Plants possess sophisticated systems to regulate gene expression, which plant synthetic biology seeks to leverage to engineer new-to-nature functions. Chemical induction systems offer a valuable tool for such efforts by enabling precise temporal, spatial, and quantitative control of gene expression. However, existing plant chemical induction systems often face challenges, such as high basal activity in the absence of the inducer, unintended activation in uncontrolled environments, or off-target effects within the plant, caused either by the system itself or its inducer. Here, a rapamycin-inducible FKBP-FRB split transcription factor system was developed for use in plants. This system employs the human FRB (hFRB) domain (amino acids 2025 to 2115 of human mTOR) fused to a DNA-binding domain and the full-length human FKBP12 (hFKBP12) protein fused to a transcriptional activation domain. Upon application of rapamycin, hFRB and hFKBP12 dimerize, forming a functional transcription factor that binds to a synthetic promoter, thereby activating the target gene. The system was systematically optimized by extensive characterization of designs with different DNA-binding domains, FRB repeats, and promoter architectures. These efforts reduced basal activity and enhanced induced activity, resulting in an 87-fold increase in target gene expression from the uninduced to the induced state, and surpassing constitutive expression under the 2×CaMV35S promoter. Simple rapamycin application methods, such as spraying onto the leaf surface or soil application, combined with the system's sensitivity to nanomolar concentrations of inducer, further highlight its practicality. This system provides a robust and precise tool for regulating gene expression in plants and offers potential for expansion with orthogonal ligands targeting FRB or FKBP mutants.
Schirmer et al. (2026) studied this question.