Figure 7.

BSS1/BOP1 Interacts with BIL1/BZR1 and Inhibits BIL1/BZR1 Transport to the Nucleus from the Cytosol.

(A) Phenotypes of the wild type, bss1-1D, bil1-1D, and the bss1-1D bil1-1D double mutant grown on medium containing 3 μM Brz in the dark for 7 d. Bar = 1 mm.

(B) Phenotypes of bss1-1D, bil1-1D, and the bss1-1D bil1-1D double mutant grown in soil in long-day conditions for 40 d.

(C) Yeast two-hybrid assay between BSS1/BOP1 and BIL1/BZR1 based on measuring β-galactosidase activity. BIL1/BZR1 was fused with the binding domain (BD) and BSS1/BOP1 was fused with the prey domain (AD). Empty vector (E) was used as a control. The results are presented as means ± sd from three independent replications.

(D) Coimmunoprecipitation (IP) of BSS1/BOP1 and BIL1/BZR1. Col-0, FLAG-BIL1, BSS1-GFP, and BSS1-GFP FLAG-BIL1 seedlings were grown on plates under light for 23 d. FLAG-BIL1 was immunoprecipitated using an anti-FLAG antibody and immunoblotted using anti-GFP and anti-FLAG antibodies

(E) BiFC analysis of the interaction between BSS1/BOP1 (nEYFP-BSS1) and BIL1/BZR1 (BIL1-cEYFP) in Arabidopsis protoplast cells. The protoplast medium contained DMSO, 100 nM BL, or 3 μM Brz, and cells were cultured for 12 h. The bottom panels show protoplasts cotransformed with nYFP-BSS1 and BSS1-cEYFP. YFP, YFP fluorescence; BF, bright field; Merge, merged image of YFP and BF. Arrows point to the nucleus. Bars = 5 μm.