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. 2017 May 9;29(5):1088–1104. doi: 10.1105/tpc.16.00908

Figure 1.

Figure 1.

Rice GCN5 Interacts with WOX11 through ADA2 in Vitro and in Vivo.

(A) Yeast two-hybrid assay of ADA2 with WOX11 and GCN5. Full-length cDNAs of GCN5, ADA2, and WOX11 were cloned into AD (the prey plasmid pGADT7) or BD (the bait plasmid pGBKT7). Yeast cells transformed with the indicated plasmids were grown on control SD-LT medium and selective SD-LTHA medium.

(B) Pull-down assay of ADA2 interaction with WOX11 and GCN5. ADA2-His fusion protein or His alone (from FET32a vector) was tested for the ability to pull down WOX11-GST (left) or GCN5-GST fusion protein (right).

(C) Confirmation of interaction of ADA2 with WOX11 in rice protoplast nuclei by BiFC. ADA2-YNE (YFP N-terminal element) and WOX11-YCE (YFP C-terminal element) interaction produced a yellow signal in the nucleus. Protoplasts transformed with ADA2-YNE and YCE, and YNE and WOX11-YCE are negative controls. Ratios of YFP signal in each group of cells are shown as histograms; significant difference between test and control groups (Student’s t tests, P value < 0.01) is denoted by double asterisks.

(D) Coimmunoprecipitation assays of ADA2 and GCN5 interaction in vivo. Immunoprecipitations were performed with GCN5-flag-HA lines (Gfh3 and Gfh4) and the wild type (ZH11) using anti-HA antibody. The precipitated proteins were analyzed by immunoblots using anti-HA and anti-ADA2 as indicated.

(E) Coimmunoprecipitation assays of sADA2 and WOX11 interaction in vivo in crown root. Immunoprecipitations were performed with WOX11 overexpression lines (OxWOX11) and wox11 mutant with anti-WOX11 antibody. The precipitated proteins were analyzed by immunoblots with anti-WOX11 and anti-ADA2.

(F) Detection of WOX11-ADA2-GCN5 complex in rice cells by coimmunoprecipitation assays. Plasmids containing WOX11-GFP or GFP alone were transformed together with those containing GCN5-2XHA and ADA2 or reverse ADA2 cDNA as a control into rice protoplasts. Immunoprecipitation assays were performed using GFP-Trap agarose beads. Samples of input and immunoprecipitation products were analyzed by immunoblots using anti-GFP, anti-ADA2, anti-HA, and/or anti-WOX11 antibody as indicated. Combinations 1, 2, and 3 of plasmids used for protoplast transformation are indicated on the bottom.