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Background: Pseudomonas syringae is a y-proteobacterium causing economically relevant diseases in practically all cultivated plants. Most isolates of this pathogen contain native plasmids collectively carrying many pathogenicity and virulence genes. However, P. syringae is generally an opportunistic pathogen primarily inhabiting environmental reservoirs, which could exert a low selective pressure for virulence plasmids. Additionally, these plasmids usually contain a large proportion of repeated sequences, which could compromise plasmid integrity. Therefore, the identification of plasmid stability determinants and mechanisms to preserve virulence genes is essential to understand the evolution of this pathogen and its adaptability to agroecosystems. Results: The three virulence plasmids of P. syringae pv. savastanoi NCPPB 3335 contain from one to seven functional stability determinants, including three highly active toxin-antitoxin systems (TA) in both pPsv48A and pPsv48C. The TA systems reduced loss frequency of pPsv48A by two orders of magnitude, whereas one of the two replicons of pPsv48C likely confers stable inheritance by itself. Notably, inactivation of the TA systems from pPsv48C exposed the plasmid to high-frequency deletions promoted by mobile genetic elements. Thus, recombination between two copies of MITEPsy2 caused the deletion of an 8.3 kb fragment, with a frequency of 3.8 ± 0.3 x 10-3. Likewise, one-ended transposition of IS801 generated plasmids containing deletions of variable size, with a frequency of 5.5 ± 2.1 x 1 0- 4, of which 80% had lost virulence gene idi. These deletion derivatives were stably maintained in the population by replication mediated by repJ, which is adjacent to IS801. IS801 also promoted deletions in plasmid pPsv48A, either by recombination or one-ended transposition. In all cases, functional TA systems contributed significantly to reduce the occurrence of plasmid deletions in vivo. Conclusions: Virulence plasmids from P. syringae harbour a diverse array of stability determinants with a variable contribution to plasmid persistence. Importantly, we showed that multiple plasmid-borne TA systems have a prominent role in preserving plasmid integrity and ensuring the maintenance of virulence genes in free-living conditions. This strategy is likely widespread amongst native plasmids of P. syringae and other bacteria. This work was funded by the Spanish Plan Nacional I + D + I grants AGL2014–53242-C2–1-R, AGL2014–53242-C2–2-R, AGL2017-82492-C2-1- R, and AGL2017-82492-C2-2-R from the Ministerio de Economía y Competitividad (MINECO), co-financed by the Fondo Europeo de Desarrollo Regional (FEDER).

BackgroundVirulence plasmids are critically exposed to genetic decay and loss, particularly inPseudomonas syringaestrains because of their high content of mobile genetic elements and their exploitation of environmental niches outside of the plant host. The demonstrated high plasticity and adaptability of P. syringae plasmids, involving the acquisition and loss of large DNA regions, contrasts with their usual high stability and the maintenance of key virulence genes in free living conditions. The identification of plasmid stability determinants and mechanisms will help to understand their evolution and adaptability to agroecosystems as well as to develop more efficient control measures.ResultsWe show that the three virulence plasmids ofP. syringaepv. savastanoi NCPPB 3335 contain diverse functional stability determinants, including three toxin-antitoxin systems (TA) in both pPsv48A and pPsv48C, whereas one of the two replicons of pPsv48C can confer stable inheritance by itself. Loss of pPsv48A increased by two orders of magnitude upon functional inactivation of its TA systems. However, inactivation of the TA systems from pPsv48C did not result in its curing but led to the recovery of diverse deletion derivatives. One type consisted in the deletion of an 8.3 kb fragment, with a frequency of 3.8 ± 0.3 × 10−3, by recombination between two copies of MITEPsy2. Likewise, IS801promoted the occurrence of deletions of variable size by one-ended transposition with a frequency of 5.5 ± 2.1 × 10−4, 80 % of which resulted in the loss of virulence geneidi. These deletion derivatives were stably maintained in the population by replication mediated byrepJ, which is adjacent to IS801. IS801also promoted deletions in plasmid pPsv48A, either by recombination or one-ended transposition. In all cases, functional TA systems contributed significantly to reduce the occurrence of plasmid deletionsin vivo.ConclusionsVirulence plasmids fromP. syringaeharbour a diverse array of stability determinants with a variable contribution to plasmid persistence. Additionally, multiple TA systems favour the long-term survival and integrity of virulence plasmids, as well as the maintenance of pathogenicity genes in free-living conditions. This strategy is likely widespread amongst native plasmids ofP. syringaeand other bacteria.

