The Immune Function Of YTH Family Members In The Arabidopsis RNA Binding Protein Interaction Network

RNA-binding proteins recognize and bind RNAs through diverse RNA-binding domains to modify the metabolism of target RNAs,such as transcription,splicing and translation.Thus they interact with each other and extensively participate in various biological processes,including growth,development,and biotic and abiotic stress responses.RBPs often have multiple family members.It remains unknown how these family members act to co-regulate biological processes,particularly during plant immune responses.YTH（YT521-B Homology）family consists of multiple members,which can recognize and bind RNAs modified with N⁶-methyladenosine（m⁶A）.Thirteen YTH proteins were identified in Arabidopsis,eleven belong to ECT（Evolutionarily Conserved C-Terminal Region）proteins.Previous studies have shown that Arabidopsis YTH proteins play an important role in leaf development,Ca²⁺signal transduction,m RNA splicing and protein degradation.However,there are few studies on YTH proteins in stress responses,especially in pathogenic microorganisms.In addition,it remains barely understood how different YTH family members regulate plant immune processes.This study used the Yeast two-Hybrid（Y2H）system to screen the interactions of Arabidopsis RBPs containing RRM（RNA Recognition Motif）,KH（K-Homology）and YTH domains.We identified some critical genes with new biological functions from the protein-protein interaction network through molecular biology,biochemistry and genetics methods.We then explored how YTH family members synergistically participated in regulating plant immunity.The main results of this study are as follows:（1）Based on the Y2H screening technique,we constructed a Protein-Protein Interaction Network(PPIN^RBP)containing 1,132 pairwise interactions among 194 RBPs.Our data showed that 33.85%of the interactions（65 pairwise interactions）could be verified in the public database,suggesting that PPIN^RBP contained more unknown RBP interactions.After further analysis of the attributes of different nodes,such as the number and strength of interactions,we identified five central molecules as candidate genes to verify protein functions.We obtained loss-of-function variants to verify their functions in abiotic stress and immune response.We found that RBP45B and RBP47A regulated SA-mediated seedling growth inhibition,and four genes,RBP-DR1,AT1G78260,ECT3 and RBP47A,were involved in response to ABA signaling.Among them,the mutation of AT1G78260 enhanced the tolerance to abscisic acid,while ECT3 had the opposite effect.We also found that the rbp-dr1 mutant showed a susceptible phenotype to Psm ES4326（Avr Rpt2）,suggesting that RBP-DR1 is a resistant-related gene,while RBP47A and ECT3 were involved in regulating plant SAR induced by SA.The above results indicate that more proteins with new biological functions would be discovered from our PPIN^RBPnetwork.（2）In the Y2H assay,we found that 35 proteins had auto-activation activity,and the YTH domain-containing family had a higher proportion than the other protein families containing the RRM and KH domains（54%vs.13%）.Further study of the YTH protein family revealed no direct relationship between the auto-activation and the subcellular localization of proteins.A member of the YTH family,ECT9,underwent liquid-liquid phase separation（LLPS）in vivo and in vitro.ECT9 exhibited a unique pattern of liquid-like puncta in the cytoplasm and nucleus.We observed that the ECT9 droplets were spherical,fluidic,and capable of fusion,typical characteristics of LLPS.We also demonstrated that the LLPS of ECT9depended on the N-terminal and C-terminal domains.（3）ECT9 collaborated with ECT1 to negatively regulate plant ETI immunity.ECT9 can interact with ECT1 in the SLCA（Split-luciferase Complementation Assay）and Co-IP（Co-Immunoprecipitation）assays.Subcellular co-localization and in vitro LLPS assays showed that ECT9 could co-condensate with LLPS-incapable ECT1.Through the bacterial growth,ion leakage and callose deposition assays,we found that ECT1 and ECT9 alone did not participate in plant immune response.The double mutant ect1/9 resisted the pathogen Psm ES4326（Avr Rpt2）but not Psm ES4326.The RNA-seq analysis showed that ECT9 regulated the plant ETI immune response by cooperating with ECT1 to jointly regulate the expression of disease-resistant genes and hypoxia stress genes during pathogen infection.In conclusion,we screened the interactions of RBPs in Arabidopsis through the Y2H assay and mapped the protein-protein interaction network PPIN^RBP.We identified new RBPs involved in plant abiotic and biotic stress responses from the network.Further studies showed that ECT9,a member of the YTH family,possessed LLPS capability in vivo and in vitro and participated in regulating plant immunity with ECT1 through a co-condensation mechanism.