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

Abstract

The catalytic activity of mitogen-activated protein kinases (MAPKs) is dynamically modified in plants. Since MAPKs have been shown to play important roles in a wide range of signaling pathways, the ability to monitor MAPK activity in living plant cells would be valuable. Here, we report the development of a genetically encoded MAPK activity sensor for use in Arabidopsis thaliana. The sensor is composed of yellow and blue fluorescent proteins, a phosphopeptide binding domain, a MAPK substrate domain and a flexible linker. Using in vitro testing, we demonstrated that phosphorylation causes an increase in the F€ orster resonance energy transfer (FRET) efficiency of the sensor. The FRET efficiency can therefore serve as a readout of kinase activity. We also produced transgenic Arabidopsis lines expressing this sensor of MAPK activity (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 in FRET efficiency. Control lines expressing a version of SOMA in which the phosphosite was mutated to an alanine did not show any substantial changes in FRET. We also expressed the sensor in a conditional loss-of-function double-mutant line for the Arabidopsis MAPK genes MPK3 and MPK6. These experiments demonstrated that MPK3/6 are necessary for the NaCl-induced FRET gain of the sensor, while other MAPKs are probably contributing to the chitin and flg22-induced increases in FRET. Taken together, our results suggest that SOMA is able to dynamically report MAPK activity in living plant cells.

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

“…To determine if the mammalian phosphoacceptor domain present in EKAREV was being targeted by CA-MPK4/6, we mutagenized the threonine residue in EKAREV-BASL thought to behave as the phosphorylation site into an alanine [ 26 , 48 , 60 ]. The resulting mutant version of the biosensor, EKAREV T48A -BASL, showed no increase in FRET efficiency when incubated with either CA-MPK4 or CA-MPK6 ( Figure 2 D, Table 1 ), similar to the results observed when the phosphorylation site of SOMA was mutagenized to an alanine ( Figure S1A,B , Table 1 ) [ 44 , 66 , 67 ]. These results demonstrate that the MAPK-induced FRET gain observed with EKAREV-BASL is dependent on the threonine present within the EKAREV phosphoacceptor domain and suggest that the increase in FRET efficiency is likely caused by phosphorylation of that threonine by CA-MPK4/6.…”

“…If so, this FRET biosensor had the potential to serve as a plant MAPK docking domain trap. To test this hypothesis, we used an in vitro assay that we previously developed for evaluating candidate plant MAPK FRET biosensors [ 44 ].…”

“…For this in vitro assay, biosensor protein and constitutively active versions of the Arabidopsis MAPKs MPK4 and MPK6 (CA-MPK4 and CA-MPK6) are expressed as recombinant proteins using Escherichia coli [ 44 , 62 ]. Recombinant biosensor protein is then incubated together with constitutively active kinase and adenosine triphosphate (ATP), and the reactions are subsequently analyzed using a scanning spectrofluorometer to measure the fluorescent emission generated by illumination of the samples at a wavelength that excites the mTurquoise-GL FRET donor.…”

“…The first plant MAPK fluorescent biosensor, the FRET-based sensor of MAPK in Arabidopsis (SOMA) was recently described [ 44 ]. SOMA has been shown to report the activities of the Arabidopsis MAPKs MPK3 and MPK6, and it is also likely to report the activities of additional MAPK isoforms in Arabidopsis , such as MPK4 [ 44 ]. As the Arabidopsis genome encodes 20 MAPK isoforms, there remains a need for additional plant MAPK biosensors that report the activities of different MAPK isoforms.…”

Properties and functions of calcium‐dependent protein kinases and their relatives inArabidopsis thaliana

“…Apart from amplitude, calcium signals also vary in duration and frequency, whose impact on kinase activation has not yet been tested. Considering that monitoring CDPK activation in vitro is tricky due to calcium release during material grinding (Seybold et al, 2018), the development of biosensors to monitor kinase activation in vivo as reported for MAPKs will be essential in the future (Zaman et al, 2019). One important limitation of this system is the identification of specific substrates for each kinase isoform.…”