John D. Taylor

Sequence variations in alleles of the avirulence gene avrPphE.R2 from Pseudomonas syringae pv. phaseolicola lead to loss of recognition of the AvrPphE protein within bean cells and a gain in cultivar-specific virulence.

The bean halo blight pathogen, Pseudomonas syringae pv. phaseolicola (Psph), is differentiated into nine races based on the presence or absence of five avirulence (avr ) genes in the bacterium, which interact with corresponding resistance genes, R1–R5, in Phaseolus vulgaris. The resistance gene R2 is matched by avrPphE, which is located adjacent to the cluster of hrp genes that are required for pathogenicity of Psph. Although only races 2, 4, 5 and 7 are avirulent on cultivars with R2 (inducing the hypersensitive response; HR), homologues of avrPphE are present in all races of Psph. DNA sequencing of avrPphE alleles from races of Psph has demonstrated two routes to virulence: via single basepair changes conferring amino acid substitutions in races 1, 3, 6 and 9 and an insertion of 104 bp in the allele in race 8. We have demonstrated that these base changes are responsible for the difference between virulence and avirulence by generating transconjugants of a virulent race harbouring plasmids expressing the various alleles of avrPphE. Agrobacterium tumefaciens‐directed expression of avrPphE from race 4 in bean leaves induced the HR in a resistance gene‐specific manner, suggesting that the AvrPphE protein is alone required for HR induction and is recognized within the plant cell. The allele from race 6, which is inactive if expressed in Psph, elicited a weak HR if expressed in planta, whereas the allele from race 1 did not. Our results suggest that the affinity of interaction between AvrPphE homologues and an unknown plant receptor mediates the severity of the plants response. Mutation of avrPphE alleles did not affect the ability to colonize bean from a low level of inoculum. The avirulence gene avrPphB, which matches the R3 resistance gene, also caused a gene‐specific HR following expression in the plant after delivery by A. tumefaciens.

Avirulence genes from Pseudomonas syringae pathovars phaseolicola and pisi confer specificity towards both host and non-host species☆

Abstract Avirulence genes from Pseudomonas syringae pathovars phaseolicola and pisi which determine specificity towards cultivars of their respective host species (bean and pea), were found also to determine specificity towards the non-host species. Transconjugants of Ps.s. pv. pisi harbouring clones containing avrPph 3, previously isolated from race 3 of Ps.s. pv. phaseolicola , induced a hypersensitive reaction (HR) in all pea cultivars tested. Clones containing avrPpi 2, previously isolated from Ps.s. pv. pisi race 2, caused Ps.s. pv. phaseolicola to induce a rapid HR in most cultivars of bean but in cv. Seafarer a markedly slower incompatible response was observed. Transconjugants of the heterologous pathovar harbouring avrPpi 2 or avrPph 3, therefore, induced resistance patterns in non-host cultivars which were unlike those of any known race of the homologous pathogen. The quantitatively different resistance responses of some bean cultivars were further characterized by analyses of phytoalexin accumulation and bacterial multiplication in pod tissue. Responses of F 2 progeny of crosses between bean cultivars towards transconjugants harbouring avrPpi 2 suggested the presence of a dominant allele at a single locus regulating the rapid HR characteristic of resistance in cv. Canadian Wonder and a dominant allele at an independent second locus regulating the slower resistance reaction of cv. Seafarer. It appears that a one gene for two genes relationship is involved in the avrPpi 2-bean interaction. Clones carrying two other avirulence genes, avrPph 2 and avrPpi 3, from Ps.s. pv. phaseolicola and Ps.s. pv. pisi , respectively, had no effect on the virulence of heterologous pathovars towards any of the cultivars tested.

