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PubMed1993

Suppression of Bean Defense Responses by Pseudomonas syringae.

We have developed a model system to examine suppression of defense responses in bean by the compatible bacterium Pseudomonas syringae pv phaseolicola. Previously, we have shown that there is a general mechanism for the induction of the bean defense genes phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), and chitinase (CHT) by incompatible, compatible, and nonpathogenic bacteria. Here, we show that bean plants infiltrated with isolates of P. s. phaseolicola failed to produce transcripts for PAL, CHS, or CHI up to 120 hr after infiltration and CHT transcript accumulation was significantly delayed when compared to the incompatible P. syringae strains. Infiltration of bean plants with 108 cells per mL of P. s. phaseolicola NPS3121 8 hr prior to infiltration with an equal concentration of incompatible P. s. pv tabaci Pt11528 significantly reduced the typical profile of defense transcript accumulation when compared to plants infiltrated with Pt11528 alone. A corresponding suppression of phytoalexin accumulation was also observed. NPS3121 also suppressed PAL, CHS, CHI, and CHT transcript accumulation and phytoalexin production induced by Escherichia coli DH5[alpha] or the elicitor glutathione. Heat-killed NPS3121 cells or cells treated with protein synthesis inhibitors lost the suppressor activity. Taken together, these experiments suggest that NPS3121 has an active mechanism to suppress the accumulation of defense transcripts and phytoalexin biosynthesis in bean.

Journal Article↗
PubMed1993

RPS2, an Arabidopsis disease resistance locus specifying recognition of Pseudomonas syringae strains expressing the avirulence gene avrRpt2.

A molecular genetic approach was used to identify and characterize plant genes that control bacterial disease resistance in Arabidopsis. A screen for mutants with altered resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) expressing the avirulence gene avrRpt2 resulted in the isolation of four susceptible rps (resistance to P. syringae) mutants. The rps mutants lost resistance specifically to bacterial strains expressing avrRpt2 as they retained resistance to Pst strains expressing the avirulence genes avrB or avrRpm1. Genetic analysis indicated that in each of the four rps mutants, susceptibility was due to a single mutation mapping to the same locus on chromosome 4. Identification of a resistance locus with specificity for a single bacterial avirulence gene suggests that this locus, designated RPS2, controls specific recognition of bacteria expressing the avirulence gene avrRpt2. Ecotype Wü-0, a naturally occurring line that is susceptible to Pst strains expressing avrRpt2, appears to lack a functional allele at RPS2, demonstrating that there is natural variation at the RPS2 locus among wild populations of Arabidopsis.

Arabidopsis↗
PubMed1995

Expression of the Tomato Pto Gene in Tobacco Enhances Resistance to Pseudomonas syringae pv tabaci Expressing avrPto.

The Pto gene encodes a serine-threonine kinase that confers resistance in tomato to Pseudomonas syringae pv tomato strains expressing the avirulence gene avrPto. We examined the ability of Pto to function in tobacco, a species that is sexually incompatible with tomato. Evidence that a heterologous Pto-like signal transduction pathway is present in tobacco was suggested by the fact that tobacco line Wisconsin-38 exhibits a hypersensitive response after infection with P. syringae pv tabaci expressing avrPto. We introduced a Pto transgene into cultivar Wisconsin-38 and assessed the ability of transformed plants to further inhibit growth of the P. s. tabaci strain expressing avrPto. The Pto-transformed tobacco plants exhibited a significant increase in resistance to the avirulent P. s. tabaci strain compared with wild-type tobacco as indicated by (1) more rapid development of a hypersensitive resistance response at high inoculum concentrations (108 colony-forming units per mL); (2) lessened severity of disease symptoms at moderate inoculum concentrations (106 and 107 colony-forming units per mL); and (3) reduced growth of avirulent P. s. tabaci in inoculated leaves. The results indicate that essential components of a Pto-mediated signal transduction pathway are conserved in tobacco and should prompt examination of resistance gene function across even broader taxonomic distances.

Journal Article↗
PubMed2001

Crystallization and preliminary X-ray analysis of clade I catalases from Pseudomonas syringae and Listeria seeligeri.

