Arabidopsis PP2C clade B members are negative feedback regulators of MPK3/MPK6 MAPK cascade in plant immunity and...

MAPK activation is a key event downstream of receptor-mediated signaling in plant immunity and development. While the activation of MAPK cascades is well-studied, their negative regulatory mechanisms remain less understood. Expression profiling of conditional gain-of-function MPK3/MPK6 plants identified the induction of clade B phosphatase genes from the PP2C family, known as AP2Cs. Overexpression of any of the 4 AP2C members suppresses MAPK activation in seedlings treated with flg22. Additionally, these plants exhibited excessive stomatal formation and clustering, phenocopying mpk3 mpk6 mutants. In contrast, single AP2C loss-of-function mutants show no discernible phenotype, suggesting functional redundancy. Mutation of all 4 AP2C members leads to enhanced MPK3/MPK6 activation and accelerated cell death upon pathogen infection. Loss of MPK6 in the ap2c1/2/3/4 quadruple mutant background confirmed that MPK6 is a key target of AP2Cs in regulating HR cell death. Further investigation revealed that the MPK3/MPK6 and CPK5/CPK6, 2 critical immune signaling pathways, along with their downstream substrate, WRKY33, are involved in the activation of AP2C expression in response to PAMP treatment and pathogen infection. We conclude that AP2C1/2/3/4 function as MAPK phosphatases, collectively forming a negative feedback regulatory loop that fine-tunes the signaling strength and duration of MPK3/MPK6 cascade to balance plant immunity and development.