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PubMed1999

Structure of the O-polysaccharide of the lipopolysaccharide of Pseudomonas syringae pv. garcae ICMP 8047.

The composition and structure of the O-polysaccharide of the lipopolysaccharide of Pseudomonas syringae pathovar garcae ICMP 8047 were studied using methylation analyses, Smith degradation, and 1H- and 13C-NMR spectroscopy, including two-dimensional correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), and H-detected 1H,13C heteronuclear multiple-quantum coherence (HMQC) experiments. The polysaccharide was found to contain L-rhamnose and 3-acetamido-3, 6-dideoxy-D-galactose (D-Fuc3NAc) in the ratio 4:1 and to consist of two types of pentasaccharide repeating units. The major (1) and minor (2) repeating units differ from each other only in the position of substitution of one of the rhamnose residues in the main chain. Similar structural heterogeneity has been reported formerly in O-polysaccharides of some other P. syringae strains having a similar monosaccharide composition. A Fuc3NAc residue is attached to the main rhamnan chain as a side chain by a (alpha1-->4) glycosidic linkage; this has not hitherto been described in P. syringae: [figure].

Carbohydrate Conformation↗
PubMedDate not supplied

[The plasmids of Pseudomonas syringae].

The paper deals with occurrence of plasmids in P. syringae strains belonging to seventeen pathovars: the strains were isolated in the USSR and other countries. One to four different plasmids having molecular weights of 20 to 90 Md have been found in various strains of the following twelve pathovars: holci, cerasi, aptata, tabaci, populi, pisi, lupini syringae, lachrymans, phaseolicola, glycinea, atrofaciens. Both virulent and avirulent P. syringae strains appear to carry such plasmids. Plasmids of incompatibility group P-2 do not occur in the strains studied.

DNA, Bacterial↗
PubMedDate not supplied

[The characteristics of the lipid A of the lipopolysaccharides in Pseudomonas syringae strains].

Component composition of lipid A of Pseudomonas syringae pv. syringae 218 and P-55, pv. syringae (holci) 8299, pv. phaseolicola 120a, pv. atrofaciens 2399 has been studied. The lipid A composition of all the strains studied includes 3-hydroxydecanoic fatty acid (3-OH-C10:0), 2-hydroxydodecanoic (2-OH-C12:0), 3-hydroxydodecanoic (3-OH-C12:0), dodecanoic (C12:0), hexadecanoic (C16:0), octadecanoioc (C18:0), hexadecenic (C16:1), octadecenic (C18:1) fatty acids. The carbohydrate part of the lipid A macromolecule of all strains after acid hydrolysis contains ethanolamine, phosphoethanolamine, glucosamine.

Amino Acids↗
PubMed1998

Occurrence of thermoregulation of genes involved in coronatine biosynthesis among various Pseudomonas syringae strains.

Several pathovars of Pseudomonas syringae produce the polyketide phytotoxin coronatine (COR). In the bacterial blight pathogen of soybean, P. syringae pv. glycinea PG4180, COR is produced at high levels at 18 degrees C whereas no toxin is synthesized at 28 degrees C. Previously, activation of three promoters inside the COR biosynthetic gene cluster by a modified two-component regulatory system was shown to influence thermoregulation of COR biosynthesis. Using phenotypic determination of COR synthesis, a transcriptional reporter gene fusion, and Western blot analysis, we screened a representative number of natural isolates of P. syringae for effects of temperature on expression of cmaA, cmaB, and cmaT, which encode enzymes involved in the biosynthesis of COR. Thermoregulation of cmaABT expression was frequent among the tested strains. However, intensities of the temperature effects varied widely. Coronatine synsthesis was found to differ at up to six-fold among COR producing strains. There was no strain which synthesized COR at 28 degrees C although some of them showed increased basal cmaABT promoter activities at this temperature. Transcriptional fusions between the cmaABT promoter and a promoterless reporter gene were found to be down regulated at 28 degrees C only in COR producing strains but not in the non-producing strains tested. The geographic origin of the bacterial strains did not influence the occurrence of temperature-dependent gene expression.

Amino Acids↗
PubMed1998

Toxin production by pathovars of Pseudomonas syringae and their antagonistic activities against epiphytic microorganisms.

