Figure 6.

Nuclear localization and DNA-binding activity of GaWRKY1. A, Subcellular localization of GFP (1 and 2) and GFP-GaWRKY1 fusion protein (3 and 4) in BY2 cells. B, Yeast one-hybrid assay using the 3×W-box as bait. Yeast cells carrying pGAD424 or pGAD-GaWRKY1 were grown for 3 d at 30°C in SD/−His/−Ura/−Leu or SD/−His/−Ura/−Leu plus 5 mm 3-amino-1,2,4-triazole (3-AT), respectively. C, EMSA performed by incubation of the purified GST-GaWRKY1 protein with ³²P-labeled DNA fragment of 3×W-box. The three lanes stand for ³²P-labeled 3×W-box (1), DNA fragment of the pBSK vector without the binding site as a nonspecific competitor (2), and 50-fold excess of the unlabeled 3×W-box as a competitor (3). The 3×W-box contained triple tandem copies of the W-box palindrome of CAD1-A promoter.