Ecdysone agonist inducible transcription in transgenic tobacco plants

Abstract

Summary A novel chemical‐induced gene regulatory system for plants consisting of two molecular components is described. The first, or regulatory, cassette comprises a chimeric receptor composed of the hinge and ligand binding domains of the Heliothis virescens ecdysone receptor and the transactivation domain of the Herpes simplex VP16 protein fused to the DNA binding domain and transactivation of a mammalian glucocorticoid receptor. The second component, a reporter cassette, contains six copies of the glucocorticoid response element (GRE) fused to the minimal 35SCaMV promoter and β‐glucuronidase. The system uses a commercially available non‐steroidal ecdysone agonist, RH5992 (tebufenozide), as an inducer. Activation of gene expression is shown in both tobacco transient protoplasts and transgenic plants. The response is ligand dependent and is modulated by the change in minimal promoter context. The system is capable of inducing transgene activity up to 420‐fold corresponding to 150% of the activity observed with positive controls (35SCaMV:GUS).

Synthetic Botany

“…Among proven orthogonal systems for modulation of gene expression are transactivation systems like the aforementioned GAL4 activator, a tTA tetracycline inducible system based on the Escherichia coli tetR repressor (Weinmann et al 1994), and an IPTG-inducible pOp/LhG4 system based on the E. coli lac operon (Moore et al 1998). The various inducible expression systems implemented in plants to date include the dexamethasone responsive rat glucocorticoid (GR) ligand-binding domain (Aoyama and Chua 1997;Craft et al 2005;Samalova et al 2005), ethanol/acetaldehyde-inducible ALCR transcription factor and alcA promoter Salter et al 1998;Roslan et al 2001), DNA-binding domain of the lexA oestrogen receptor (Bruce et al 2000;Zuo et al 2000), copper-inducible ace1 promoter (Mett et al 1993), and ecdysone receptor (EcR) ligand binding-domain inducible by insecticide methoxyfenozide (Martinez et al 1999;Padidam et al 2003;Koo et al 2004). Other useful circuits comprise the Cre-Lox recombinase system that can be used for induction of transcription (Hoff et al 2001) or transgene excision (Chakraborti et al 2008), and a catalytically inactive version of Cas9 fused to regulatory domains.…”

Characterisation of novel target promoters for the dexamethasone-inducible/tetracycline-repressible regulator TGV using luciferase and isopentenyl transferase as sensitive reporter genes

“…A number of chemically inducible expression systems are available, namely tetracycline (tc)-regulated systems (Gatz et al 1992;Weinmann et al 1994), a copperinducible system (Mett et al 1993), a dexamethasone (dx)-inducible system (Aoyama and Chua 1997), an ethanol-inducible system (Caddick et al 1998), an ecdysone agonist-inducible system (Martinez et al 1999) and a dx-inducible/tc-repressible system (BoÈ hner et al 1999, see below).…”

A Versatile and Reliable Two-Component System for Tissue-Specific Gene Induction in Arabidopsis

“…These features make it difficult to produce distinct sectors of induced and uninduced gene expression essential to determine the cell autonomy of a phenotype and to address hitherto intractable problems encountered during reproductive developmental studies (where gene activation can prevent production of viable seeds). Some systems, based on nonvolatile inducers, have shown leaky activity (Martinez et al, 1999;De Veylder et al, 2000) or an inability to reliably activate responder T-DNAs randomly inserted in the genome (Baroux et al, 2005). This prohibits their use in precise activation-tagging screens.…”