Isolation and characterization of cloned DNA conferring specific avirulence in Pseudomonas syringae pv. pisi to pea (Pisum sativum) cultivars, which possess the resistance allele, R2

Abstract A partial gene library of Pseudomonas syringae pv. pisi race 2 strain 203 was constructed in the broad host range cosmid pLAFR3. Individual clones from this library were transferred by a replica plate mating procedure to P. syringae pv. pisi race 1 strain PF247. Mobilization of the clones from Escherichia coli involved triparental mating with a conjugative helper plasmid. Transconjugants bearing the library clones were stem-inoculated into pea ( Pisum sativum ) cultivar Early Onward (susceptible to race 1, but resistant to race 2). A single clone, designated pAV270, conferred race-specific avirulence towards a single dominant host gene R2, segregating in an F 2 population of the crosses Kelvedon Wonder (susceptible to both race 1 and 2) × Early Onward and Kelvedon Wonder × Fortune (also carrying R2 in addition to other resistance genes). Subcloning of pAV270 in pLAFR3 indicated that the gene (designated avrAspi 1) responsible for the observed phenotype was located on a 4·0 kb Eco RI fragment of strain 203 DNA.

Excision from tRNA genes of a large chromosomal region, carrying avrPphB, associated with race change in the bean pathogen, Pseudomonas syringae pv. phaseolicola

Pseudomonas syringae pv. phaseolicola (Pph) race 4 strain 1302A carries avirulence gene avrPphB. Strain RJ3, a sectoral variant from a 1302A culture, exhibited an extended host range in cultivars of bean and soybean resulting from the absence of avrPphB from the RJ3 chromosome. Complementation of RJ3 with avrPphB restored the race 4 phenotype. Both strains showed similar in planta growth in susceptible bean cultivars. Analysis of RJ3 indicated loss of > 40 kb of DNA surrounding avrPphB. Collinearity of the two genomes was determined for the left and right junctions of the deleted avrPphB region; the left junction is ≈ 19 kb and the right junction > 20 kb from avrPphB in 1302A. Sequencing revealed that the region containing avrPphB was inserted into a tRNALYS gene, which was re‐formed at the right junction in strain 1302A. A putative lysine tRNA pseudogene (ΨtRNALYS) was found at the left junction of the insertion. All tRNA genes were in identical orientation in the chromosome. Genes near the left junction exhibited predicted protein homologies with gene products associated with a virulence locus of the periodontal pathogen Actinobacillus actinomycetemcomitans. Specific oligonucleotide primers that differentiate 1302A from RJ3 were designed and used to demonstrate that avrPphB was located in different regions of the chromosome in other strains of Pph. Deletion of a large region of the chromosome containing an avirulence gene represents a new route to race change in Pph.

Multiphasic approach for the identification of the different classification levels of Pseudomonas savastanoi pv. phaseolicola

The relationships between strains of Pseudomonas savastanoi pv. phaseolicola (P. sav. phaseolicola), P. syringae pv. tabaci (P. syr. tabaci) and P. syr. syringae which all cause disease on bean; the related species P. sav. glycinea and P. syr. actinidiae, and reference bacteria, were evaluated by studying the phenotypic and genetic diversity of a collection of 62 strains. All the P. sav. phaseolicola strains tested produced characteristic watersoaked lesions on bean pods. Other pathovars produced varying combinations of symptoms including necrotic lesions, with or without watersoaked centres and sunken tissue collapse of the lesion (P. syr. tabaci) and necrotic lesions with or without sunken collapse (P. syr. syringae). At the genomospecies level, all the strains of P. sav. phaseolicola, P. sav. glycinea and P. syr. tabaci, belonging to genomospecies 2, could be separated from P. syr. syringae strains (genomospecies 1) and P. syr. actinidiae strains (unknown genomospecies) by BOX-PCR and DNA/DNA hybridisation. To distinguish P. sav. phaseolicola, within genomospecies 2, from P. sav. glycinea and P. syr. tabaci, it was necessary to perform nutritional characterisations myo-inositol negative and p-hydroxy benzoate positive for P. sav. phaseolicola strains), PCR with specific primers designed from the tox region (positive for all of the P. sav. phaseolicola strains) and serotyping, as 71% of the P. sav. phaseolicola strains reacted as O-serogroup PHA1. Important intrapathovar variation was seen by genomic fingerprinting with REP and ERIC primers, as well as with RAPD primers (AE7 and AE10) and esterase profilings. While RAPD fingerprinting detected variability correlated with two race-associated evolutionary lines, REP, ERIC and esterase profiles revealed intrapathovar variation linked to some host origins, that separated the kudzu isolates, and the mungbean isolates, from the other P. sav. phaseolicola strains.