75 strains of 21 various Pseudomonas syringae (P.) pathovars were investigated in different tests for their toxin production. Data from literature about the production of the known phytotoxins phaseolotoxin (pv. phaseolicola), tabtoxin (pv. coronafaciens, pv. tabaci), coronatine (pv. atropurpurea, pv. glycinea, pv. maculicola, pv. morsprunorum, pv. tomato), and toxins of the lipodepsipeptide group (pv. aptata, pv. atrofaciens, pv. syringae) could be confirmed. Besides, a production of the phytohormone ethylene was detected for P. phaseolicola isolates from kudzu (Pueraria lobata) and for all tested P. glycinea and P. cannabina strains. Strains of P. apii, P. aptata, P. atrofaciens, and P. tomato produced antimetabolic toxins which could be detected with an agar diffusion assay with Escherichia coli as indicator strain. These antimetabolites inhibit a step in the arginine/ornithine biosynthesis. P. maculicola strains caused inhibition zones in this assay which could not be reversed by the tested amino acids. All strains with inhibitory effect against E. coli and Geotrichum candidum were also proved for their antagonistic activity against a selection of typical phyllosphere microorganisms. Most of the phytotoxins possess antimicrobial activity with different spectrum and efficiency. Only the lipodepsipeptide-producers showed antifungal activities. Our results show that the production of toxins is a widespread property among Pseudomonas syringae pathovars, and that some pathovars can produce more than one toxin. This characteristic and the antimicrobial activity of most toxins could be of advantage for the toxin-producing bacteria to adapt to different habitats.

Amino Acids↗
PubMed2005

Cis-trans isomerase gene in psychrophilic Pseudomonas syringae is constitutively expressed during growth and under conditions of temperature and solvent stress.

In a recent study, we established that psychrophilic Pseudomonas syringae (Lz4W) requires trans-monounsaturated fatty acid for growth at higher temperatures (Kiran et al. in Extremophiles, 2004). It was also demonstrated that the cti gene was highly conserved and exhibited high sequence identity with cti of other Pseudomonas spp. (Kiran et al. in Extremophiles, 2004). Therefore it would be interesting to understand the expression of the cti gene so as to unravel the molecular basis of adaptation of microorganisms to high temperature. In the present study, the expression of cti was monitored by RT-PCR analysis during different growth stages and under conditions of high temperature and solvent stress in P. syringae. Results indicated that the cti gene is constitutively expressed during different stages of growth and the transcript level is unaltered even under conditions of temperature and solvent stress implying that the observed increase in trans-monounsaturated fatty acids (Kiran et al. in Extremophiles, 2004) is not under transcriptional control. A putative promoter present in the intergenic region of the metH and cti gene has also been characterized. The translation start site ATG, the Shine-Dalgarno sequence AGGA and the transcription start site "C" were also identified. These results provide evidence for the first time that the cti gene is constitutively expressed under normal conditions of growth and under conditions of temperature and solvent stress thus implying that the Cti enzyme is post-transcriptionally regulated.

Base Sequence↗
PubMed2006

Suppression by ABA of salicylic acid and lignin accumulation and the expression of multiple genes, in Arabidopsis infected with Pseudomonas syringae pv. tomato.

Abscisic acid (ABA) has been implicated in determining the outcome of interactions between many plants and their pathogens. We had previously shown that increased concentrations of ABA within leaves of Arabidopsis induced susceptibility towards an avirulent strain of Pseudomonas syringae pathovar (pv.) tomato. We now show that ABA induces susceptibility via suppression of the accumulation of components crucial for a resistance response. Lignin and salicylic acid concentrations in leaves were increased during a resistant interaction but reduced when plants were treated with ABA. The reduction in lignin and salicylic acid production was independent of the development of the hypersensitive response (HR), indicating that, in this host-pathogen system, HR is not required for resistance. Genome-wide gene expression analysis using microarrays showed that treatment with ABA suppressed the expression of many defence-related genes, including those important for phenylpropanoid biosynthesis and those encoding resistance-related proteins. Together, these results show that resistance induction in Arabidopsis to an avirulent strain of P. syringae pv. tomato is regulated by ABA.

Abscisic Acid↗
PubMed2005

Phytohormones mediate volatile emissions during the interaction of compatible and incompatible pathogens: the role of ethylene in Pseudomonas syringae infected tobacco.

