Highlighter - a Synthetic Light-Gated Gene Expression Switch for Plants

In the process of applying optogenetic sensors in plants to study phytohormone patterning it has become clear that high-resolution sensing alone is not sufficient - we also need to be able to perturb phytohormone patterning in high-resolution to distinguish correlation from causation. Optogenetic actuators are ideal tools for performing high-resolution perturbations in a minimally invasive manner with maximal spatiotemporal resolution. However, a major challenge for designing optogenetic actuators for plants is that plants are conventionally grown in mixed light environments, cycling between changing broad spectrum light regimes and darkness. Consequently, an optogenetic actuator for plants would ideally adopt one target gene expression state during light-dark cycling and an altered target gene expression state following treatment with light spectra that are not found in the horticultural environment.We have now developed an orthogonal synthetic light-gated gene expression system for plants which is fully genetically encoded and functions without the need for exogenously supplied chromophores. Our system, called Highlighter, was developed by repurposing the cyanobacterial CcaS-CcaR system through semi-rational engineering. When tested by transiently transfecting N. benthamiana, the system was inactive under blue light conditions and active in the dark and under green, red and white light conditions. Furthermore, Highlighters activity could be tuned by modulating the red-to-blue ratio of white light. We are presently adapting Highlighter for use in stably transformed plants and continue to optimize the system, engineer new spectral variants and envision using the system to perturb hormone dynamics in single cells of stably transformed plants with rapid induction and reversion kinetics using minimally invasive light signals.