We have evaluated the interaction that bacterial genotypes and plant hosts have with the loss of pathogenicity in tumors, using seven Agrobacterium tumefaciens strains inoculated on 12 herbaceous and woody hosts. We performed a screening of the agrobacteria present inside the tumors, looking for nonpathogenic strains, and found a high variability of those strains in this niche. To verify the origin of the putative nonpathogenic mutant bacteria, we applied an efficient, reproducible, and specific randomly amplified polymorphic DNA analysis method. In contrast with previous studies, we recovered a very small percentage (0.01%) of nonpathogenic strains that can be considered true mutants. Of 5,419 agrobacterial isolates examined, 662 were nonpathogenic in tomato, although only 7 (from pepper and tomato tumors induced by two A. tumefaciens strains) could be considered to derive from the inoculated strain. Six mutants were affected in the transferred DNA (T-DNA) region; one of them contained IS426 inserted into the iaaM gene, whereas the whole T-DNA region was apparently deleted in three other mutants, and the virulence of the remaining two mutants was fully restored with the T-DNA genes as well. The plasmid profile was altered in six of the mutants, with changes in the size of the Ti plasmid or other plasmids and/or the acquisition of new plasmids. Our results also suggest that the frequent occurrence of nonpathogenic clones in the tumors is probably due to the preferential growth of nonpathogenic agrobacteria, of either endophytic or environmental origin, but different from the bacterial strain inducing the tumor. This research was supported partially by project INIA (grant SC93- 117) of the Spanish Ministry of Agriculture and by COST Action 873 (European Union). Pablo Llop has a contract from the Ministry of Education and INIA, with funding from the European Union (Social European Funding).

Erwinia amylovora, the causal agent of fire blight, is genetically very homogeneous, and current methodologies provide insufficient or contradictory information about the probable dispersal routes of the pathogen. With the final aim to obtain specific and reliable molecular markers for different lineages of the pathogen, we studied the molecular basis of rep-polymerase chain reaction (PCR) polymorphism using seven different arbitrary primers to fingerprint 93 E. amylovora strains from different countries, including Spain. Polymorphism was very low, and was displayed by only 11 E. amylovora strains, which produced 22 polymorphic bands. Five of 11 polymorphic bands cloned contained DNA that was present in more than 85% of the strains, whereas six bands were due to DNA present exclusively in the strains producing the rep-PCR polymorphism. Also, five of the polymorphic bands were due to the possession of either the ubiquitous plasmid pEA29, of plasmid pEU30, which was exclusively found in strains from North America, or of a 35-kb cryptic plasmid, present only in 28 strains from Northern Spain. We designed primer pairs from several cloned polymorphic bands that allowed the specific identification of the strains producing the polymorphism. Our results indicate that rep-PCR is not adequate for constructing genealogies of E. amylovora, although the strategy illustrated here, as well as the designed primers, can be used effectively in epidemiological studies with this pathogen.

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.

The presence of genetic determinants homologous to rulAB genes for ultraviolet (UV) radiation resistance was determined in a collection of Pseudomonas syringae pv. syringae strains isolated from mango. The potential role of these plasmids in UV tolerance and ecological fitness in the mango phyllosphere was also evaluated. Nearly all of the 62-kb plasmids present in the P. syringae pv. syringae strains hybridized with a rulAB probe, but these 62-kb plasmids showed differences in restriction patterns. In vitro assays of tolerance to UV radiation of P. syringae pv. syringae strains showed a higher survival of the strains harboring the 62-kb plasmids compared to strains lacking plasmids when exposed to UVC or UVA+B fractions. Similar results were observed when transconjugants harboring the 62-kb plasmid were tested. Survival assays were carried out under field conditions, and a higher survival of P. syringae pv. syringae strains harboring 62-kb plasmids under direct solar radiation on the adaxial surface of leaves was also observed. When the assays were carried out in shady areas or on the abaxial surface of leaves, survival time was comparable for all the assayed strains, whether or not they contained a 62-kb plasmid hybridizing to rulAB. Our results indicate that P. syringae pv. syringae strains harboring 62-kb plasmids show an increase in ecological fitness when colonizing the mango phyllosphere.

