Submitted to: Plant Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/28/2005
Publication Date: 2/1/2006
Citation: Tavva, V.S., Dinkins, R.D., Palli, S.R., Collins, G.B. 2006. Development of a methoxyfenozide-responsive gene switch for applications in plants. Plant Journal. 45:457-469.
Interpretive Summary: Expression of transgenes is becoming a powerful tool in many biotechnology applications including food product improvement. next generation of transgenic crops will require the expression of multiple transgenes for pest and disease resistance, improvement of nutritional quality, as well as for the production of alternative products such as biopharmaceutical compounds. Constitutive promoters, those that express all the time in all tissues, are presently the primary means used to express transgenes in plants. Metabolic energy waste, negative pleiotropic effects and potential gene escape are some fo the disadvantages associated with the use of these constitutive promoters. To counter these problems, gene switches that can regulate the expression of transgenes through hormone analogues and antibiotics have been developed. However, most of the compounds that are used to induce gene expression are impractical or inappropriate for regulation of transgenes in large-scale settings such as field or greenhouse applications. Compounds that can serve as a ligand that are suitable for regulation of transgenes for large-scale production and field use are the commercially available non-steroidal insect ecdysone agonists, tebufenozide, methoxyfenozide, halofenozide and chromafenozide. Tebufenozide, in conjunction with an ecdysone receptor switch has been shown to regulate reporter gene activity in transgenic tobacco, Arabidopsis and corn. We have developed an ecdysone gene switch that is induce in the presence of methoxyfenoxide in low concentrations that would be suitable for field-use applications.
Technical Abstract: The ecdysone receptor (EcR) from Choristoneura fumiferana (CfEcR) has been used to develop gene switches for conditional regulation of target gene expression in plants and humans. All EcR-based gene switches developed to date for use in plants are monopartate and require 'M concentration of ligand for activation of the target gene, and this has limited the use of this gene switch for large-scale applications. We developed a CfEcR-based two-hybrid gene switch that requires only nanomolar concentrations of the chemical ligand, methoxyfenozide for the induction of target gene expression. The chemical ligand, methoxyfenozide is already registered for field use with an excellent safety profile and it has strong potential as a gene switch ligand for large scale applications in the field. A two-hybrid gene switch requires three expression cassettes, two receptor cassettes and one target gene expression cassette for the regulation of target gene expression. The two-hybrid gene switch works through the formation of a functional heterodimer between EcR and the retinoid X receptor (RXR) upon application of chemical ligand. The receptor constructs were prepared by fusing DEF domains of CfEcR to the GAL4 DNA binding domain and EF domains of Choristoneura fumiferana ultraspiracle (CfUSP) or Locusta migratoria retinoid X receptor (LmRXR) or Mus musculus retinoid X receptor (MmRXR) or Homo sapiens retinoid X receptor (HsRXR) to the VP16 activation domain. These receptor constructs were tested for their ability to induce expression of the luciferase reporter gene placed under the control of 5X GAL4 response elements and –46 35S minimal promoter in protoplasts isolated from tobacco, corn and soybean and in transgenic Arabidopsis and tobacco plants. By adopting the two-hybrid format, the sensitivity of the CfEcR gene switch has been improved from micromolar to nanomolar concentrations of methoxyfenozide. The sensitivity of the G:CfE(DEF):V:LmR(EF) two-hybrid switch is 25 to 625 times greater than the monopartate gene switch, based on the plant species tested.