Interaction of GmRR1 and GmHP1. A) Subcellular localization of 35S:GmRR1:GFP fusion protein in cells of Nicotiana benthamiana leaves. GFP, green fluorescence; BF, bright field; DAPI, nuclei were stained with DAPI. Scale bars, 50 µm. B) Yeast two-hybrid assay. Positive control (BD-53 and AD-T7), negative control (BD-lam and AD-T7). Sequences of GmRR1 and GmHP1 were fused to both the pGADT7 activation domain (AD, prey) and the pGBKT7 binding domain (BD, bait). The SD/-Trp/-Leu/X-α-gal (Upper) and SD/-Trp/-Leu/-His/-Ade/X-α-gal (Lower) plates were incubated at 30 °C for 3 days and then visualized. C) Bimolecular fluorescence complementation assay. GmRR1 and GmHP1 were both fused with the N-terminal half of yellow fluorescent protein (nYFP) and the C-terminal half of YFP (cYFP). YFP, yellow fluorescence; BF, bright field. Scale bars, 50 µm. D) Luciferase reporter assay confirmed the interaction between GmRR1 and GmHP1. E) Analysis of relative expression of GmHP1 in transgenic plants and mutants (n = 3). ko-1, ko-2, and ko-3 represent three different knockout GmRR1 lines; OE-1, OE-2, and OE-3 represent three different GmRR1 overexpression lines. Significant differences according to two-sided t test (***P < 0.001). Data are means ± standard error of the mean (n = 3).