Alan Vivian

Cultivar-specific avirulence and virulence functions assigned to avrPphF in Pseudomonas syringae pv. phaseolicola, the cause of bean halo-blight disease

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 roles of plasmids in phytopathogenic bacteria: mobile arsenals?

Plasmids are extrachromosomal elements of finite size, usually stably inherited within a bacterial cell line and potentially capable of transfer between strains, species or genera. The most widely used systems of grouping for plasmids rely on incompatibility between closely related replicons. This review surveys our current knowledge of plasmids among members of six Gram-negative genera of phytopathogenic bacteria (Burkholderia, Erwinia, Pantoea, Pseudomonas, Ralstonia, Xanthomonas), but excludes Agrobacterium. A major aspect concerns the growing interest in the role of plasmids in pathogenicity and host specificity and the possible advantages of plasmid-borne locations for the genes involved. A range of plasmid-borne phenotypes, including toxin and hormone production, and resistance to bactericides, are reviewed. The role of mobile elements and their association with pathogenicity islands on plasmids and in the bacterial chromosome provide indicators of possible evolutionary routes to the acquisition of disease-inducing capabilities. The paucity of knowledge concerning almost all aspects of plasmid biology among phytopathogenic bacteria is implicit : we argue the need for more work in this somewhat neglected area, to provide a clearer understanding of the molecular strategies adopted by bacteria that enable them to cause disease and evade host defences among a wide range of important crop plants.

Avirulence genes in plant-pathogenic bacteria : signals or weapons ?

Phytopathogenic bacteria generally have limited host ranges, often confined to members of a single plant species or genus. This appears to result from negative factors restricting the host range ra.ther than from positive factors which allow the pathogen to infect its hosts. These negative factors are avirulence genes present in the pathogen, which interact with matching resistance genes in the host. When a pathogen carrying an avirulence gene attacks a plant with the corresponding resistance gene, a cascade of responses (the hypersensitive response, HR ; Klement, 1982) is triggered in the plant and this results in localized host-cell death, preventing spread of the pathogen and the onset o f disease (the resistant response, described as an ‘incompatible’ interaction). In the absence of either one or both of the matching gene pair, the plant fails to recognize the bacterium as a pathogen, the H R is not triggered and disease will usually follow (a susceptible response, resulting from a ‘ compatible:’ interaction).

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.

A tellurite-resistance genetic determinant from phytopathogenic pseudomonads encodes a thiopurine methyltransferase: evidence of a widely-conserved family of methyltransferases.

A tellurite-resistance genetic determinant was isolated from the pea blight pathogen Pseudomonas syringae pathovar pisi by a shotgun strategy involving a tellurite-selective screening in Escherichia coli. A 1.65 kb tellurite resistance insert was obtained and analysed. It harbours a single complete and functional ORF encoding a deduced protein of 24, 445 Da. The deduced AA sequence shows significant similarities with the complete human thiopurine methyltransferase enzyme, a methyltransferase from Synechocystis and a methyltransferase-like sequence from Bordetella pertussis. The encoded thiopurine methyltransferase activity was demonstrated using a radiochemical microassay for the methylation of 6-mercaptopurine. This gene was detected in most P. syringae legume pathogens.

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.

Replication regions from plant-pathogenic Pseudomonas syringae plasmids are similar to ColE2-related replicons

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 897 %, 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 CoIE2-related plasmids. However, both peptides were 100 residues longer than any of the known CoIE2-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 ru/AB 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.

Isolation and Partial Characterization of Three Classes of Mutant in Pseudomonas syringae Pathovar pisi with Altered Behaviour towards Their Host, Pisum sativum

SUMMARY: Mating with Escherichia coli strain SM10 carrying the Tn5 vector pSUP2011 was used to mutagenize Pseudomonas syringae pv. pisi strain 299A. The resulting transconjugants were each tested by stem-inoculation into several pea (Pisum sativum) cultivars. Three classes of mutant, which probably resulted from insertion of part or all of RP4-2-Tc:: Mu into the genome of strain 299A, showed reduced virulence towards one or more pea cultivars. The single class I mutant was avirulent on all pea cultivars tested and had lost the ability to induce a hypersensitive response in tobacco (Nicotiana tabacum) cv. White Burley; the single class II mutant induced a hypersensitive response on all pea cultivars and tobacco; class III mutants showed reduced virulence towards pea cv. Early Onward, while remaining fully virulent towards other normally susceptible pea cultivars, and inducing a hypersensitive response in tobacco.

Specific oligonucleotide primers for the rapid identification and detection of the agent of tomato pith necrosis, Pseudomonas corrugata, by PCR amplification : evidence for two distinct genomic groups

Unique DNA bands from strains representative of two groups of Pseudomonas corrugata, as shown by amplification of their genomic DNA by polymerase chain reaction using short random sequence oligonucleotide primers (RAPD-PCR), were isolated, cloned and sequenced. Two pairs of specific primer sequences, based on the ends of the cloned unique DNA bands from strains IPVCT10.3 and IPVCT8.1, were used in multiplex PCR with a range of P. corrugata strains. All strains produced one of the two specific bands, 1100 bp (from the IPVCT10.3-based primers) and 600 bp (from the IPVCT8.1-based primers), representing groups designated I and II, respectively. The primers were also tested on a wider range of Pseudomonas species, including the closely-related fluorescent Pseudomonas genomospecies FP1, FP2 and FP3: none of these bacteria produced any bands following amplification by PCR with these primers. The primer sets detected P. corrugata in tomato pith necrosis-infected plants providing a useful tool for rapid identification and epidemiological studies.