria, respectively. Levels of ABA increased rapidly during bacterial colonisation. Exogenous ABA application enhanced susceptibility, whereas colonisation was reduced in an ABA biosynthetic mutant. Expression of the bacterial effector AvrPtoB in planta modified host ABA signalling. Our data suggest that a major virulence strategy is effector-mediated manipulation of plant hormone homeostasis, which leads to the suppression of defence responses.     

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.     

Layered basal defenses underlie non-host resistance of Arabidopsis to Pseudomonas syringae pv. phaseolicola

Arabidopsis is a non-host for Pseudomonas syringae pv. phaseolicola NPS3121 (Pph), a bacterial pathogen of bean. Pph does not induce a hypersensitive response in Arabidopsis. Here we show that Arabidopsis instead resists Pph with multi-layered basal defense. Our approach was: (i) to identify defense readouts induced by Pph; (ii) to determine whether mutations in known Arabidopsis defense genes disrupt Pph-induced defense signaling; (iii) to determine whether heterologous type III effectors from pathogens of Arabidopsis suppress Pph-induced defense signaling, and (iv) to ascertain how basal defenses contribute to resistance against Pph by individually or multiply disrupting defense signaling pathways with mutations and heterologous type III effectors. We demonstrate that Pph elicits a minimum of three basal defense-signaling pathways in Arabidopsis. These pathways have unique readouts, including PR-1 protein accumulation and morphologically distinct types of callose deposition. Further, they require distinct defense genes, including PMR4, RAR1, SID2, NPR1, and PAD4 . Finally, they are suppressed differentially by heterologous type III effectors, including AvrRpm1 and HopM1. Pph growth is enhanced only when multiple defense pathways are disrupted. For example, mutation of NPR1 or SID2 combined with the action of AvrRpm1 and HopM1 renders Arabidopsis highly susceptible to Pph. Thus, non-host resistance of Arabidopsis to Pph is based on multiple, individually effective layers of basal defense.     

Indigenous plasmids in Pseudomonas syringae pv. tomato: conjugative transfer and role in copper resistance

Twenty strains of Pseudomonas syringae pv. tomato were examined for the presence of plasmid DNA. P. syringae pv. tomato plasmids were grouped into five size classes: class A ranged from 95 to 103 kilobases (kb); class B ranged from 71 to 83 kb; class C ranged from 59 to 67 kb; class D ranged from 37 to 39 kb; and class E was 29 kb. All strains contained at least two plasmids in classes A and B. The conjugative ability of P. syringae pv. tomato plasmids in three strains was demonstrated by mobilization of the nonconjugative plasmid RSF1010 into Pseudomonas syringae pv. syringae recipients. Plasmids from the three conjugative strains were labeled with Tn5. Four conjugative plasmids were identified by their repeated transfer to P. syringae pv. syringae recipients. P. syringae pv. tomato strains varied in sensitivity to copper sulfate (CuSO4): MICs were 0.4 to 0.6 mM for sensitive strains, 1.2 mM for moderately resistant strains, and 1.6 to 2.0 mM for very resistant strains. One very resistant strain, PT23, functioned as a donor of copper resistance. Recipient P. syringae pv. syringae strains PS51 and PS61 were inhibited by 0.1 mM CuSO4, whereas the CuSO4 MICs for transconjugant strains PS51(pPT23A) and PS61(pPT23C) were 1.8 and 2.6 mM, respectively. P. syringae pv. tomato strains PT12.2 and PT17.2 were inhibited by 0.6 mM copper sulfate, but their copper sulfate MICs were 2.6 and 1.8 mM, respectively, when they acquired pPT23C. Therefore, copper resistance in PT23 was controlled by two conjugative plasmids, designated pPT23A (101 kb) and pPT23C (67 kb).