TY - JOUR T1 - Phosphatase PP2C6 negatively regulates phosphorylation status of Pti1b kinase, a regulator of flagellin-triggered immunity in tomato JF - bioRxiv DO - 10.1101/613612 SP - 613612 AU - Fabian Giska AU - Gregory B. Martin Y1 - 2019/01/01 UR - http://biorxiv.org/content/early/2019/04/18/613612.abstract N2 - 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 PP2C6 acts as a negative regulator by dephosphorylating the Pti1b kinase, thereby interfering with its ability to activate plant immune responses.ANXRanxur receptor-like kinaseBAK1brassinosteroid insensitive 1-associated kinase 1BIK1botrytis-induced kinase1CERK1chitin elicitor receptor kinase 1CORECold shock protein receptorcspcold shock proteinCWIcell wall integrityEGFepidermal growth factorflgflagellinFLS2flagellin sensing 2KAPPkinase associated protein phosphataseLRRleucine-rich repeatMAMPsmicrobe-associated molecular patternsMAPKsmitogen-activated protein kinasesNADPHnicotinamide adenine dinucleotide phosphateOxi1oxidative signal-inducible1PBLavrPphB susceptible-likePP2Cprotein phosphatase 2CPRRspattern recognition receptorsPTIpattern-triggered immunityPti1bPto interactor 1bRBOHDrespiratory burst oxidase homolog protein DRLCKsreceptor-like cytoplasmic kinasesROSreactive oxygen speciesXB15XA21 binding protein 15. ER -