Figure 9.

In vivo transactivation of SlARF2 promoters by SlERF.D7. A and B, Schematic diagrams of the reporter and effector constructs. The GUS and GFP reporter plasmids contain the promoters of SlARF2A and SlARF2B. The effector plasmids contain the SlERF.D7 CDS under the control of the CaMV35S promoter. C, Confirmation of the activation of SlARF2A and SlARF2B promoters by SlERF.D7 via the GUS complementation assay in N. benthamiana leaves. GUS assay was performed after 48 h of infiltration. GUS activity is expressed in nmole 4-methylumelliferone mg⁻¹ protein min⁻¹. Activation of the SlACS2 promoter by RIN-: positive control, Activation of the SlARF3 promoter by SlERF.D7-: negative control. Error bars represent ±sd of three biological replicates. Asterisks indicate the statistical significance using Student’s t test: *, 0.01 < P-value < 0.05; **, 0.001 < P-value < 0.01. D and E, Transient expression assay shows that SlERF.D7 activates the expression of the GFP reporter gene. The bottom panel indicates the combination of reporter and effector plasmids infiltrated. The GFP reporter gene is driven by ARF2A and ARF2B promoters. Representative images of N. benthamiana leaves 48 h after infiltration captured via NightSHADE Plant Imaging System are shown. Activation of the SlACS2 promoter by RIN-: positive control, activation of the SlARF3 promoter by SlERF.D7-: negative control. E, Quantitative analysis of fluorescence intensity in three independent determinations was assessed. Error bars represent ±sd of three biological replicates. Asterisks indicate the statistical significance using Student’s t test: *, 0.01 < P-value < 0.05; **, 0.001 < P-value < 0.01.