RNAi Antiviral Pathway In Recilia Dorsalis Modulates Vector Competence For Rice Dwarf Virus

In nature,one species of insect vector may transmit one or more plant viruses.The transmission efficiency of different insect vector is varying.The divergence of acquirement and transmission is termed as vector competent,which is influenced by genetic background,barriers of tissue or membrane,symbiotic bacteria or RNAi antiviral immune pathway in insect vector,and so on.Previous study demonstrated that the RNAi antiviral immune pathway in its potential vector Laodelphax striatellus modulates vector competence for Southern rice black-streaked dwarf virus(SRBSDV)Rice dwarf virus(RDV),a member of in the family of Reovididae,is mainly transmitted by leafhopper Nephotettix cincticeps in a persistent-propagative manner,and other leafhopper like in an inefficiency manner.It is known that nonstructural protein Pns10,which assembles into virion-containing tubule structures for RDV spread,is unable to interact with cytoplasmic actin of R.dorsalis in the molecular level,causing the RDV fail to efficiently spread in the insect vector,which finally cause the inefficiency of R.dorsalis transmitting RDV.Recently,it is found that the infection of Rice gall dwarf virus(RGDV)triggers the siRNA(small interfering)antiviral pathway in cultured cells and competent vector R.dorsalis,confirming that siRNA pathway modulates viral infection in its insect vector.It is unknown that whether the siRNA antiviral pathway in R.dorsalis modulates the vector competence for RDV.Based on the system of RDV-R.dorsalis,this study perform some investigation as follows.(1)The sequential route of RDV infection and the barriers encountered by RDV in its insect vector were identified by performing the microinjection,immunolabeling and traditional biological assay.It was confirmed that the barriers of midgut is the key in blocking viral efficient infection,proliferation and dissemination in R.dorsalis.(2)The small RNA sequencing and deep sequencing of viruliferous cultured cells of R.dorsalis revealed the distribution characteristics of viral-derivered small interfering RNA(vsiRNA)and the expression profile of genes involving in RNAi pathway,including Argonautel(AGO1),Argonaute2(AG02),Dicerl(DCR1),Dicer2(DCR2),systemic RNA interference defective-1(SID)and P-element induced wimpy testis(PIW1).(3)The application of RNAi induced by double-strands RNA(dsRNA)synthesized in vitro revealed that DCR2 and AG02 in siRNA pathway were the key genes modulating RDV infection in RNAi pathway.The RNAi induced by dsRNA microinjection in vivo revealed that knockdown of DCR2 and AGO2 significantly improved the viral infection,replication and transmission by R.dorsalis.Thus,it was confirmed that siRNA pathway modulated the R.dorsalis competence for RDV.In conclusion,this study demonstrates that the RNAi innate immunity,except the tissue system and membrane barriers,blocked the RDV infection in R.dorsalis,based on the results of the cultured cells assay,RNAi experiments,immunolabeling,microinjection and western blot.It was verified that DCR2 and AGO2 in the siRNA pathway were the key genes in the RNAi antiviral immunity.Knockdown of the genes of DCR2 or AG02,RDV infection and proliferation in vector cell monolayers(VCMs)of R.dorsalis was promoted.Knockdown of DCR2 gene significantly increased the capability of acquisition and transmission by R.dorsalis,suggesting that RNAi pathway modulated R.dorsalis competence for RDV.This study provided a further understanding about the interaction between R.dorsalis and RDV,and this may helpful to explore new strategy and methods to block the transmission of RDV by its insect vector providing theoretical basis for control of rice dwarf disease.