Haem-containing catalases are homotetrameric molecules that degrade hydrogen peroxide. Phylogenetically, the haem-containing catalases can be grouped into three main lines or clades. The crystal structures of seven catalases have been determined, all from clades II and III. In order to obtain a structure of an enzyme from clade I, which includes all plant, algae and some bacterial enzymes, two bacterial catalases, CatF from Pseudomonas syringae and Kat from Listeria seeligeri, have been crystallized by the hanging-drop vapour-diffusion technique, using PEG and ammonium sulfate as precipitants, respectively. Crystals of P. syringae CatF, with a plate-like morphology, belong to the monoclinic space group P2(1), with unit-cell parameters a = 60.6, b = 153.9, c = 109.2 A, beta = 102.8 degrees. From these crystals a diffraction data set to 1.8 A resolution with 98% completeness was collected using synchrotron radiation. Crystals of L. seeligeri Kat, with a well developed bipyramidal morphology, belong to space group I222 (or I2(1)2(1)2(1)), with unit-cell parameters a = 74.4, b = 121.3, c = 368.5 A. These crystals diffracted beyond 2.2 A resolution when using synchrotron radiation, but presented anisotropic diffraction, with the weakest direction perpendicular to the long c axis.

Catalase↗
PubMed1993

Homology between a genetic locus (mdoA) involved in the osmoregulated biosynthesis of periplasmic glucans in Escherichia coli and a genetic locus (hrpM) controlling pathogenicity of Pseudomonas syringae.

Membrane-derived oligosaccharides (MDO) of Escherichia coli are representative members of a family of glucans found in the periplasmic space of Gram-negative bacteria. The two genes forming the mdoGH operon are necessary for the synthesis of MDO. The nucleotide sequence (4759 bp) and the transcriptional start of this operon were determined. Both gene products were further characterized by gene fusion analysis. MdoG is a 56 kDa periplasmic protein whose function remains to be determined. MdoH, whose presence was shown to be necessary for normal glucosyl transferase activity, is a 97 kDa protein spanning the cytoplasmic membrane. To our surprise, these proteins are not homologous to the periplasmic glucan biosynthetic enzymes previously characterized in the Rhizobiaceae family. However, a considerable homology (69% identical nucleotides out of 2816) was discovered between mdoGH and the two genes present at the hrpM locus of the phytopathogenic bacterium Pseudomonas syringae pv. syringae. Functions of these genes remain mysterious but they are known to be required for both the expression of disease symptoms on host plants and the development of the hypersensitive reaction on non-host plants (Mills and Mukhopadhyay, 1990). These results confirm the importance of periplasmic glucans for the physiological ecology of Gram-negative bacteria.

Amino Acid Sequence↗
PubMed1990

Production of monoclonal antibodies to Pseudomonas syringae pv. phaseolicola and Xanthomonas campestris pv. phaseoli.

The production of monoclonal antibodies (MAbs) to ethylenediamine tetraacetic acid (sodium salt) soluble antigens of Pseudomonas syringae pv. phaseolicola and Xanthomonas campestris pv. phaseoli (fuscans strain) is described. MAbs A6-1 and A6-2 produced to Ps. syringae pv. phaseolicola are pathovar specific. Although MAb XP2 produced to X. campestris pv. phaseoli recognized surface antigens of all strains of this pathovar (including fuscans strains) it cross-reacted specifically with X. campestris pv. malvacearum; it did not react with any other known bacteria or unidentified epiphytes from navy bean seed or leaves. The isotype of both MAbs XP2 and A6-1 is IgG3 whereas that of MAb A6-2 is IgG2a. The reactive antigens are thermostable, but their chemical nature has not been determined.

Antibodies, Bacterial↗
PubMed1998

Histidine utilisation operon (hut) is upregulated at low temperature in the antarctic psychrotrophic bacterium Pseudomonas syringae.

