Figure 3.

OsBAG4 Interacts with OsMYB106 In Vivo and In Vitro.

(A) Immunoblot analysis of the coimmunoprecipitates from the co-IP assay. The 35S_pro:OsBAG4-GFP (OsBAG4-GFP) or 35S_pro:GFP (GFP) construct was cotransfected with 35S_pro:FLAG-OsMYB106 (FLAG-OsMYB106) into rice protoplasts. co-IP was performed using anti-FLAG antibody, and coimmunoprecipitated proteins were detected using anti-GFP antibody. Three biological repeats were performed, yielding similar results.

(B) Results of BiFC assay. OsBAG4, OsMYB106, and OsDjC51 were fused to the N- or C-terminal half of Venus (OsBAG4-nV, OsBAG4-cV, OsMYB106-nV, OsMYB106-cV, OsDjC51-nV, and OsDjC51-cV). OsBAG4-nV and OsMYB106-cV, OsBAG4-cV and OsMYB106-nV, OsBAG4-nV and OsDjC51-cV, or OsBAG4-cV and OsDjC51-nV were coexpressed with NLS-RFP (nuclear marker). Similar results were observed in at least 50 cells from three independent experiments. Bars = 10 μm.

(C) In vivo split firefly luciferase complementation assay to test the interaction between OsBAG4 and OsMYB106. OsBAG4 fused with FLucN (OsBAG4-FLucN) was coexpressed with OsMYB106 fused with the C-terminal half (OsMYB106-FLucC) in NIP protoplasts. FLucC and FLucN vectors were used as negative controls. Luciferase activities were measured after 8- and 12-h incubation. Data represent means ± sd (n = 3, transfection experiments were performed three times). Individual values (black circles) are shown.

(D) In vitro pull-down assay to detect the direct interaction between OsBAG4 and OsMYB106. GST-OsBAG4 or GST was incubated with His-OsMYB106 or His-GFP and pulled down with glutathione–agarose beads, followed by immunoblotting with anti-His and anti-GST antibodies. GST and His-GFP were used as negative controls. Three biological repeats were performed, yielding similar results. Red asterisks indicate nonspecific bands; blue asterisks indicate broken bands.

(E) Schematic representation of domain structures of OsMYB106.

(F) Pull-down assay to identify the domains of OsMYB106 that interact with OsBAG4. GFP, OsMYB106-R2R3, and OsMYB106-T were fused with the N-terminal His epitope and incubated with GST or GST-OsBAG4, and pulled down with glutathione–agarose beads followed by immunoblotting with anti-His and anti-GST antibodies. GST and His-GFP were used as negative controls. Blue asterisks indicate broken bands.