Figure 6.

CaWRKY50 subcellular localization in plant cell and yeast one-hybrid assays. (A) CaWRKY50-EYFP fusion protein localizes to onion cell nucleus. The CaWRKY50-EYFP fusion and NLS-mCherry nuclear marker genes were transiently expressed in onion cells and visualized after 2 days. Bar is 20 µm for EYFP panel and 50 µm for CaWRKY50-EYFP panel. (B) CaWRKY50 transactivates reporter gene by its C-terminal region in yeast one-hybrid system. Full-length and four truncated forms of CaWRKY50 were fused with GAL4 DNA-binding domain in pGBKT7 vector. Yeast transformation and β-gal activity (in miller units) were quantified to check the strength of transcativation. (C) Sequences of pChn5 oligonucleotides cloned in pHis2.1 vector, underlined are two W-boxes and their mutated mW-box forms. (D) Yeast one-hybrid analysis of CaWRKY50 against its own 1.2 kb promoter (proW50) and pChn5 W-boxes. Positive (pHis2.1-pro53 + pGADT7-p53) and negative (pHis2.1 + pGADT7-p53; pHis2.1-mW-box + pGADT7-CaWRKY50) controls are also included. The growth of yeast cells co-transformed with positive controls, proW50, and W-box clones suggests interaction of CaWRKY50 with own promoter and W-box. This figure is available in black and white in print and in colour at DNA Research online.