Pseudomonas syringae exchangeable effector loci: sequence diversity in representative pathovars and virulence function in P. syringae pv. syringae B728a

Pseudomonas syringae is a plant pathogen whose pathogenicity and host specificity are thought to be determined by Hop/Avr effector proteins injected into plant cells by a type III secretion system. P. syringae pv. syringae B728a, which causes brown spot of bean, is a particularly well-studied strain. The type III secretion system in P. syringae is encoded by hrp (hypersensitive response and pathogenicity) and hrc (hrp conserved) genes, which are clustered in a pathogenicity island with a tripartite structure such that the hrp/hrc genes are flanked by a conserved effector locus and an exchangeable effector locus (EEL). The EELs of P. syringae pv. syringae B728a, P. syringae strain 61, and P. syringae pv. tomato DC3000 differ in size and effector gene composition; the EEL of P. syringae pv. syringae B728a is the largest and most complex. The three putative effector proteins encoded by the P. syringae pv. syringae B728a EEL--HopPsyC, HopPsyE, and HopPsyV--were demonstrated to be secreted in an Hrp-dependent manner in culture. Heterologous expression of hopPsyC, hopPsyE, and hopPsyV in P. syringae pv. tabaci induced the hypersensitive response in tobacco leaves, demonstrating avirulence activity in a nonhost plant. Deletion of the P. syringae pv. syringae B728a EEL strongly reduced virulence in host bean leaves. EELs from nine additional strains representing nine P. syringae pathovars were isolated and sequenced. Homologs of avrPphE (e.g., hopPsyE) and hopPsyA were particularly common. Comparative analyses of these effector genes and hrpK (which flanks the EEL) suggest that the EEL effector genes were acquired by horizontal transfer after the acquisition of the hrp/hrc gene cluster but before the divergence of modern pathovars and that some EELs underwent transpositions yielding effector exchanges or point mutations producing effector pseudogenes after their acquisition.     

Chemotaxis of Pseudomonas syringae subsp. savastanoi Virulence Mutants

Several mutants of Pseudomonas syringae subsp. savastanoi were tested for their ability to sense and respond to a chemotactic gradient in low concentrations of yeast extract. The mutants were deficient in one or both of the genes coding for the synthesis of the plant hormones indole-3-acetic acid (IAA) and isopentenyl adenosine. Mutations which resulted in the loss of IAA production were due to the loss of the entire plasmid containing the iaa operon or to an 18-kb deletion of the iaa region. Additional mutants tested were deficient in their ability to produce isopentenyl adenosine as a result of the loss of the ptz-bearing plasmid. In all cases, strains which had lost the ability to produce IAA exhibited enhanced motility of up to 2.5 times that of the wild type (IAA⁺) in medium containing 0.01% yeast extract. No differences in motility were observed on medium containing lower concentrations of yeast extract. The presence or absence of the cytokinin plasmid and the presence or absence of inorganic nitrogen in the medium had no effect on the relative mobility of the strains.     

Fatty Acid Composition of Pseudomonas syringae pv. savastanoi

Over 85% of total cellular fatty acids of 30 strains of P. syringae pv. savastanoi, grown for one day at 28 °C on King's medium B (KB) agar, were 12:0 (5.0%), 16:0 (27.5%), 16:1 (36.7%) and 18:1 (16.8%). Three hydroxy-substituted fatty acids comprised 7.2% of the total and 22 other minor components, each occurring at concentrations of less than 1%, comprised an additional 4%. Three percent were unidentified components. Cells grown for 3 and 6 days on KB agar contained lower concentrations of the unsaturated 16:1 (30.4 and 21.1%, respectively), and higher concentrations of branched-chain and cyclopropane fatty acids than one-day old cells. No consistent differences in fatty acid composition could be detected between virulent and avirulent strains, nor between pv. savastanoi and other pathovars of P. syringae. However, when cells were grown on a chemically-defined medium for 6 days, concentrations of 16:0 and a tentatively-identified 17-carbon hydroxy fatty acid were higher, and those of 12:0 and 16:1 were lower in strains from Fraxinus than from Olea. P. fluorescens (7 strains) and P. viridiflava (6 strains) could be differentiated from each other but not from P. syringae.     

Distribution of Pseudomonas syringae pathovars into twenty-three O serogroups.

