The largest plasmid (pPT23A, 100 kb) from Pseudomonas syringae pv.tomato PT23, carries the gene cluster for the synthesis of the phytotoxin coronatine and most of its DNA sequences are repeated in a co-residing plasmid, pPT23B (83 kb). Some DNA fragments from pPT23A are also repeated elsewhere in the genome. To investigate the role in virulence of these plasmids, avoiding the effects of gene redundancy, we constructed strain UPN2, which was derived by curing the four native plasmids from PT23. The necrotic lesions induced in tomato by UPN2 were four-fold smaller than those produced by PT23. This phenotype was only partially restored by pPT23A, and the production of lesions of wild type size was dependent on the concomitant presence in UPN2 of pPT23A and pPT23B. Mutants defective in the production of coronatine, but which still produced coronafacic acid, induced lesions of an intermediate size between PT23 and UPN2. The virulence determinants present on pPT23B could be modifying the amount, structure or uptake rate by the plant of the coronafacic acid molecules produced by genes present on pPT23A. Therefore, the production of necrotic lesions on tomato plants by strain PT23 is due substantially to the action of compounds whose determinants are located on plasmids pPT23A and pPT23B, including, but not limited to coronatine. The involvement of the coronafacic acid gene cluster in virulence in the absence of the cluster for the synthesis of coronamic acid, may help explain its maintenance in the bacterial population before being incorporated into the coronatine cluster. Likewise, the physical separation of the virulence determinants in the two independent replicons pPT23A and pPT23B, may have driven the evolution of strain PT23 so that it maintains two potentially incompatible plasmids.