The antarctic psychrotrophic bacterium Pseudomonas syringae was mutagenised using a transposon Tn5-OT182 which facilitates identification of promoter fusions expressing the reporter gene (lacZ) for beta-galactosidase. Most mutants expressed beta-galactosidase both at optimal growth temperature (20-22 degrees C) and at low temperature (4 degrees C). But a small percentage of the mutants (approximately 5%) were unique in that they expressed beta-galactosidase activity predominantly at low temperature. One such mutant was found to have an insertion in the gene for urocanase (hutU) of the histidine utilisation (hut) operon. Direct assay of urocanase and histidase activity in wild-type cells of various antarctic psychrotrophic strains including P. syringae, P. fluorescens and P. putida also suggested that the hut operon is expressed at an elevated level at low temperature.

Amino Acid Sequence↗
PubMed1999

Studies on the cytoplasmic protein tyrosine kinase activity of the Antarctic psychrotrophic bacterium Pseudomonas syringae.

The Antarctic psychrotrophic bacterium Pseudomonas syringae contains a 66-kDa cytoplasmic protein which was found to by phosphorylated on a tyrosine residue [Ray, M.K. et al. (1994) FEMS Microbiol. Lett. 122, pp. 49-54]. To investigate the nature of the cytoplasmic protein tyrosine kinase and its role in the bacterial physiology, we carried out some biochemical studies of the enzyme in vitro in the presence of exogenous peptide substrates and expression studies in vivo at low and high temperature during various phases of growth. The results suggest that the protein tyrosine kinase associated with the cytoplasmic fraction of the bacterium has certain similarities and dissimilarities with the known eukaryotic tyrosine kinases. The protein tyrosine kinase could phosphorylate exogenous substrate corresponding to the N-terminal peptide of p34cdc2 kinase but could not do so on poly(Glu:Tyr). The enzyme could not be inhibited by genistein, staurosporine and dimethyl aminopurine, but could be inhibited by piceatannol which is a known competitive inhibitor of the peptide binding site of mammalian protein tyrosine kinases. The enzyme activity in the cytoplasm is uniquely inhibited by sodium orthovanadate (IC50 = 20 microM) which is a known protein tyrosine phosphatase inhibitor. The expression studies show that the enzyme is produced more at a higher temperature (22 degrees C) of growth than at lower temperature (4 degrees C) and during the stationary phase of growth of P. syringae.

Amino Acid Sequence↗
PubMed2000

Phosphorylation of CorS and CorR, regulatory proteins that modulate production of the phytotoxin coronatine in Pseudomonas syringae.

Production of the phytotoxin coronatine (COR) in Pseudomonas syringae pv. glycinea PG4180 is controlled by a modified two-component regulatory system consisting of three genes, corR, corP, and corS. CorR and CorP show similarity to response regulators, and CorS is related to histidine protein kinases that function as environmental sensors. In this study, CorR, CorP and the cytoplasmic portion of CorS, designated CorSDelta, were overproduced in P. syringae as translational fusions to the maltose-binding protein and purified by affinity chromatography. Autophosphorylation of CorSDelta was demonstrated when [gamma-(32)P]ATP was used as a phosphodonor. Phosphorylated CorSDelta (CorSDelta approximately P) was stable under basic conditions, but extremely sensitive when the pH was reduced, which is consistent with phosphorylation at a histidine residue. CorSDelta approximately P transferred its phosphoryl group to CorR but not to CorP, which correlates with the presence of a receiver aspartate residue in the former but not the latter protein. These results indicate that CorS functions as a histidine protein kinase and transphosphorylates CorR, a positive activator of cor gene transcription.

Amino Acids↗
PubMed2002

A functional screen for the type III (Hrp) secretome of the plant pathogen Pseudomonas syringae.

Type III secreted "effector" proteins of bacterial pathogens play central roles in virulence, yet are notoriously difficult to identify. We used an in vivo genetic screen to identify 13 effectors secreted by the type III apparatus (called Hrp, for "hypersensitive response and pathogenicity") of the plant pathogen Pseudomonas syringae. Although sharing little overall homology, the amino-terminal regions of these effectors had strikingly similar amino acid compositions. This feature facilitated the bioinformatic prediction of 38 P. syringae effectors, including 15 previously unknown proteins. The secretion of two of these putative effectors was shown to be type III--dependent. Effectors showed high interstrain variation, supporting a role for some effectors in adaptation to different hosts.

