Optimization of a Modular Steroid-Inducible Gene Expression System for Use in Rice

SUMMARY Chemically inducible systems that provide both spatial and temporal control of gene expression are essential tools, with many applications in plant biology. 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 six hours 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 0.01μM 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). The system is thus suitable for general use in rice, when the method of DEX application and relevant controls are tailored appropriately for each specific application. Key words: Golden Gate; dexamethasone; Oryza sativa, pOp6, LhGR