A molecular typing system for Agrobacterium strains based on the polymerase chain reaction–random amplified polymorphic DNA (PCR–RAPD) procedure was developed. It employs one to four different 10-mer primers and the results are highly reproducible. The band patterns obtained with the four primers for each of the 39 Agrobacterium strains analysed were different enough to differentiate the strains from each other. Strains with similar chromosomal background but different plasmid content, e.g. strains C58 and A281, gave the same band pattern with all the primers. Ten host plants were inoculated with eight Agrobacterium strains and the isolates obtained from the resulting tumours were analysed using the RAPD system developed here. The procedure allowed rapid identification of isolates recovered from tumours by comparison of their band patterns with band patterns of strains used as inoculum. The procedure also discriminated the various strains analysed. Purified bacterial cell suspensions, used for RAPD analyses, produced the same results as purified DNA, and greatly simplified the procedure. This system can be applied for rapid screening of Agrobacterium-like colonies isolated from plant tumours for epidemiological and genetic diversity studies.

The largest plasmid (pPT23A, 100 kb) from Pseudomonas syringae pv.tomato PT23, carries the gene cluster for the synthesis of the phytotoxin coronatine and most of its DNA sequences are repeated in a co-residing plasmid, pPT23B (83 kb). Some DNA fragments from pPT23A are also repeated elsewhere in the genome. To investigate the role in virulence of these plasmids, avoiding the effects of gene redundancy, we constructed strain UPN2, which was derived by curing the four native plasmids from PT23. The necrotic lesions induced in tomato by UPN2 were four-fold smaller than those produced by PT23. This phenotype was only partially restored by pPT23A, and the production of lesions of wild type size was dependent on the concomitant presence in UPN2 of pPT23A and pPT23B. Mutants defective in the production of coronatine, but which still produced coronafacic acid, induced lesions of an intermediate size between PT23 and UPN2. The virulence determinants present on pPT23B could be modifying the amount, structure or uptake rate by the plant of the coronafacic acid molecules produced by genes present on pPT23A. Therefore, the production of necrotic lesions on tomato plants by strain PT23 is due substantially to the action of compounds whose determinants are located on plasmids pPT23A and pPT23B, including, but not limited to coronatine. The involvement of the coronafacic acid gene cluster in virulence in the absence of the cluster for the synthesis of coronamic acid, may help explain its maintenance in the bacterial population before being incorporated into the coronatine cluster. Likewise, the physical separation of the virulence determinants in the two independent replicons pPT23A and pPT23B, may have driven the evolution of strain PT23 so that it maintains two potentially incompatible plasmids.

It was previously shown that most Pseudomonas syringae strains contain one or more plasmids with cross-hybridizing replication regions and other areas of homology, and these plasmids were designated the pPT23A-like family. The majority of these plasmids encode genes conferring epiphytic fitness or resistance to antibacterial compounds and those investigated in this study are essential for pathogenicity or increased virulence. The phylogeny of 14 pPT23A-like plasmids from five P. syringae pathovars was studied by comparing a fragment of the sequence of their repA genes (encoding a replicase essential for replication). In the phylogenetic tree obtained, four groups (≤88·8% identity between their members) could be identified. The first group contained the plasmids from three P. syringae pv. tomato strains, a P. syringae pv. apii strain and five out of the seven P. syringae pv. syringae strains, with identity ranging between 88·8 and 100%. The clustering of the pv. syringae strains did not reflect host specialization or previously reported phylogenetic relationships. The second group contained the plasmids from two strains of pv. glycinea and pv. tomato (95·5% identity), and it also included the previously sequenced replicon of a pathogenicity plasmid from P. syringae pv. phaseolicola. The plasmids from the remaining two pv. syringae strains were distantly related to the other plasmid sequences. Hybridization experiments using different genes or transposable elements previously described as plasmid-borne in P. syringae, showed that the gene content of highly related plasmids could be dissimilar, suggesting the occurrence of major plasmid reorganizations. Additionally, the phylogeny of the different native plasmids did not always correlate with the phylogeny of their harbouring strains, as determined by the analysis of extragenic repetitive consensus (ERIC) and arbitrarily primed PCR (AP-PCR) products. Collectively, these results suggest that pPT23A-like plasmids were, in most cases, acquired early during evolution.

