Y2H and bimolecular fluorescence complementary Imaging Assays
The full-length CDS of TaUBC24 was cloned into the DNA-binding domain vector pGBKT7 (BD). The full-length CDS of TaICE1 was amplified and cloned into the activation domain fusion vector pGADT7 (AD). Yeast strain AH109 was cotransformed with TaICE1-AD and BD-CsUBC24. Positive clones were selected on a synthetic dropout medium (SD/-Trp/-Leu). The interactions were tested on SD/-Trp/-Leu/-His/-Ade medium containing X-agalactosidase. Activation was observed after 3 d of growth on selection plates.
To generate TaUBC24 -GFPN andTaICE1 -GFPC, the full-length CDS ofTaUBC24 and TaICE1 genes were cloned into the upstream of GFPN and GFPC sequences in pCMBIA2300-BiFC vector. The resulted constructs were transiently coexpressed into N. benthamiana leaves for 2 d to generate split GFP proteins for bimolecular fluorescence complementation (BiFC) assays. The GFP (excitation wavelength at 488 nm) signals resulted from protein-protein interactions in leaves were observed under the fluorescent microscope.