An improved biolistic-based method (BPIS) for identifying protein interactions of transcription factors in plants

Transcription factors (TFs) play a crucial role in gene regulation. They drive chromatin remodeling, transcription, mRNA splicing, and RNA processing via dynamic protein interactions. However, their low abundance and complex binding networks complicate the study of TF partners. Here, we present biolistic protein interaction screening (BPIS) as a method for identifying TF partners. BPIS uses biotin-labeled DNA probes (5' end) and cyanine 3 (Cy3, 3' end) to bind the target TF and form DNA-protein complexes. This complex is introduced into plant cells via biolistic transformation, enabling interaction with target protein partners to form larger complexes. The complexes are purified with streptavidin beads, separated by non-denaturing gel electrophoresis, and the target bands are excised under Cy3 fluorescence for mass spectrometry. We employed BPIS to identify BpLOB1 partners in Betula platyphylla and AtAST1 partners in Arabidopsis thaliana, confirming interactions with specificity rates of 73.17% and 77.27%, respectively, demonstrating high reliability. BPIS features two key improvements: dual-labeled DNA probes facilitate two rounds of purification to minimize background noise, and introducing TF-DNA complexes into cells enhances TF abundance for isolating interacting proteins in their native environment. It could serve as a robust platform for future research in gene regulation and protein interactions.