Fig. 3: GmILPA1 interacts with GmGA2ox-like and GmUBL1. | Nature Communications

Fig. 3: GmILPA1 interacts with GmGA2ox-like and GmUBL1.

From: UV-B irradiation-activated E3 ligase GmILPA1 modulates gibberellin catabolism to increase plant height in soybean

Fig. 3

a Yeast two-hybrid assays showing the interactions among GmILPA1, GmGA2ox-like, and GmUBL1. GmILPA1 and GmUBL1 were fused to GAL4-BD, and GmGA2ox-like was fused to GAL4-AD when encoded by the pGADT7 and pGBKT7 vectors. Serial dilutions of equivalent amounts of yeast were plated on synthetic defined (SD) medium without Leu (L), Trp (T) (SD-L-T), or without Leu (L), Trp (T), and His (H) (SD-L-T-H) containing 1 mM 3-amino-1,2,4-triazole (3-AT), with growth on triple dropout media indicating an interaction between the tested proteins. b A BiFC assay used to verify the interactions among GmILPA1, GmGA2ox-like, and GmUBL1 in vivo in N. benthamiana leaves. The non-interacting proteins, mGmGA2ox-like (mutated construct of D-Box motif) and GmPUB21 (a U-Box E3 Ubiquitin Ligases), were used as a negative control. Scale bars, 50 μm. c–e Co-IP of GmILPA1, GmGA2ox-like, and GmUBL1. Immunoprecipitation was performed with anti-GFP-agarose beads using N. benthamiana leaves co-infiltrated with the constructs GmGA2ox-like-GFP and GmILPA1-MYC (c), GmUBL1-GFP and GmILPA1-MYC (d), or GmUBL1-GFP and GmGA2ox-like-MYC (e). f BiFC assays indicating that UV-B treatment promotes the interaction between GmILPA1 and GmGA2ox-like. N. benthamiana leaves were co-infiltrated with GmGA2ox-like–nYFP and GmILPA1–cYFP and exposed to 1 h of UV-B (21 μmol m−2 s−1) or kept in white light before imaging. Scale bars, 50 μm. g Relative YFP fluorescence intensity in the cytoplasm from the images in (f), (n = 30 cells, P = 1.30 × 10−5), the relative fluorescence intensities of cytoplasm and whole cells were quantified and the cytoplasm-to- background ratios are plotted. Data are presented as mean values ± SD, Student’s t-test was used for the significance test, ***P < 0.001. h Co-IP assays showed that UV-B increased the interaction between GmILPA1 and GmGA2ox-like. N. benthamiana were co-transformed with GmGA2ox-like-GFP and GmILPA1-MYC, and treated with or without 12 h UV-B. Then, they were used in the co-immunoprecipitation assay. Immunoprecipitation was performed using GFP-Trap agarose beads, and the immunoblots were probed using anti-myc and anti-GFP antibodies. Source data are provided as a Source Data file.

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