The effect of the plasmid-encoded rulAB (resistance to ultraviolet radiation) determinant on responses of Pseudomonas syringae to ultraviolet-B (UV-B) radiation and the distribution of rulAB among pathovars of P. syringae were determined. The cloned rulAB determinant and the native rulAB+ plasmid pPSR1 both conferred approximately a 10-fold increase in survival on P. syringae pv. syringae FF5 following increasing doses of UV-B radiation. rulAB+ P. syringae strains also maintained significantly larger epiphytic populations on leaf surfaces irradiated with UV-B. rulAB-insertional mutants, constructed in two native rulAB+ strains, were from 10- to 100-fold more sensitive to UV-B radiation. The UV tolerance phenotype and the rulAB genes were widely distributed among P. syringae pathovars isolated from varied plant hosts throughout the world and within a broad range of genotypic backgrounds of P. syringae pv. syringae. With one exception, the rulAB determinant was harboured on pPT23A-like plasmids; these replicons are indigenous residents of the species P. syringae and also tend to encode determinants of importance in host-pathogen interactions.

Strain PT23 of Pseudomonas syringae pv. tomato contains four native plasmids, designated A, B, C, and D. By DNA hybridization of genomic and plasmid DNA digests from the wild type and a plasmid-cured strain, we determined that c. 61 kb (c. 74%) of pPT23B is repeated in pPT23 A and only c. 17 kb (c. 21%) is in single copy in strain PT23. pPT23B also contains DNA repeated in the chromosome that occurs in three DNA fragments of 0.6, 4.6, and 9.6 kb that might be transposable elements. Additionally, the 9.6 kb fragment also shares sequences with the three other plasmids of strain PT23. By DNA hybridization with the origin of replication from a native plasmid of P. syringae pv. syringae and in vivo replication tests, we identified the origins of replication of plasmids A, B, and D and showed that they cross-hybridize. The putative par region from pPT23 A has also been identified and is not conserved in the other three native plasmids from strain PT23. By using the defined minimal origin of replication from pPT23 A as a probe, we showed that it is highly conserved in 14 strains belonging to nine different pathovars of P. syringae and that as many as five different native plasmids with closely related origins of replication coexist in the same cell. The duplication and reorganization of plasmids might therefore occur at high frequency and could be responsible for the existence of large numbers of native plasmids in P. syringae strains.

Avirulence gene D (avrD) in strain PT23 of Pseudomonas syringae pv. tomato (Pst) specifies the production of syringolides, which are elicitors of plant defense reactions. An 83-kb indigenous plasmid (pPT23B) that carries avrD has been mapped and characterized and a putative par region was identified. pPT23B contains a large amount of DNA that is repeated in other native plasmids in PT23. A putative mobile insertion element that occurs on plasmid pPT23A as well as on the chromosome was also identified in strain PT23. New broad-host-range expression vectors that functioned in Pst were constructed for overexpression of the cloned avrD gene and high-level production of the syringolides. Introduction of an avrD overexpression plasmid into PT23 or plasmid-cured strains led to identical syringolide peaks on HPLC with no new peaks observed. These results suggested that neither pPT23B nor other indigenous plasmids in Pst carry additional genes required for syringolide production or metabolism. Pst strains lacking pPT23B were not impaired in virulence on tomato plants.