Serological reactions of Pseudomonas syringae and Pseudomonas viridiflava were studied by Ouchterlony double diffusion. A total of 55 polyclonal antisera, containing anti-lipopolysaccharide (anti-LPS) precipitating antibodies, were cross-tested against antigenic suspensions of 51 strains. Twenty-three O serogroups were defined, primarily on the reaction of the type strains. Two families of O serogroups showed antigenic crossreactivities (PHA, MOP1, MOP2, MOP3, HEL1, HEL2, and SYR1; PERSAVTOM1, PERSAVTOM2, DEL, POR, and SYR2). Ten O serogroups showed a clearcut specificity: APTPIS, TAB, VIR1, VIR2, VIR3, SYR3, SYR4, SYR5, HUS, and LAC. The last serogroup (RIB) contained strains with rough colony morphology and side chain-deficient LPSs, as evidenced by sodium dodecyl sulfate-polycrylamide gel electrophoresis. The LPS basis of the O serogroups was demonstrated by immunoblotting. Serological reference strains were designated for all of the O serogroups and correspondence was established between the O serogroups studied and seven previous serogroups (L. T. Pastushenko and I.D. Simonovich, Mikrobiol, Zh. 41:222-229 and 330-339, 1979). A total of 355 strains of P. syringae (sensu lato) belonging to 15 pathovars, not including pathovar syringae, were typed into the 23 described O serogroups. O serogroups were assigned after double-diffusion reactions, with each strain compared with serological references. The utility of O serogrouping to study P. syringae pathovar structure and diversity is discussed.     

Pathogenicity of Pseudomonas syringae subsp. savastanoi Mutants Defective in Phytohormone Production

The present study compares the pathogenicity on olive and oleander plants of three wild-type strains of Pseudomonas syringae subsp. savastanoi (ITM317 and PBa230 from olive and ITM519 from oleander) and three phytohormone-deficient mutants of ITM519: ITM519-41 (Iaa⁺/cytokinins-), ITM519-7 (Iaa⁻/cytokinins⁺), ITM519-6 (Iaa⁻/cytokinins⁻), Mutants not producing IAA (ITM519-7and ITM519-6) only induced necrosis of the inoculated tissues (ITM519-,6) or swellings on the stems attributed to cytokinin production accompanied by necrosis (ITM519-7). By contrast, the Iaa⁺/cytokinins⁻ mutant (ITM519-41) induced attenuated symptoms on stems and knots similar to those obtained with the parent strain on oleander leaves. Olive strains induced necrosis of oleander leaves and were virulent and avirulent, respectively, on olive and oleander stems.
Strain ITM519 and its three mutants were all able to multiply in oleander leaves at similar rates, reaching the same final populations. By contrast, the two olive strains multiply poorly, reaching populations c. 10²-fold lower.
These results confirm that expression of IAA genes alone is sufficient to initiate the development of knots on oleander while cytokinins are necessary for the full expression of the disease symptoms (determining knot size). The findings also indicate that the plant tissues (stems and leaves) react differently to the various strains of the bacterium and, furthermore, suggest that, besides phytohormones, other pathogenetic factors could be involved in this host-pathogen interaction. The necrotic reaction of oleander leaves heavily inoculated with olive strains was interpreted as a possible form of hypersensitivity reaction.     

Genetic diversity and relationships of two pathovars of Pseudomonas syringae

To determine genetic relationships within and between two pathovars of Pseudomonas syringae, strains typical of P. syringae pv. tomato (P. s. tomato) and selected strains of P. syringae pv. syringae (P. s. syringae) were characterized by three methods. DNA-DNA hybridization experiments showed that strains of P. s. tomato and P. s. syringae were, respectively, 86-100% and 37-47% homologous to DNA from a P. s. tomato reference strain when tested under stringent conditions. An analysis of electrophoretic variation in enzymes encoded by 26 loci placed 17 P. s. tomato strains studied in a group of four electrophoretic types, and these strains had a mean genetic diversity per locus of 0.076. Six P. s. syringae strains formed a second group of six electrophoretic types, which had a higher mean genetic diversity per locus of 0.479. The mean genetic distance separating P. s. tomato from P. s. syringae (D = 0.94) was unexpectedly large for strains of a single species. An analysis of restriction fragment length polymorphisms (RFLPs) with three cloned hybridization probes demonstrated that each of the P. s. tomato and P. s. syringae strains was unique. A method was developed to quantify the RFLP difference between pairs of strains, and cluster analysis revealed relationships among P. s. tomato, but not among P. s. syringae, that were similar to those based on enzyme polymorphisms. Implications of these findings for bacterial systematics and epidemiology are discussed.     

A cytokinin from the culture filtrate of Pseudomonas syringae pv. savastanoi

The structure of a new cytokinin, isolated from the culture filtrate of Pseudomonas syringae pv. savastanoi, is assigned on the basis of spectroscopic data including its tetracetyl derivative and comparison with related adenine derivatives. It was identified as 6-(4-hydroxy-1,3-dimethylbut-trans-2-enylamino-9-β-D-ribofuranosyl)purine.     

