Proc. Natl. Acad. Sci. USA Vol. 96, pp. 10875–10880, September 1999 Microbiology 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 (plant disease resistance͞hypersensitive reaction͞signal transduction) ROBERT W. JACKSON*, EVANGELOS ATHANASSOPOULOS†‡,GEORGE TSIAMIS†‡,JOHN W. MANSFIELD†§,ANE SESMA¶, ʈ DAWN L. ARNOLD*, MARJORIE J. GIBBON*, JESUS MURILLO¶,JOHN D. TAYLOR , AND ALAN VIVIAN* *Department of Biological and Biomedical Sciences, University of the West of England, Coldharbor Lane, Bristol BS16 1QY, United Kingdom; †Department of Biological Sciences, Wye College, Wye, Ashford, Kent TN25 5AH, United Kingdom; ¶Escuela Te´cnica Superior de Ingenieros Agro´nomos, Universidad Pu´blica de Navarra, 31006 Pamplona, Spain; and ʈHorticulture Research International, Wellesbourne, Warwick CV35 9EF, United Kingdom Communicated by Noel T. Keen, University of California, Riverside, CA, July 7, 1999 (received for review April 2, 1999) ABSTRACT The 154-kb plasmid was cured from race 7 Certain avr genes, although recognized by their ability to strain 1449B of the phytopathogen Pseudomonas syringae pv. activate plant defenses (the HR), also may have a role in phaseolicola (Pph). Cured strains lost virulence toward bean, pathogenicity in the absence of the interacting R gene in the causing the hypersensitive reaction in previously susceptible host plant. In some cases there is a clear, qualitative effect on cultivars. Restoration of virulence was achieved by complemen- pathogenicity of mutations in avr genes, as with avrBs2 in tation with cosmid clones spanning a 30-kb region of the plasmid certain races of Xanthomonas campestris pv. vesicatoria in that contained previously identified avirulence (avr) genes avrD, pepper and avrRpm1 in P. syringae pv. maculicola in Arabi- avrPphC, and avrPphF. Single transposon insertions at multiple dopsis (9, 10). However, the effect of avr gene mutations often sites (including one located in avrPphF) abolished restoration of is incomplete; for example, the avrE locus has a quantitative virulence by genomic clones. Sequencing 11 kb of the comple- role in virulence in P. syringae pv. tomato strain PT23 but is menting region identified three potential virulence (vir) genes dispensable in strain DC3000 (11). If the avr gene products do that were predicted to encode hydrophilic proteins and shared have a role in pathogenicity they may contribute to a redun- the hrp-box promoter motif indicating regulation by HrpL. One dancy of pathogenicity factors so that loss of a single avr gene gene achieved partial restoration of virulence when cloned on its product may not lead to total loss of ability to cause disease. own and therefore was designated virPphA as the first (A) gene It has been proposed that gene-for-gene interactions are su- from Pph to be identified for virulence function. In soybean, perimposed on an established basic, species-specific parasitism virPphA acted as an avr gene controlling expression of a rapid (12). Genes controlling such fundamental aspects of infection are cultivar-specific hypersensitive reaction. Sequencing also re- located on well-characterized pathogenicity islands (PAIs) in vealed the presence of homologs of the insertion sequence IS100 animal pathogens (13, 14). There is increasing support for the from Yersinia and transposase Tn501 from P. aeruginosa. The hypothesis that bacteria pathogenic to animals have evolved from proximity of several avr and vir genes together with mobile their nonpathogenic ancestors after acquisition of PAIs that ϩ elements, as well as G؉C content significantly lower than that typically contain large fragments of DNA that differ in G C expected for P. syringae, indicates that we have located a plasmid- content from the rest of the genome (13). PAIs have been located borne pathogenicity island equivalent to those found in mam- on plasmids and also mapped to the chromosome. Examples of malian pathogens. the former include the pCD1 plasmid in Yersinia pestis and of the latter, the islands SPI-1 and SPI-2 of Salmonella typhimurium (15, 16). In Salmonella, SPI-1 and SPI-2 both contain components of Varietal resistance to halo-blight disease of bean (Phaseolus type III secretion systems (17, 18). This specialized protein vulgaris L.) caused by Pseudomonas syringae pv. phaseolicola delivery system is also the main component of the common hrp (Pph) is determined by gene-for-gene interactions involving cluster found in plant pathogenic bacteria, which also may be five resistance (R) genes in the host and five matching aviru- considered a PAI. Mutations in hrp genes cause loss of the ability lence (avr) genes in the pathogen. Depending on the presence to elicit the HR in resistant plants (whether host or nonhost) and or absence of functional avr genes, nine races of Pph have been to cause disease symptoms (i.e., to be pathogenic) in susceptible distinguished (1, 2). The avr genes matching R1, R2, and R3 cultivars. In Ralstonia solanacearum, hrp genes are located on a