Screening of Populus simonii×Populus nigra WRKY70-interactive proteins by the yeast two-hybrid system

Populus simonii×Populus nigra WRKY70 (PsnWRKY70) is a salt stress responsive transcription factor. In order to further identify the proteins that interact with PsnWRKY70 in salt stress response pathway, we used P. simonii×P. nigra leaves treated by 140 mmol/L NaCl solution as materials to construct a homogeneous pGADT7-DEST yeast two-hybrid cDNA library by DSN. PsnWRKY70 gene was subcloned to pGBKT7 vector to construct BD-WRKY recombinant plasmid, and then the BD-WRKY plasmid was used as a bait to screen the yeast two-hybrid cDNA library of P. simonii×P. nigra. After twice screening tests and a rotary experiment, five proteins that interact with PsnWRKY70 were detected. We analyzed the conserved domains of the five proteins and found that there were two hypothetical proteins (HP1 and HP2, HP1 contains a ClpP domain), one cyclase associated protein (CAP1), one RNA recognition motif-containing family protein (RRM) and one Ulp1 protease family protein (Ulp1) among them. The cis-elements that exist in 2 000 bp upstream of the orthologous Populus trichocarpa genes of CAP1, HP1, RRM, HP2 and Ulp1 were also analyzed, and the results suggest that the promoter regions of all the five orthologous P. trichocarpa genes contained plenty of W-box which can specifically bind to WRKY transcription factors. GST-pull down technology was utilized to validate the direct interaction between the complete CAP1/HP1/RRM/HP2/Ulp1 proteins and PsnWRKY70. An equal amount of His-X (X: CAP1/HP1/RRM/HP2/Ulp1) fusion proteins was dropped into normal glutathione agarose resin and GST or GST-WRKY protein-bound glutathione agarose resin respectively, then the interactive proteins were separated by SDS-PAGE. The final Western blot analysis indicated that HP1, RRM and Ulp1 can directly interact with PsnWRKY70 in vitro, while CAP1 or HP2 cannot.