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

Abstract

Inducible systems for transgene expression activated by a chemical inducer or an inducer of non-plant origin are desirable tools for both basic plant research and biotechnology. Although, the technology has been widely exploited in dicotyledonous model plants such as Arabidopsis, it has not been optimised for use with the monocotyledonous model species, namely rice. We have adapted the dexamethasone-inducible pOp6/LhGR system for rice and the results indicated that it is fast, sensitive and tightly regulated, with high levels of induction that remain stable over several generations. Most importantly, we have shown that the system does not cause negative growth defects in vitro or in soil grown plants. Interestingly in the process of testing, we found that another steroid, triamcinolone acetonide, is a more potent inducer in rice than dexamethasone. We present serious considerations for the construct design to avoid undesirable effects caused by the system in plants, leakiness and possible silencing, as well as simple steps to maximize translation efficiency of a gene of interest. Finally, we compare the performance of the pOp6/LhGR system with other chemically inducible systems tested in rice in terms of the properties of an ideal inducible system.

Optogenetic and Chemical Induction Systems for Regulation of Transgene Expression in Plants: Use in Basic and Applied Research

“…In another study, the LhGR2 transcription factor with codon-optimized Gal4 AD sequence [93] under the control of adjusted maize ubiquitin promoter and the pOpIn2 bidirectional reporter cassette, which is non-toxic for monocots [94], were used to induce the β-glucuronidase gene expression in transgenic rice Oryza sativa spp. japonica [95]. Dexamethasone-induced reporter gene activity was doseand time-dependent.…”

“…Additionally, the induction of transgene expression with another glucocorticoid derivative, triamcinolone acetonide (TA), was comparable, if not higher, than with dexamethasone. The system was functional at later stages of plant development in soil-grown plants; long-term induction has no negative effects on the development and growth of rice [95].…”

A procedure for Dex-induced gene transactivation in Arabidopsis ovules

“…Prior to induction, LhGR is retained in the cytoplasm by the HSP90 chaperone but once in the nucleus, it binds to the pOP6 promoter at optimally-spaced repeats in [ 3 ]. The pOP/LhGR-inducible system had been demonstrated to induce tight and reliable gene expression control and has been widely used in Arabidopsis, Cardamine, tobacco and rice [ 4 – 12 ] and references therein. Reports of the successful use of the system include the induction of transcription factors, toxins for cell ablations, the cre/lox system for recombination, RNAi for gene downregulation and other cellular components, in leaves, roots, cambium tissue, zygote, embryo and apical meristems (see for instance [ 9 , 10 , 13 – 19 ] inter alia ) .…”