PP2A-4 Negatively Regulates Innate Immune Responses By Dephosphorylating MKK5 In Arabidopsis

Plants live in a very complex circumstance and constantly theatened by bacteria,fungi,viruses,nematodes,aphids and other potential pathogens thoughout their life cycles.Plants have evolved robust innate immune systems to protect them from pathogen invasions.Plant innate immunity is composed of PAMP-triggered immunity(PTI)and effector-triggered immunity(ETI).PAMP-triggered immunity plays an important role in plant defense against a broad spectrum of potential pathogens.Intracellular PTI responses include ROS burst,MAPK cascades activation,rapid increases in expressions of pathogenesis related genes and deposition of callose.MAPK cascades mediate signal transduction process after the sense of pathogens.It has been reported that flg22,a conserved 22 amino-acid peptide of flagellin,recognized by the receptor-like kinase FLS2(fagellin-sensing 2),activates the MEKK1/MEKKs-MKK4/MKK5-MPK3/MPK6 casecade and thereby leads to a variety of innate immune responses in the host cells.The results of GUS histochemical staining showed that PP2A-4 gene was expressed in almost all organs and development stages of Arabidopsis.The transcript levels of defense related genes were also detected by quantitative real-time PCR,and the results showed that transcript levels of PR1 and PR2 in pp2a-4 mutant were much higher than wild type,which implied that PP2A-4 might negatively regulated the defense responses in Arabidopsis thaliana.The flg22-triggered increase of callose deposition was more remarkable in pp2a-4 than Col-0,which revealed that the knock-out of PP2A-4 promoted the deposition of callose.Psm ES4326 is a kind of gram-negative bacteria,whose flagellin can activate the innate immune defense in Arabidopsis.The statistical analysis of colony number in 0 and 3 days post-inoculation with Psm ES4326 indicated that the growth of bacteria was inhibited in pp2a-4 mutant while increased in PP2A-4 trangentic plants.These data indicated that PP2A-4 modulated innate immunity negatively.Reversible protein phosphorylation represents a major form of post-translational modifications,which controls many regulatory circuits in eukaryotes by modulating the conformation,activity,localization and stability of substrate proteins.To investigate whether PP2A-4 paticipates in innate immunity via MKK5,the interaction of PP2A-4 and MKK5 was tested by Y2H,pull-down,BiFC and Co-IP experiments,which concluded that PP2A-4 interacted with MKK5 in vitro and in vivo.Further more,the dephosphorylation of MKK5 by PP2A-4 was verified.PP2A-4 inhibited the shift of phosphorylated MKK5 in E.coli.The activation of MPK3/MPK6 triggered by flg22 was modulated by phosphatases to inactivation in Col-0 while it remained highly active in pp2a-4 treated by flg22 for 30 min,which indicated that PP2A-4 participated in the deactivation of MPK3/MPK6.The phosphorylation of NtWIPK/NtSIPK which were activated by △MEKK1-MKK5 cascade was decreased while coexpressing with PP2A-4 in Nicotiana tabacum.These data suggested that MKK5 can be dephosphorylated and inactivated by PP2A-4.In conclusion,we proposed that PP2A-4 negatively regulated innate immune responses,probably by dephosphorylating MKK5 and inhibiting its kinase activity.