Extended Data Fig. 9: ZnF interacts with TaBRI1 and TaBKI1, and ZnF mediates TaBKI1 ubiquitination and degradation.
From: Reducing brassinosteroid signalling enhances grain yield in semi-dwarf wheat
a,b, BKI1 (a) and BRI1 (b) protein sequence alignment between Arabidopsis thaliana and Triticum aestivum. Point mutations in putative phosphorylation sites of BKI1 in Arabidopsis or wheat is marked with red boxes. The sequence of kinase ___domain is underlined by the red dashed-arrows. c, The protein levels of TaBRI1 in Fielder and a znf-aabbdd mutant (#2) were quantified by ImageJ software. Actin served as a loading control (n = 3 independent experiments). d, The firefly luciferase (LUC) complementation imaging (LCI) assay shows no interaction between ZnF and TaBAK1. e, LCI assays confirmed that the co-expression of TaBRI1 with TaBKI1 and ZnF enhanced the interaction between TaBKI1 and ZnF. The combination ratios of A. tumefaciens strains harboring different expression vectors were indicated (n = 4 independent experiments in upper panels, n = 3 independent experiments in lower panels). f, Comparison of the His-TaBKI1 degradation rates in the cell extracts between Fielder and znf-aabbdd (#2) in a cell-free degradation assay (n = 3 independent experiments). g, In vitro self-ubiquitination activity of the MBP-tagged ZnF-CT314–473aa containing the intact RING ___domain. Ubn stands for ubiquitin conjugates. h, In vitro ubiquitination of His-TaBKI1 by ZnF-CT. Asterisk indicates the nonspecific bands. i, Differential interaction intensities of ZnF with TaBKI1 and its mutant forms. The wild type TaBKI1-nLUC, the mutant TaBKI1Y153D-nLUC (Tyr-to-Asp mutation at the 153 residue), and TaBKI1Y153F-nLUC (Tyr-to-Phe mutation at the 153 residue) were separately co-expressed with cLUC-ZnF, and the interaction intensities represented by LUC signals were analyzed with IndiGo software (n = 3 independent experiments). In c,e,f, asterisks indicate the significant differences (* P < 0.05; ** P < 0.01; ns, no significant difference) from two-tailed Student’s t-tests. Different letters in i indicate significant differences (P < 0.05, one-way ANOVA, Tukey’s HSD test). In d,g,h, all experiment was repeated independently at least twice with similar results. Data are mean ± s.d. (for P values, see Source Data).
