Pseudomonas Syringae Strains Research Articles

Pea aphid as both host and vector for the phytopathogenic bacterium Pseudomonas syringae.

Aphids are widespread agricultural pests that are capable of disseminating plant viral diseases; however, despite coming into frequent contact with epiphytic bacteria, aphids are considered to have no role in bacterial transmission. Here, we demonstrate the ability of pea aphids to vector the phytopathogen Pseudomonas syringae pv. syringae B728a (PsyB728a). While feeding on plants colonized by epiphytic bacteria, aphids acquire the bacteria, which colonize the digestive tract, multiply, and are excreted in the aphid honeydew, resulting in inoculation of the phyllosphere with up to 10(7) phytopathogenic bacteria per cm(2). Within days of ingesting bacteria, aphids succumb to bacterial sepsis, indicating that aphids serve as an alternative, nonplant host for PsyB728a. The related strain Pseudomonas syringae pv. tomato DC3000 is >1,000-fold less virulent than PsyB728a in the pea aphid, suggesting that PsyB728a possesses strain-specific pathogenicity factors that allow it to exploit aphids as hosts. To identify these factors, we performed a mutagenesis screen and recovered PsyB728a mutants that were hypovirulent, including one defective in a gene required for flagellum formation and motility. These interactions illustrate that aphids can also vector bacterial pathogens and that even seemingly host-restricted pathogens can have alternative host specificities and lifestyles.

A Draft Genome Sequence of Pseudomonas syringae pv. tomato T1 Reveals a Type III Effector Repertoire Significantly Divergent from That of Pseudomonas syringae pv. tomato DC3000

Diverse gene products including phytotoxins, pathogen-associated molecular patterns, and type III secreted effectors influence interactions between Pseudomonas syringae strains and plants, with additional yet uncharacterized factors likely contributing as well. Of particular interest are those interactions governing pathogen-host specificity. Comparative genomics of closely related pathogens with different host specificity represents an excellent approach for identification of genes contributing to host-range determination. A draft genome sequence of Pseudomonas syringae pv. tomato T1, which is pathogenic on tomato but nonpathogenic on Arabidopsis thaliana, was obtained for this purpose and compared with the genome of the closely related A. thaliana and tomato model pathogen P. syringae pv. tomato DC3000. Although the overall genetic content of each of the two genomes appears to be highly similar, the repertoire of effectors was found to diverge significantly. Several P. syringae pv. tomato T1 effectors absent from strain DC3000 were confirmed to be translocated into plants, with the well-studied effector AvrRpt2 representing a likely candidate for host-range determination. However, the presence of avrRpt2 was not found sufficient to explain A. thaliana resistance to P. syringae pv. tomato T1, suggesting that other effectors and possibly type III secretion system-independent factors also play a role in this interaction.

Diversity among Pseudomonas syringae strains originating from fruit trees in Serbia

Pseudomonas syringae is a widespread and economically important plant pathogen, one found on a number of hosts, including fruit trees, field crops, vegetables, and ornamental plants. This bacterium has been experimentally identified as a parasite of pear, apple, apricot, peach, cherry, sour cherry, plum, and raspberry. The present study was designed to establish differences between strains isolated from fruit trees in Serbia. The pathogenic and biochemical characteristics of isolates were studied. The BOX-PCR method was used to generate genomic fingerprints of Pseudomonas syringae isolates and to identify strains that were previously not distinguishable by other classification methods. Different Bacillus sp. strains were tested for in vitro inhibitory activity against Pseudononas syringae isolates. Bacillus sp. strains show inhibitory activity only against P. syringae isolates that originated from peach. The obtained results demonstrate that the population of the bacterium Pseudomonas syringae from the fruit trees in Serbia is very diverse.

