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.     

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.     

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.     

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.     

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 MgCl₂ 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°C, which is 5°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 × 10⁶ 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.     

Evidence for a correlation between auxin production and host plant species among strains of Pseudomonas syringae subsp. savastanoi.

Auxin production by 131 strains of Pseudomonas syringae subsp. savastanoi was investigated with the aim of looking for correlations among this characteristic and the origin of the strains, the types of symptoms, and the host plant. Most of the P.syringae subsp. savastanoi strains, except those isolated from ash, produced auxin and harbored iaa genes. Among ash strains, which were pathogenic only on ash, only 2 out of 33 were found to produce auxin and to harbor iaa genes.     

Evidence for a correlation between auxin production and host plant species among strains of Pseudomonas syringae subsp. savastanoi.

Auxin production by 131 strains of Pseudomonas syringae subsp. savastanoi was investigated with the aim of looking for correlations among this characteristic and the origin of the strains, the types of symptoms, and the host plant. Most of the P.syringae subsp. savastanoi strains, except those isolated from ash, produced auxin and harbored iaa genes. Among ash strains, which were pathogenic only on ash, only 2 out of 33 were found to produce auxin and to harbor iaa genes.     

Role of indoleacetic acid-lysine synthetase in regulation of indoleacetic acid pool size and virulence of Pseudomonas syringae subsp. savastanoi.

The phytopathogen Pseudomonas syringae subsp. savastanoi incites the production of galls on olive and oleander plants. Gall formation is dependent upon the bacterial synthesis of the phytohormone indole-3-acetic acid (IAA). Strains isolated from oleander galls are capable of further metabolizing IAA to an amino acid conjugate, 3-indoleacetyl-epsilon-L-lysine (IAA-lysine); bacterial olive gall isolates lack this activity. In this study, the cloned gene for IAA-lysine synthetase (iaaL+) was introduced into strains isolated from olive and oleander galls to determine its effect on the regulation of IAA pool size and virulence. IAA-lysine was synthesized by isolates from olive galls when iaaL+ was introduced by conjugation, but the amount of IAA which accumulated in culture by the transconjugant was reduced by one-third. When the iaaL+ locus of an oleander gall isolate was inactivated by Tn5 mutagenesis, the resulting mutant did not convert IAA to IAA-lysine; however, it accumulated fivefold more IAA in culture than the wild type did. When inoculated into oleander plants, the iaaL mutant did not cause typical gall symptoms, nor did it replicate within host tissue similarly to the wild type.     

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.     

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.     

Global genomic analysis of Pseudomonas savastanoi pv. savastanoi plasmids

Pseudomonas savastanoi pv. savastanoi strains harbor native plasmids belonging to the pPT23A plasmid family (PFPs) which are detected in all pathovars of the related species Pseudomonas syringae examined and contribute to the ecological and pathogenic fitness of their host. However, there is a general lack of information about the gene content of P. savastanoi pv. savastanoi plasmids and their role in the interaction of this pathogen with olive plants. We designed a DNA macroarray containing 135 plasmid-borne P. syringae genes to conduct a global genetic analysis of 32 plasmids obtained from 10 P. savastanoi pv. savastanoi strains. Hybridization results revealed that the number of PFPs per strain varied from one to four. Additionally, most strains contained at least one plasmid (designated non-PFP) that did not hybridize to the repA gene of pPT23A. Only three PFPs contained genes involved in the biosynthesis of the virulence factor indole-3-acetic acid (iaaM, iaaH, and iaaL). In contrast, ptz, a gene involved in the biosynthesis of cytokinins, was found in five PFPs and one non-PFP. Genes encoding a type IV secretion system (T4SS), type IVA, were found in both PFPs and non-PFPs; however, type IVB genes were found only on PFPs. Nine plasmids encoded both T4SSs, whereas seven other plasmids carried none of these genes. Most PFPs and non-PFPs hybridized to at least one putative type III secretion system effector gene and to a variety of additional genes encoding known P. syringae virulence factors and one or more insertion sequence transposase genes. These results indicate that non-PFPs may contribute to the virulence and fitness of the P. savastanoi pv. savastanoi host. The overall gene content of P. savastanoi pv. savastanoi plasmids, with their repeated information, mosaic arrangement, and insertion sequences, suggests a possible role in adaptation to a changing environment.     

