Development of dual-visible reporter assays to determine the DNA-protein interaction.
Huayu SunSining WangKebin YangChenglei ZhuYan LiuZhimin GaoPublished in: The Plant journal : for cell and molecular biology (2023)
DNA-protein interaction reporter assays application for relational or ratiometric measurements within an experimental system are popular in biology research. However, the existing reporter-based interaction assays always require special equipment, expensive chemicals, and complicated operation. Here, we developed a DNA-protein interactions technology integrating two visible reporters, RUBY and UV-visible GFP (eYGFPuv), that allows the expression of the cassette reporter contained cis-acting DNA element (DE) fused upstream of TATA box and RUBY, and a constitutive promoter regulating eYGFPuv in the same construct. The interaction of transcription factor (TF) and the DE can be detected by co-expressed the cassette reporter and TF in tobacco leaves where the cassette reporter alone serves as a control. We also revealed that eight function-unknown bamboo AP2/ERFs interacted with the DE of ANT-AP2R1R2 (ABE), DRE (DBE), GCC-box (EBE), and RAV1 binding element (RBE) respectively, which are consistent with the results by dual-luciferase reporter assays. Thus, the dual-visible reporters offer a convenient, visible, and cost-saving way alternative to other existing techniques for DNA-protein interaction in plants.