Interactions between the phytohormones ethylene, salicylic acid (SA), and jasmonic acid (JA) are thought to regulate the specificity of induced plant defenses against microbial pathogens and herbivores. However, the nature of these interactions leading to induced plant volatile emissions during pathogen infection is unclear. We previously demonstrated that a complex volatile blend including (E)-beta-ocimene, methyl salicylate (MeSA), and numerous sesquiterpenes was released by tobacco plants, Nicotiana tabacum K326, infected with an avirulent/incompatible strain of Pseudomonas syringae pv. tomato (Pst DC3000). In contrast, a volatile blend, mainly consisting of MeSA and two unidentified sesquiterpenes, was released by plants infected with P. syringae pv. tabaci (Pstb) in a virulent/compatible interaction. In this study, we examined the interaction of multiple pathogen stresses, phytohormone signaling, and induced volatile emissions in tobacco. Combined pathogen infection involved the inoculation of one leaf with Pst DC 3000 and of a second leaf, from the same plant, with Pstb. Combined infection reduced emissions of ocimene and MeSA compared to plants infected with Pst DC 3000 alone, but with no significant changes in total sesquiterpene emissions. In the compatible interaction, Pstb elicited a large ethylene burst with a peak emission occurring 3 days after inoculation. In contrast, the incompatible interaction involving Pst DC3000 displayed no such ethylene induction. Pstb-induced ethylene production was not significantly altered by Pst DC3000 in the combined infection. We postulated that Pstb-induced ethylene production may play a regulatory role in altering the typical volatile emission in tobacco in response to Pst DC3000 infection. To clarify the role of ethylene, we dynamically applied ethylene to the headspace of tobacco plants following infection with Pst DC3000. Consistent with Pstb-induced ethylene, exogenous ethylene reduced both ocimene and MeSA emissions, and selectively altered the ratios and amounts of induced sesquiterpene emissions. Our findings suggest that ethylene can regulate the magnitude and blend of induced volatile emissions during pathogen infection.

Cyclopentanes↗
PubMed2005

Calmodulin-like proteins from Arabidopsis and tomato are involved in host defense against Pseudomonas syringae pv. tomato.

Complex signal transduction pathways underlie the myriad plant responses to attack by pathogens. Ca(2+) is a universal second messenger in eukaryotes that modulates various signal transduction pathways through stimulus-specific changes in its intracellular concentration. Ca(2+)-binding proteins such as calmodulin (CaM) detect Ca(2+) signals and regulate downstream targets as part of a coordinated cellular response to a given stimulus. Here we report the characterization of a tomato gene (APR134) encoding a CaM-related protein that is induced in disease-resistant leaves in response to attack by Pseudomonas syringae pv. tomato. We show that suppression of APR134 gene expression in tomato (Solanum lycopersicum), using virus-induced gene silencing (VIGS), compromises the plant's immune response. We isolated APR134-like genes from Arabidopsis, termed CML42 and CML43, to investigate whether they serve a functionally similar role. Gene expression analysis revealed that CML43 is rapidly induced in disease-resistant Arabidopsis leaves following inoculation with Pseudomonas syringae pv. tomato. Overexpression of CML43 in Arabidopsis accelerated the hypersensitive response. Recombinant APR134, CML42, and CML43 proteins all bind Ca(2+ )in vitro. Collectively, our data support a role for CML43, and APR134 as important mediators of Ca(2+)-dependent signals during the plant immune response to bacterial pathogens.

Amino Acid Sequence↗
PubMed2004

Intragenomic heterogeneity of the 16S rRNA-23S rRNA internal transcribed spacer among Pseudomonas syringae and Pseudomonas fluorescens strains.

The 16S-23S rRNA internal transcribed spacer regions (ITS1) from 14 strains of Pseudomonas syringae and P. fluorescens were sequenced. ITS1 exhibited significant sequence variability among different operons within a single genome. From 1 to 4 types of ITS1 were found in individual genomes of the P. syringae and P. fluorescens strains. A total of eight ITS1 types were identified among strains studied. The ITS1 nucleotide sequences consisted of conserved blocks including, among others, a stem-forming region of box B, tRNAIle and tRNAAla genes and several variable blocks. The differences in the variable regions were mostly due to insertions and/or deletions of nucleotide blocks. The intragenomic heterogeneity of ITS1 was brought about by different combinations of variable blocks, which possibly have resulted from recombination and horizontal transfer.

Base Sequence↗
PubMed2004

Arabidopsis CYP86A2 represses Pseudomonas syringae type III genes and is required for cuticle development.

Pseudomonas syringae relies on type III secretion system to deliver effector proteins into the host cell for parasitism. Type III genes are induced in planta, but host factors affecting the induction are poorly understood. Here we report on the identification of an Arabidopsis mutant, att1 (for aberrant induction of type three genes), that greatly enhances the expression of bacterial type III genes avrPto and hrpL. att1 plants display enhanced disease severity to a virulent strain of P. syringae, suggesting a role of ATT1 in disease resistance. ATT1 encodes CYP86A2, a cytochrome P450 monooxygenase catalyzing fatty acid oxidation. The cutin content is reduced to 30% in att1, indicating that CYP86A2 plays a major role in the biosynthesis of extracellular lipids. att1 has a loose cuticle membrane ultrastructure and shows increased permeability to water vapor, demonstrating the importance of the cuticle membrane in controlling water loss. The enhanced avrPto-luc expression is specific to att1, but not another cuticle mutant, wax2. The results suggest that certain cutin-related fatty acids synthesized by CYP86A2 may repress bacterial type III gene expression in the intercellular spaces.

