Figure 4.

OsBUL1 interacts with LO9-177. A, Yeast two-hybrid systems demonstrated that OsBUL1 interacts with LO9-177, a small protein containing the KxDL motif. Also, OsBUL1 forms a homodimer and interacts with OsBUL3 and OsIBH1 but not with OsBU1, OsBUL2, and ILI1 (OsILI1). B, His pull-down assays with bacterial recombinant proteins.OsBUL1-GST fusion proteins were pulled down only with LO-177:His (6x His residues) fusion proteins. C, BiFC assays showed yellow florescent signals from reconstructed YFP in both the cytoplasm and nucleus of rice protoplasts containing YFPn:OsBUL1 and YFPc:LO9-177. The blue color is autofluorescence from chloroplasts, and nuclear localization signal (NLS)-RFP marker was used for nuclear labeling. Merged fluorescence signals include reconstructed YFP by BiFC, RFP by NLS-RFP, and autofluorescence in chloroplasts. The negative controls that were conducted by cotransfecting unfused YFP fragments together with a single complementary BiFC interactor-protein resulted in no detectable signals (Supplemental Fig. S9C). Bar = 5 µm.