Strains of the plant-pathogenic bacterium Xanthomonas axonopodis pv. citri are differentiated into two groups with respect to aggressiveness (normal and weak) on Citrus grandis cultivars but not on other Citrus species such as Citrus sinensis. Random mutagenesis using the transposon Tn5 in X. axonopodis pv. citri strain KC21, which showed weak aggressiveness on a C. grandis cultivar, was used to isolate mutant KC21T46, which regained a normal level of aggressiveness on the cultivar. The gene inactivated by the transposon, hssB3.0, was shown to be responsible for the suppression of virulence on C. grandis. Sequence analysis revealed it to be a new member of the pthA homologs, which was almost identical in sequence to the other homologs except for the number of tandem repeats in the central region of the gene. hssB3.0 appears to be a chimera of other pthA homologs, pB3.1 and pB3.7, and could have been generated by recombination between these two genes. Importantly, in X. axonopodis pv. citri, hssB3.0 was found in all of the tested isolates belonging to the weakly aggressive group but not in the isolates of the normally aggressive group. Isolation of the virulence-deficient mutant KC21T14 from KC21, in which the pathogenicity gene pthA-KC21 was disrupted, showed that hssB3.0 induces a defense response on the host but partially interrupts canker development elicited by the pathogenicity gene in this bacterium.