High-resolution melting analysis as a powerful tool to discriminate and genotype Pseudomonas savastanoi pathovars and strains

Pseudomonas savastanoi is a serious pathogen of Olive, Oleander, Ash, and several other Oleaceae. Its epiphytic or endophytic presence in asymptomatic plants is crucial for the spread of Olive and Oleander knot disease, as already ascertained for P. savastanoi pv. savastanoi (Psv) on Olive and for pv. nerii (Psn) on Oleander, while no information is available for pv. fraxini (Psf) on Ash. Nothing is known yet about the distribution on the different host plants and the real host range of these pathovars in nature, although cross-infections were observed following artificial inoculations. A multiplex Real-Time PCR assay was recently developed to simultaneously and quantitatively discriminate in vitro and in planta these P. savastanoi pathovars, for routine culture confirmation and for epidemiological and diagnostical studies. Here an innovative High-Resolution Melting Analysis (HRMA)-based assay was set up to unequivocally discriminate Psv, Psn and Psf, according to several single nucleotide polymorphisms found in their Type Three Secretion System clusters. The genetic distances among 56 P. savastanoi strains belonging to these pathovars were also evaluated, confirming and refining data previously obtained by fAFLP. To our knowledge, this is the first time that HRMA is applied to a bacterial plant pathogen, and one of the few multiplex HRMA-based assays developed so far. This protocol provides a rapid, sensitive, specific tool to differentiate and detect Psv, Psn and Psf strains, also in vivo and against other related bacteria, with lower costs than conventional multiplex Real-Time PCR. Its application is particularly suitable for sanitary certification programs for P. savastanoi, aimed at avoiding the spreading of this phytopathogen through asymptomatic plants.     

Comparison of strains of Pseudomonas syringae pv. savastanoi from olive leaves and knots

A collection of strains of Pseudomonas syringae pv. savastanoi was subjected to numeric phenetic analysis of 60 characters using unweighted average linkage on the simple matching coefficient. Most strains recovered by washing random leaves in April and October shared lower similarity values between themselves than with the majority of those isolated from 6-month-old knots in October and April, respectively.     

The molecular basis of host specialization in bean pathovars of Pseudomonas syringae

Biotrophic phytopathogens are typically limited to their adapted host range. In recent decades, investigations have teased apart the general molecular basis of intraspecific variation for innate immunity of plants, typically involving receptor proteins that enable perception of pathogen-associated molecular patterns or avirulence elicitors from the pathogen as triggers for defense induction. However, general consensus concerning evolutionary and molecular factors that alter host range across closely related phytopathogen isolates has been more elusive. Here, through genome comparisons and genetic manipulations, we investigate the underlying mechanisms that structure host range across closely related strains of Pseudomonas syringae isolated from different legume hosts. Although type III secretion-independent virulence factors are conserved across these three strains, we find that the presence of two genes encoding type III effectors (hopC1 and hopM1) and the absence of another (avrB2) potentially contribute to host range differences between pathovars glycinea and phaseolicola. These findings reinforce the idea that a complex genetic basis underlies host range evolution in plant pathogens. This complexity is present even in host-microbe interactions featuring relatively little divergence among both hosts and their adapted pathogens.     

The Grouping of Strains of Pseudomonas syringae subsp. savastanoi by DNA Restriction Fingerprinting

A selected group of strains of Pseudomonas syringae subsp. savastanoi from olive, oleander and ash were compared with pathogenicity tests and with DNA restriction fingerprinting using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. The strains from each host were distinguishable by their pathogenicity to the same host and to the other two plant species. A division into the same groups was obtained with unweighted pair-group method with averages (UPGMA) clustering of the data from genomic fingerprinting, even though high overall similarity between the strains also indicated that they formed a single, well characterized taxon. It seems clear that the subspecies savastanoi of P. syringae comprises at least 3 groups of strains that differ in their precise host range, in the nature of the symptoms induced on the individual hosts, and in their genomic profile.     

