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. 2018 Mar 21;14(3):e1007248. doi: 10.1371/journal.pgen.1007248

Fig 5. MeJA greatly enhanced FIN219 and GUS-CCT1 interaction under continuous blue light.

Fig 5

(A) Schematic diagrams of different constructs corresponding to the full-length FIN219 (GST-FIN219), the N terminus and C terminus of FIN219 (GST-FIN219N and GST-FIN219C) and the full-length cryptochrome CRY1 (CBP-CRY1), the N terminus and C terminus of CRY1 (CBP-CNT1 and CBP-CCT1). (B) Pull-down assay of FIN219 interacting with CRY1 via its C terminus. Recombinant proteins GST, GST-FIN219, GST-FIN219N or GST-FIN219C were mixed with CBP-CRY1 and underwent protein pull-down assays (left panel). The asterisk (*) indicates nonspecific bands. The arrowhead represents GST-FIN219C. Right panel: Recombinant proteins CBP-CRY1, CBP-CNT, or CBP-CCT1 were mixed with GST-FIN219 and underwent protein pull-down assays. The mixtures were immunoprecipitated with glutathione sepharose for GST-tag, then probed with antibodies against CBP-tag. Upper and lower arrowheads represent CBP-CRY1 and CBP-CCT1, respectively. (C) BiFC assays showing FIN219 and CRY1 or COP1 interaction in the dark (top panel) and blue light (BL) (bottom panel). The protoplasts isolated from short-day grown Col-0 were transfected with YN-CRY1 or COP1 and YC-FIN219 without (–) or with (+) 50 μM MeJA treatment. Blue light (BL): 2.2 μmol•m-2•s-1. (D) Co-immunoprecipitation assay showing FIN219 and GUS-CCT1 interaction greatly enhanced by MeJA under blue light. GUS-CCT1 transgenic seedlings were grown in the dark and blue light for 4 days with (+) or without (–) MeJA. Total proteins 2 mg extracted from seedlings were immunoprecipitated with FIN219 monoclonal antibodies, then probed with GUS and COP1 polyclonal antibodies. (E) Co-immunoprecipitation assay showing FIN219 and CRY1 interaction greatly enhanced by MeJA under the dark. Wild-type Col-0 and fin219-2 seedlings were grown in the dark and blue light for 4 days with (+) or without (–) MeJA. Total proteins 2 mg extracted from seedlings were immunoprecipitated with FIN219 monoclonal antibodies, and then probed with CRY1 polyclonal antibodies.