Fig. 1
Interaction of OsIMα1a and OsIMα1b with OsWRKY76. a Schematic diagrams of OsWRKY76.1 (W76.1), OsIMα1a (IMα1a), OsIMα1b (IMα1b), and their deletion mutants. NLS, nuclear localization signal; WD, WRKY domain; ARM, armadillo repeats; IBB, importin-β-binding domain; XB, exportin binding domain. b W76.1 and its deletion mutants were fused to the Gal4 DNA-binding domain (BD). IMα1a, IMα1b, IMαΔIBB1a, and IMαΔIBB1b were fused to the Gal4 activation domain (AD). Yeast cells with serial dilutions (100, 10–1, and 10–2) were incubated in synthetic dropout medium lacking Leu and Trp (left panel) or Leu, Trp, His, and Ade (right panel) and photographed 3 days after plating. c Pull-down assays of OsIMαΔIBB1a and OsIMαΔIBB1b interacting with OsWRKY62 and OsWRKY76. All proteins were purified with their N-termini fused with GST and their C-termini fused with 3×flag or 3×myc tag. Each protein (about 1 µg) with its corresponding tag combination was incubated at 4 °C for 3 h in the immunoprecipitation (IP) buffer. The protein complexes were precipitated with anti-Flag affinity gel, washed five times with the IP buffer, and separated on 10% SDS-PAGE gels. The proteins were detected by western blots with anti-Myc and anti-Flag antibodies. Similar results were obtained from three repeats. NLS, nuclear localization signal; WD, WRKY domain