UPNa. Departamento de Producción Agraria. Laboratorio de Patología Vegetal Mobile genetic elements are widespread in Pseudomonas syringae, and often associate with virulence genes. Genome reannotation of the model bean pathogen P. syringae pv. phaseolicola 1448A identified seventeen types of insertion sequences and two miniature inverted-repeat transposable elements (MITEs) with a biased distribution, representing 2.8% of the chromosome, 25.8% of the 132-kb virulence plasmid and 2.7% of the 52-kb plasmid. Employing an entrapment vector containing sacB, we estimated that transposition frequency oscillated between 2.6 x 10(-5) and 1.1 x 10(-6), depending on the clone, although it was stable for each clone after consecutive transfers in culture media. Transposition frequency was similar for bacteria grown in rich or minimal media, and from cells recovered from compatible and incompatible plant hosts, indicating that growth conditions do not influence transposition in strain 1448A. Most of the entrapped insertions contained a full-length IS801 element, with the remaining insertions corresponding to sequences smaller than any transposable element identified in strain 1448A, and collectively identified as miniature sequences. From these, fragments of 229, 360 and 679-nt of the right end of IS801 ended in a consensus tetranucleotide and likely resulted from one-ended transposition of IS801. An average 0.7% of the insertions analyzed consisted of IS801 carrying a fragment of variable size from gene PSPPH_0008/PSPPH_0017, showing that IS801 can mobilize DNA in vivo. Retrospective analysis of complete plasmids and genomes of P. syringae suggests, however, that most fragments of IS801 are likely the result of reorganizations rather than one-ended transpositions, and that this element might preferentially contribute to genome flexibility by generating homologous regions of recombination. A further miniature sequence previously found to affect host range specificity and virulence, designated MITEPsy1 (100-nt), represented an average 2.4% of the total number of insertions entrapped in sacB, demonstrating for the first time the mobilization of a MITE in bacteria. This work was funded with the Spanish Plan Nacional I+D+I grant AGL2008-55311-CO2-01 (Ministerio de Ciencia e Innovación; http://www.micinn.es/) co financed by FEDER.

Pseudomonas savastanoi pv. savastanoi NCPPB 3335 is a model for the study of the molecular basis of disease production and tumor formation in woody hosts, and its draft genome sequence has been recently obtained. Here we closed the sequence of the plasmid complement of this strain, composed of three circular molecules of 78,357 nt (pPsv48A), 45,220 nt (pPsv48B), and 42,103 nt (pPsv48C), all belonging to the pPT23A-like family of plasmids widely distributed in the P. syringae complex., This research has been supported by the Spanish Plan Nacional I+D+I grants AGL2008-55311-CO2-01 and AGL2008-55311-CO2-02 (Ministerio de Ciencia e Innovación; http://www.micinn.es/), co-financed by FEDER, and by grants ref. 57/2007 from the Departamento de Educación y Cultura, Gobierno de Navarra, Spain (http://www.educacion.navarra.es/portal/) and ref. P08-CVI-03475 from the Junta de Andalucía, Spain (http://www.juntadeandalucia.es)

Bacterial apical necrosis of mango, elicited by Pseudomonas syringae pv. syringae, limits fruit production in southern Spain and Portugal. Examination of a collection of P. syringae pv. syringae isolates for copper resistance showed that 59% were resistant to cupric sulfate. The survey of a mango orchard revealed an increase in frequencies of copper-resistant bacteria after repeated treatments with Bordeaux mixture. These data suggest that selection of copper-resistant strains could be a major reason for control failures following management with copper bactericides. Most copper-resistant isolates harbored plasmids, although the majority of them contained a 62-kb plasmid that also was present in copper-sensitive strains. The 62-kb plasmids were differentiated by restriction enzyme analysis and hybridization to copABCD DNA. The most frequently found copper-resistant plasmid type (62.1) was transferable by conjugation. Southern blot hybridizations showed that genetic determinants partially homologous to copABCD were present in all the copper-resistant strains examined, and usually were associated with plasmids; these determinants were not detected in copper-sensitive strains. The selective pressure exerted by copper bactericide sprays on the diversity of copper resistance determinants in bacterial populations of mango is discussed.