Bioactive lipopeptides of ice-nucleating snow bacteriumPseudomonas syringaestrain 31R1

The production of secondary metabolite lipopeptides by ice-nucleating Pseudomonas syringae strain 31R1 was investigated. Pseudomonas syringae strain 31R1 is a rifampicin-resistant derivative of P. syringae no. 31 used for the commercial production of snow. It is shown that P. syringae strain 31R1 produces antifungal lipodepsipeptides, syringomycins E and G, and, in addition, a novel and unique lipopeptide, peptin31. Spectroscopic and spectrometric analyses revealed that peptin31 is a linear undecalipopeptide with sequence identities to N- and C-terminal portions but lacking 11 amino acids of known lipodepsipeptide syringopeptin SPPhv. Peptin31 displayed antifungal activities against Rhodotorula pilimanae, Rhizoctonia solani, and Trichoderma harzianum and also hemolytic and antibacterial activities. Extracts of P. syringae strain 31R1 grown in medium with chloride were fungicidal, but not when grown without chloride. The latter extracts lacked peptin 31 and contained des-chloro forms of syringomycins E and G with low antifungal activities. Thus, the three lipopeptides account for the fungicidal properties of P. syringae 31R1 extracts. The occurrence of these bioactive metabolites should be considered when P. syringae no. 31 and its derivatives are used in products for making artificial snow.

Evaluation of Pseudomonas syringae strain ESC-11 for biocontrol of crown rot and anthracnose of banana

Pseudomonas syringae strain ESC-11 and 250 μg/ml each of thiabendazole (TBZ) and imazalil reduced crown rot of banana caused by Fusarium aff. sacchari by 30–36% and 83–86%, respectively, in laboratory experiments. Four field trials performed in Costa Rica varied in treatment combinations. In field trials 1 and 2, 125 and 250 μg/ml each of TBZ and imazalil + 0.5% or 1% alum (aluminum ammonium sulfate) and ESC-11, and 250 μg/ml each of TBZ and imazalil + 1% alum reduced rot and mold. ESC-11 alone or with 0.5% alum significantly reduced rot and mold in field trial 2. In trial 3, 50 and 100 μg/ml of TBZ alone and with ESC-11 reduced mold. In trial 4, 125 μg/ml each of TBZ and imazalil and ESC-11, and 300 μg/ml each of TBZ and imazalil reduced rot, and 50 and 125 μg/ml each of TBZ and imazalil and ESC-11, and 300 μg/ml each of TBZ and imazalil reduced mold. In three field trials, there was no significant difference among treatments for latex staining. In field trial 2 only, combinations of TBZ, imazalil, and alum with or without ESC-11, reduced anthracnose, caused by Colletotrichum musae. The complex of crown rot fungi, order of treatment application, effect of alum and fungicides on ESC-11, concentration of ESC-11, and level of disease may contribute to the variation in crown rot and anthracnose control by ESC-11. Though ESC-11 alone was not effective in reducing disease, further testing in combination with low rates of fungicide should be done.

Roadmap to New Virulence Determinants in Pseudomonas syringae: Insights from Comparative Genomics and Genome Organization

Systematic comparison of the current repertoire of virulence-associated genes for three Pseudomonas syringae strains with complete genome sequences, P. syringae pv. tomato DC3,000, P. syringae pv. phaseolicola 1448A, and P. syringae pv. syringae B728a, is prompted by recent advances in virulence factor identification in P. syringae and other bacteria. Among these are genes linked to epiphytic fitness, plant- and insect-active toxins, secretion pathways, and virulence regulators, all reflected in the recently updated DC3,000 genome annotation. Distribution of virulence genes in relation to P. syringae genome organization was analyzed to distinguish patterns of conservation among genomes and association between genes and mobile genetic elements. Variable regions were identified on the basis of deviation in sequence composition and gaps in syntenic alignment among the three genomes. Mapping gene location relative to the genome structure revealed strong segregation of the HrpL regulon with variable genome regions (VR), divergent distribution patterns for toxin genes depending on association with plant or insect pathogenesis, and patterns of distribution for other virulence genes that highlight potential sources of strain-to-strain differences in host interaction. Distribution of VR among other sequenced bacterial genomes was analyzed and future plans for characterization of this potential reservoir of virulence genes are discussed.

