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. 2021 Jan 16;185(4):1875–1893. doi: 10.1093/plphys/kiab013

Figure 3.

Figure 3

BR enhances PuBZR1–PuACO1 interaction. A, B, Interaction of PuBZR1 and PuACO1 in a BiFC assay. Nicotiana benthamiana leaves were co-infiltrated with PuBZR1-cYFP and PuACO1-nYFP constructs and kept in the dark for 48 h, and then EBR was injected into the infiltrated leaves. Nicotiana benthamiana leaves were visualized by confocal microscopy 4 h after EBR injection (A). These leaves were also visualized at 0, 4, and 12 h after EBR injection (B). NF-YA4-mCherry was used as a nuclear marker. PuBZR1C-cYFP with PuACO1-nYFP, PuBZR1-cYFP with nYFP, and cYFP with PuACO1-nYFP, were used as negative controls. Scale bars, 50 μM. C, A firefly Luc complementation imaging assay showing that EBR treatment enhanced the interaction between PuBZR1 and PuACO1 in N. benthamiana leaves. Agrobacterium tumefaciens strain EHA105 harboring different constructs was infiltrated into N. benthamiana leaves. Untreated, N. benthamiana leaves not receiving any treatment; EBR, N. benthamiana leaves treated with EBR. Luc activities were recorded in these regions 3 d after infiltration. Bar, 1 cm. The black lines were used to indicate the different regions on each leaf. D, A firefly Luc complementation imaging assay showing that Brz treatment weakened the interaction between PuBZR1 and PuACO1 in N. benthamiana leaves. Bar, 1 cm. E, A co-IP assay showing that EBR treatment enhanced the interaction between PuBZR1 and PuACO1. PuBZR1 fused to a Myc tag (PuBZR1-Myc) and PuACO1 fused to a GFP tag (PuACO1-GFP) were overexpressed in N. benthamiana leaves, and a Myc antibody was used for immunoprecipitation analysis. Myc and GFP antibodies were used in an immunoblot analysis. The band detected by the GFP antibody in the precipitated protein sample indicates the interaction between PuBZR1 and PuACO1 (Lane 5) and EBR treatment enhances the interaction (Lane 7). F, A co-IP assay showing that Brz treatment weakened the interaction between PuBZR1 and PuACO1. The co-IP assay was performed as in (E). The band detected by the GFP antibody in the precipitated protein sample indicates the interaction between PuBZR1 and PuACO1 (Lane 5) and Brz treatment weakens the interaction (Lane 7).