Controlling heterologous protein synthesis through a plant RNA ThermoSwitch.

Abstract

BACKGROUND: Plants are far from passive in the face of changing temperatures and have evolved transcriptional, post-transcriptional, and post-translational strategies to stay one step ahead of the environment. Among the most exciting recent discoveries are RNA ThermoSwitches, embedded within 5' untranslated regions (UTR) of several mRNAs. These cis-acting RNA elements sense temperature shifts and instantly tune translational output, acting as molecular thermostats inside the cell. First uncovered in Arabidopsis thaliana, ThermoSwitches are now emerging as a powerful new frontier for biotechnology, offering a programmable way to modulate protein production through temperature adjustments. RESULTS: Using a dual fluorescence reporter construct introduced by Agrobacterium-mediated transient expression, we have demonstrated that the native ThermoSwitch within the 5' UTR of the Arabidopsis phytochrome-interacting factor 7 (PIF7) mRNA functions as a potent temperature-responsive module in Nicotiana benthamiana leaves. A shift from 17 to 27 °C triggered a pronounced increase in reporter translation, delivering ~ 70% higher output within one day. This magnitude of enhancement mirrors the rise in endogenous PIF7 translation observed in Arabidopsis following the same temperature shift. The response remained even when the native upstream 5' UTR context was replaced with an unrelated sequence, demonstrating that the ThermoSwitch operates autonomously. Crucially, locking the hairpin into a strengthened, rigid conformation abolished the temperature response entirely. CONCLUSIONS: This work provides the first in vivo evidence that a native plant RNA ThermoSwitch functions effectively in an Agrobacterium-mediated transient expression system, establishing a homogeneous, temperature-responsive gene regulation system, free from chemical inducers or repressors. Demonstrating that the Arabidopsis PIF7 ThermoSwitch operates autonomously in Nicotiana benthamiana highlights its value as a versatile plug-and-play module that can be readily deployed across plant systems for broad biotechnological applications.