The tomato (Solanum lycopersicum) protein kinase Pto confers resistance to Pseudomonas syringae pv. tomato bacteria expressing the AvrPto and AvrPtoB effector proteins. Pto specifically recognizes both effectors by direct physical interactions triggering activation of immune responses. Here, we used a chemical-genetic approach to sensitize Pto to analogs of PP1, an ATP-competitive small molecule inhibitor. By using PP1 analogs in combination with the sensitized Pto (Ptoas), we examined the role of Pto kinase activity in effector recognition and signal transduction. Strikingly, while PP1 analogs efficiently inhibited kinase activity of Ptoas in vitro, they enhanced interactions of Ptoas with AvrPto and AvrPtoB in a yeast two-hybrid system. In addition, in the presence of PP1 analogs, Ptoas bypassed mutations either at an autophosphorylation site critical for the PtoAvrPto interaction or at catalytically essential residues and interacted with both effectors. Moreover, in the presence of the PP1 analog 3MB-PP1, a kinase-deficient form of Ptoas triggered an AvrPto-dependent hypersensitive response in planta. These findings suggest that, rather than phosphorylation per se, a conformational change likely triggered by autophosphorylation in Pto and mimicked by ligand binding in Ptoas is a prerequisite for recognition of bacterial effectors. Following recognition, kinase activity appears to be dispensable for Pto signaling in planta. The chemical-genetic strategy applied here to develop specific small molecule inhibitors of Pto represents an invaluable tool for the study of biological functions of other plant protein kinases in vivo.