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 approximately 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 (PsitRNALYS) 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.