Dissecting The Molecular Mechanism Of CPK18-Mediated Regulation Of Rice Ethylene Signaling And Disease Resistance

As one of the most important crops,rice is the staple food for half of the world’s population.Rice blast,caused by Magnaporthe Oryzae,is the most devasting disease,which is threatening the world’s food security.During plant interaction with pathogens,plants have evolved complicated innate immune system and sophisticated gene networks to balance the defense and development.However,the molecular mechanism by which plant disease signaling pathway crosstalk with developmental signaling pathways is not fully understood.Dissecting the molecular mechanism of rice defense will provide novel insight into plant-pathogen interactions and facilitate enhancing rice disease resistance via gene editing.As a calcium-dependent protein kinase(CDPK),the CDPK family is the pivotal player to transduce and amplify pathogen invasion signals from the cell surface to downstream components.It is also the convergent point of disease,developmental,and abiotic stress signaling pathways.Previous studies indicate CPK18 and rice mitogen-activated protein kinase(MAPK)gene MPK5 interact and phosphorylate each other,but the biological function of such reciprocal phosphorylation is poorly understood.Here,we combined biochemical,genetic,and multi-omic approaches to characterize the function of CPK18.Our results reveal how MPK5-CPK18 phosphorylation balances rice defense and development.The main findings are summarized as follows.(1)The growth and development phenotype analysis and rice blast resistance analysis of CPK18 silenced rice plants(CPK18-RNAi)and transgenic rice plants expressing constitutively activated CPK18(CPK18-AC)showed that CPK18 negatively regulates rice resistance but positively regulates rice growth and development.At the same time,the resistance of CPK18 knock-out plants(CPK18-KO)to rice blast is also significantly improved,but the plant height and tiller number are not significantly different from the wild type,which is inconsistent with the growth phenotype of CPK18-RNAi rice.Further analysis found that mutated CPK18 in CPK18-KO rice has at least 3 splicing variants.Sequence analysis suggests these mutated CPK18 variants have an open reading frame encoding the calcium-binding domain.Whether this ORF was expressed and functional in CPK18-KO plants requires further validation.(2)We analyzed the transcriptome,proteome,and phosphorylation-proteome of CPK18-RNAi lines.The transcriptome analysis indicated that 868 genes were changed more than 2 folds in CPK18-RNAi plants comparing to wild type.GO and KEGG analysis of these differentially expressed genes revealed defense-related secondary metabolism,MAPK,and phytohormone signaling were up-regulated,while developmental-related genes were down-regulated.Further analyses indicated that defense-related transcription factors like ERF,WRKY,NAC,were up-regulated,while the GRF transcription factor,which is related to GA signaling,was down-regulated.Quantitative proteome analysis also revealed Chitinase,PR protein were increased,while phosphorylated EIN2 protein was decreased in CPK18-RNAi plants.These data imply that CPK18 is likely involved in a complex gene network to regulate rice development,hormone response,and defense.(3)Silencing CPK18 significantly impairs rice ethylene production and signaling.In comparison with the wild type,the ethylene production was significantly decreased in CPK18-RNAi and increased in CPK18 AC plants.Exogenous applying ACC restored the height of CPK18-RNAi plants to normal,suggesting CPK18 regulate rice development in an ethylene dependent way.The Rice blast infection induces comparable ethylene production between CPK18-AC and wild type.Unexpectedly,CPK18-RNAi plants produced 7 times higher ethylene than wild type plants at 96 hours post rice blast infection.We speculated that CPK18 differentially regulates ethylene biosynthesis/signaling under normal conditions and pathogen invasion.(4)Finally,the biological function of MPK5-CPK18 phosphorylation was analyzed.In vitro and in vivo assays demonstrated MPK5 phosphorylated the Thr505 of CPK18.CPK18 with phosphorylation mimic mutation(Thr505-to-Asp)exhibited distinct biochemical features than wild type CPK18 protein,suggesting that MPK5 modulate CPK18 kinase activity via phosphorylating Thr505.Gene editing rice(CPK18-GE)was generated by precisely disrupting the T505 site using CRISPR/Cas9 technologies.Similar to CPK18-RNAi and CPK18-KO lines,CPK18-GE plants have higher resistance against rice blast than wild type.Interestingly,the height and effectively tiller number of CPK18-GE were the same as wild type,but the seed size and 1000-seeds weight increase significantly,which ultimately increases the yield per plant by 21.3%-26.6%.These data strongly suggest that CPK18 Thr505 phosphorylation is important to balance rice defense and growth.More importantly,precise editing of the Thr505 site of CPK18 can simultaneously improve disease resistance and yield.In summary,this study characterized CPK18 function by integrating biochemical,genetic,and multi-omics approaches.Our results reveal the dual regulatory roles of CPK18 in defense and growth.Particularly,CPK18 may modulate ethylene production in different pathways under normal and pathogen infection conditions.We found MPK5 phosphorylated CPK18 Thr505 and this phosphorylation plays an important role to balance rice defense and growth.Targeted editing Thr505 site can increase rice disease resistance and yield.Together,our study presents novel insights into the rice defense signaling pathway and provides a new strategy for applying the gene-editing method to simultaneously improve disease resistance and other agronomic traits.