OsGSK2 interacts with and phosphorylates OsOFP8.
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(A) Yeast two-hybrid analysis for the interaction between OsOFP8 and OsGSK2, DLT, and OsBZR1. Co-transformed yeast clones were placed on SD dropout plates to detect the interactions. SD-Leu-Trp: synthetic complete medium lacking Leu and Trp for co-transformation detection. SD-Leu-Trp-His-Ade: synthetic complete medium lacking Trp, Leu, His and Ade for interaction detection. (B) Immunoprecipitation (IP) assay shows that OsOFP8 is in association with OsGSK2. Arabidopsis protoplasts expressing either YFP and 3HA-OsGSK2 or OsOFP8-YFP and 3HA-OsGSK2 were subjected to protein extraction. The Input (cell lysate) and IP were immunoblotted with indicated antibodies. WB: GFP indicates western blotting with GFP antibody. WB: HA indicates western blotting with HA antibody. (C) BiFC assay shows the interaction between OsOFP8 and OsGSK2. Chl means chlorophyll. (D) OsOFP8 phosphorylation analysis. Arabidopsis protoplast cells expressing OsOFP8-YFP only or OsOFP8-YFP with 3HA-OsGSK2 were subjected to protein extraction and then immunoprecipitated with either GFP antibody (WB: ant-GFP) or HA antibody (WB: anti-HA). Phosphorylation was detected with biotin-pendant Zn2+-Phos-tag (BTL-111). Black and white arrowheads indicate OsOFP-YFP and 3HA-OsGSK2, respectively. Black and white arrows indicate phosphorylated OsOFP8-YFP-P and 3HA-OsGSK2-P, respectively. * represents the internally phosphorylated OsOFP8-YFP.