Isolation and Partial Characterization of Bacteriophages of the Phytopathogen Pseudomonas syringae

Bacteriophages isolated from culture supernatants of Pseudomonas syringae pv. syringae and from sewage were identified. The DNA from each phage was isolated and digested with the restriction endonuclease EcoRI. Eight isolates were determined to be different, with two phage isolates from sewage having restriction patterns identical to two phages from culture supernatants. The sizes of the phage DNA ranged from 24 to49 kilobases for isolates from sewage and from 39 to 52.5 kilobases for the isolates from culture supernatants. Buoyant densities of phage particles in CsCl varied from 1.498 to 1.507 g/cm³ for isolates from sewage and from 1.506 to 1.516 g/cm³ for isolates from culture supernatants. Electron microscopy revealed four morphological types. Based on plaque-forming ability of culture supernatants, 31 out of 47 strains of P. syringae are probably lysogenic.     

Identification of Bacteriophages for Biocontrol of the Kiwifruit Canker Phytopathogen Pseudomonas syringae pv. actinidiae

Pseudomonas syringae pv. actinidiae is a reemerging pathogen which causes bacterial canker of kiwifruit (Actinidia sp.). Since 2008, a global outbreak of P. syringae pv. actinidiae has occurred, and in 2010 this pathogen was detected in New Zealand. The economic impact and the development of resistance in P. syringae pv. actinidiae and other pathovars against antibiotics and copper sprays have led to a search for alternative management strategies. We isolated 275 phages, 258 of which were active against P. syringae pv. actinidiae. Extensive host range testing on P. syringae pv. actinidiae, other pseudomonads, and bacteria isolated from kiwifruit orchards showed that most phages have a narrow host range. Twenty-four were analyzed by electron microscopy, pulse-field gel electrophoresis, and restriction digestion. Their suitability for biocontrol was tested by assessing stability and the absence of lysogeny and transduction. A detailed host range was performed, phage-resistant bacteria were isolated, and resistance to other phages was examined. The phages belonged to the Caudovirales and were analyzed based on morphology and genome size, which showed them to be representatives of Myoviridae, Podoviridae, and Siphoviridae. Twenty-one Myoviridae members have similar morphologies and genome sizes yet differ in restriction patterns, host range, and resistance, indicating a closely related group. Nine of these Myoviridae members were sequenced, and each was unique. The most closely related sequenced phages were a group infecting Pseudomonas aeruginosa and characterized by phages JG004 and PAK_P1. In summary, this study reports the isolation and characterization of P. syringae pv. actinidiae phages and provides a framework for the intelligent formulation of phage biocontrol agents against kiwifruit bacterial canker.     

Closely Related Plasmid Replicons Coexisting in the Phytopathogen Pseudomonas syringae Show a Mosaic Organization of the Replication Region and Altered Incompatibility Behavior

Many Pseudomonas syringae strains contain native plasmids that are important for host-pathogen interactions, and most of them contain several coexisting plasmids (pPT23A-like plasmids) that cross-hybridize to replication sequences from pPT23A, which also carries a gene cluster coding for the phytotoxin coronatine in P. syringae pv. tomato PT23. In this study, three functional pPT23A-like replicons were cloned from P. syringae pv. glycinea race 6, suggesting that the compatibility of highly related replicons is a common feature of P. syringae strains. Hybridization experiments using three separate incompatibility determinants previously identified from pPT23A and the rulAB (UV radiation tolerance) genes showed that the organization of the replication region among pPT23A-like plasmids from several P. syringae pathovars is poorly conserved. The putative repA gene from four pPT23A-like replicons from P. syringae pv. glycinea race 6 was amplified by using specific primers. The restriction profiles of the resulting PCR products for the race 6 plasmids were more similar to each other than they were to that of pPT23A. These data, together with the existence of other cross-hybridizing DNA regions around the replicon among the race 6 pPT23A-like plasmids, suggest that some of these plasmids may have originated from duplication events. Our results also imply that modifications of the repA sequences and the poor conservation of putative maintenance determinants contribute to the suppression of incompatibility among members of the pPT23A-like family, thus enhancing the genomic plasticity of P. syringae.     

