Development of Tightly Regulated Ecdysone Receptor-Based Gene Switches

Genetic engineering of plants through trans gene technology is being used to enhance agronomic performance or improve quality traits in a wide variety of plant species, and has become a fundamental tool for basic research in plant biotechnology. Constitutive promoters, those that are expressed all the time in all tissues, are presently the primary means used to express trans genes in plants. However, such an always-on mechanism sometimes severely obstructs both practical applications and basic research. Metabolic energy waste, negative pleiotropic effects and potential gene escape are some of the disadvantages associated with the use of constitutive promoters. To counter these problems, gene switches that can regulate the expression of trans genes through hormone analogs and antibiotics have been developed. While most of the gene switches developed to date are useful for regulation of variety of genes for research applications, most of the ligands are impractical or inappropriate for regulation of trans genes for large-scale applications in green house and field. Ligands that are suitable for regulation of transgenes for large-scale field use are the commercially available non-steroidal ecdysone agonists. Ecdysone agonist and ecdysone receptor (EcR) have been shown to regulate gene expression in tobacco, Arabidopsis and com. These initial versions of EcR gene switches are leaky and require high concentrations (in the micromolar range) ofligand for gene activation. Our recent work solved one of the limitations. Recently, we developed a highly sensitive EcR gene switch that can support the induction oftransgenes with nanomolar concentration of ecdysone agonist in both Arabidopsis and tobacco plants. Consultations with our industrial partners revealed that highly sensitive and tight EcR gene switch will have several applications such as regulated expression of genes for pest and disease resistance, improvement in nutrition quality of food, as well as for production of products such as biopharmaceuticals in plants. Therefore, the current proposal aims to solve the second limitation, leakiness i.e. expression of trans genes in the absence of ligand. We also propose to evaluate the performance of EcR gene switch in functional genomics studies and to rescue the phenotype in Arabidopsis mutants. The major objective ofthis proposal is to develop tight ecdysone receptor (EcR)-based chemical inducible gene regulation systems (gene switches) that can be used for regulation of transgenes in plants.