Bacterial apical necrosis of mango, produced by Pseudomonas syrin- gae pv. syringae (Pss), is the main disease affecting mango production in the Mediterranean area. Surveys carried out in the main areas of cultivation ascertained the presence of Pss strains and resulted in a collection of Pss strains from different seasons and locations (including mainland Spain and Canary Islands, Portugal, Italy and Israel). To study the diversity relationships among these Pss strains, different phenotypic and genetic techniques were evaluated by using a selection of repre- sentative Pss strains isolated from mango tissues. The use of physiological tests were based on conventional identification techniques (API tests), toxins production based on biological tests, and analysis of copper resistance. The genetic diversity studies were mainly based on repetitive PCR fingerprinting using ERIC, BOX and REP primers set with UPGMA analysis, and 16S rDNA gene sequencing and ARDRA analysis. Additionally, the native plasmid profiles of these representative strains were determined, and the presence of some genes of interest were detected by hybridization analysis in the most abundant plasmid (62 kb). Preliminary results indicate a considerable phenotypic diversity. Analysis of genetic techniques resulted in repetitive PCR fingerprintings using some primers sets, showing higher diversity than the other techniques used.

Pseudomonas syringae pv. phaseolicola is the causal agent of halo blight disease of beans ( Phaseolus vulgaris L.), which is characterized by water-soaked lesions surrounded by a chlorotic halo resulting from the action of a non-host-specific toxin known as phaseolotoxin. This phytotoxin inhibits the enzyme ornithine carbamoyltransferase involved in arginine biosynthesis. Different evidence suggested that genes involved in phaseolotoxin production were clustered. Two genes had been previously identified in our laboratory within this cluster: argK , which is involved in the immunity of the bacterium to its own toxin, and amtA , which is involved in the synthesis of homoarginine. We sequenced the region around argK and amtA in P. syringae pv. phaseolicola NPS3121 to determine the limits of the putative phaseolotoxin gene cluster and to determine the transcriptional pattern of the genes comprising it. We report that the phaseolotoxin cluster (Pht cluster) is composed of 23 genes and is flanked by insertion sequences and transposases. The mutation of 14 of the genes within the cluster lead to a Tox − phenotype for 11 of them, while three mutants exhibited low levels of toxin production. The analysis of fusions of selected DNA fragments to uidA , Northern probing, and reverse transcription-PCR indicate the presence of five transcriptional units, two monocistronic and three polycistronic; one is internal to a larger operon. The site for transcription initiation has been determined for each promoter, and the putative promoter regions were identified. Preliminary results also indicate that the gene product of phtL is involved in the regulation of the synthesis of phaseolotoxin.

The pPT23A plasmid family of Pseudomonas syringae contains members that contribute to the ecological and pathogenic fitness of their P. syringae hosts. In an effort to understand the evolution of these plasmids and their hosts, we undertook a comparative analysis of the phylogeny of plasmid genes and that of conserved chromosomal genes from P. syringae . In total, comparative sequence and phylogenetic analyses were done utilizing 47 pPT23A family plasmids (PFPs) from 16 pathovars belonging to six genomospecies. Our results showed that the plasmid replication gene ( repA ), the only gene currently known to be distributed among all the PFPs, had a phylogeny that was distinct from that of the P. syringae hosts of these plasmids and from those of other individual genes on PFPs. The phylogenies of two housekeeping chromosomal genes, those for DNA gyrase B subunit ( gyrB ) and primary sigma factor ( rpoD ), however, were strongly associated with genomospecies of P. syringae . Based on the results from this study, we conclude that the pPT23A plasmid family represents a dynamic genome that is mobile among P. syringae pathovars.

Virulence for bean and soybean is determined by effector genes in a plasmid-borne pathogenicity island (PAI) in race 7 strain 1449B of Pseudomonas syringae pv. phaseolicola. One of the effector genes, avrPphF , confers either pathogenicity, virulence, or avirulence depending on the plant host and is absent from races 2, 3, 4, 6, and 8 of this pathogen. Analysis of cosmid clones and comparison of DNA sequences showed that the absence of avrPphF from strain 1448A is due to deletion of a continuous 9.5-kb fragment. The remainder of the PAI is well conserved in strains 1448A and 1449B. The left junction of the deleted region consists of a chimeric transposable element generated from the fusion of homologs of IS 1492 from Pseudomonas putida and IS 1090 from Ralstonia eutropha . The borders of the deletion were conserved in 66 P. syringae pv. phaseolicola strains isolated in different countries and representing the five races lacking avrPphF . However, six strains isolated in Spain had a 10.5-kb deletion that extended 1 kb further from the right junction. The perfect conservation of the 28-nucleotide right repeat of the IS 1090 homolog in the two deletion types and in the other 47 insertions of the IS 1090 homolog in the 1448A genome strongly suggests that the avrPphF deletions were mediated by the activity of the chimeric mobile element. Our data strongly support a clonal origin for the races of P. syringae pv. phaseolicola lacking avrPphF .

