MicroRNA-based gene silencing is a potent transgenic technique for plant trait improvement. (14-16) In addition, regulation strategies at the post-transcriptional level are being developed to layer gene regulation. (12, 13) Engineered DNA binding proteins-such as zinc fingers, transcription activator-like effectors, and more recently the inactivated CRISPR/CAS9 system (dCAS9) fused to a repressor domain (e.g., SRDX)-allow specific transcription inhibition. In tobacco plants, when the bacterial LacO and TetO operator sequences were inserted into plant promoters, the downstream reporter expression was repressed up to 10-fold, and to 100-fold in the presence of repressor proteins LacI and TetR, respectively. In bacterial operons, repressor proteins bind to operator sequences and repress transcription. (10, 11) Robust and versatile tools that function as “switch off” devices to repress transgene expression are still lacking. (6, 7) Few natural and synthetic promoters have been developed to drive transgene expression in response to environmental, (5, 8) metabolic, (9) or chemical stimuli. In plant research, most efforts to control transgene expression are limited to a small number of tissue-specific promoters that have been characterized and documented. Using a bioinformatics approach, we identified 54 orthologous systems from various bacteria, and then validated in planta the activity for a few of those systems, demonstrating the potential diversity of such a two-component repressor system. In addition to tissue-specific transgene repression, this system offers stimuli-dependent expression control. We demonstrated that this regulatory device was functional in monocotyledonous and dicotyledonous plant species, and showed that it can be used to repress transgene expression by >400-fold and to synchronize transgene repression. By employing the endoribonuclease Csy4 and its recognition sequence from Pseudomonas aeruginosa and manipulating 5′UTR of mRNA, we developed a two-component expression–repression system to tightly control synthesis of transgene products. Meeting these challenges will facilitate transgene expression regulation and support the fine-tuning of metabolic pathways to avoid the accumulation of undesired intermediates. Tight control and multifactorial regulation of gene expression are important challenges in genetic engineering and are critical for the development of regulatory circuits.
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