Couto, Daniel Niebergall, Roda Liang, Xiangxiu Bücherl, Christoph A. Sklenar, Jan P. Macho, Alberto Ntoukakis, Vardis Derbyshire, Paul Altenbach, Denise Maclean, Dan Robatzek, Silke Uhrig, Joachim Menke, Frank Zhou, Jian-Min Zipfel, Cyril PP2C38 is phosphorylated in response to PAMP perception. <p>(A) PAMP perception induces PP2C38 band shift. PPC2C38-FLAG protein expressed in <i>N</i>. <i>benthamiana</i> leaves and treated (+) or not (-) with 100 nM elf18 for the indicated times. Upper band corresponds to phosphorylated PP2C38 form (pPP2C38). 12% bisacrylamide gels were used for better protein separation. Experiment repeated two times with similar results. (B) PP2C band shift is due to phosphorylation. Immunoprecipitated PP2C38-FLAG proteins from <i>N</i>. <i>benthamiana</i> leaves co-expressing EFR-GFP treated with 100 nM elf18 were incubated with calf intestine phosphatase (CIP) in the presence or absence of the phosphatase inhibitor NaF. (C) PP2C38 is phosphorylated on S77 <i>in vivo</i>. Immunoprecipitated PP2C38-FLAG proteins from <i>N</i>. <i>benthamiana</i> were submitted for LTQ-Orbitrap MS/MS analysis. The DpSGPQATFVGVY phosphopeptide was identified as a doubly charged precursor (m/z 660.78) with fragmentation pattern consisting of singly and doubly charged b- and y- ions. Modified peptide sequence and fragmentation pattern shown above spectrum. (D) S77 phosphorylation is required for BIK1 trans-phosphorylation of PP2C38 <i>in vitro</i>. Recombinant GST-BIK1 was incubated with [<sup>32</sup>P]γ-ATP to promote auto-phosphorylation, followed by addition of recombinant MBP-PP2C38. <i>In vitro</i> PP2C38 trans-phosphorylation is revealed by autoradiography. CBB staining shown as loading control. Experiment repeated two times with similar results. (E-F) S77 is required for flg22-induced PP2C38 band shift. Phospho-dead PP2C38<sup>S77A</sup>-FLAG variant transiently expressed in <i>N</i>. <i>benthamiana</i> (E) or in <i>Arabidopsis</i> Col-0 protoplasts (F) does not exhibit a band shift after 20 min 100 nM flg22 treatment. 12% bisacrylamide gels were used for better protein separation. Experiments repeated at least three times with similar results.</p> BIK 1-mediated immunity;NADPH oxidase RBOHD;PRRs FLS 2;protein phosphatase PP 2C;PP 2C;PTI;EFR;Arabidopsis cytoplasmic kinase BIK 1;Arabidopsis Protein Phosphatase PP 2C Negatively Regulates;BIK 1 activity;co-receptor BAK 1. PP 2C;Central Immune Kinase BIK 1 Plants;BIK 1 turnover;cell surface-localized pattern recognition receptors;BIK 1 activation;PAMP-induced BIK 1 phosphorylation 2016-08-05
    https://plos.figshare.com/articles/figure/PP2C38_is_phosphorylated_in_response_to_PAMP_perception_/3870777
10.1371/journal.ppat.1005811.g005