The type III secretion system (TTSS) proteins form a needle-like structure injecting effector proteins into eukaryotic target cells. Although the TTSS forms an important pathway for bacterium-host interaction, its assembly process in vivo is poorly understood. The process is thought to include the opening of a pore before TTSS proteins are inserted into the bacterial cell wall. The proteins that break the bacterial cell wall have not yet been identified. We hypothesize that a hypersensitive response and pathogenicity (hrp) gene functions to digest the bacterial cell wall because it contains a conserved protein sequence similar to lytic transglycosylase. In this study, we cloned hrp-associated 2 (hpa2) genes from the bacteria Xanthomonas oryzae pathovars. We show in vitro that expressed Hpa2 protein has a lytic activity against bacterial cell walls. The analysis of a loss-of-function mutant of the hpa2 gene suggests that the hpa2 affects bacterial proliferation in host plants and a hypersensitive response in nonhost plants. As this is the first of such enzyme activity identified in the Hrp protein family, we speculate that the Hpa2 contributes to the assembly of the TTSS by enlarging gaps in the peptidoglycan meshwork of bacterial cell walls.