Tomato 14-3-3 Protein 7 Positively Regulates Immunity-associated Programmed Cell Death by Enhancing Protein Abundance and Signaling Ability of MAPKKK {alpha}

Programmed cell death (PCD) is triggered when Pto, a Ser-Thr protein kinase, recognizes either the AvrPto or AvrPtoB effector from Pseudomonas syringae pv tomato. This PCD requires mitogen-activated protein kinase kinase kinase (MAPKKK alpha ) as a positive regulator in tomato (Solanum lycopersicum) and Nicotiana benthamiana. To examine how PCD-eliciting activity of the tomato MAPKKK alpha protein is regulated, we screened for MAPKKK alpha -interacting proteins in tomato and identified a 14-3-3 protein, TFT7. Virus-induced gene silencing using the TFT7 gene in N. benthamiana compromised both Pto- and MAPKKK alpha -mediated PCD, and coexpression of TFT7 with tomato MAPKKK alpha enhanced MAPKKK alpha -mediated PCD. TFT7 was also required for PCD associated with several other disease resistance proteins and contributed to resistance against P. syringae pv tomato. Coexpression of TFT7 with MAPKKK alpha in vivo caused increased accumulation of the kinase and enhanced phosphorylation of two MAP kinases. TFT7 protein contains a phosphopeptide binding motif that is present in human 14-3-3 epsilon, and substitutions in this motif abolished interaction with MAPKKK alpha in vivo and also the PCD-enhancing activity of TFT7. A 14-3-3 binding motif, including a putative phosphorylated Ser-535, is present in the C-terminal region of MAPKKK alpha. An S535A substitution in MAPKKK alpha reduced interaction with TFT7 and both PCD-eliciting ability and stability of MAPKKK alpha. Our results provide new insights into a role for 14-3-3 proteins in regulating immunity-associated PCD pathways in plants.