Improvement of a monopartite ecdysone receptor gene switch and demonstration of its utility in regulation of transgene expression in plants.

In plants, regulation of transgene expression is typically accomplished through the use of inducible promoter systems. The ecdysone receptor (EcR) gene switch is one of the best inducible systems available to regulate transgene expression in plants. However, the monopartite EcR gene switches developed to date require micromolar concentrations of ligand for activation. We tested several EcR mutants that were generated by changing one or two amino acid residues in the highly flexible ligand-binding domain of Choristoneura fumiferana EcR (CfEcR). Based on the transient expression assays, we selected a double mutant, V395I + Y415E (VY), of CfEcR (CfEcR(VY)) for further testing in stable transformation experiments. The CfEcR(VY) mutant only slightly improved the induction characteristics of the two-hybrid gene switch, whereas the CfEcR(VY) mutant significantly improved the induction characteristics of the monopartite gene switch (VGCfE(VY)). The ligand sensitivity of the VGCfE(VY) switch was improved by 125-15 625-fold in different transgenic lines analyzed, compared to the VGCfE(Wt) switch. The utility of the VGCfE(VY) switch was tested by regulating the expression of an Arabidopsis zinc finger protein gene (AtZFP11) in both tobacco and Arabidopsis plants. These data showed that the VGCfE(VY) switch efficiently regulated the expression of AtZFP11 and that the phenotype of AtZFP11 could be induced by the application of ligand. In addition, the affected plants recovered after withdrawal of the ligand, demonstrating the utility of this gene switch in regulating the expression of critical transgenes in plants.