A yeast library-hybrid assay to screen maize-Rhizoctonia transcription factors and protein-protein interactions in one experimental pipeline

Yeast one- (Y1H) and two-hybrid (Y2H) assays are widely used to study transcription factors (TFs) and protein-protein interactions (PPIs), respectively. Here we combined the Y1H and Y2H into a yeast library-hybrid (YLH) assay, which can systematically screen transcription factors (TFs) and PPIs in one experimental pipeline. In typical TFs, the DNA-binding domain (DBD) and activation domain (AD) evolved separately, but were covalently linked. In the YLH assay, TFs are identified based on functionally conserved ADs, whereas in various Y1H assays TFs are identified based on DBDs. Using the YLH method, we isolated 51 pairs of maize-Rhizoctonia PPIs and 38 novel Rhizoctonia solani TFs. TFs and PPIs related to pathogen virulence and plant resistance responses were isolated by the YLH assay. Our results show that 57.75% of isolated TFs contain typical DBDs such as Zn2Cys6, nucleic acid-binding OB-fold, winged helix repressor DNA-binding, and zinc finger CCHC-type. Key PPI pairs related to major functional categories such as metabolism and cellular signalling were obtained. The percentage of verified PPIs is 69.39%. We proved that common TFs have nonspecific or broad-spectrum activities in the yeast plasmid gene expression system. YLH screening can be conducted on library scales to systematically reveal possible TFs and PPIs at the same time.