Discovery of ADP-Ribosylation and Other Plant Defense Pathway Elements Through Expression Profiling of Four Different Arabidopsis–Pseudomonas R-avr Interactions

A dissection of plant defense pathways was initiated through gene expression profiling of the responses of a single Arabidopsis thaliana genotype to isogenic Pseudomonas syringae strains expressing one of four different cloned avirulence (avr) genes. Differences in the expression profiles elicited by different resistance (R)-avr interactions were observed. A role for poly(ADP-ribosyl)ation in plant defense responses was suggested initially by the upregulated expression of genes encoding NUDT7 and poly(ADP-ribose) glycohydrolase in multiple R-avr interactions. Gene knockout plant lines were tested for 20 candidate genes identified by the expression profiling, and Arabidopsis NUDT7 mutants allowed less growth of virulent P. syringae (as previously reported) but also exhibited a reduced hypersensitive-response phenotype. Inhibitors of poly(ADP-ribose) polymerase (PARP) disrupted FLS2-mediated basal defense responses such as callose deposition. EIN2 (ethylene response) and IXR1 and IXR2 (cellulose synthase) mutants impacted the FLS2-mediated responses that occur during PARP inhibition, whereas no impacts were observed for NPR1, PAD4, or NDR1 mutants. In the expression profiling work, false-positive selection and grouping of genes was reduced by requiring simultaneous satisfaction of statistical significance criteria for each of three separate analysis methods, and by clustering genes based on statistical confidence values for each gene rather than on average fold-change of transcript abundance.

The Pseudomonas syringae Type III Effector HopAM1 Enhances Virulence on Water-Stressed Plants

Pseudomonas syringae strains deliver diverse type III effector proteins into host cells, where they can act as virulence factors. Although the functions of the majority of type III effectors are unknown, several have been shown to interfere with plant basal defense mechanisms. Type III effectors also could contribute to bacterial virulence by enhancing nutrient uptake and pathogen adaptation to the environment of the host plant. We demonstrate that the type III effector HopAM1 (formerly known as AvrPpiB) enhances the virulence of a weak pathogen in plants that are grown under drought stress. This is the first report of a type III effector that aids pathogen adaptation to water availability in the host plant. Expression of HopAM1 makes transgenic Ws-0 Arabidopsis hypersensitive to abscisic acid (ABA) for stomatal closure and germination arrest. Conditional expression of HopAM1 in Arabidopsis also suppresses basal defenses. ABA responses overlap with defense responses and ABA has been shown to suppress defense against P. syringae pathogens. We propose that HopAM1 aids P. syringae virulence by manipulation of ABA responses that suppress defense responses. In addition, host ABA responses enhanced by type III delivery of HopAM1 protect developing bacterial colonies inside leaves from osmotic stress.

Plastid ω3‐fatty acid desaturase‐dependent accumulation of a systemic acquired resistance inducing activity in petiole exudates of Arabidopsis thaliana is independent of jasmonic acid

Systemic acquired resistance (SAR) is an inducible defense mechanism that is activated throughout the plant, subsequent to localized inoculation with a pathogen. The establishment of SAR requires translocation of an unknown signal from the pathogen-inoculated leaf to the distal organs, where salicylic acid-dependent defenses are activated. We demonstrate here that petiole exudates (PeXs) collected from Arabidopsis leaves inoculated with an avirulent (Avr) Pseudomonas syringae strain promote resistance when applied to Arabidopsis, tomato (Lycopersicum esculentum) and wheat (Triticum aestivum). Arabidopsis FATTY ACID DESATURASE7 (FAD7), SUPPRESSOR OF FATTY ACID DESATURASE DEFICIENCY1 (SFD1) and SFD2 genes are required for accumulation of the SAR-inducing activity. In contrast to Avr PeX from wild-type plants, Avr PeXs from fad7, sfd1 and sfd2 mutants were unable to activate SAR when applied to wild-type plants. However, the SAR-inducing activity was reconstituted by mixing Avr PeXs collected from fad7 and sfd1 with Avr PeX from the SAR-deficient dir1 mutant. Since FAD7, SFD1 and SFD2 are involved in plastid glycerolipid biosynthesis and SAR is also compromised in the Arabidopsis monogalactosyldiacylglycerol synthase1 mutant we suggest that a plastid glycerolipid-dependent factor is required in Avr PeX along with the DIR1-encoded lipid transfer protein for long-distance signaling in SAR. FAD7-synthesized lipids provide fatty acids for synthesis of jasmonic acid (JA). However, co-infiltration of JA and methylJA with Avr PeX from fad7 and sfd1 did not reconstitute the SAR-inducing activity. In addition, JA did not co-purify with the SAR-inducing activity confirming that JA is not the mobile signal in SAR.