Amino Acid Sequence↗
PubMed1985

Construction of a cosmid clone library of Pseudomonas syringae pv. phaseolicola and isolation of genes by functional complementation.

A genomic library constructed from a wild-type strain of Pseudomonas syringae pv. phaseolicola in the broad-host-range cosmid vector pVK102 was used to isolate wild-type genes by complementation of Tn5-induced auxotrophic mutants. Selection pressure was required for maintenance of the vector and members of the library in strains of P. syringae.

Cloning, Molecular↗
PubMed1986

Generation and Characterization of Tn5 Insertion Mutations in Pseudomonas syringae pv. tomato.

Tn5-induced insertion mutations were generated in the Pseudomonas syringae pv. tomato genome by mating this plant pathogen with an Escherichia coli strain carrying the suicide plasmid vector for Tn5, pGS9. Km transconjugants occurred at frequencies ranging from 2 x 10 to 9 x 10; approximately 5.5% of these transconjugants were also Cm, indicating the presence of additional pGS9 DNA sequences. Approximately 1% of the Km Cm mutants were auxotrophic. Southern blot analysis revealed that the Tn5 element had inserted into one unique site on the chromosome for each Km Cm transconjugant examined. Physical and genetic tests of Tn5-induced auxotrophs showed that Tn5 mutations in P. syringae pv. tomato were very stable and that secondary transposition of Tn5 or its insertion sequence IS50 was a rare event. Nine of 920 Km Cm transconjugants screened on tomato seedlings either were avirulent or produced very mild symptoms. Each of the virulence mutants was the result of a unique single-site Tn5 insertion. Five mutants also failed to induce a hypersensitivity reaction on tobacco.

Journal Article↗
PubMed1986

Isolation and Characterization of an Fe(III)-Chelating Compound Produced by Pseudomonas syringae.

The phytopathogenic bacterium Pseudomonas syringae produces a fluorescent pigment when it is grown in iron-deficient media. This pigment forms a very stable Fe(III) complex that was purified in this form by using a novel procedure based on ultrafiltration and column chromatography. The Fe(III) complex has a molecular weight of 1,100 and contains 1 mol of Fe(III). The pigment is composed of an amino acid moiety with three threonines, three serines, one lysine, delta-N-hydroxyornithine, and a quinoline-type fluorescent chromophore. These features and its stability constant (in the range of 10) suggest that the fluorescent pigment of P. syringae is related to the siderophores produced by another Pseudomonas species.

Journal Article↗
PubMed1988

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 MgCl(2) 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 degrees C, which is 5 degrees 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 x 10 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.

Journal Article↗
PubMed1989

Survival of Ice Nucleation-Active and Genetically Engineered Non-Ice-Nucleating Pseudomonas syringae Strains after Freezing.

The survival after freezing of ice nucleation-active (INA) and genetically engineered non-INA strains of Pseudomonas syringae was compared. Each strain was applied to oat seedlings and allowed to colonize for 3 days, and the plants were subjected to various freezing temperatures. Plant leaves were harvested before and after freezing on two consecutive days, and bacterial populations were determined. Populations of the INA wild-type strain increased 15-fold in the 18 h after the oat plants incurred frost damage at -5 and -12 degrees C. Plants colonized by the non-INA strain were undamaged at -5 degrees C and exhibited no changes in population size after two freeze trials. As freezing temperatures were lowered (-7, -9, and -12 degrees C), oat plants colonized by the non-INA strain suffered increased frost damage concomitant with bacterial population increases following 18 h. At -12 degrees C, both strains behaved identically. The data show a relationship between frost damage to plants and increased bacterial population size during the following 18 h, indicating a potential competitive advantage of INA strains of P. syringae over non-INA strains in mild freezing environments.

Journal Article↗
PubMed1990

Fate of Ice Nucleation-Active Pseudomonas syringae Strains in Alpine Soils and Waters and in Synthetic Snow Samples.

