pOp6/LhGR: a stringently regulated and highly responsive dexamethasone‐inducible gene expression system for tobacco

Abstract

SummaryWe describe pOp/LhGR, a dexamethasone-inducible derivative of the pOp/LhG4 transcription activation system, and its use in tobacco to regulate expression of uidA (encoding b-glucuronidase; GUS) and the cytokinin-biosnythetic gene ipt. The pOp/LhGR system exhibited stringent regulation and strong induced phenotypes in soil and tissue culture. In conjunction with an improved target promoter, pOp6, that carries six copies of an optimized lac operator sequence the pOp6/LhGR system directed induced GUS activities that exceeded those obtained with pOp/LhG4 or the CaMV 35S promoter but without increased uninduced activity. A single dose of dexamethasone was sufficient to direct cytotoxic levels of ipt expression in soil-grown plants although uninduced plants grew normally throughout a complete life cycle. In vitro, induced transcripts were detectable within an hour of dexamethasone application and 1 nM dexamethasone was sufficient for half maximal induction of GUS activity. Various methods of dexamethasone application were successfully applied under tissue culture and greenhouse conditions. We observed no inhibitory effects of dexamethasone or LhGR on plant development even with the highest concentrations of inducer, although tobacco seedlings were adversely affected by ethanol used as a solvent for dexamethasone stock solutions. The pOp/LhGR system provides a highly sensitive, efficient, and tightly regulated chemically inducible transgene expression system for tobacco plants.

MultiSite Gateway-Compatible Cell Type-Specific Gene-Inducible System for Plants

“…High levels of ethanol used in some inducible systems (Roslan et al, 2001;Claassens et al, 2005;Craft et al, 2005;Samalova et al, 2005;Camargo et al, 2007) were shown to be variably toxic when used experimentally, while dexamethasone (Craft et al, 2005;Moore et al, 2006) or 17-b-estradiol (Zuo et al, 2000;Tornero et al, 2002;Vilarrasa-Blasi et al, 2014) have exhibited little or no toxicity when tested at physiological concentrations by different laboratories.…”

Targeted manipulation of leaf form via local growth repression

“…Although the depth of sinus indentation was clearly increased towards the base of the leaves, a smaller change was observed towards the leaf tip, suggesting that the basic pattern of lobbing/serration was not altered by these manipulations. The pOp synthetic promoter used in these experiments (Craft et al, 2005;Samalova et al, 2005) resulted in simultaneous transcription of AtKRP1 and a GUS reporter gene after Dex induction, allowing verification that target gene expression was localized to the expected region of the leaf flank ( Figure S1d-g). Analysis of leaf histology showed that the cells in the region of CUC2-directed AtKRP1 expression were greatly enlarged relative to adjacent cells (Figure 2e-g) and relative to cells in equivalent positions in wild-type leaves (Figure 2c).…”

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.…”