Amino Acid Sequence↗
PubMed2003

The Pseudomonas syringae type III-secreted protein HopPtoD2 possesses protein tyrosine phosphatase activity and suppresses programmed cell death in plants.

The bacterial plant pathogen Pseudomonas syringae possesses a type III protein secretion system that delivers many virulence proteins into plant cells. A subset of these proteins (called Avr proteins) is recognized by the plant's innate immune system and triggers defences. One defence-associated response is the hypersensitive response (HR), a programmed cell death (PCD) of plant tissue. We have previously identified HopPtoD2 as a type III secreted protein from P. s. pv. tomato DC3000. Sequence analysis revealed that an N-terminal domain shared homology with AvrPphD and a C-terminal domain was similar to protein tyrosine phosphatases (PTPs). We demonstrated that purified HopPtoD2 possessed PTP activity and this activity required a conserved catalytic Cys residue (Cys(378)). Interestingly, HopPtoD2 was capable of suppressing the HR elicited by an avirulent P. syringae strain on Nicotiana benthamiana. HopPtoD2 derivatives that lacked Cys(378) no longer suppressed the HR indicating that HR suppression required PTP activity. A constitutively active MAPK kinase, called NtMEK2DD, is capable of eliciting an HR-like cell death when transiently expressed in tobacco. When NtMEK2DD and HopPtoD2 were co-delivered into plant cells, the HR was suppressed indicating that HopPtoD2 acts downstream of NtMEK2DD. DC3000 hopPtoD2 mutants were slightly reduced in their ability to multiply in planta and displayed an enhanced ability to elicit an HR. The identification of HopPtoD2 as a PTP and a PCD suppressor suggests that the inactivation of MAPK pathways is a virulence strategy utilized by bacterial plant pathogens.

Apoptosis↗
PubMed2004

Identification of Pseudomonas syringae type III effectors that can suppress programmed cell death in plants and yeast.

The Pseudomonas syringae pv. tomato DC3000 type III secretion system (TTSS) is required for bacterial pathogenicity on plants and elicitation of the hypersensitive response (HR), a programmed cell death (PCD) that occurs on resistant plants. Cosmid pHIR11 enables non-pathogens to elicit an HR dependent upon the TTSS and the effector HopPsyA. We used pHIR11 to determine that effectors HopPtoE, avirulence AvrPphEPto, AvrPpiB1Pto, AvrPtoB, and HopPtoF could suppress a HopPsyA-dependent HR on tobacco and Arabidopsis. Mixed inoculum and Agrobacterium-mediated transient expression experiments confirmed that suppressor action occurred within plant cells. These suppressors, with the exception of AvrPpiB1Pto, inhibited the expression of the tobacco pathogenesis-related (PR) gene PR1a. DC3000 suppressor mutants elicited an enhanced HR consistent with these mutants lacking an HR suppressor. Additionally, HopPtoG was identified as a suppressor on the basis of an enhanced HR produced by a hopPtoG mutant. Remarkably, these proteins functioned to inhibit the ability of the pro-apoptotic protein, Bax to induce PCD in plants and yeast, indicating that these effectors function as anti-PCD proteins in a trans-kingdom manner. The high proportion of effectors that suppress PCD suggests that suppressing plant immunity is one of the primary roles for DC3000 effectors and a central requirement for P. syringae pathogenesis.

Apoptosis↗
PubMed2005

Exoribonuclease R interacts with endoribonuclease E and an RNA helicase in the psychrotrophic bacterium Pseudomonas syringae Lz4W.

Endoribonuclease E, a key enzyme involved in RNA decay and processing in bacteria, organizes a protein complex called degradosome. In Escherichia coli, Rhodobacter capsulatus, and Streptomyces coelicolor, RNase E interacts with the phosphate-dependent exoribonuclease polynucleotide phosphorylase, DEAD-box helicase(s), and additional factors in an RNA-degrading complex. To characterize the degradosome of the psychrotrophic bacterium Pseudomonas syringae Lz4W, RNase E was enriched by cation exchange chromatography and fractionation in a glycerol density gradient. Most surprisingly, the hydrolytic exoribonuclease RNase R was found to co-purify with RNase E. Co-immunoprecipitation and Ni(2+)-affinity pull-down experiments confirmed the specific interaction between RNase R and RNase E. Additionally, the DEAD-box helicase RhlE was identified as part of this protein complex. Fractions comprising the three proteins showed RNase E and RNase R activity and efficiently degraded a synthetic stem-loop containing RNA in the presence of ATP. The unexpected association of RNase R with RNase E and RhlE in an RNA-degrading complex indicates that the cold-adapted P. syringae has a degradosome of novel structure. The identification of RNase R instead of polynucleotide phosphorylase in this complex underlines the importance of the interaction between endo- and exoribonucleases for the bacterial RNA metabolism. The physical association of RNase E with an exoribonuclease and an RNA helicase apparently is a common theme in the composition of bacterial RNA-degrading complexes.

