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Arabidopsis MITOGEN-ACTIVATED PROTEIN KINASE3 (MAPK3 or MPK3) and MPK6 play important signaling roles in plant immunity and growth/development. MAPK KINASE4 (MKK4) and MKK5 function redundantly upstream of MPK3 and MPK6 in these processes. YDA, also known as MAPKKK4, is upstream of MKK4/MKK5 and forms a complete MAPK cascade (YDA–MKK4/MKK5–MPK3/MPK6) in regulating plant growth and development. In plant immunity, MAPKKK3 and MAPKKK5 function redundantly upstream of the same MKK4/MKK5–MPK3/MPK6 module. However, the residual activation of MPK3/MPK6 in the mapkkk3 mapkkk5 double mutant in response to flg22 PAMP treatment suggests the presence of additional MAPKKK(s) in this MAPK cascade in signaling plant immunity. To investigate whether YDA is also involved in plant immunity, we attempted to generate mapkkk3 mapkkk5 yda triple mutants. However, it was not possible to recover one of the double mutant combinations (mapkkk5 yda) or the triple mutant (mapkkk3 mapkkk5 yda) due to a failure of embryogenesis. Using the CRISPR-Cas9 approach, we generated weak, N-terminal deletion alleles of YDA, yda-del, in a mapkkk3 mapkkk5 background. PAMP-triggered MPK3/MPK6 activation was further reduced in the mapkkk3 mapkkk5 yda-del mutant, and the triple mutant was more susceptible to pathogen infection, suggesting YDA also plays an important role in plant immune signaling. In addition, MAPKKK5 and, to a lesser extent, MAPKKK3 were found to contribute to gamete function and embryogenesis, together with YDA. While the double homozygous mapkkk3 yda mutant showed the same growth and development defects as the yda single mutant, mapkkk5 yda double mutant and mapkkk3 mapkkk5 yda triple mutants were embryo lethal, similar to the mpk3 mpk6 double mutants. These results demonstrate that YDA, MAPKKK3, and MAPKKK5 have overlapping functions upstream of the MKK4/MKK5–MPK3/MPK6 module in both plant immunity and growth/development. 

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.