Fig. 5
Protein–protein and protein–DNA interactions in the cells of the poplar leaves. Protein–protein interactions were illustrated with various methods using transient co-transformation in the poplar leaves by syringe infiltration under the optimal experimental parameters described above in the Results section. a. BiFC assay of AtWRKY40-YFPN and AtWRKY40-YFPC. The combination of AtWRKY40-YFPN and YFPC was used as a negative control. At 5 dpi, the infiltrated leaves were detached, and the YFP fluorescent signals were observed under a Nikon inverted fluorescence microscope TE2000-E. YFP signals and DAPI fluorescence overlapped in the nucleus. b. Split luciferase assay of AtNRT3.1-Nluc and AtNRT2.1-Cluc, showing stronger LUC activity compared to the negative control combination of AtNRT3.1-Nluc and Cluc. At 5 dpi, the transformed leaves were infiltrated with 2 mM luciferin by using a syringe without a needle, left in dark for 6 min to quench the fluorescence, and then detached for the luminescence intensity assay. The color scale shows the luminescence intensity, with blue indicating the lowest and red the highest. c. Images of the co-immunoprecipitation assay show the interaction of PtoUBC34s with PtoMYB221. PtoUBC34s-Flag was co-expressed with PtoMYB221-Myc, and protein extracts were incubated with anti-Flag coupled agarose. Immunoprecipitates (IP) and input proteins were analyzed by immunoblotting using anti-Flag and anti-Myc antibodies. The uncropped images can be found in Fig. S7. d. In vivo Förster resonance energy transfer (FRET-FLIM) measurement of co-expressing YFP-PtoUBC34s and PtoMYB221-RFP, with YFP-PtoUBC34s as the donor. The YFP-PtoUBC34s donor alone used as a negative control. The data are presented as means ±SE (n = 3). *P < 0.05, Student’s t-test. e. Dual LUC assay detected the repression ability of the EAR-like motif repression domain of SUPERMAN (SUPRD) [48, 54]. Relative LUC activities were measured after co-transformation with the reporter and effectors, where pGreen-SK was used as a control vector. The data are presented as means ±SE (n = 6). ** P < 0.01, Student’s t-test