Chemical-regulated, site-specific DNA excision in transgenic plants

We have developed a chemical-inducible, site-specific DNA excision system in transgenic Arabidopsis plants mediated by the Cre/loxP DNA recombination system. Expression of the Cre recombinase was tightly controlled by an estrogen receptor-based fusion transactivator XVE. Upon induction by beta-estradiol, sequences encoding the selectable marker, Cre, and XVE sandwiched by two loxP sites were excised from the Arabidopsis genome, leading to activation of the downstream GFP (green fluorescent protein) reporter gene. Genetic and molecular analyses indicated that the system is tightly controlled, showing high-efficiency inducible DNA excision in all 19 transgenic events tested with either single or multiple T-DNA insertions. The system provides a highly reliable method to generate marker-free transgenic plants after transformation through either organogenesis or somatic embryogenesis.     

Technical advance: stringent control of transgene expression in Arabidopsis thaliana using the Top10 promoter system

We show that the tightly regulated tetracycline-sensitive Top10 promoter system (Weinmann et al. Plant J. 1994, 5, 559-569) is functional in Arabidopsis thaliana. A pure breeding A. thaliana line (JL-tTA/8) was generated which expressed a chimeric fusion of the tetracycline repressor and the activation domain of Herpes simplex virus (tTA), from a single transgenic locus. Plants from this line were crossed with transgenics carrying the ER-targeted green fluorescent protein coding sequence (mGFP5) under control of the Top10 promoter sequence. Progeny from this cross displayed ER-targeted GFP fluorescence throughout the plant, indicating that the tTA-Top10 promoter interaction was functional in A. thaliana. GFP expression was repressed by 100 ng ml-1 tetracycline, an order of magnitude lower than the concentration used previously to repress expression in Nicotiana tabacum. Moreover, the level of GFP expression was controlled by varying the concentration of tetracycline in the medium, allowing a titred regulation of transgenic activity that was previously unavailable in A. thaliana. The kinetics of GFP activity were determined following de-repression of the Top10:mGFP5 transgene, with a visible ER-targeted GFP signal appearing from 24 to 48 h after de-repression.     

A chemical-regulated inducible RNAi system in plants

Constitutive expression of an intron-containing self-complementary 'hairpin' RNA (ihpRNA) has recently been shown to efficiently silence target genes in transgenic plants. However, this technique cannot be applied to genes whose silencing may block plant regeneration or result in embryo lethality. To obviate these potential problems, we have used a chemical-inducible Cre/loxP (CLX) recombination system to trigger the expression of an intron-containing inverted-repeat RNA (RNAi) in plants. A detailed characterization of the inducible RNAi system in transgenic Arabidopsis thaliana and Nicotiana benthamiana plants demonstrated that this system is stringently controlled. Moreover, it can be used to induce silencing of both transgenes and endogenous genes at different developmental stages and at high efficiency and without any detectable secondary affects. In addition to inducing complete silencing, the RNAi can be produced at various times after germination to initiate and obtain different degrees of gene silencing. Upon induction, transgenic plants with genetic chimera were obtained as demonstrated by PCR analysis. Such chimeric plants may provide a useful system to study signaling mechanisms of gene silencing in Arabidopsis as well as other cases of long-distance signaling without grafting. The merits of using the inducible CLX system for RNAi expression are discussed.