AvrPto-dependent Pto-interacting proteins and AvrPto-interacting proteins in tomato

The plant-intracellular interaction of the avirulence protein AvrPto of Pse udomonas syringae pathovar tomato, the agent of bacterial speck disease, an d the corresponding tomato resistance protein Pto triggers responses leadin g to disease resistance. Pto, a serine/threonine protein kinase, also inter acts with a putative downstream kinase, Pto-interactor 1, as well as with m embers of a family of transcription factors Pto-interactors 4, 5, and 6. Th ese proteins are likely involved, respectively, in a phosphorylation cascad e resulting in hypersensitive cell death, and in defense gene activation, T he mechanism by which the interaction of AvrPto and Pto initiates defense r esponse signaling is not known, To pursue the hypothesis that tertiary inte ractions are involved, we modified the yeast two-hybrid protein interaction trap and conducted a search for tomato proteins that interact with Pto onl y in the presence of AvrPto. Five classes of AvrPto-dependent Pto interacto rs were isolated, and their interaction specificity confirmed. Also, to she d light on a recently demonstrated virulence activity of AvrPto, we conduct ed a standard two-hybrid screen for tomato proteins in addition to Pto that interact with AvrPto: i.e., potential virulence targets or modifiers of Av rPto. By constructing an N-terminal rather than a C-terminal fusion of AvrP to to the LexA DNA binding domain, we were able to overcome autoactivation by AvrPto and identify four classes of specific AvrPto-interacting proteins .