Yeast Two-Hybrid Assay

The yeast two-hybrid screening assays were conducted according to the instructions for the Matchmaker Gold yeast two

hybrid system (Clontech Laboratories, Mountain View, CA,United States). Gene-specifific primers was designed to amplify sequence corresponding to the open reading frame. As the full-length cry1Ab/c gene selfactivates, it was cleaved into three segments according to its three difffferent domains as described previously (Fu et al., 2020). The three fragments of Cry1Ab/c  were cloned into the pGBKT7 vector  having the Gal4 DNA-binding domain (BD; Clontech, Madison, WI, USA) as a bait. Complementary DNA (cDNA) was prepared from total RNA, extracted from rice plants mixed developmental stages using Trizol (Invitrogen, Carlsbad, CA, USA). The library was cloned into the pGADT7 vector having the Gal4 transcriptional activation domain (AD) as a prey.  The vectors harboring the cDNA library and the Cry1Ab/c were transformed into the yeast cells Y2H Gold and Y187, respectively, which were examined using double-defificiency SD media lacking tryptophan and leucine (SD-TL) and quadruple-defificiency SD media lacking tryptophan, leucine, histidine and adenine (SD-TLHA) with X-α-gal and Abar. Positive colonies on the quadruple-defificiency screening plates were selected as templates and PCR amplifification and sequencing were conducted to detect the inserted fragments. The NCBI database was consulted for gene identifification. Five of the 60 cDNAs encoded Hd3a and were represented by almost full-length Hd3a open reading frames. 

         In a  yeast  two-hybrid  assay,  the yeast plasmids, PGBKT7-Hd3a and PGADT7-Cry1Ab/c were co-transformed into  competent Y2H Gold yeast cells. Centrifuge to pellet yeast cells, discard the supernatant and resuspend in 0.9% (w/v) NaCl solution. Transformants were culturing on SD-TL/X-α-gal, SD-TLHA/ X-α-gal/Abar plates for blue colour development at 30℃ .