Ser360 and Ser364 in the Kinase Domain of Tomato SlMAPKKKα are Critical for Programmed Cell Death Associated with Plant Immunity

SlMAPKKKα, a tomato (Solanum lycopersicum) mitogen-activated protein kinase kinase kinase, is a positive regulator of Pto-mediated effector-triggered immunity, which elicits programmed cell death (PCD) in plants. In this study, we examined whether putative phosphorylation sites in the conserved activation segment of the SlMAPKKKα kinase domain are critical for eliciting PCD. Three amino acids, threonine(353), serine(360) (Ser(360)), or serine(364) (Ser(364)), in the conserved activation segment of SlMAPKKKα kinase domain were substituted to alanine (T353A, S360A, or S364A), and these variants were transiently expressed in tomato and Nicotiana benthamiana plants. Two alanine substitutions, S360A and S364A, completely abolished SlMAPKKKα PCD-eliciting activity in both plants, while T353A substitution did not affect its PCD-eliciting activity. SlMAPKKKα wild type and variant proteins accumulated to similar levels in plant leaves. However, SlMAPKKKα protein with the largest size was missed when either S360A or S364A substitutions were expressed, whereas proteins with the smaller masses were more accumulated than those of full-length of SIMAPKKKα and T353A. These results suggest that phosphorylation of SlMAPKKKα at Ser(360) and Ser(364) is critical for PCD elicitation in plants.