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. 2022 Jul 21;34(10):4088–4104. doi: 10.1093/plcell/koac209

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TCH3 targets the autoinhibitory region (AIR) of CPK5 and promotes CDPK-dependent CBP60g phosphorylation. A, CPK4^CA/CPK5^CA-induced phosphorylation of CBP60g remains intact in the cml^quad mutant. B, TCH3-induced phosphorylation of CBP60g is significantly compromised in the cpk4 cpk5 cpk6 cpk11 mutant. Protoplasts isolated from WT or mutant plants as indicated were transfected with Pro35S:CBP60g-FLAG alone or together with CPK4^CA/CPK5^CA-HA or TCH3-HA, treated with or without 2 µM flg22 for 5 min. Protein samples extracted from protoplasts were separated by Phos-tag SDS-PAGE and subjected to anti-FLAG or anti-HA immunoblot. C, TCH3 interacts with the C-terminal of CPK5. Nicotiana benthamiana leaves infiltrated with Pro35S:NLuc-TCH3, Pro35S:CLuc-CPK5, Pro35S:CLuc-CPK5CT, Pro35S:CLuc-CPK5CA, Pro35S:CLuc-XLG2, or Pro35S:NLuc-BIK1 as indicated were subjected to luciferase complementation assay. D, TCH3 interacts with CPK5-AIR (AIR) in N. benthamiana. Nicotiana benthamiana leaves infiltrated with Pro35S:NLuc-AIR, Pro35S:CLuc-TCH3, Pro35S:CLuc-XLG2, or Pro35S:NLuc-BIK1 as indicated were subjected to luciferase complementation assay. E, TCH3 interacts with CPK5-CRD (CRD) in N. benthamiana. Nicotiana benthamiana leaves infiltrated with Pro35S:NLuc-CRD, Pro35S:CLuc-TCH3, Pro35S:CLuc-XLG2, or Pro35S:NLuc-BIK1 as indicated were subjected to luciferase complementation assay. F, TCH3 impairs the interaction between CPK5-CRD (CRD) and AIR. N.b. leaves infiltrated with Pro35S:NLuc-AIR, Pro35S:CLuc-CRD, Pro35S:TCH3-FLAG, Pro35S:CLuc-XLG2, or Pro35S:NLuc-BIK1 as indicated were subjected to luciferase complementation assay. Infiltrated N. benthamiana leaves were sliced into strips 2 d post infiltration, and relative luminescence was determined by Microplate Luminometer. Error bars indicate SD of three biological repeats. Student’s t test was carried out to determine the significance of difference. Different letters indicate significant differences. G, TCH3 enhances CPK5 activity towards CBP60g in vitro. MBP-CBP60g was incubated with His-CPK5 or together with GST-TCH3 as indicated and subjected to in vitro phosphorylation assay. CBB staining indicates loading of the protein. The ratio of phosphorylation signal over protein amount is shown above the phosphor band. The experiments were repeated 3 times with similar results.