Cloning And Functional Study On The Key Genes Of RLR Signaling Pathway In The Pacific Oyster

RIG-I-like receptors(RLRs)are cytoplasmic pattern recognition receptors(PRRs)which are known to play an important role in sensing RNA virus pathogen-associated molecular patterns(PAMPs).Firstly,RLRs recognize the virus RNA.Then RLRs bind to the mitochondrial-membrane-located protein VISA,trigger the host immune response and ultimately activate the transcription factor NF-?B and IRFs.Much more in-depth study has been executed on mammals and even bony fishes' RLR immune pathways.However,there are few researches on invertebrates RLR signaling pathway.In this study,we set up identification and functional study on key genes of the Pacific oyster RLR signaling pathway through molecular biology,bioinformatics and immunology technologies.Firstly,we cloned oyster CgRIG-I-1 and CgVISA genes.Sequence alignments and homology analysis showed that CgRIG-I-1 and CgVISA are new members of invertebrate RIG-I family and VISA family respectively.Considering our previous studies,CgRIG-I-1 and CgVISA mRNA were expressed in all oyster tissues,and their expression were induced by poly(I:C),poly(dA:dT)and oyster herpesvirus 1(OsHV-1)challenges.CgRIG-I-1 protein,as a pattern recognition receptor,can bind to poly(I:C)directly and interact with the CgVISA protein through the N-terminal CARD domains.The transmembrane domain of CgVISA plays an important role in mediating CgRIG-I-1_CARD and CgVISA interaction.Furthermore,CgVISA protein could dimerize through its transmembrane domain,which may be essential for the recruitment of downstream signaling moleculars.Secondly,we cloned and characterized CgTRAF2,CgTRAF3 and CgTRAF6 from C.gigasa for the first time.The results of qRT-PCR showed that,the mRNA expression of CgTRAF2,CgTRAF3 and CgTRAF6 was all induced by the virus PAMP stimulation.The mRNA expression pattern of CgTRAFs after the RNAi of CgVISA showed the similar expression style with that of CgVISA,indicating that CgTRAF2/3/6 might be a signal molecule involved in Crassostrea gigas RLR/VISA immune pathway.And yeast two-hybrid and co-immunoprecipitation result proposed that CgTRAF6 could interact with CgVISA protein.In addition,CgTRAF2 located in the cytoplasm;CgTRAF3 presences an alternative splicing form;CgTRAF6 can activate the NF-?B promoter.Finally,we obtained the full-lengh cDNA sequences of oyster CgIRF2 and CgIRF8 by RACE.CgIRF2 and CgIRF8 mRNA expression were found in all tested tissues and induced by poly(I:C)and OsHV-1 challenges.CgIRF2 and CgIRF8 might function downstream of CgVISA.And Overexpression of CgIRF2 and CgIRF8 protein can significantly activate IFN? and ISRE promoters.Furthermore,we found that CgIRF2 and CgIRF8 could locate both in cell nucleus and in cytoplasmic.In summary,we firstly confirmed the presence of VISA-dependent RLR signaling pathways in a invertebrate C.gigas.We identified key genes of oyster RLR pathway such as CgRIG-I-1,CgVISA,CgTRAF2/3/6,CgIRF2 and CgIRF8 and studied the roles they played in oyster innate immunity.Our studies would broad people's understanding of invertebrate RLR immune pathway and would facilitate the further research on invertebrates RLR signaling.