Virulence of the phytopathogen Pseudomonas syringae pv. maculicola is rpoN dependent

We cloned the rpoN (ntrA and glnF) gene encoding sigma(54) from the phytopathogen Pseudomonas syringae pv. maculicola strain ES4326. The P. syringae ES4326 rpoN gene complemented Pseudomonas aeruginosa, Escherichia coli, and Klebsiella aerogenes rpoN mutants for a variety of rpoN mutant phenotypes, including the inability to utilize nitrate as sole nitrogen source. DNA sequence analysis of the P. syringae ES4326 rpoN gene revealed that the deduced amino acid sequence was most similar (86% identity; 95% similarity) to the sigma(54) protein encoded by the Pseudomonas putida rpoN gene. A marker exchange protocol was used to construct an ES4326 rpoN insertional mutation, rpoN::Km(r). In contrast to wild-type ES4326, ES4326 rpoN::Km(r) was nonmotile and could not utilize nitrate, urea, C(4)-dicarboxylic acids, several amino acids, or concentrations of ammonia below 2 mM as nitrogen sources. rpoN was essential for production of the phytotoxin coronatine and for expression of the structural genes encoding coronamic acid. In addition, ES4326 rpoN::Km(r) did not multiply or elicit disease symptoms when infiltrated into Arabidopsis thaliana leaves, did not elicit the accumulation of several Arabidopsis defense-related mRNAs, and did not elicit a hypersensitive response (HR) when infiltrated into tobacco (Nicotiana tabacum) leaves. Furthermore, whereas P. syringae ES4326 carrying the avirulence gene avrRpt2 elicited an HR when infiltrated into Arabidopsis ecotype Columbia leaves, ES4326 rpoN::Km(r) carrying avrRpt2 elicited no response. Constitutive expression of ES4326 hrpL in ES4326 rpoN::Km(r) partially restored defense-related mRNA accumulation, showing a direct role for the hrp cluster in host defense gene induction in a compatible host-pathogen interaction. However, constitutive expression of hrpL in ES4326 rpoN::Km(r) did not restore coronatine production, showing that coronatine biosynthesis requires factors other than hrpL.     

Inhibition by Agrobacterium tumefaciens and Pseudomonas savastanoi of development of the hypersensitive response elicited by Pseudomonas syringae pv. phaseolicola.

Injection into tobacco leaves of biotype 1 Agrobacterium tumefaciens or of Pseudomonas savastanoi inhibited the development of a visible hypersensitive response to the subsequent injection at the same site of Pseudomonas syringae pv. phaseolicola. This interference with the hypersensitive response was not seen with injection of bacterial growth medium or Escherichia coli cells. Live A. tumefaciens cells were required for the inhibitory effect. Various mutants and strains of A. tumefaciens were examined to determine the genes involved. Known chromosomal mutations generally had no effect on the ability of A. tumefaciens to inhibit the hypersensitive response, except for chvB mutants which showed a reduced (but still significant) inhibition of the hypersensitive response. Ti plasmid genes appeared to be required for the inhibition of the hypersensitive response. The bacteria did not need to be virulent in order to inhibit the hypersensitive response. Deletion of the vir region from pTi had no effect on the inhibition. However, the T region of the Ti plasmid was required for inhibition. Studies of transposon mutants suggested that the tms but not tmr or ocs genes were required. These genes were not acting after transfer to plant cells since they were effective in strains lacking vir genes and thus unable to transfer DNA to plant cells. The results suggest that the expression of the tms genes in the bacteria may inhibit the development of the hypersensitive response by the plant. An examination of the genes required in P. savastanoi for the inhibition of the hypersensitive response suggested that bacterial production of auxin was also required for the inhibition of the hypersensitive response by these bacteria.