Adenosine Triphosphate↗
PubMed2003

Efficiency of procedures for induction and cultivation of Pseudomonas syringae pv. pisi L-form.

The L-form of Pseudomonas syringae pv. phaseolicola has been proved to induce resistance to bean halo blight. Various procedures were tested to induce the L-form of Pseudomonas syringae pv. pisi for its potential use as biocontrol agent of pea bacterial blight. Cell-wall deficient cells were induced in a liquid medium with penicillin following a protocol described for P. s. pv. phaseolicola. Cell growth on solid induction medium developed as typical granular and vacuolated structures, and characteristic colonies were observed in the first transfer. However, there was poor growth in subsequent transfers and some reversion to the parental type. To improve the induction procedure, the following new procedures were applied: (1) viability of cells was monitored during induction. The optimum induction time in liquid medium with penicillin was lower for pv. pisi than for pv. phaseolicola. Viability of L-forms in solid induction medium with penicillin was low and decreased in time. (2) the inducer ticarcillin was combined with clavulanic acid, which prevented the reversion to the parental type and (3) a range of concentrations of penicillin and ticarcillin/clavulanic acid was applied by the spiral gradient endpoint method for calculation of minimum inhibitory concentrations (MIC). Based on the results from these tests an induction method for P. s. pv. pisi L-form is proposed and the relevance of L-form is discussed for practice.

Anti-Bacterial Agents↗
PubMed2005

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

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

Amino Acid Sequence↗
PubMed2005

Proposed guidelines for a unified nomenclature and phylogenetic analysis of type III Hop effector proteins in the plant pathogen Pseudomonas syringae.

Pathovars of Pseudomonas syringae interact with their plant hosts via the action of Hrp outer protein (Hop) effector proteins, injected into plant cells by the type III secretion system (TTSS). Recent availability of complete genome sequences for a number of P. syringae pathovars has led to a significant increase in the rate of effector discovery. However, lack of a systematic nomenclature has resulted in multiple names being assigned to the same Hop, unrelated Hops designated by the same alphabetic character, and failure of name choices to reflect consistent standards of experimental confirmation or phylogenetic relatedness. Therefore, specific experimental and bioinformatic criteria are proposed for proteins to be designated as Hops. A generic Hop name structure, HopXY#pv strain, also is proposed, wherein family membership is indicated by the alphabetic characters, subgroup membership numerically, and source pathovar and strain in subscript. Guidelines are provided for phylogenetic characterization and name selection for Hops that are novel, related to previously characterized Hops, chimeras, pseudogenes, truncations, or nonexpressed alleles. Phylogenetic analyses of previously characterized Hops are described, the results of which have been used to guide their integration into the proposed nomenclature.

Bacterial Proteins↗
PubMed2005

The Pseudomonas syringae avrRpt2 gene contributes to virulence on tomato.

In order to cause disease on plants, gram-negative phytopathogenic bacteria introduce numerous virulence factors into the host cell in order to render host tissue more hospitable for pathogen proliferation. The mode of action of such bacterial virulence factors and their interaction with host defense pathways remain poorly understood. avrRpt2, a gene from Pseudomonas syringae pv. tomato JL1065, has been shown to promote the virulence of heterologous P. syringae strains on Arabidopsis thaliana. However, the contribution of avrRpt2 to the virulence of JL1065 has not been examined previously. We show that a mutant derivative of JL1065 that carries a disruption in avrRpt2 is impaired in its ability to cause disease on tomato (Lycopersicon esculentum), indicating that avrRpt2 also acts as a virulence gene in its native strain on a natural host. The virulence activity of avrRpt2 was detectable on tomato lines that are defective in either ethylene perception or the accumulation of salicylic acid, but could not be detected on a tomato mutant insensitive to jasmonic acid. The enhanced virulence conferred by the expression of avrRpt2 in JL1065 was not associated with the suppression of several defense-related genes induced during the infection of tomato.

Arabidopsis↗