We have initiated a project to identify protein-protein interactions involved in the pathogenicity of the bacterial plant pathogen Xanthomonas axonopodis pv. citri. Using a yeast two-hybrid system based on Gal4 DNA-binding and activation domains, we have focused on identifying interactions involving subunits, regulators, and substrates of the type III secretion system coded by the hrp (for hypersensitive response and pathogenicity), hrc (for hrp conserved), and hpa (for hrp associated) genes. We have identified several previously uncharacterized interactions involving (i) HrpG, a two-component system response regulator responsible for the expression of X. axonopodis pv. citri hrp operons, and XAC0095, a previously uncharacterized protein encountered only in Xanthomonas spp.; (ii) HpaA, a protein secreted by the type III secretion system, HpaB, and the C-terminal domain of HrcV; (iii) HrpB1, HrpD6, and HrpW; and (iv) HrpB2 and HrcU. Homotropic interactions were also identified for the ATPase HrcN. These newly identified protein-protein interactions increase our understanding of the functional integration of phytopathogen-specific type III secretion system components and suggest new hypotheses regarding the molecular mechanisms underlying Xanthomonas pathogenicity.A number of species of gram-negative bacteria are able to infect and cause disease in, or establish symbiotic relationships with, specific plant hosts (2). These phytopathogenic bacteria use a full barrage of molecular strategies by which to enter and colonize host tissues. This invasion eventually modifies, and in many cases compromises, plant homeostasis at the tissue level or at the level of the entire plant. Biochemical, genetic, and cellular studies of phytopathogenic bacteria have revealed that these mechanisms involve a variety of factors such as adhesins, pili, bacterial signaling factors, receptors of external and plantderived factors, proteins involved in signal transduction, specialized transcription factors, alternate sigma factors, and proteins which generate, assemble, and regulate specific macromolecular secretion systems that transport bacterial macromolecular pathogenicity factors (1,2,8,15,25,32,53,67). Only a few of these processes are understood fully at the molecular level and, in the few cases where they have been elucidated for more than one system, significant differences have been observed (32, 39)-differences that must eventually be attributed to the specific biologies of the interacting bacterium-plant pair.During the infective process, a large variety of pathogenic gram-negative bacteria inject macromolecular pathogenic factors into their animal or plant host cells. Bacteria may use one of two systems to accomplish this: the type III (6,12,20,25,26,32,46) and type IV (3,10,11,14,18,19,60,65,69) secretion systems. Recently, the genomes of a number of bacterial phytopathogens have been sequenced, including Xylella spp. (52, 58), Ralstonia solanacearum (49), Xanthomonas spp. (21), Agrobacterium tumefaciens (66), and Pseudomonas s...