Figure 6. CsJAZ6 regulates catechin biosynthesis through physically interacting with CsEGL3 and CsTTG1.
(a) Yeast two-hybrid assay to confirm the interaction of CsJAZ6 and CsEGL3/CsTTG1. The co-transfected yeast competent cells were grown on SD/-LT medium with or without ABA. The empty vector pGADT7 was used as a negative control. (b) BiFC analysis of the interaction between CsJAZ6 and CsEGL3/CsTTG1 in vivo . Leaves fromtobacco were infiltrated with agrobacteria as indicated. The N- and C-terminal fragments of YFP (nYFP and cYFP) were fused to the C-terminus of CsJAZ6 and CsEGL3/CsTTG1, respectively. Combinations of nYFP or cYFP with the corresponding CsJAZ6 or CsEGL3/CsTTG1 constructs were used as negative controls. Bars, 20 μm. (c-d) Co-IP assay of CsJAZ6-CsEGL3/CsTTG1 binding. Recombinant CsEGL3/CsTTG1-Flag protein was incubated with total proteins isolated from tobacco leaves expressing CsJAZ6-Myc driven by the 35s promoter. Anti-Flag antibody was used to detect CsEGL3 or CsTTG1 fusion proteins in solutions before (Crude) and after (Pull-down) immunoprecipitation. KD, Kilodalton.(e) Yeast three-hybrid assay investigating the influence of CsJAZ6 on the CsAN1-CsEGL3 interaction. CsAN1-CsEGL3 binding activity is represented by β-galactosidase activity, and the promoter drivingCsJAZ6 expression was suppressed by increasing Met concentration. Error bars indicate the SD of three technical replicates; the results were consistent across the three biological replicates. Error bars indicate the SD of three biological replicates. (f) Structural schematics of the effector (35S:CsHSFA1b and 35S:CsHSFA2 ) and reporter (CsJAZ6Pro::LUC ) constructs used for the dual-luciferase transient expression assay. (g) Relative firefly luciferase (LUC) to Renilla luciferase (REN) ratios from transient expression assays. These represent the activity of theCsANS promoter in the absence/presence of MBW (CsAN1-CsEGL3-CsTTG1) complex and the combination of MBW-CsJAZ6. Error bars indicate the SD of three biological replicates. Different letters above the bars indicate significant differences between groups (P<0.05; ANOVA with Fisher’s LSD test). Error bars indicate the SD of three biological replicates.