PP2C38 is phosphorylated in response to PAMP perception.
(A) PAMP perception induces PP2C38 band shift. PPC2C38-FLAG protein expressed in N. benthamiana 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 N. benthamiana 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 in vivo. Immunoprecipitated PP2C38-FLAG proteins from N. benthamiana 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 in vitro. Recombinant GST-BIK1 was incubated with [32P]γ-ATP to promote auto-phosphorylation, followed by addition of recombinant MBP-PP2C38. In vitro 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 PP2C38S77A-FLAG variant transiently expressed in N. benthamiana (E) or in Arabidopsis 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.