Interaction Between RSV And RNAi Systems Of Vector Insect

Rice stripe disease is one of the the most diseases of rice diseases in China.In nature,RSV is mainly transmitted by the small brown planthopper in a persistent-proliferational manner.The small brown planthopper can not only spread RSV to rice plant by piercing-sucking feeding behavior,but take viral particles into eggs with the nymph development.In the viral infection to the planthoppers,RSV camouflages the immune response,which is often conflit to viral replication and transmission.Small RNA silencing pathways,especially to the small interfering RNA(siRNA)and microRNA(miRNA)pathways,plays an important role in defending against viruses in plants and insects.However,how insect-transmitted phytoviruses regulate the RNAimediated antiviral response in vector insects has barely been uncovered.In this study,we explored the interaction between rice stripe virus(RSV)and the miRNA/siRNA pathway of the small brown planthopper.The main research results are as follows:The response of miRNA pathway and siRNA pathway in L.striatellus against RSV infection.By using the genome of L.striatellus,eight miRNA-related and siRNA-related genes in the small brown planthopper were identified.The nonviruliferous insects were inoculated with RSV by the artificial method,which is performed by injecting RSV crude extracts in an intrapleural injection way.The expression profiles of these eight target genes were detected at the transcriptional level and expressional level by using real-time PCR and Western blotting before or after inoculation.We found that during the RSV infection,the expression of the eight genes in the miRNA and siRNA pathways did not have significant difference at the transcriptional level and expressional level.The results shows that the miRNA and siRNA pathways of vector insects do not respond to RSV infection.The role of miRNA and siRNA pathways in controlling RSV replication.In order to explore the functions of miRNA and siRNA pathways in RSV replication,silenced the expression of Ago1,Ago2 or translin by injecting the crude RSV extract and double strand RNA(dsRNA)into the nonviruliferous insects at a ratio of 1:1.Knockdown of Ago2 promoted the replication of RSV,while interfering with Ago1 did not affect the replication of RSV.When the translin transcriptional level of translin was knocked down,The RSV proliferation was inhibited in the high RSV carrying viruliferous planthoppers.Similarly,RSV replication was dramtically repressed in the nonvirulifersous insects which were co-injected with the dsRNA of RSV crude extract as well as dstranslin.These results indicate that inhibition of siRNA pathway promotes RSV replication,while inhibition of miRNA pathway has no significant effect on RSV replication,but activation of miRNA pathway by interfering with translin will limit RSV replication in L.striatellus.The interaction between the viral protein NS2 and Ago2 inhibited the activation of the siRNA pathway of L.striatellus,and the interaction of the RdRp2 protein and translin regulated the anti-virus function of the miRNA pathway of L.striatellus.In order to verify whether RSV affects virus replication through the interaction of viral proteins with the small RNA pathway of L.striatellus,found that the NS2 protein of RSV interacts with Ago2,and the RdRp2 protein of RSV interacts with translin by immunoprecipitation experiments.In addition,when the RSV crude extract and NS2 dsRNA were injected into the nonviruliferous insects at a ratio of 1:1 the transcripts of NS2 was down-regulated and the replication amount of RSV was significantly reduced.These results prove that the interaction of NS2 protein and Ago2 may inhibit the siRNA pathway to promote RSV replication,and insect translin acts as an inhibitor of miRNA during RSV infection.MiRNA screening and target gene prediction of translin regulation in Laodelphax striatellus.The total RNA was extracted at 4 days and 6 days after the interference with translin in viruliferous L.striatellus.Three replicates were selected for control group and treatment group respectively.The miRNA and transcriptome were sequenced by RNA-seq to obtain the differentially expressed miRNA and the differentially expressed gene.Eight miRNAs regulated by translin were found,and the target gene binding sites of these eight up-regulated miRNAs were predicted by RNAhybrid and mi Randa algorithms.In summary,our research reveals that the viral proteins NS2 and RdRp of the plant virus RSV regulate the miRNA pathway and siRNA pathway of the vector insect L.striatellus.This protein binding regulation of insect RNAi systems reflects a complicated and diverse coevolution of viruses with their vector insects.The results of the study provide a new understanding of the interaction mechanism between L.striatellus and rice stripe virus,as well as new strategies for controlling the spread of arboviruses.