Bacterial non‐host resistance: interactions of Arabidopsis with non‐adapted Pseudomonas syringae strains

Although interactions of plants with virulent and avirulent host pathogens are under intensive study, relatively little is known about plant interactions with non-adapted pathogens and the molecular events underlying non-host resistance. Here we show that two Pseudomonas syringae strains for which Arabidopsis is a non-host plant, P. syringae pathovar (pv.) glycinea (Psg) and P. syringae pv. phaseolicola (Psp),induce salicylic acid (SA) accumulation and pathogenesis-related gene expression at inoculation sites, and that induction of these defences is largely dependent on bacterial type III secretion. The defence signalling components activated by non-adapted bacteria resemble those initiated by host pathogens, including SA, non-expressor of PR-1, non-race specific disease resistance 1, phytoalexin-deficient 4 and enhanced disease susceptibility 1. However, some differences in individual defence pathways induced by Psg and Psp exist, suggesting that for each strain, distinct sets of type III effectors are recognized by the plant. Although induction of SA-related defences occurs, it does not directly contribute to bacterial non-host resistance, because Arabidopsis mutants compromised in SA signalling and other classical defence pathways do not permit enhanced survival of Psg or Psp in leaves. The finding that numbers of non-adapted bacteria in leaf extracellular spaces rapidly decline after inoculation suggests that they fail to overcome toxic or structural defence barriers preceding SA-related responses. Consistent with this hypothesis, rapid, type III secretion system-independent upregulation of the lignin biosynthesis genes, PAL1 and BCB, which might contribute to an early induced, cell wall-based defence mechanism, occurs in response to non-adapted bacteria. Moreover, knockout of PAL1 permits increased leaf survival of non-host bacteria. In addition, different survival rates of non-adapted bacteria in leaves from Arabidopsis accessions and mutants with distinct glucosinolate composition or hydrolysis exist. Possible roles for early inducible, cell wall-based defences and the glucosinolate/myrosinase system in bacterial non-host resistance are discussed.

Cell Signalling: The Merry Lives of BAK1

Plant receptor-like kinases characterised by leucine-rich repeats have been shown to play dual roles in seemingly unrelated biological processes, inviting comparison with TOLL-like receptors of animals.

Management of Sclerotium rolfsii with integration of non-conventional chemicals, vermicompost and Pseudomonas syringae

Two non-conventional chemicals, ZnSO4 (10−4 mM) and oxalic acid (4 mM) were tested (alone as well as in combination with seeds bacterized with Pseudomonas syringae strain PUR46 and vermicompost substitution in the potting soil), for their ability to suppress collar rot of chickpea (Cicer arietinum) caused by Sclerotium rolfsii under greenhouse conditions. ZnSO4 and oxalic acid were applied as pre-inoculation foliar spray on chickpea and subsequently challenged with S. rolfsii. Both the chemicals provided significant protection to chickpea compared to control (100% plant mortality) when used alone as well as in combination with PUR46 and vermicompost. However, ZnSO4 was more effective than oxalic acid against S. rolfsii. Amongst the treatments tried, plant mortality was least when ZnSO4 was used in combination with seed bacterization with PUR46 and 25% vermicompost substitution. The findings indicate the utility of integration of the above factors in managing collar rot efficiently.

Competitive index in mixed infections: a sensitive and accurate assay for the genetic analysis of Pseudomonas syringae–plant interactions

Mixed infections have been broadly applied to the study of bacterial pathogens in animals. However, the application of mixed infection-based methods in plant pathogens has been very limited. An important factor for this limitation is the different dynamics that mixed infections have been reported to show in the different types of models. Reports in systemic animal infections have shown that any bacterium has the same probability of multiplying within a mixed infection than in a single infection. However, in plant pathogens, bacterial growth in a mixed infection does not seem to reflect growth in a single infection, as growth interference takes place between the co-inoculated strains. Here we show that growth interference in mixed infection between different Pseudomonas syringae strains is not intrinsic to growth within a plant host, but dependent on the dose of inoculation. We also show that the minimal inoculation dose required to avoid interference depends on the aggressiveness of the pathogen as well as the type of virulence factor that differentiates the co-inoculated strains. This study establishes the basis for the use of mixed infection-based applications to the study of phytopathogenic bacteria. Analysis of the virulence of a type III effector mutant and an hrp regulatory mutant illustrate the increased accuracy and sensitivity of competitive index assays vs. regular growth assays. Several applications of this assay are addressed, and potential implications for this and other mixed infection-based methods are discussed.

