Fig. 1

Copper-inducible reporter gene activation in transient expression in Nicotiana benthamiana. a Schematic representation of the copper-inducible gene expression system. The CUP2 fused to an activation domain such as Gal4 changes its conformation upon copper binding and binds a Copper Binding Site (CBS) operator allowing transcription of the downstream gene (green arrow). b Copper-mediated activation of Firefly Luciferase (FLuc) reporter gene by different activation domains (Gal4, VP16, VPR, and TV) fused to CUP2 after 5 mM copper sulfate application. c Copper-mediated activation of FLuc by different concentrations of copper sulfate. The construct CUP2:Gal4 was used as the copper-dependent transcriptional factor. d Pictures of leaves 2 days after treatment with different concentrations of copper sulfate. For b and c, activation is expressed as Normalized FLuc/RLuc ratios of N. benthamiana leaves transiently expressing the genetic constructs. All FLuc/RLuc ratios were normalized using the FLuc/RLuc ratios of N. benthamiana leaves expressing a constitutive pNOS:Fluc reporter. Error bars indicate SD (n ≥ 3). Statistical analyses were performed using one-way ANOVA (Tukey’s multiple comparisons test, P-Value ≤ 0.05). Variables within the same statistical groups are marked with the same letters. The figure includes images from Biorender (biorender.com)