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. 1999 Sep 14;96(19):10875-80.
doi: 10.1073/pnas.96.19.10875.

Identification of a pathogenicity island, which contains genes for virulence and avirulence, on a large native plasmid in the bean pathogen Pseudomonas syringae pathovar phaseolicola

R W Jackson  1 , E Athanassopoulos, G Tsiamis, J W Mansfield, A Sesma, D L Arnold, M J Gibbon, J Murillo, J D Taylor, A Vivian
Affiliations

Identification of a pathogenicity island, which contains genes for virulence and avirulence, on a large native plasmid in the bean pathogen Pseudomonas syringae pathovar phaseolicola

R W Jackson et al. Proc Natl Acad Sci U S A. .

Abstract

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 IS100 from Yersinia and transposase Tn501 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.

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Figures

Figure 1
Figure 1
Curing the 154-kb plasmid from Pph race 7 strain 1449B. (A) Plasmid profiles from races of Pph; M gives profile from E. coli 39R861 that contains plasmids of known size (44). (B) The 154-kb plasmid pAV511 was cured from 1449B by using the rep gene cloned in pPPY51. Both RW50 and RW60 have been cured of pAV511, but RW50 retains pPPY51. The smaller plasmid in 1449B, RW50, and RW60 is designated pAV512. Chr, chromosomal DNA.
Figure 2
Figure 2
Reaction phenotypes in bean pods and soybean leaves inoculated with Pph. (A) Pods of bean cultivars (from left to right): A43, Canadian Wonder, Red Mexican, and Tendergreen 3 days after stab-inoculation with (top to bottom site): Pph race 7 strain 1449B, cured strain RW60, complemented strains RW60 (pAV521) and RW60 (pAV518), and RW60 (pLAFR3) vector control. Note that pAV521 carries avrPphF, the avr gene matching resistance gene R1 in Red Mexican. (B) Time course of HR development in pods of cv. Red Mexican photographed 1, 2, 3, and 5 days after inoculation with 1449B (Upper) and RW60 (Lower); note the more orange lesion caused by the plasmid-cured strain. (C) Leaves of soybean cv. Osumi 10 days after inoculation with (left to right) 1449B, RW60, and RW60 (pAV518).
Figure 3
Figure 3
Development of the HR and susceptible tissue collapse in leaves of cvs. Red Mexican and Tendergreen inoculated with suspensions of 2 × 108 cells/ml of race 7 strain 1449B (black bars), cured strain RW60 (white bars), complemented strains RW60 (pAV521) (light gray bars) and RW60 (pAV518) (dark gray bars). Symptom scores assigned to infiltrated tissue were: 0, no reaction; 1, partial glazing; 2, 100% glazing over the inoculation site; 3, up to 50% tissue collapse; 4, between 50 and 100% collapse, 5, 100% collapse; 6, 100% collapse and browning, as observed during the HR. Data are the mean scores from sites in two leaves. Note that pAV521 carries avrPphF, the avr gene matching resistance gene R1 in Red Mexican.
Figure 4
Figure 4
Characterized region of Pph plasmid pAV511 found to contain the PAI. The DNA common to genomic clones pAV518 and pAV521 is marked by the underlying line. The positions of single Tn3 gus insertions that abolished restoration of virulence by pAV521 (gray arrows) and pAV518 (black arrows) are marked. Note that insertions that did not affect virulence were not mapped.
Figure 5
Figure 5
Location of ORFs for ORF1 (designated virPphA), ORF2, ORF3, and ORF4 within the 11 kb of DNA sequenced. The directions of transcription are indicated by horizontal arrowheads. Sites of Tn3 gus insertions abolishing restoration of virulence by pAV518 are indicated by vertical arrows. Sequences found to have similarity to IS100 and Tn501 are indicated. Clone pAV530 containing the 6.7-kb EcoRI fragment restored virulence almost as effectively (+++) as the genomic clone pAV518 (scored as ++++); pAV533 and pAV536 were slightly less effective. Clone pAV535, containing ORF4, consistently delayed the onset of the HR caused by RW60, a reaction scored as +.
Figure 6
Figure 6
Possible mechanisms by which virulence (Vir) factors block the HR in bean caused by avr gene activity, which is masked in wild-type Pph. The masked avr genes are designated β. Routes to virulence are via 1) suppression of β avr gene expression; 2) blocking delivery of the encoded β avr protein, or 3) interference with generation of the HR after recognition. By contrast, α avr genes, such as avrPphE, are not affected.
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References

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