Arabidopsis Actin-Depolymerizing Factor AtADF4 Mediates Defense Signal Transduction Triggered by the Pseudomonas syringae Effector AvrPphB

The actin cytoskeleton has been implicated in plant defenses against pathogenic fungi and oomycetes with limited, indirect evidence. To date, there are no reports linking actin with resistance against phytopathogenic bacteria. The dynamic behavior of actin filaments is regulated by a diverse array of actin-binding proteins, among which is the Actin-Depolymerizing Factor (ADF) family of proteins. Here, we demonstrate that actin dynamics play a role in the activation of gene-for-gene resistance in Arabidopsis (Arabidopsis thaliana) following inoculation with the phytopathogenic bacterium Pseudomonas syringae pv tomato. Using a reverse genetics approach, we explored the roles of Arabidopsis ADFs in plant defenses. AtADF4 was identified as being specifically required for resistance triggered by the effector AvrPphB but not AvrRpt2 or AvrB. Recombinant AtADF4 bound to monomeric actin (G-actin) with a marked preference for the ADP-loaded form and inhibited the rate of nucleotide exchange on G-actin, indicating that AtADF4 is a bona fide actin-depolymerizing factor. Exogenous application of the actin-disrupting agent cytochalasin D partially rescued the Atadf4 mutant in the AvrPphB-mediated hypersensitive response, demonstrating that AtADF4 mediates defense signaling through modification of the actin cytoskeleton. Unlike the mechanism by which the actin cytoskeleton confers resistance against fungi and oomycetes, AtADF4 is not involved in resistance against pathogen entry. Collectively, this study identifies AtADF4 as a novel component of the plant defense signaling pathway and provides strong evidence for actin dynamics as a primary component that orchestrates plant defenses against P. syringae.The actin cytoskeleton has been implicated in plant defenses against pathogenic fungi and oomycetes (Hardham et al., 2007). Evidence largely comes from studies using actin cytoskeleton-disrupting agents, such as cytochalasins. Treatments with a variety of cytochalasins were shown to increase the penetration rate of both adapted and nonadapted pathogens in multiple plant-pathogen systems, thereby implicating the actin cytoskeleton as having a role in basal defenses and nonhost resistance (Kobayashi et al., 1997; Yun et al., 2003; Shimada et al., 2006; Miklis et al., 2007). The actin cytoskeleton may also play a role in race-specific resistance (Skalamera and Heath, 1998). To date, no reports linking actin dynamics with resistance against phytopathogenic bacteria have been published.While the actin cytoskeleton as a virulence target of plant pathogens has not been documented, it was well characterized in mammalian pathosystems, particularly in studies investigating macrophage interactions with the pathogenic bacterium Yersinia pestis (Mattoo et al., 2007). Yersinia species deliver a suite of effectors into the target host cell, and at least four of them (YopE, YpkA/YopO, YopT, and YopH) are involved in rearrangement of the actin cytoskeleton (Aepfelbacher and Heesemann, 2001). YopT, a Cys protease, targets a plasma membrane-localized Rho GTPase in affected phagocytes (Aepfelbacher and Heesemann, 2001). Cleavage of the GTPase by YopT releases the prenylated protein from the plasma membrane and disrupts the actin cytoskeleton, effectively shutting down phagocytosis, preventing elimination of the pathogen (Iriarte and Cornelis, 1998; Shao et al., 2002). Similarly, microbial pathogens also usurp host processes for the benefit of infection, disease, and death. Listeria species hijack the host''s cytoskeleton to move around inside the infected cell through the induction of directed polymerization of actin (Pistor et al., 1994). Salmonella injects into host cells two actin-binding proteins (SipA and SipC) as well as other regulators of actin dynamics to enhance phagocytic uptake and intracellular propagation (Galan and Zhou, 2000). In short, either by preventing polymerization or by promoting it, pathogens have evolved strategies to modify the host actin cytoskeleton for purposes of evading detection or eliciting disease and death.Dynamic actin cytoskeleton rearrangements are regulated by a pool of actin-binding proteins, which sense environmental changes and modulate the cytoskeleton through various biochemical activities (Hussey et al., 2006; Staiger and Blanchoin, 2006). Among the proteins that regulate these dynamic processes are the Actin-Depolymerizing Factor (ADF) family of proteins (Maciver and Hussey, 2002). In general, ADFs bind both monomeric (G-) and filamentous (F-) actin to increase actin dynamics. They function by severing F-actin to generate more ends for polymerization and by increasing the dissociation rate of actin monomers from the pointed ends (Maciver, 1998; Maciver and Hussey, 2002). Plant ADFs play roles in pollen tube growth (Chen et al., 2003), root formation (Thomas and Schiefelbein, 2002), and cold acclimation (Ouellet et al., 2001). There is also one report linking ADFs with plant defenses (Miklis et al., 2007). In that study, ectopic expression of barley (Hordeum vulgare) HvADF3 and several isovariants of Arabidopsis (Arabidopsis thaliana) ADFs in barley epidermal cells was shown to compromise penetration resistance to powdery mildew fungi (Miklis et al., 2007).The Arabidopsis-Pseudomonas syringae interaction provides an ideal model plant-pathogen system to study plant defense signaling. Like Yersinia species, P. syringae delivers effector proteins into the host cells via the type III secretion system and relies on these proteins for pathogenesis (Alfano and Collmer, 2004). However, once these proteins (Avr) are recognized either directly or indirectly by plant resistance (R) proteins, plant immune responses are activated (Jones and Dangl, 2006). Exciting progress has been made toward understanding the indirect recognition of several pairs of Avr-R proteins; the best examples include AvrB/AvrRPM1-RPM1, AvrRpt2-RPS2, and AvrPphB-RPS5. During activation of defense mediated by AvrB/AvrRPM1-RPM1 and AvrRpt2-RPS2, the phosphorylation or elimination of a third protein, RIN4, is essential (Mackey et al., 2002; Axtell and Staskawicz, 2003). In the case of AvrPphB-RPS5 recognition, the AvrPphB Cys protease of the same family as YopT (Shao et al., 2002) cleaves the plant protein kinase PBS1, inducing a conformational change in RPS5, which in turn leads to the activation of resistance (Ade et al., 2007). Although these studies have greatly enhanced our understanding of how pathogen effectors initiate plant defense responses, the ultimate signaling processes associated with the activation of resistance remain largely unknown, due to the limited number of genetic loci identified in these pathways. In this work, we hypothesize that actin-binding proteins play a role during plant-bacteria interactions based on the functional and structural similarity between AvrPphB and YopT.There are 11 ADFs in the Arabidopsis genome (Ruzicka et al., 2007). We utilized a reverse genetics approach to identify the putative roles these proteins play in plant resistance against the bacterial pathogen P. syringae pv tomato (Pst). AtADF4 was identified as a novel signaling component in the AvrPphB-RPS5-mediated defense signal transduction pathway. Loss of AtADF4 confers on Arabidopsis enhanced susceptibility to P. syringae expressing AvrPphB. Further subcellular localization and biochemical analyses, as well as pharmacological studies, suggest that AtADF4 functions as a bona fide actin-depolymerizing factor through modifying the actin cytoskeleton. Unlike the documented mechanism by which the actin cytoskeleton plays roles in resistance against fungi and oomycetes, the resistance against P. syringae mediated by AtADF4 is not involved in hindering pathogen entry.     

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.     

