The Pseudomonas syringae Hrp pathogenicity island has a tripartite mosaic structure composed of a cluster of type III secretion genes bounded by exchangeable effector and conserved effector loci that contribute to parasitic fitness and pathogenicity in plants James R. Alfano* † , Amy O. Charkowski †‡§ , Wen-Ling Deng ‡ , Jorge L. Badel ‡ , Tanja Petnicki-Ocwieja*, Karin van Dijk*, and Alan Collmer ‡¶ *Department of Biological Sciences, University of Nevada, Las Vegas, NV 89154-4004; and ‡ Department of Plant Pathology, Cornell University, Ithaca, NY 14853-4203 Communicated by Eugene W. Nester, University of Washington, Seattle, WA, February 16, 2000 (received for review December 9, 1999) The plant pathogenic bacterium Pseudomonas syringae is divided into pathovars differing in host specificity, with P. syringae pv. syringae (Psy) and P. syringae pv. tomato (Pto) representing par- ticularly divergent pathovars. P. syringae hrphrc genes encode a type III protein secretion system that appears to translocate Avr and Hop effector proteins into plant cells. DNA sequence analysis of the hrphrc regions in Psy 61, Psy B728a, and Pto DC3000 has revealed a Hrp pathogenicity island (Pai) with a tripartite mosaic structure. The hrphrc gene cluster is conserved in all three strains and is flanked by a unique exchangeable effector locus (EEL) and a conserved effector locus (CEL). The EELs begin 3 nt downstream of the stop codon of hrpK and end, after 2.5–7.3 kb of dissimilar intervening DNA with tRNA Leu –queA–tgt sequences that are also found in Pseudomonas aeruginosa but without linkage to any Hrp Pai sequences. The EELs encode diverse putative effectors, includ- ing HopPsyA (HrmA) in Psy 61 and proteins similar to AvrPphE and the AvrBAvrCAvrPphC and AvrBsTAvrRxvYopJ protein fam- ilies in Psy B728a. The EELs also contain mobile genetic element sequences and have a G  C content significantly lower than the rest of the Hrp Pai or the P. syringae genome. The CEL carries at least seven ORFs that are conserved between Psy B728a and Pto DC3000. Deletion of the Pto DC3000 EEL slightly reduces bacterial growth in tomato, whereas deletion of a large portion of the CEL strongly reduces growth and abolishes pathogenicity in tomato. T he plant pathogenic bacterium Pseudomonas syringae is noted for its diverse and host-specific interactions with plants (1). A specific strain may be assigned to one of at least 40 pathovars on the basis of its host range among different plant species and then further assigned to a race on the basis of differential interactions among cultivars of the host. In host plants the bacteria typically grow to high population levels in leaf intercellular spaces and then produce necrotic lesions. In non- host plants or in host plants with race-specific resistance, the bacteria elicit the hypersensitive response (HR), a rapid, de- fense-associated programmed death of plant cells in contact with the pathogen (2). The ability to produce either of these reactions in plants appears to be directed by hrp (HR and pathogenicity) and hrc (HR and conserved) genes that encode a type III protein secretion pathway and by avr (avirulence) and hop (Hrp- dependent outer protein) genes that encode effector proteins injected into plant cells by the pathway (2). These effectors may also betray the parasite to the HR-triggering R-gene surveillance system of potential hosts (hence the avr designation), and plant breeding for resistance based on such gene-for-gene (avr–R) interactions may produce complex combinations of races and differential cultivars (3). hrphrc genes are probably universal among necrosis-causing Gram-negative plant pathogens, and they have been sequenced in P. syringae pv. syringae (Psy) 61, Erwinia amylovora Ea321, Xanthomonas campestris pv. vesicato- ria (Xcv) 85–10, and Ralstonia solanacearum GMI1000 (2). On the basis of their distinct gene arrangements and regulatory components, the hrphrc gene clusters of these four bacteria can be divided into two groups: I (Pseudomonas and Erwinia) and II (Xanthomonas and Ralstonia). The discrepancy between the distribution of these groups and the phylogeny of the bacteria provides some evidence that hrphrc gene clusters have been horizontally acquired and therefore may represent pathogenicity islands (Pais) (2). Hacker et al. (4) have defined Pais as gene clusters that (i) include many virulence genes, (ii) are selectively present in pathogenic strains, (iii) have different G + C content compared with host bacteria DNA, (iv) occupy large chromosomal regions, (v) are often flanked by direct repeats, (vi) are bordered by tRNA genes andor cryptic mobile genetic elements, and (vii) are unstable. Some Pais have inserted into different genomic locations in the same species (5). Others reveal a mosaic structure indicative of multiple horizontal acquisitions (6). Genes encoding type III secretion systems are present in Pais in animal-pathogenic Salmonella spp. and Pseudomonas aeruginosa and on large plasmids in Yersinia and Shigella spp. Genes encoding effectors secreted by the pathway in these organisms are commonly linked to the pathway genes (7), although a noteworthy exception is sopE, which is carried by a temperate phage without apparent linkage to SPI1 in certain isolates of Salmonella typhimurium (8). Three avrhop genes have already Abbreviations: Psy, Pseudomonas syringae pv. syringae; Pto, P. syringae pv. tomato; Pph, P. syringae pv. phaseolicola; HR, hypersensitive response; hrp, HR and pathogenicity; hrc, HR and conserved; Pai, pathogenicity island; EEL, exchangeable effector locus; CEL, conserved effector locus; Hop, Hrp-dependent outer protein; Avr, avirulence; cfu, colony-forming units. Data deposition: The nucleotide sequences reported in this paper have been deposited in the GenBank database (accession nos. AF232003 for Psy 61 EEL, AF232004 for Pto DC3000 EEL, AF232005 for Psy B728a EEL, and AF232006 for Pto DC3000 CEL). † J.R.A. and A.O.C. contributed equally to this work. § Present address: United States Department of Agriculture, ARS, WRRC, 800 Buchanan St., Albany, CA 94710. ¶ To whom reprint requests should be addressed. E-mail: arc2@cornell.edu. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact. 4856 – 4861 | PNAS | April 25, 2000 | vol. 97 | no. 9