Using Pseudomonas spp. for Integrated Biological Control

ABSTRACT Pseudomonas spp. have been studied for decades as model organisms for biological control of plant disease. Currently, there are three commercial formulations of pseudomonads registered with the U.S. Environmental Protection Agency for plant disease suppression, Bio-Save 10 LP, Bio-Save 11 LP, and BlightBan A506. Bio-Save 10 LP and Bio-Save 11 LP, products of Jet Harvest Solutions, Longwood, FL, contain Pseudomonas syringae strains ESC-10 and ESC-11, respectively. These products are applied in packinghouses to prevent postharvest fungal diseases during storage of citrus, pome, stone fruits, and potatoes. BlightBan A506, produced by NuFarm Americas, Burr Ridge, IL, contains P. fluorescens strain A506. BlightBan A506 is applied primarily to pear and apple trees during bloom to suppress the bacterial disease fire blight. Combining BlightBan A506 with the antibiotic streptomycin improves control of fire blight, even in areas with streptomycin-resistant populations of the pathogen. BlightBan A506 also may reduce fruit russet and mild frost injury. These biocontrol products consisting of Pseudomonas spp. provide moderate to excellent efficacy against multiple production constraints, are relatively easy to apply, and they can be integrated with conventional products for disease control. These characteristics will contribute to the adoption of these products by growers and packinghouses.

A high‐performance, small‐scale microarray for expression profiling of many samples in Arabidopsis–pathogen studies

Studies of the behavior of biological systems often require monitoring of the expression of many genes in a large number of samples. While whole-genome arrays provide high-quality gene-expression profiles, their high cost generally limits the number of samples that can be studied. Although inexpensive small-scale arrays representing genes of interest could be used for many applications, it is challenging to obtain accurate measurements with conventional small-scale microarrays. We have developed a small-scale microarray system that yields highly accurate and reproducible expression measurements. This was achieved by implementing a stable gene-based quantile normalization method for array-to-array normalization, and a probe-printing design that allows use of a statistical model to correct for effects of print tips and uneven hybridization. The array measures expression values in a single sample, rather than ratios between two samples. This allows accurate comparisons among many samples. The array typically yielded correlation coefficients higher than 0.99 between technically duplicated samples. Accuracy was demonstrated by a correlation coefficient of 0.88 between expression ratios determined from this array and an Affymetrix GeneChip, by quantitative RT-PCR, and by spiking known amounts of specific RNAs into the RNA samples used for profiling. The array was used to compare the responses of wild-type, rps2 and ndr1 mutant plants to infection by a Pseudomonas syringae strain expressing avrRpt2. The results suggest that ndr1 affects a defense-signaling pathway(s) in addition to the RPS2-dependent pathway, and indicate that the microarray is a powerful tool for systems analyses of the Arabidopsis disease-signaling network.

Ethylene Chemotaxis in Pseudomonas aeruginosa and Other Pseudomonas Species

Pseudomonas aeruginosa strain PAO1 was attracted by ethylene. Assays of a set of mutants containing deletions in 26 potential methyl-accepting chemotaxis protein genes revealed that tlpQ (PA2654) encodes the chemoreceptor for positive chemotaxis to ethylene. Pseudomonas fluorescens, Pseudomonas putida, and Pseudomonas syringae strains also exhibited positive chemotaxis toward ethylene.

Pseudomonas syringae Diseases of Fruit Trees: Progress Toward Understanding and Control.

Pseudomonas syringae Diseases of Fruit Trees: Progress Toward Understanding and Control.

