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. 2021 Sep 1;188(1):540–559. doi: 10.1093/plphys/kiab420

Figure 5.

Figure 5

MdBBX7 interacts with MdMIEL1 in vivo and in vitro. A, Schematic diagrams of MdBBX7 deletions and MdMIEL1 deletions. F3 contains MdMIEL1 aa 1–198, whereas F4 contains MdMIEL1 aa 141–267. B, Y2H assays showing the domains responsible for the interaction between MdBBX7 and MdMIEL1. Yeast cells were grown on SD–Ade–His–Leu–Trp and subjected to x-gal assay. C, Interaction of MdBBX7N (N terminus of MdBBX7 containing the two B-box motifs) with full-length MdMIEL1 by Y2H analysis. D, Firefly luciferase complementation imaging assay for the MdBBX7 and MdMIEL1 interaction in tobacco (N. benthamiana). E, Pull-down assay analyzing the interaction of MdBBX7-N214 with MdMIEL1 in vitro. MBP-MdMIEL1 and GST-MdBBX7-N214 proteins expressed in E. coli were purified and used in this analysis. F, BiFC assay showing that MdBBX7 and MdMIEL1 interact in tobacco. Bars = 25 μm. G, In vivo interaction of MdBBX7 and MdMIEL1 detected by Co-IP assay. Proteins were extracted from 4-week-old MdBBX7 OE plants leaves, and immunoprecipitated with anti-MdBBX7 antibody. Western blotting was performed with anti-MdBBX7 or anti-MdMIEL1.