Running title: PP2C6 phosphatase and plant immunityAbbreviations: ANXR, anxur receptor-like kinase; BAK1, brassinosteroid insensitive 1-associated kinase 1; BIK1, botrytis-induced kinase1; CERK1, chitin elicitor receptor kinase 1; CORE, Cold shock protein receptor; csp, cold shock protein; CWI, cell wall integrity; EGF, epidermal growth factor; flg, flagellin; FLS2, flagellin sensing 2; KAPP, kinase associated protein phosphatase; LRR, leucine-rich repeat; MAMPs, microbe-associated molecular patterns; MAPKs, mitogen-activated protein kinases; NADPH, nicotinamide adenine dinucleotide phosphate, Oxi1, oxidative signal-inducible1; PBL, avrPphB susceptible-like; PP2C, protein phosphatase 2C; PRRs, pattern recognition receptors; PTI, patterntriggered immunity; Pti1b, Pto interactor 1b; RBOHD, respiratory burst oxidase homolog protein D; RLCKs, receptor-like cytoplasmic kinases; ROS, reactive oxygen species; XB15, XA21 binding protein 15. PP2C6 phosphatase and plant immunity 2 Abstract Plant immune responses, including the production of reactive oxygen species (ROS), are triggered when pattern recognition receptors (PRR) become activated upon detection of microbe-associated molecular patterns (MAMPs). Receptor-like cytoplasmic kinases are key components of PRR-dependent signaling pathways. In tomato two such kinases, Pti1a and Pti1b, are important positive regulators of the plant immune response. However, it is unknown how these kinases control plant immunity at the molecular level, and how their activity is regulated. To investigate these issues, we used mass spectrometry to search for interactors of Pti1b in Nicotiana benthamiana leaves and identified a protein phosphatase, PP2C6. An in vitro pull-down assay and in vivo split luciferase complementation assay verified this interaction. Pti1b was found to autophosphorylate on threonine-233 and this phosphorylation was abolished in the presence of PP2C6. An arginine-to-cysteine substitution at position 240 in the Arabidopsis MARIS kinase was previously reported to convert it into a constitutive-active form. The analogous substitution in Pti1b made it resistant to PP2C6 phosphatase activity, although it still interacted with PP2C6. Treatment of N. benthamiana leaves with the MAMP flg22 induced threonine phosphorylation of Pti1b. Expression of PP2C6, but not a phosphatase-inactive variant of this protein, in N. benthamiana leaves greatly reduced ROS production in response to treatment with MAMPs flg22 or csp22. The results indicate that PP2C6acts as a negative regulator by dephosphorylating the Pti1b kinase, thereby interfering with its ability to activate plant immune responses.
PP2C6 phosphatase and plant immunity