The plant-intracellular interaction of the avirulence protein AvrPto of Pseudomonas syringae pathovar tomato, the agent of bacterial speck disease, and the corresponding tomato resistance protein Pto triggers responses leading to disease resistance. Pto, a serine/threonine protein kinase, also interacts with a putative downstream kinase, Pto-interactor 1, as well as with members of a family of transcription factors Pto-interactors 4, 5, and 6. These proteins are likely involved, respectively, in a phosphorylation cascade resulting in hypersensitive cell death, and in defense gene activation. The mechanism by which the interaction of AvrPto and Pto initiates defense response signaling is not known. To pursue the hypothesis that tertiary interactions are involved, we modified the yeast two-hybrid protein interaction trap and conducted a search for tomato proteins that interact with Pto only in the presence of AvrPto. Five classes of AvrPto-dependent Pto interactors were isolated, and their interaction specificity confirmed. Also, to shed light on a recently demonstrated virulence activity of AvrPto, we conducted a standard two-hybrid screen for tomato proteins in addition to Pto that interact with AvrPto: i.e., potential virulence targets or modifiers of AvrPto. By constructing an N-terminal rather than a C-terminal fusion of AvrPto to the LexA DNA binding domain, we were able to overcome autoactivation by AvrPto and identify four classes of specific AvrPto-interacting proteins.