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Title: A Förster resonance energy transfer sensor for live‐cell imaging of mitogen‐activated protein kinase activity in A rabidopsis

The catalytic activity of mitogen‐activated protein kinases (MAPKs) is dynamically modified in plants. SinceMAPKs have been shown to play important roles in a wide range of signaling pathways, the ability to monitorMAPKactivity in living plant cells would be valuable. Here, we report the development of a genetically encodedMAPKactivity sensor for use inArabidopsis thaliana. The sensor is composed of yellow and blue fluorescent proteins, a phosphopeptide binding domain, aMAPKsubstrate domain and a flexible linker. Usingin vitrotesting, we demonstrated that phosphorylation causes an increase in the Förster resonance energy transfer (FRET) efficiency of the sensor. TheFRETefficiency can therefore serve as a readout of kinase activity. We also produced transgenic Arabidopsis lines expressing this sensor ofMAPKactivity (SOMA) and performed live‐cell imaging experiments using detached cotyledons. Treatment with NaCl, the synthetic flagellin peptide flg22 and chitin all led to rapid gains inFRETefficiency. Control lines expressing a version ofSOMAin which the phosphosite was mutated to an alanine did not show any substantial changes inFRET. We also expressed the sensor in a conditional loss‐of‐function double‐mutant line for the ArabidopsisMAPKgenesMPK3andMPK6. These experiments demonstrated thatMPK3/6 are necessary for the NaCl‐inducedFRETgain of the sensor, while otherMAPKs are probably contributing to the chitin and flg22‐induced increases inFRET. Taken together, our results suggest thatSOMAis able to dynamically reportMAPKactivity in living plant cells.

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Author(s) / Creator(s):
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Publisher / Repository:
Date Published:
Journal Name:
The Plant Journal
Page Range / eLocation ID:
p. 970-983
Medium: X
Sponsoring Org:
National Science Foundation
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