Fig. 3
Interaction of OsIMαΔIBB1a and OsIMαΔIBB1b with the WD of OsWRKY62. a WD sequences of OsWRKY62 and OsWRKY76. The basic amino acids in red are mutated to Alanine. The amino acid differences between W62WD and W76WD are underlined in W76WD. b Pull-down assays of OsIMαΔIBB1a and OsIMαΔIBB1b interacting with W62WD and its mutants. The W62WD and its mutants were sandwiched with GST and 3myc tags and expressed. Each protein (about 1 µg) was incubated with GST-IMαΔIBB1a-3flag or GST-IMαΔIBB1b-3flag at 4 °C for 3 h in the IP buffer. The protein complexes were precipitated, washed five times with the IP buffer, separated on 10% SDS-PAGE gels, and detected by western blots with anti-Myc and anti-Flag antibodies. GST-3myc was used as a negative control. Similar results were obtained from three repeats. WDAA, WDAAA, and WD5A for RK, KKK, and both of them in W62WD, respectively, were all mutated to Alanines. c The concatenated basic amino acids in W62WD were important for nuclear localization. W62WD and W62WD5A were cloned in frame with GFP-GUS chimeric gene, respectively. The generated plasmid in combination with 35S::IMαΔIBB1a-dsRED were introduced into the leaf cells of N. benthamiana. Confocal images were taken 72 h after the agroinfiltration. From left panel to right: GFP images (GFP), dsRED images (RED), and combined GFP and RED in the bright field (Merged). d Interactions of IMαΔIBB1a with W62WD and AvrPib. W62WD, AvrPib, and their mutants were fused in frame with YFP N-terminal region (YFPN), and IMαΔIBB1a was fused with YFP C-terminal region (YFPC). The plasmids indicated were transformed into N. benthamiana leaves. Confocal images were taken at 72 h after the treatments. Red fluorescence (35S::dsREDNLS) shows nuclear localization. From left panels to right: YFP images (YFP), dsRED images (RED), and combined YFP and RED in the bright field (Merged). Bar = 20 µm