The molecular analysis of pathogenicity and virulence in Pseudomonas syringae is hampered by functional gene redundancy and by the usually small contribution of each individual virulence gene. In consequence, classical techniques, such as transposon mutagenesis, have proved of limited use for the identification of virulence genes. Curing of native plasmids, however, has allowed the identification of diverse virulence genes and pathogenicity islands in several P. syringae pathovars. In this paper, we summarise the strategies and techniques that we have used successfully for the eviction of native plasmids and the analysis of the resulting strains.

The avrPphF gene was cloned from Pseudomonas syringae pathovar phaseolicola (PPH:) races 5 and 7, based on its ability to confer avirulence towards bean cultivars carrying the R1 gene for halo-blight resistance, such as Red Mexican. avrPphF comprised two open reading frames, which were both required for function, and was located on a 154 kb plasmid (pAV511) in PPH: Strain RW60 of PPH:, lacking pAV511, displayed a loss in virulence to a range of previously susceptible cultivars such as Tendergreen and Canadian Wonder. In Tendergreen virulence was restored to RW60 by avrPphF alone, whereas subcloned avrPphF in the absence of pAV511 greatly accelerated the hypersensitive resistance reaction caused by RW60 in Canadian Wonder. A second gene from pAV511, avrPphC, which controls avirulence to soybean, was found to block the activity of avrPphF in Canadian Wonder, but not in Red Mexican. avrPphF also conferred virulence in soybean. The multiple functions of avrPphF illustrate how effector proteins from plant pathogens have evolved to be recognized by R gene products and, therefore, be classified as encoded by avirulence genes.

The 154-kb plasmid was cured from race 7 strain 1449B of the phytopathogen Pseudomonas syringae pv. phaseolicola ( Pph ). Cured strains lost virulence toward bean, causing the hypersensitive reaction in previously susceptible cultivars. Restoration of virulence was achieved by complementation with cosmid clones spanning a 30-kb region of the plasmid that contained previously identified avirulence ( avr ) genes avrD , avrPphC , and avrPphF . Single transposon insertions at multiple sites (including one located in avrPphF ) abolished restoration of virulence by genomic clones. Sequencing 11 kb of the complementing region identified three potential virulence ( vir ) genes that were predicted to encode hydrophilic proteins and shared the hrp -box promoter motif indicating regulation by HrpL. One gene achieved partial restoration of virulence when cloned on its own and therefore was designated virPphA as the first ( A ) gene from Pph to be identified for virulence function. In soybean, virPphA acted as an avr gene controlling expression of a rapid cultivar-specific hypersensitive reaction. Sequencing also revealed the presence of homologs of the insertion sequence IS 100 from Yersinia and transposase Tn 501 from P. aeruginosa . The proximity of several avr and vir genes together with mobile elements, as well as G+C content significantly lower than that expected for P. syringae , indicates that we have located a plasmid-borne pathogenicity island equivalent to those found in mammalian pathogens.

Summary: Many strains of the phytopathogen Pseudomonas syringae contain mutually compatible plasmids that share extensive regions of sequence homology and essential replication determinants. The replication regions of two compatible large plasmids involved in virulence or pathogenicity, pPT23A from P. syringae pv. tomato strain PT23 and pAV505 from P. syringae pv. phaseolicola strain HRI1302A, were isolated. DNA sequencing of the origins of replication revealed homologous ORFs, designated ORF-Pto and ORF-Pph, respectively. Both ORFs are 1311 bp long and encode peptides of 437 amino acids with predicted molecular masses of 48259 (Pto) and 48334 (Pph) Da. Expression of the two ORFs in Escherichia coli produced peptides of 50 kDa (Pto) and 56 kDa (Pph). The predicted peptides showed an overall identity of 89·7%, being highly conserved from residues 1 to 373, but showing considerable variation in their C-terminal regions (50% identity over the last 64 aa). The two ORFs had significant similarity with the putative replication protein from plasmid pTiK12 of Thiobacillus intermedius and other ColE2-related plasmids. However, both peptides were 100 residues longer than any of the known ColE2-related rep sequences. Subcloning of fragments from the replication region of pPT23A revealed the presence of at least three incompatibility determinants, designated IncA, IncB and IncC. Partial sequencing of the region downstream of ORF-Pto revealed homology to the rulAB genes, involved in UV resistance, from plasmid pPSR1. It is proposed that the replication origin of pPT23A serves as the prototype of a family of related plasmids.