Changes in Cell Size and Flagellation in the Phytopathogen Pseudomonas syringae pv. tabaci Cultured in vitro and in planta: A Comparative Electron Microscope Study

In parallel experiments, cells of Pseudomonas syringae pv. tabaci were infiltrated into tobacco leaves (to determine bacterial changes occurring in planta) and inoculated into nutrient broth (to make comparative observations on cells cultured in vitro). In each case, details of surface structure, bacterial size and flagellation were determined in a sequence of samples by transmission electron microscopy of whole mount stained and unstained preparations. In both in planta and in vitro environments, bacterial population showed a clear phase of exponential increase. In each case, bacterial size was highest during the early part of the multiplication phase, then decreased during the rest of the multiplication period. In each case also, the proportion of cells with flagella showed a similar trend - with an initial decrease after infiltration, followed by a major increase during the phase of bacterial multiplication. These results suggest that changes in bacterial size and flagellation in planta relate directly to the growth phase of the population, and are therefore determined primarily by internal cellular (endogenous) factors - rather than by external factors within the leaf environment.     

Generalized Transduction in Corynebacterium renale

A phage, designated RP28, was obtained which carried out generalized transduction in Corynebacterium renale, and 11 different auxotrophic markers were all transduced.     

Generalized Transduction in CAULOBACTER CRESCENTUS

Two closely related bacteriophage, ϕCr30 and ϕCr35, are the first bacteriophage shown to mediate generalized transduction in Caulobacter crescentus. Unlike most other transducing phage, they are virulent and do not form any sort of lysogenic relationship with their host. However, they are rather inefficient at adsorption, so that transductants have a good chance of survival. The phage particles have a head 80 nm in diameter and a contractile tail 140 nm in length. Procedures for growth and transduction with ϕCr30 are relatively simple; thus, it will be of great value for the genetic analysis of C. crescentus.     

Periplasmic glucans of Pseudomonas syringae pv. syringae.

We report the initial characterization of glucans present in the periplasmic space of Pseudomonas syringae pv. syringae (strain R32). These compounds were found to be neutral, unsubstituted, and composed solely of glucose. Their size ranges from 6 to 13 glucose units/mol. Linkage studies and nuclear magnetic resonance analyses demonstrated that the glucans are linked by beta-1,2 and beta-1,6 glycosidic bonds. In contrast to the periplasmic glucans found in other plant pathogenic bacteria, the glucans of P. syringae pv. syringae are not cyclic but are highly branched structures. Acetolysis studies demonstrated that the backbone consists of beta-1,2-linked glucose units to which the branches are attached by beta-1,6 linkages. These periplasmic glucans were more abundant when the osmolarity of the growth medium was lower. Thus, P. syringae pv. syringae appears to synthesize periplasmic glucans in response to the osmolarity of the medium. The structural characteristics of these glucans are very similar to the membrane-derived oligosaccharides of Escherichia coli, apart from the neutral character, which contrasts with the highly anionic E. coli membrane-derived oligosaccharides.     

Chromosome mapping in Pseudomonas syringae pv. syringae strain PS224

A conjugation system for mapping the chromosome of Pseudomonas syringae pv. syringae PS224 has been developed using the IncP-10 plasmid R91-5; pMO22, a Tn501-loaded derivative of R91-5; and pMO75, R91-5 loaded with Tn5. Nine different donor origins were identified with R91-5 and pMO22. By insertion of Tn5 into various sites of the chromosome, an additional six donor origins were available using pMO75 as the donor plasmid. In all, 36 markers were located on three linkage groups. Many donor strains were unstable and the limited availability of stable donor strains has limited the extent to which markers have been located. This instability of donor strains is in marked contrast to the highly stable donor strains found in P. putida using the same plasmids. As in P. aeruginosa and P. putida, auxotrophic markers in P. syringae do not show the clustering of related markers found in enterobacteria.     

Next-generation genomics of Pseudomonas syringae

The first wave of Pseudomonas syringae next-generation genomic studies has revealed insights into host-specific virulence and immunity, genome dynamics and evolution, and genetic and metabolic specialization. These studies have further enhanced our understanding of type III effector diversity, identified an atypical type III secretion system (T3SS) in a new clade of nonpathogenic P. syringae, identified metabolic pathways common to pathogens of woody hosts and revealed extensive genomic diversity among strains that infect common hosts. In general, these discoveries have illustrated the utility of draft genome sequencing for quickly and economically identifying candidate loci for more refined genetic and functional analyses.     