Genome‐based discovery, structure prediction and functional analysis of cyclic lipopeptide antibiotics in Pseudomonas species

Analysis of microbial genome sequences have revealed numerous genes involved in antibiotic biosynthesis. In Pseudomonads, several gene clusters encoding non-ribosomal peptide synthetases (NRPSs) were predicted to be involved in the synthesis of cyclic lipopeptide (CLP) antibiotics. Most of these predictions, however, are untested and the association between genome sequence and biological function of the predicted metabolite is lacking. Here we report the genome-based identification of previously unknown CLP gene clusters in plant pathogenic Pseudomonas syringae strains B728a and DC3000 and in plant beneficial Pseudomonas fluorescens Pf0-1 and SBW25. For P. fluorescens SBW25, a model strain in studying bacterial evolution and adaptation, the structure of the CLP with a predicted 9-amino acid peptide moiety was confirmed by chemical analyses. Mutagenesis confirmed that the three identified NRPS genes are essential for CLP synthesis in strain SBW25. CLP production was shown to play a key role in motility, biofilm formation and in activity of SBW25 against zoospores of Phytophthora infestans. This is the first time that an antimicrobial metabolite is identified from strain SBW25. The results indicate that genome mining may enable the discovery of unknown gene clusters and traits that are highly relevant in the lifestyle of plant beneficial and plant pathogenic bacteria.

Closing the Circle on the Discovery of Genes Encoding Hrp Regulon Members and Type III Secretion System Effectors in the Genomes of Three Model Pseudomonas syringae Strains

Pseudomonas syringae strains translocate large and distinct collections of effector proteins into plant cells via the type III secretion system (T3SS). Mutations in T3SS-encoding hrp genes are unable to elicit the hypersensitive response or pathogenesis in nonhost and host plants, respectively. Mutations in individual effectors lack strong phenotypes, which has impeded their discovery. P. syringae effectors are designated Hop (Hrp outer protein) or Avr (avirulence) proteins. Some Hop proteins are considered to be extracellular T3SS helpers acting at the plant-bacterium interface. Identification of complete sets of effectors and related proteins has been enabled by the application of bioinformatic and high-throughput experimental techniques to the complete genome sequences of three model strains: P. syringae pv. tomato DC3000, P. syringae pv. phaseolicola 1448A, and P. syringae pv. syringae B728a. Several recent papers, including three in this issue of Molecular Plant-Microbe Interactions, address the effector inventories of these strains. These studies establish that active effector genes in P. syringae are expressed by the HrpL alternative sigma factor and can be predicted on the basis of cis Hrp promoter sequences and N-terminal amino-acid patterns. Among the three strains analyzed, P. syringae pv. tomato DC3000 has the largest effector inventory and P. syringae pv. syringae B728a has the smallest. Each strain has several effector genes that appear inactive. Only five of the 46 effector families that are represented in these three strains have an active member in all of the strains. Web-based community resources for managing and sharing growing information on these complex effector arsenals should help future efforts to understand how effectors promote P. syringae virulence.

The Arabidopsis MAP kinase kinase MKK1 participates in defence responses to the bacterial elicitor flagellin

Plants sense pathogens through both pathogen-associated molecular patterns and recognition of race-specific virulence factors, which induce basal defence or an accelerated defence (often manifest in the form of local cell death), respectively. A mitogen-activated protein kinase (MAPK) module in Arabidopsis was previously proposed to signal from perception of the bacterial elicitor flagellin to the activation of basal defence-related genes. Here, we present evidence for a parallel MAPK-signalling pathway involved in the response to flg22, a peptide corresponding to the most conserved domain of flagellin. The endogenous Arabidopsis MAP kinase kinase MKK1 is activated in cells treated with flg22, phosphorylates the MAPK MPK4 in vitro, and activates it in vivo in protoplasts. In mkk1 mutant plants, the activation by flg22 of MPK4 and two other flg22-induced MAPKs (MPK3 and MPK6) is impaired. In the mkk1 mutant, a battery of both flg22-induced and flg22-repressed genes show altered expression, indicating that MKK1 negatively regulates the activity of flagellin-responsive genes. Intriguingly, in contrast to the mpk4 mutant, mkk1 shows no morphological anomalies and is compromised in resistance to both virulent and avirulent Pseudomonas syringae strains. Thus, the MKK1 signalling pathway modulates the expression of genes responding to elicitors and plays an important role in pathogen defence.