Fig. 2
In planta heterodimerization of WRKY18, WRKY25, and WRKY53 in N. benthamiana, and transactivation assays in Arabidopsis protoplasts for the effect of WRKY18/WRKY25 heterodimer effect on the PWRKY53. (A) Leaves of N. benthamiana were transformed with pBiFCt2in1-NN constructs containing the possible combinations for the heterodimerization of the three WRKYs of the subnetwork. The transformed leaves were analyzed under a confocal laser scanning microscope: yellow fluorescence (YFP) indicates interaction (BiFC), and red fluorescence (RFP) serves as a transformation control. Boxplots representing the relative fluorescence ratio (%YFP/RFP) are presented. Sample size (n) is indicated in the figure and represents independent biological replicates. (B) Arabidopsis protoplasts were transformed with fragments of the promoters of WRKY18 (3000 bp), WRKY25 (3000 bp), and WRKY53 (2759 bp), each fused to the GUS reporter gene, along with 35S:WRKY18 (W18), 35S:WRKY25 (W25), 35S:WRKY53 (W53), or 35S:WRKY18/WRKY25 (W18 + W25) as effector constructs. Values relative to values of the empty vector control are presented as boxplots, with sample sizes (n) shown for each group within the plot representing independent biological replicates. In both (A) and (B) one-way ANOVA followed by Tukey’s HSD post-hoc test was performed. Lowercase letters indicate statistically significant differences between groups (p ≤ 0.05).
