Thr38 and Ser198 are Pto autophosphorylation sites required for the AvrPto-Pto-mediated hypersensitive response

The tomato Pto kinase confers Pseudomonas syringae pv, tomato expressing th e AvrPto protein. To elucidate the role of Pto autophosphorylation in disea se resistance, eight sites autophosphorylated by Pto in vitro were identifi ed by a combination of HPLC purification of tryptic phosphopeptides, MALDI- TOF/MS analysis and Edman degradation. Mutational analysis of the autophosp horylation sites revealed that Pto residues Thr38 and Ser198 are required f or AvrPto-Pto-mediated elicitation of a hypersensitive response in the plan t. Thr38, which is the main Pto autophosporylation site and is located outs ide the kinase catalytic domain, was also required for Pto kinase activity and its physical interaction with AvrPto, the Pti1 kinase and the transcrip tion factor Pti4, Ser198, located in the Pto activation domain, was dispens able for kinase activity and for interaction with AvrPto, However, a mutati on at this site resulted in altered Pto interactions with the Pti1 kinase a nd the Pto interactors of unknown function Pti3 and Pti10. These results su ggest that autophosphorylation events at Pto Thr38 and Ser198 are required for signal transduction by Pto and participate in distinct molecular mechan isms.