Evaluation of determinative tests for pathovars of Pseudomonas syringae van Hall 1902

The utility of 36 presumptive determinative tests for 32 pathovars of Pseudomonas syringae was investigated. A total of 395 strains was examined. Most strains of 12 of these pathovars ( Ps. syringae pv. cannabina, Ps. syr. delphinii, Ps. syr. glycinea, Ps. syr. helianthi, Ps. syr. lachrymans, Ps. syr. mori, Ps. syr. morsprunorum, Ps. syr. phaseolicola, Ps. syr. 'porri', Ps. syr. papulans, Ps. syr. savastanoi and Ps. syr. tabaci ) formed clusters when test data were compared by centroid analysis. Pseudomonas syr. syringae, Ps. syr. aptata, Ps. syr. atrofaciens, Ps. syr. dysoxyli and Ps. syr. japonica formed a single cluster, indicating their possible synonymy. Strains of Ps. syr. antirrhini and Ps. syr. tomato were indistinguishable, as were those of Ps. syr. garcae and Ps. syr. oryzae. Strains of Ps. syr. berberidis, Ps. syr. coronafaciens, Ps. syr. eriobotryae, Ps. syr. maculicola, Ps. syr. passiflorae, Ps. syr. pisi and Ps. syr. striafaciens and Ps. syr. tagetis did not form distinguishable clusters.
The tests which reliably differentiated pathovars are recorded in a determinative scheme.     

Serological and molecular size characterization of flagellins of Pseudomonas syringae pathovars and related bacteria

Flagella from a total of 118 strains representing mostly pathovars of the phytopathogenic group Pseudomonas syringae, but also P. chlororaphis, P. cichorii, P. corrugata, P. fluorescens, P. fuscovaginae, P. stutzeri, P. viridiflava, as well as related phytopathogenic genera (Burkholderia cepacia and Ralstonia solanacearum) were characterized by immuno-fluorescent staining, SDS-PAGE, and immunoblotting. Eighty-six strains of the P. syringae group pathovars, P. cichorii and P. viridiflava were shown to possess flagella of serotypes H1 or H2, composed of a unique flagellin, whose molecular size varied between 31 and 31.5 kDa. Similarities between the P. syringae flagellin and a 31 kDa surface protein involved in pathogenicity are pointed out. The distribution of H1 and H2 antigens in the nine recently described genomospecies of P. syringae-P. viridiflava group suggested that flagellin would represent a phylogenetic marker within the arginin-dihydrolase-negative fluorescent pseudomonads. The characterization of flagellin was proposed as an identification tool at a level situated between genus and species.     

Pseudomycins, a family of novel peptides from Pseudomonas syringae possessing broad-spectrum antifungal activity.

A family of peptide antimycotics, termed pseudomycins, has been isolated from liquid cultures of Pseudomonas syringae, a plant-associated bacterium. These compounds were purified using Amberlite XAD-2 and reverse-phase liquid chromatography. Pseudomycin A, the predominant peptide in a family of four, showed selective phytotoxicity, and had impressive activity against the human pathogen Candida albicans. Amino acid, mass spectroscopic, and comparative electrophoretic and chromatographic analyses revealed that the pseudomycins are different from previously described antimycotics from P. syringae, including syringomycin, syringotoxin and syringostatins. Pseudomycins A-C contain hydroxyaspartic acid, aspartic acid, serine, arginine, lysine and diaminobutyric acid. The molecular masses of pseudomycins A-C, as determined by plasma desorption mass spectrometry, are 1224, 1208 and 1252 Da, respectively. Pseudomycin D, on the other hand, has a molecular mass of 2401 Da and is more complex than pseudomycins A-C.     

Isolation and Characterization of Pseudomonas syringae subsp. savastanoi Mutants Defective in Hypersensitive Response Elicitation and Pathogenicity

Olive strain ITM317 of Pseudomonas syringae subsp. savastanoi , the causal agent of 'Olive and Oleander knot disease' was mutagenized by random transposon (Tn5) insertion. Of the 1 400 transconjugants, four were altered in their ability to induce a hypersensitive reaction (HR) on tobacco; Southern blot analysis showed that a single copy of the Tn5 element was present in their chromosomes. In particular, mutants ITM317–69, ITM317–1010 and ITM317–1194 did not elicit HR whereas mutant ITM317–916 induced a variable response. When assayed for pathogenicity on olive, mutants ITM317–916 and ITM317–1010 induced knots comparable both in size and morphology to those caused by the parental strain. Prototrophic mutant ITM317–1194, still able to produce indole-3-acetic acid and cytokinins, did not cause any knot formation on olive; furthermore, it was unable to multiply in host tissue. Auxotrophic mutant ITM317–69 caused the formation of smaller-sized knots and its prototrophic revertant fully regained the parental phenotypes, suggesting that a single Tn5 insertion had a pleiotropic effect on the mutated phenotypes. Tn5-containing Eco RI fragments from mutants ITM317–69, ITM317–916, ITM317–1010 and ITM317–1194 were cloned into the plasmid vector pBR322. Hybridization of these clones with the hrp gene cluster of P. s. pv. syringae strain 61 was not detected. These results suggest that genes different from those of the above gene cluster might be involved in the interaction of P. s. subsp. savastanoi with olive and with the non-host plant tobacco.