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

Proteolysis, a ubiquitous process in living organisms, is driven by proteases that regulate numerous signaling pathways through the hydrolysis of peptide bonds in protein substrates. Understanding the temporal and spatial dynamics of proteolysis and the activation of proteases is crucial for elucidating their roles in biological pathways. Here, we introduce a suite of genetically encoded FRET reporters designed to detect various proteolytic activities in plants. These sensors effectively reported in planta the specific activity of both Tobacco Etch Virus protease and caspase-3. Furthermore, we developed sensors for detecting plant metacaspase activity, validated through both in vitro and in planta experiments. These experiments revealed the spatial dynamics of proteolysis triggered by metacaspase activation following wounding and programmed cell death in roots. The implementation of these tools in plant biology research opens new avenues for investigating proteolytic mechanisms, significantly enhancing the potential for in-depth studies. Our work demonstrates the feasibility of using these sensors to detect diverse protease activities in vivo with high spatiotemporal resolution. These plant proteolytic biosensors hence represent a valuable toolbox for understanding protease functions within their natural context, paving the way for future advancements in plant biology research.