A Chemical-Induced, Seed-Soaking Activation Procedure for Regulated Gene Expression in Rice

Abstract

Inducible gene expression has emerged as a powerful tool for plant functional genomics. The estrogen receptor-based, chemical-inducible system XVE has been used in many plant species, but the limited systemic movement of inducer β-estradiol in transgenic rice plants has prohibited a wide use of the XVE system in this important food crop. Here, we constructed an improved chemical-regulated, site-specific recombination system by employing the XVE transactivator in combination with a Cre/loxP-FRT system, and optimized a seed-soaking procedure for XVE induction in rice. By using a gus gene and an hpRNAi cassette targeted for OsPDS as reporters, we demonstrated that soaking transgenic seeds with estradiol solution could induce highly efficient site-specific recombination in germinating embryos, resulting in constitutive and high-level expression of target gene or RNAi cassette in intact rice plants from induced seeds. The strategy reported here thereby provides a useful gene activation approach for effectively regulating gene expression in rice.

Targeted genome editing inNicotiana tabacumusing inducible CRISPR/Cas9 system

“…The GUS gene was amplified and introduced into the XVE vector pER8 [18] to generate the estrogen-inducible expression construct pER-GUS (Figure 1a). The GUS reporter is a reliable and extremely senesitive system that allows histochemical assessment of gene activity in plants [16,38]. After transformation of N. tabacum , hygromycin-resistant plants that can develop roots on hygromycin-containing medium (Figure S1) were selected as candidates for PCR identification.…”

“…Though the XVE system can be efficiently activated by a low concentration (8 nM) of estradiol, varying inducer concentrations were reported in different plant species [16,18,19,35]. In Arabidopsis, a relatively low concentration (∼5 µM) of estradiol was used for treatment [18,19], and in rice, however, the inducer concentration increased to 20 µM [16,35]. A long incubation resulted in reduced transcript level of target gene due to instability of the inducer [18].…”

“…Moreover, transient expression of Cas9 is sufficient to generate targeted mutagenesis [35,45]. Considering the low inducibility of target gene in leaves of intact transgenic plants treated with estradiol through root absorption [16,32], the explants used in this study were therefore cultured on estradiol-containing (20 µM) medium for a long time (28 d). Optimization of the inducer concentrations and induction time, of course, would help to improve the expression of Cas9 upon induction.…”

“…Inducible gene expression systems allowing temporal expression of target genes are employed as powerful tools for research in plant functional genomics [16]. Chemical-inducible systems have been widely used in plants, and inducible systems responding to different chemical inducers, such as estradiol [17,18,19], ethanol [20,21,22,23], glucocorticoid [24,25], ecdysone [26], and tetracycline [27] have been successfully developed.…”

“…The application of glucocorticoid-inducible system, however, was found to cause growth defects in several plant species, including Arabidopsis, tobacco and rice [24,29,30]. The estradiol-inducible system, based on transcriptional activator XVE [18], has been applied to multiple plant species, and no physiological or morphological effect was observed on treated plants [16,18,19,31,32]. Inducible systems are previously used to remove selectable markers or investigate expression patterns of target genes in transgenic plants [19,32,33].…”