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.