Expanding the Toolkit of Fluorescent Biosensors for Studying Mitogen Activated Protein Kinases in Plants.

Mitogen-activated protein kinases (MAPKs) are key regulators of numerous biological processes in plants. To better understand the mechanisms by which these kinases function, high resolution measurement of MAPK activation kinetics in different biological contexts would be beneficial. One method to measure MAPK activation in plants is via fluorescence-based genetically-encoded biosensors, which can provide real-time readouts of the temporal and spatial dynamics of kinase activation in living tissue. Although fluorescent biosensors have been widely used to study MAPK dynamics in animal cells, there is currently only one MAPK biosensor that has been described for use in plants. To facilitate creation of additional plant-specific MAPK fluorescent biosensors, we report the development of two new tools: an in vitro assay for efficiently characterizing MAPK docking domains and a translocation-based kinase biosensor for use in plants. The implementation of these two methods has allowed us to expand the available pool of plant MAPK biosensors, while also providing a means to generate more specific and selective MAPK biosensors in the future. Biosensors developed using these methods have the potential to enhance our understanding of the roles MAPKs play in diverse plant signaling networks affecting growth, development, and stress response.