EAGER: Developing a Fluorescent Biosensor to Enable Real-Time Analysis of MAP Kinase Signaling Pathways in Living Arabidopsis Plants

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Intellectual Merit. Understanding the molecular mechanisms of signal transduction constitutes an important research goal for biological scientists. Work with the model plant Arabidopsis thaliana has revealed that a given signaling molecule has the potential to process information in response to a wide range of different stimuli. For example, the MAP kinase protein MPK6 from Arabidopsis is known to be involved in signaling pathways related to biotic stress, abiotic stress, cell fate determination, and cell division. This observation raises the important experimental question of how one protein can properly interpret and process information from a variety of different inputs. One proposed mechanism for achieving this type of pathway specificity invokes the concept of spatial compartmentalization. In this model, molecules of the MPK6 kinase that are activated by a biotic stressor would be restricted to one subcellular location, while MPK6 molecules responding to cell division cues would be located in another. To directly test the role that spatial regulation plays in MAP kinase signaling, it is necessary to have some type of tool for visualizing the spatial and temporal dynamics of MAP kinase activity within living cells. Fluorescent biosensor molecules that monitor MAP kinase activity provide one solution to this challenge, and they have recently been developed for monitoring human MAP kinases. For this project a fluorescent biosensor that will be able to measure the activity of the Arabidopsis MAP kinase MPK6 will be developed. With Arabidopsis, it is possible to observe with a confocal microscope an intact, living organism. In addition, with Arabidopsis the full power of genetic analysis can brought to bear on future experiments involving the type of kinase activity sensor that will be developed. An MPK6 biosensor will, for the first time, provide researchers with a tool for monitoring at the subcellular level the spatial and temporal dynamics of protein kinase activity in a living Arabidopsis plant. Because MPK6 plays a role in a large number of signaling pathways, such a tool would be of wide interest to the scientific community.Broader Impacts. The MPK6 biosensor that it is planned to produce has the potential to be widely used by members of the plant signal transduction community. In addition, this project will provide training opportunities for two post-doctoral fellows, one PhD student, and one undergraduate. The postdocs hired to work on this project will serve as the mentors for the undergraduate student, which will provide the postdocs with valuable mentoring experience.