Molecular genetics of Pseudomonas syringae pathovar pisi : plasmid involvement in cultivar-specific incompatibility

A mutant (PF24) of the race 1 strain, 299A, of Pseudomonas syringae pv. pisi has been characterized in terms of its interactions with pea (Pisum sativum) cultivars. The mutant showed a changed reaction (avirulence to virulence) with a group of pea cultivars, including cvs. Belinda and Puget, previously thought to contain resistance genes R1 and R3. Avirulence towards cv. Puget was restored by transfer of any one of five cosmid clones from a race 3 (strain 870A) gene library to a rifampicin-resistant derivative of PF24. These observations were in agreement with a revised race-specific resistance genotype for Belinda and similar cultivars comprising a single resistance gene, R3. An incompatible interaction was observed between strain PF24 and cvs. Vinco (postulated to harbour race-specific resistance genes R1, R2, R3 and R5) and Hursts Greenshaft (R4 and possibly R1), indicating that the mutant retains at least one avirulence gene (A1 or A1 and A4). Mutant PF24 showed loss of a cryptic plasmid (pAV212) compared with its progenitor, strain 299A. A subclone (pAV233) of one of the race 3 restoration clones showed strong hybridization with similar-sized digestion fragments in race 3 plasmid DNA, confirming the A3 gene to be plasmid-borne. Strong cross-hybridization was also observed with a single 3.27 kb EcoRI fragment of plasmid DNA present in strain 299A but absent from strain PF24. This is consistent with the corresponding A3 determinant being located on pAV212 in the race 1 strain 299A. The novel avirulence gene corresponding to A3 in strain 870A is provisionally designated avrPpi3.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential Responses to Pea Bacterial Blight in Stems, Leaves and Pods Under Glasshouse and Field Conditions

Resistance to pea bacterial blight (Pseudomonas syringae pv. pisi) in different plant parts was assessed in 19 Pisum sativum cultivars and landraces, carrying race-specific resistance genes (R-genes) and two Pisum abyssinicum accessions carrying race-nonspecific resistance. Stems, leaves and pods were inoculated with seven races of P. s. pv. pisi under glasshouse conditions. For both race-specific and nonspecific resistance, a resistant response in the stem was not always associated with resistance in leaf and pod. Race-specific genes conferred stem resistance consistently, however, there was variability in the responses of leaves and pods which depended on the matching R-gene and A-gene (avirulence gene in the pathogen) combination. R2 generally conferred resistance in all plant parts. R3 or R4 singly did not confer complete resistance in leaf and pod, however, R3 in combination with R2 or R4 enhanced leaf and pod resistance. Race-nonspecific resistance conferred stem resistance to all races, leaf and pod resistance to races 2, 5 and 7 and variable reactions in leaves and pods to races 1, 3, 4 and 6.Disease expression was also studied in the field under autumn/winter conditions. P. sativum cultivar, Kelvedon Wonder (with no R genes), and two P. abyssinicum accessions, were inoculated with the most frequent races in Europe under field conditions (2, 4 and 6). Kelvedon Wonder was very susceptible to all three races, whereas P. abyssinicum was much less affected. The combination of disease resistance with frost tolerance in P. abyssinicum enabled plants to survive through the winter. A breeding strategy combining race-nonspecific resistance derived from P. abyssinicum with race-specific R-genes should provide durable resistance under severe disease pressure.

The Molecular Basis of Specificity in the Interaction Between Pseudomonas Syringae pv. pisi and Cultivars of Pisum sativum.

We review progress in the isolation and characterization of a avirulence genes from Pseudomonas syringae pv. pisi, which are responsible for the pattern of racecuitivar interactions with pea (Pisum sativum). The avirulence gene, avrPpiA1.R2 was obtained from the race 2 isolate 203 and was found to be chromosomally located in all race 2 strains examined, whereas homologues in races 5 and 7 were on plasmids. The gene, avrPpiB1.R3 was obtained from the race 3 isolate 870A and together with homologues from races 1 and 7 was shown to be plasmid-borne. Both genes have been sequenced and their products shown to share many features of other P. syringae avr genes. The recent isolation of a cosmid clone apparently harbouring the race 5 gene, avrPpiD, has confirmed the existence of the fifth avirulence gene postulated in the gene-for-gene scheme.