A MAPK Cascade Regulates Disease Resistance Through Endogenous Peptide Regulation

Mitogen-activated protein kinase（MAPK）cascades are highly conserved signaling modules in eukaryotes,typically consist of three protein kinases:MAPK,MAPK kinase（MAPKK）,and MAPKK kinase（MAPKKK）.MAPK mainly functions in receiving the signals from the receptors directly or indirectly,then transmiting and amplifying the signals.A large number of studies reveal that MAPK cascades play an important role in various stages of plant growth,development,and plant immunity.To date,two MAPK cascades,MEKK1-MKK1/MKK2-MPK4 and MAPKKK3/MAPKKK5-MKK4/MKK5-MPK3/MPK6,have been implicated in plant immunity.Plant elicitor peptides（Peps）are endogenous peptides derived from plants that can induce innate immune responses in plants.It was reported that the Peps are involved in amplifying the flg22-induced immunity in Arabidopsis.Under normal growth condition,the Peps exist in the form of precursor protein PROPEPs,and the C-terminal region was processed to form the 23-26 amino acid Peps that are released into apoplast when cell damage occurs.Current studies have shown that exogenous application of Peps inhibits the growth of primary root and enhances plant resistance against different pathogens,insects and herbivores.In this study,we demonstrated that the expression of PROPEPs is highly induced in MKK4^DD after dexamethasone treatment,the conditional gain-of-function MPK3/MPK6system.The flg22-induced PROPEPs expression is significantly reduced in MPK3SR and MPK6SR,the conditional loss-of-function double mpk3 mpk6 mutants.These results suggest that the MKK4/MKK5-MPK3/MPK6 cascade is involved in the regulation of PROPEPs gene expression.WRKY33 is an important transcription factor directly downstream of MPK3/MPK6.Both the MPK3/MPK6 gain-of-function induction and flg22-induced PROPEPs expression are significantly reduced in wrky33 mutant,demonstrating that WRKY33 is required for PROPEPs regulation after MPK3/MPK6 activation.We also confirmed that exogenous application of Pep2 induces the phosphorylation of MPK3/MPK6 in the low concentration within short time in Arabidopsis.The Pep2-induced MPK3/MPK6 activation is mostly abolished in the Peps receptor mutant pepr1 pepr2.Taken together,it is possible that a regulation loop of PAMPs-PRRs-MKK4/MKK5-MPK3/MPK6-WRKY33-PROPEPs-Peps-PEPR1/2-MKK4/MKK5-MPK3/MPK6-PROPEPs may involve in the regulation of immunity in Arabidopsis.Fluorescent reporter analysis shows that the subcellular localizations of PROPEP1,PROPEP2,and PROPEP3 are similar,including nucleus,cytoplasm and cell membrane.However,they are differentially expressed in roots.PROPEP1 and PROPEP3 are localized in the endodermis of maturation zone,while PROPEP2 is localized in the epidermis,cortex,and endodermis of elongation and meristematic zones.These results suggest that the PROPEPs may have tissue specificity in plant immune responses.Our results illustrate that the MPK3/MPK6 cascade contributes to PROPEPs regulation through WRKY33.And the amplication of Peps triggers the MPK3/MPK6 activation.Therefore,the MAPK cascade contributes to plant immunity may through WRKY33-PROPEPs regulation.