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

Abstract

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 four 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 four 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, two 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.