Abstract
Protein-protein interactions are fundamental components in the life activities of each cell. They play a pivotal role in various biological processes, including replication, transcription, translation, cell cycle regulation, and signal transduction. Distinct interaction networks are present in every species, individual, and cell. Various technical methods have been confirmed to map these interactions and to identify proteins that interact directly or indirectly. Yeast two-hybrid (Y2H) is an extensively employed system for determining the interaction sites or domains between two known proteins with physiological effects. However, the yeast dual hybrid method has certain limitations, as the autoactivation of bait proteins often lead to false positive outcomes. In this study, we optimized the assembly of bait proteins by introducing a transcriptional silencing motif (EAR inhibitory motif of SUPERMAN gene SRDX) to suppress the autoactivation. We selected five bait proteins with autoactivation activity, including ApGNAT12, ApCPP5, ApVOZ1, ApMYB2, and ApWRKY41. Notably, by introducing SDRX inhibitory motifs at the C-terminus of these proteins, the autoactivation activity of these proteins was effectively suppressed. In addition, we conducted a yeast two-hybrid library screening experiment coupled with high-throughput sequencing, using ApMYB2 as an example, and the outcomes revealed the reliability of this method. Together, our findings indicate that the inhibitory motif can effectively inhibit autoactivation in yeast two-hybrid systems, suggesting broad applications in the protein-protein interaction research.