Constitutive Expresser Of Pathogenesisrelated Genes 5 Is An Rnabinding Protein Controlling Plant Immunity Via An RNA Processing Complex

Plants have two ways of responding to the invading pathogenic microorganism:one is pathogen-associated molecular pattern(PAMP)-triggered immunity(PTI)and effector-triggered immunity(ETI)that are respectively mediated by cell-surface pattern-recognition receptors(PRRs)and intracellular nucleotide-binding /leucine-rich repeat receptor(NLRs).Plant innate immunity is capable of fighting against a variety of evolving pathogens while ETI is the primary mode of plant immunity.CPR5 is a key negative immune regulator.The cpr5 mutuant has the phenotype of heightened resistance,such as early senescence of cotyledon,increased concentration of salicylic acid and constitutive expression of disease resistance gene(PRs).Previous studies have revealed that CPR5 protein is localized in nuclear pores and invovled in plant immunity through regulating the nucleocytoplasmic transportation and cell cycle.Intriguingly,one of the two CPR5 genes of African rice got a copy mutation,which improved plant disease resistance.Besides,it is noteworthy that the cpr5 mutant has an autonomous disease-resistant phenotype,which provides a theoretical basis for cultivating and improving new varieties of crops.Certainly,this kind of characteristic is of paramount importance to agricultural production.Here we report a novel immune regulatory mechanism of CPR5 protein,which is involved in the regulation of plant immunity through two RNA processing complexes,NTC and CPSF.Our main findings are as follows:(1)In order to further dissect the function of CPR5 protein,we treated cpr5 mutants with EMS mutagens.As a consequence,we successfully screened two suppressors,cpr5 scpr44 and cpr5 scpr57.The suppressor genes PRL1 and FIP1 were successfully cloned by DNA-seq analysis and gene complementation verification.(2)We got the triple mutant cpr5 prl1 fip1 by mutant hybridization.Furthermore,the results of ion leakage,programmed cell death(PCD),PR gene expression and pathogen infection proved that double mutants prl1 fip1 suffered more than single mutants prl1 and fip1 in plant immune response.What we mentioned above indicated that PRL1 and FIP1,belonging to the NTC and CPSF complexes respectively,were localized downstream of CPR5 protein,coordinately activate plant PCD and immunity response.(3)The result of RNA-seq analysis suggested that there were only 36.82%overlaps between differentially expressed genes(DEGs)in cpr5 mutants and that induced by cpr5 prl1 fip1,in this case,which meant that CPR5-regulated DEGs were dependent on PRL1 and FIP1.Accordingly,these data showed that PRL1 and FIP1 proteins coordinatelty participated in CPR5 signaling pathway and activated the plant immune system.(4)The results of the Yeast two-hybrid(Y2H),Co-Immunoprecipitation(C o-IP),Bimolecular fluorescence complementation(Bi FC)and Fluorescence Reso nance Energy Transfer-Fluorescence Life-time Imaging Microscopy(FRET-FLIM) experiments identified that CPR5 protein could interact with PRL1 and FIP1 proteins on the nucleus and co-localized with PRL1 and FIP1 in nuclear speckl es,a membraneless organelle with RNA processing function.(5)The sequential analysis of CPR5 protein revealed that there was a Serine/Arginine-rich(SR)domain in the N-terminus.By retrieving the database,we found that CPR5 protein and RNPS1,SR45,SR45 a protein all had a SR domain.In addition,by comparing the homology domain of different proteins,we found that CPR5 protein was homologous to dhomorosophila Tra2,human RNPS1,arabidopsis SR45 and SR45 a.They all had two conserved RNA-recognition domains RNP1 and RNP2.What’s more,we also found that the SR-RRM domain of RNPS1,SR45 and SR45 a could be completely replaceble by CPR5 proteins after performing the functional substitution and complement experiment of conserved domain SR-RRM.To sum up,these data demonstated that CPR5 protein was a RNA binding protein of the SR family.(6)Mining deeper into the data of RNA-seq,we found that the alternative splicing of RNA in cpr5 mutants was significantly different from that in WT.Meanwhile,we conducted the RT-PCR experiment to corroborate the significant differences of these RNA splicing,indicating that CPR5 protein could modulate the alternative splicing of pre-m RNA.(7)Finally,we confirmed the RNA-binding activity of the CPR5 protein b y perfoming Electrophoretic Mobility Shift Assay(EMSA)and RNA immunopr-ecipitation(RIP).The above studies suggest that CPR5 protein is a RNA-binding protein belonging to the SR family,which can modulate plant immunity through RNA splicing factor NTC and polyadenylation factor CPSF.Combined with the previous findings of our team,our research reveals that CPR5 integrates three fundamental life processes:nucleocytoplasmic transport,cell cycle and RNA processing to modulate plant immunity,which provides a fresh idea for our exploration of the mechanism of plant immunity and a novel scientific basis for cultivating new crop varieties.