have been cloned and sequenced. Their full designations are megaplasmid whereas they are chromosomal in Erwinia, Pseudo- avrPphF.R1, avrPphE.R2, and avrPphB.R3; the terminal R gene monas, and Xanthomonas (for recent reviews see refs. 19–21). designation will not be used here (3–5). Both avrPphE and The proteins encoded by avr genes appear to be delivered via avrPphB are chromosomal, whereas avrPphF is located on a the type III secretion system into plant cells, where they act as large plasmid in those races that cause the hypersensitive elicitors of the HR. Evidence for this has been obtained by the reaction (HR) in cultivars of bean with the matching R1 gene. transient expression of avr genes in plants, production of the The HR is a resistance response recognized by the rapid death Avr protein leading to activation of a rapid HR in an R of plant cells at inoculation sites and the restriction of micro- gene-specific manner; examples include avrPphB and avrPphE bial colonization (2). Additional avr genes located on the (22). In animal pathogens, the type III secretion system is well plasmid in Pph determine ability to elicit the HR in nonhost plants, avrPphC and a homolog of avrD (soybean interactors), Abbreviations: avr, avirulence gene; vir, virulence gene; HR, hyper- and avrPphD that interacts with pea (6–8). sensitive reaction; PAI, pathogenicity island; Pph, Pseudomonas sy- ringae pv. phaseolicola; R, resistance gene; dsp, disease-specific gene. The publication costs of this article were defrayed in part by page charge Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AF141883). payment. This article must therefore be hereby marked ‘‘advertisement’’ in ‡E.A. and G.T. contributed equally to this work. accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. E-mail: j.mansfield@ PNAS is available online at www.pnas.org. wye.ac.uk. 10875 Downloaded by guest on September 26, 2021 10876 Microbiology: Jackson et al. Proc. Natl. Acad. Sci. USA 96 (1999) recognized as a route for export of pathogenicity factors such 4–5 h at 100 V, or for preparation of probe DNA in 0.5% as Yops in Yersinia, invasion plasmid antigens in Shigella, Sips ultra-pure low-melting point agarose (GIBCO͞BRL). A gene and Sops in Salmonella, and Esps in enteropathogenic Esch- library was constructed in the cosmid vector pLAFR3 (2). erichia coli (23–25). Circumstantial evidence from the exis- Hybridization. Separated DNA was transferred to Hybond tence of the type III system and activity of Avr proteins inside Nϩ nylon membrane (ICN) by vacuum blotter (Appligene). plant cells suggests that similar pathogenicity determinants or Colony blotting was as described by Sambrook et al. (33), using virulence factors, as encoded by PAIs in pathogens of animals, BIOTRANS nylon colony blot. Probe DNA usually was puri- also should be present in phytopathogenic bacteria (19). fied by using a QIAEX II gel extraction kit (Qiagen), but In addition to pathogenicity factors thought to be secreted pAV511 was excised from low-melting point gels. Restrict- through the type III hrp-dependent system and to establish ed͞amplified DNA was radiolabeled by using a High Prime basic parasitism, degradative enzymes, extracellular polysac- radiolabeling kit (Boeringher Mannheim), and pAV511 was charides, and low molecular weight toxins are produced by radiolabeled (1 h) in agarose by using Ready-to-Go beads many plant pathogens (26). Genes encoding the pathogenicity (Amersham Pharmacia). Blots were hybridized (65°C, 16 h) determinants that are hrp independent often are clustered, but with the labeled probes in hybridization solution (33), and then they are not generally recognized as PAIs. An example is the washed to high stringency (34). cluster of genes involved in the production of phaseolotoxin by Plasmid Transfer Procedures and Curing. Plasmid constructs Pph. Secretion of phaseolotoxin causes the yellow haloes that were transferred from E. coli to rifampicin-resistant recipient develop around lesions in bean leaves (27, 28). strains of Pseudomonas by using a replica plate triparental mating Races of Pph harbor plasmids ranging in size from 25 to 160 procedure (35). Electroporations were carried out with a Gene kb (29). The large native plasmid (designated pAV511 in race Pulser (Bio-Rad) (36). Genomic clones were mutagenized by 7 strain 1449B) is known to carry several avr genes. To examine transposon insertion using Tn3 gus (37), and marker exchange the function of pAV511 we attempted to cure the plasmid by mutants were obtained as described (in ref. 2). expression of its origin of replication in trans. The successfully Plasmid pPPY51 (which carries the replication gene from cured strains were found to be compromised in virulence pAV505, a ca. 140-kb plasmid from race 4 strain 1302A; ref. toward bean. Here we report the identification of a region on 38) was electroporated into cells of 1449B to cure the homol- pAV511 that controls virulence, which we describe as a PAI ogous plasmid pAV511.