Shedding light on plant proteolysis: genetically encoded fluorescent sensors as tools for profiling protease activities

Proteolysis is a universal process, as proteases play a pivotal role in modulating numerous signaling pathways. Proteases control the fate and function of their target proteins by hydrolyzing peptide bonds within these proteins. Understanding the temporal and spatial dynamics of proteolytic events, including the proteases that execute them, is crucial for elucidating their particular roles across diverse biological processes. In this study, we developed and characterized a set of genetically encoded Förster resonance energy transfer (FRET)-based reporters for the detection of various proteolytic activities in plants. Our sensors reliably reported the activity of specific proteases, exhibiting a performance comparable to previously established detection systems. In addition, we engineered variants capable of detecting the spatial dynamics of metacaspase-triggered proteolysis after wounding and during programmed cell death in roots. We demonstrated the feasibility of these FRET-based sensors for detecting various activities in vivo with high spatiotemporal resolution. The implementation of these tools in plant research opens opportunities to explore proteolytic mechanisms with enhanced precision. Overall, these biosensors constitute a versatile toolbox for probing protease function within its native cellular context, paving the way for deeper insights into plant biology and signaling.