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

Figure 5.

Figure 5

Ser450 and Ser495 are required for PAMP-induced full phosphorylation of CBP60g. A, Ser450 is required for CBP60g full phosphorylation induced by flg22 in protoplasts. Protoplasts isolated from WT plants were transfected with Pro35S:CBP60g-FLAG or Pro35S:CBP60gS450A-FLAG, treated with or without flg22. B, Ser495 is required for CBP60g full phosphorylation induced by flg22 in protoplasts. Protoplasts isolated from WT plants were transfected with Pro35S:CBP60g-FLAG or Pro35S:CBP60gS495A-FLAG, treated with or without flg22. C, Double mutation of Ser450 and Ser495 largely impaired flg22-induced full phosphorylation of CBP60g in protoplasts. Protoplasts were transfected with Pro35S:CBP60g-FLAG or Pro35S:CBP60gSSAA-FLAG, treated with or without flg22. D-E, Double mutation of Ser450 and Ser495 largely impaired flg22-induced (D) and chitin-induced (E) full phosphorylation of CBP60g in transgenic plants. Pro35S:CBP60g-FLAG or Pro35S:CBP60gSSAA-FLAG transgenic plants were treated with or without 2 µM flg22, or 250 µg/mL chitin. Total protein was extracted from protoplasts or leaves of transgenic plants. Protein samples were subjected to Phos-tag SDS-PAGE followed by anti-FLAG immunoblot. CBB staining of the filter indicates loading of the protein. SSAA stands for double mutation of Ser450A and Ser495A residues. The experiments were repeated 3 times with similar results.