The stability of the ice nucleation activity (INA) and viability of INA Pseudomonas syringae 31a, used as an ice nucleator in the manufacture of synthetic snow, was determined in snow. The viability of P. syringae 1-2b, a rifampin-resistant mutant selected from strain 31a to improve recovery from test samples, was determined in laboratory tests of three alpine soil and water samples from three different sources. Snow samples were exposed to environmental conditions or held in darkness at -20 degrees C. Samples of soil and water were maintained in darkness at 0, 7.5, or 15 degrees C. Parent strain 31a INA decreased significantly (>99.0%) in snow exposed to sunlight and freeze-thaw, while the INA of the cell population in snow held in darkness at -20 degrees C remained essentially unchanged. No viable strain 31a was detected in snow exposed to the environment after 7 days, while the viability of strain 31a in snow held in darkness at -20 degrees C decreased to <3% of the original inoculation at the test conclusion. Mutant strain 1-2b viability was undetectable or had decreased significantly 19 days postinoculation in soil samples held at 0 or 15 degrees C. In contrast, 1-2b viability remained detectable at low levels for the duration of the test in soils held at 7.5 degrees C. The 1-2b population demonstrated a significantly longer half-life in peatlike soil than in the loam soils tested. The rate of decrease in 1-2b viability was essentially the same in the three alpine water samples tested with respect to water temperature and sample location.

Journal Article↗
PubMed1993

DNA sequence and transcriptional analysis of the tblA gene required for tabtoxin biosynthesis by Pseudomonas syringae.

The tblA gene of Pseudomonas syringae is required for tabtoxin biosynthesis and is under the control of a regulatory gene, lemA. We have determined the nucleotide sequence of the tblA gene and identified the 5' end of the tblA gene transcript. The sequence of the tblA gene was identified to that of the recently reported open reading frame 1 gene of the tabA region of the BR2 chromosome. The open reading frame of the tblA gene potentially encodes a protein of 231 amino acids. mRNA from the tblA gene was detected at all phases of cells grown in minimal medium. This result is correlated with the constitutive production of tabtoxinine-beta-lactam (the biologically active part of the toxin) by P. syringae BR2R in minimal medium, as quantitated by a phenylisothiocyanate derivatization method.

Base Sequence↗
PubMed1993

Dynamics, spread, and persistence of a single genotype of Pseudomonas syringae relative to those of its conspecifics on populations of snap bean leaflets.

A rifampin-resistant strain of Pseudomonas syringae (R10) was introduced onto bean plants grown in field plots to examine the processes of growth, spread, and survival of a single genotype relative to the dynamics of its conspecifics on populations of individual leaflets. R10 was applied to four plots (400, 200, 100, and 50 m2), each of which was centered in a quadrant of a bean field (90 by 90 m). Population sizes of the species P. syringae and of R10 were determined on each of 25 individual leaflets collected from the largest plot (A) at approximately weekly intervals during a 10-week period following application. The spread of R10 from all plots was monitored by leaf imprinting of individual leaflets collected at sites along four transects, each of which bisected two of the plots. The introduced strain was a dominant component of the species for about 5 weeks postinoculation on leaflets from plot A. Although the population sizes of R10 remained at about 5.0 to 5.5 log10 CFU per leaflet, the strain became a progressively minor component of the species as the population sizes of its conspecifics continued to increase during the latter part of the growing season. In general, a positive correlation was found for the population sizes of R10 and its conspecifics on individual leaflets collected throughout the growing season. This result suggests that large numbers of R10 early in the growing season did not exclude the colonization of bean leaflets by its conspecifics. It is apparent that the species pool comprised genotypes that were more fit than R10 under the conditions that prevailed during the latter part of the growing season. By 6 weeks postinoculation, R10 was detected at all sites sampled within the unsprayed areas of the field. However, it was present as a minor component of the species. The persistence of R10 throughout the winter and into the following growing season was monitored in plot A, which was plowed and replanted with wheat in the fall. R10 was detected on some of the samples (wheat seedlings or soil) taken at approximately monthly intervals from November to June of the following year. In June, the field was plowed and replanted with beans. We could not detect R10 on emerging bean seedlings in plot A. The results demonstrate that the successful spread and persistence of an introduced bacterium do not necessarily lead to the establishment of large populations of the bacterium in adjacent untreated areas or on its host plant in subsequent growing seasons.

Ecology↗