Many Pseudomonas syringae strains contain native plasmids that are important for host-pathogen interactions, and most of them contain several coexisting plasmids (pPT23A-like plasmids) that cross-hybridize to replication sequences from pPT23A, which also carries a gene cluster coding for the phytotoxin coronatine in P. syringae pv. tomato PT23. In this study, three functional pPT23A-like replicons were cloned from P. syringae pv. glycinea race 6, suggesting that the compatibility of highly related replicons is a common feature of P. syringae strains. Hybridization experiments using three separate incompatibility determinants previously identified from pPT23A and the rulAB (UV radiation tolerance) genes showed that the organization of the replication region among pPT23A-like plasmids from several P. syringae pathovars is poorly conserved. The putative repA gene from four pPT23A-like replicons from P. syringae pv. glycinea race 6 was amplified by using specific primers. The restriction profiles of the resulting PCR products for the race 6 plasmids were more similar to each other than they were to that of pPT23A. These data, together with the existence of other cross-hybridizing DNA regions around the replicon among the race 6 pPT23A-like plasmids, suggest that some of these plasmids may have originated from duplication events. Our results also imply that modifications of the repA sequences and the poor conservation of putative maintenance determinants contribute to the suppression of incompatibility among members of the pPT23A-like family, thus enhancing the genomic plasticity of P. syringae .

The genetic determinants for H2-uptake (hup genes) of R. lequminosarum are clustered in a region of about 15 kb of the symbiotic plasmid and are organized in six transcriptional units designated regions hupI to hupVI, all of which are transcribed in the same direction. When DNA restriction fragments containing these hup regions were expressed in E. coli cells under the control of a phage T7 promoter, ten proteins were shown to be specifically expressed by the hup DNA. A 7.3 kb KpnI fragment contaning regions hupI, hupII and part of region hupIII was sequenced. The analysis of the nucleotide sequence of the DNA covering regions hupI/hupII revealed the presence of a putative operon containing the genes for the structural subunits of the hydrogenase and three more open reading frames. A second putative operon containing two ORFs was identified in the DNA corresponding to region hupIII. Both operons have NtrA-binding consensus sequences in the upstream non-coding regions.

The bean (Phaseolus spp.) plant pathogen Pseudomonas syringae pv. phaseolicola is characterized by the ability to produce phaseolotoxin (Tox+). We recently reported that the majority of the Spanish P. syringae pv. phaseolicola population is unable to synthesize this toxin (Tox−). These Tox− isolates appear to lack the entire DNA region for the biosynthesis of phaseolotoxin (argK-tox gene cluster), as shown by PCR amplification and DNA hybridization using DNA sequences specific for separated genes of this cluster. Tox+ and Tox− isolates also showed genomic divergence that included differences in ERIC-PCR and arbitrarily primed-PCR profiles. Tox+ isolates showed distinct patterns of IS801 genomic insertions and contained a chromosomal IS801 insertion that was absent from Tox− isolates. Using a heteroduplex mobility assay, sequence differences were observed only among the intergenic transcribed spacer of the five rDNA operons of the Tox− isolates. The techniques used allowed the unequivocal differentiation of isolates of P. syringae pv. phaseolicola from the closely related soybean (Glycine max) pathogen, P. syringae pv. glycinea. Finally, a pathogenicity island that is essential for the pathogenicity of P. syringae pv. phaseolicola on beans appears to be conserved among Tox+, but not among Tox− isolates, which also lacked the characteristic large plasmid that carries this pathogenicity island. It is proposed that the results presented here justify the separation of the Tox+ and Tox− P. syringae pv. phaseolicola isolates into two distinct genetic lineages, designated Pph1 and Pph2, respectively, that show relevant genomic differences that include the pathogenicity gene complement.