Ice crystallization by Pseudomonas syringae

Several bacterial species can serve as biological ice nuclei. The best characterized of these is Pseudomonas syringae, a widely distributed bacterial epiphyte of plants. These biological ice nuclei find various applications in different fields, but an optimized production method was required in order to obtain the highly active cells which may be exploited as ice nucleators. The results presented here show that P. syringae cells reduce supercooling of liquid or solid media and enhance ice crystal formation at sub-zero temperatures, thus leading to a remarkable control of the crystallization phenomenon and a potential for energy savings. Our discussion focuses on recent and future applications of these ice nucleators in freezing operations, spray-ice technology and biotechnological processes. Received: 21 December 1999 / Received revision: 29 February 2000 / Accepted: 6 March 2000     

The pyoverdins of Pseudomonas syringae and Pseudomonas cichorii

The structure elucidation of the cyclic (lactonic) forms of the pyoverdins with a succinamide side chain originally produced by the closely related species Pseudomonas syringae and P. cichorii is reported. Mass spectrometry and nuclear magnetic resonance analyses as well as the determination of the configuration of the amino acids after degradation indicate that these two pyoverdins differ only by the replacement of the first in-chain serine by glycine. The pyoverdins of P. syringae and P. cichorii and the dihydropyoverdin of P. syringae can be used by both species as siderophores.     

Phytoalexin Accumulation in Arabidopsis thaliana during the Hypersensitive Reaction to Pseudomonas syringae pv syringae

Inoculation of leaves of Arabidopsis thaliana (L.) Heynh. with the wheat pathogen, Pseudomonas syringae pv syringae, resulted in the expression of the hypersensitive reaction and in phytoalexin accumulation. No phytoalexin accumulation was detected after infiltration of leaves with a mutant of P. s. syringae deficient in the ability to elicit a hypersensitive reaction; with the crucifer pathogen, Xanthomonas campestris pv campestris; or with 10 millimolar potassium phosphate buffer (pH 6.9). Phytoalexin accumulation was correlated with the restricted in vivo growth of P. s. syringae. A phytoalexin was purified by a combination of reverse phase flash chromatography, thin layer chromatography, followed by reverse phase high performance liquid chromatography. The Arabidopsis phytoalexin was identified as 3-thiazol-2′-yl-indole on the basis of ultraviolet, infrared, mass spectral, ¹H-nuclear magnetic resonance, and ¹³C-nuclear magnetic resonance data.     

Characterization of alginate lyase from Pseudomonas syringae pv. syringae

The gene encoding alginate lyase (algL) in Pseudomonas syringae pv. syringae was cloned, sequenced, and overexpressed in Escherichia coli. Alginate lyase activity was optimal when the pH was 7.0 and when assays were conducted at 42 degrees C in the presence of 0.2 M NaCl. In substrate specificity studies, AlgL from P. syringae showed a preference for deacetylated polymannuronic acid. Sequence alignment with other alginate lyases revealed conserved regions within AlgL likely to be important for the structure and/or function of the enzyme. Site-directed mutagenesis of histidine and tryptophan residues at positions 204 and 207, respectively, indicated that these amino acids are critical for lyase activity.     

Extracytoplasmic function sigma factors in Pseudomonas syringae

Genome analyses of the plant pathogens Pseudomonas syringae pv. tomato DC3000, pv. syringae B728a and pv. phaseolicola 1448A reveal fewer extracytoplasmic function (ECF) sigma factors than in related Pseudomonads with different lifestyles. We highlight the presence of a P. syringae-specific ECF sigma factor that is an interesting target for future studies because of its potential role in the adaptation of P. syringae to its specialized phytopathogenic lifestyle.