A modular steroid-inducible gene expression system for use in rice

Abstract

Background Chemically inducible systems that provide both spatial and temporal control of gene expression are essential tools, with many applications in plant biology, yet they have not been extensively tested in monocotyledonous species. Results Using Golden Gate modular cloning, we have created a monocot-optimized dexamethasone (DEX)-inducible pOp6/LhGR system and tested its efficacy in rice using the reporter enzyme β-glucuronidase (GUS). The system is tightly regulated and highly sensitive to DEX application, with 6 h of induction sufficient to induce high levels of GUS activity in transgenic callus. In seedlings, GUS activity was detectable in the root after in vitro application of just 0.01 μM DEX. However, transgenic plants manifested severe developmental perturbations when grown on higher concentrations of DEX. The direct cause of these growth defects is not known, but the rice genome contains sequences with high similarity to the LhGR target sequence lacO, suggesting non-specific activation of endogenous genes by DEX induction. These off-target effects can be minimized by quenching with isopropyl β-D-1-thiogalactopyranoside (IPTG). Conclusions Our results demonstrate that the system is suitable for general use in rice, when the method of DEX application and relevant controls are tailored appropriately for each specific application.

Mitigation of emerging implications of climate change on food production systems

“…Phase III ran from 2015 to 2019 and was co-ordinated by Jane Langdale at the University of Oxford. Advances in Phase III were sufficient to secure funding for a fourth phase that aims to develop a prototype for C4 metabolism (Vlad et al, 2019).…”

The steroid-inducible pOp6/LhGR gene expression system is fast, sensitive and does NOT cause plant growth defects in rice (Oryza sativa)

“…The pOp6/LhGR system is very sensitive, 0.01 M Dex was sufficient to induce GUS activity in painted leaves (Fig. 3B) and in roots of in vitro grown seedlings of the pOp6/rcoLhGR (Vlad et al, 2019). Interestingly, in some lines maximum levels of induction were reached with 0.1 M Dex, while others required 10 M Dex to reach levels similar to the constitutive pZmUbi promoter (Fig.…”

“…It is a widely used system for which a comprehensive library of celltype specific activator lines was created in Arabidopsis (Schurholz et al, 2018); the system was combined with artificial micro-RNA (amiRNA) to knockdown multigene expression (Goth et al, 2012;Samalova et al, 2020) and hairpin RNAi molecules to silence gene expression (Wielopolska et al, 2005;Liu and Yoder, 2016). Apart from Arabidopis (Craft et al, 2005) and tobacco (Samalova et al, 2005) the system was tested in various other species including citrus plant (Rossignol et al, 2014) and Medicago truncatula (Liu and Yoder, 2016) and most recently in rice (Vlad et al, 2019). However, the specific modifications made to the original version of the pOp6/LhGR system caused undesirable effects in plants.…”

“…The XVE system was tested in rice (Sreekala et al, 2005;Okuzaki et al, 2011) but its use remains very limited due to the lack of systemic movement of the estradiol inducer (Hirose et al, 2012, Chen et al, 2017. Other two steroid-inducible systems tested, GVG and a modular gene expression system (Vlad et al, 2019) derived from the pOp6/LhGR system met with a mixed success in rice, as they caused severe growth and developmental defects of the plants (Ouwerkerk et al, 2001;Vlad et al, 2019).…”

A heat-shock inducible system for flexible gene expression in cereals

“…Temperature-independent systems have also been established successfully, including the use of the barley drought-responsive promoter HvDhn4s [15]. Recently, a dexamethasone-inducible system was established in rice, though application of dexamethasone was found to also cause non-specific activation of endogenous rice genes [16].…”