Fig. 3: Interaction of NPH3 and 14-3-3 proteins is triggered by blue light irradiation and abolished by mutation of the third last NPH3 residue. | Nature Communications

Fig. 3: Interaction of NPH3 and 14-3-3 proteins is triggered by blue light irradiation and abolished by mutation of the third last NPH3 residue.

From: Light-triggered and phosphorylation-dependent 14-3-3 association with NON-PHOTOTROPIC HYPOCOTYL 3 is required for hypocotyl phototropism

Fig. 3

a Yeast two-hybrid interaction analysis of the Arabidopsis 14-3-3 isoform omega with NPH3 variants (upper panel). Expression of the diverse NPH3 fusion proteins was confirmed by anti-HA immunoblot of yeast extracts (lower panel). AD, activating domain, BD, binding domain. b In vivo interaction of 14-3-3 omega:mEGFP and either mCherry:NPH3 or mCherry:NPH3-S744A in transiently transformed N. benthamiana leaves. Expression was driven by the 35S promoter. Freshly transformed tobacco plants were kept under constant light for 42 h. The crude extract was immunoprecipitated using GFP beads. Input and immunoprecipitate (IP) were separated on 11% SDS-PAGE gels, followed by immunoblotting with anti-GFP and anti-RFP antibodies, respectively. c Arabidopsis 14-3-3 epsilon interactors were identified by mass spectrometry analysis of anti-GFP immunoprecipitations from etiolated seedlings expressing 14-3-3 epsilon:GFP and either maintained in darkness or irradiated with blue light (BL) (1 μmol m−2 s−1) for 30 min (two biological replicates). Expression was driven by the native promoter. Protein intensities of 14-3-3 client proteins were normalized to relative abundance of the bait protein (Supplementary Table 1). Fold changes in relative abundance (mean ± SD, logarithmic scale) of blue light treatment vs. darkness are given. AHA1, AHA2, Arabidopsis H+-ATPase; CINV1, cytosolic invertase 1; EIN2, ethylene insensitive 2; PhyA, phytochrome A; SPS1, sucrose phosphate synthase 1. d In vivo interaction of 14-3-3 omega:mEGFP and mCherry:NPH3 in transiently transformed N. benthamiana leaves. Expression was driven by the 35S promoter. Dark-adapted tobacco plants were either kept in darkness (D) or treated with BL (10 μmol m−2 s−1) for 40 min. The crude extract was immunoprecipitated using GFP beads. Input, flowthrough (FT) and IP were separated on 11% SDS-PAGE gels, followed by immunoblotting with anti-GFP and anti-RFP antibodies, respectively. Experiments in a, b, and d were performed at least three times with similar results.

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