Engineering Rapamycin-Induced Dimerization for Control of Gene Expression in Plants

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.