Functional Study Of JAK-STAT And RNAi Pathways In Laodelphax Striatellus During Virus Infection

Insects are one of the most abundant group of animals with a vast number of species on earth.More than 75% of known plant viruses are transmitted by aphids,whiteflies,leafhoppers,planthoppers and other insects.Although insects are small organisms with a short life cycle,they have formed efficient antiviral defense systems through long-term evolution.Currently,insects mainly rely on RNA interference(RNAi),Toll,Immune deficiency(IMD),Janus kinase/signal transducers and activators of transcription(JAK-STAT),apoptosis,and autophagy for immune response against viruses.The small brown planthopper(SBPH,Laodelphax striatellus)is an agricultural pest.Previous studies have shown that the RNAi pathway,Toll pathway,JNK pathway and AMP-activated protein kinase(AMPK)pathway can be activated by plant virus and participate in the persistent viral infection in SBPH.In this study,we used molecular biology,transcriptomics,and s RNA analysis to clone and analyze the key genes of apoptosis,JAK-STAT,and RNAi immune pathways in planthopper.The functions of these pathways were subsequently investigated.The main results are listed below:1.Under transmission electron microscopy(TEM),abundant apoptotic cells were observed in RSV-infected cells of SBPH.These virus-infected epithelial cells displayed apoptotic characteristics,including disappearance or invagination of nuclear membrane,condensed nuclei,marginalized chromatin,vacuolated mitochondria and irregular cristae.Furthermore,TUNEL assay revealed that green(FITC)apoptotic signals were detected in the midgut and salivary glands of RSV-infected SBPH and these signals overlapped with the nucleus.Meanwhile,RSV infection up-regulated the protein expression of pro-apoptotic genes and down-regulated the protein expression of anti-apoptotic genes.Inhibiting the expression of Caspase genes significantly decreased the accumulation of RSV.Our results indicating that RSV infection induces apoptosis to benefit virus accumulation in SBPH.2.Yeast two-hybrid system and MBP pull-down assay indicated that suppressor of cytokine signaling 5(SOCS5)interact with anti-apoptotic gene BCL2 via the SH2 domain.The 26 S proteasome pathway is involved in SOCS5-induced degradation of BCL2.Yeast onehybrid and EMSA(electrophoretic mobility shift assay)assays showed that the signal transducer and activator of transcription 5B(STAT5B)direct binds to the three promoters of SOCS5 and to regulate its expression.RSV infection activates the insect JAK-STAT pathway.When the expression of STAT5 B and SOCS5 was suppressed,a significant decrease in viral accumulation of SBPHs was observed,as well as fewer RSV virions in salivary glands and ovaries.Furthermore,ds STAT5B-or ds SOCS5-treated SBPHs had drastically reduced virus acquisition after injection with RSV crude extracts or feeding on RSV-infected rice plants.The protein levels of pro-apoptotic genes were substantially reduced and the accumulation of antiapoptotic gene BCL2 was significantly increased when STAT5 B or SOCS5 was knockdown.These results indicated that RSV infection activated the JAK-STAT pathway,and this pathway can regulate apoptosis to promote viral infection in SBPH.RBSDV,another SBPH-transmitted plant reovirus,also can activate the JAK-STAT pathway and apoptosis.Knockdown of STAT5 B or SOCS5 significantly reduced RBSDV acquisition by SBPHs similar to RSV.3.Silencing of miRNA pathway genes AGO1 or DCL1 significantly reduced the survival rate,egg production and hatching rate of SBPH.Silencing of piRNA pathway genes Piwi or AGO3 significantly decreased the egg production and hatching rate,while extended the development period from the second to the fifth instar of the insects.Silencing with siRNA pathway genes AGO2,DCL2 and R2D2 did not affect the egg production and hatching rate of SBPH.Moreover,knockdown of AGO2 significantly extended the development period of the second and fifth instar,while inhibition of R2D2 shortened the development period of the fourth instar of SBPHs.These results suggested that key genes of RNAi pathway are involved in regulating the growth and development of SBPHs,especially for the genes of miRNA and piRNA pathways.4.q RT-PCR and s RNA analysis shown that miRNA and piRNA pathways were not involved in antiviral immunity in SBPH.Inhibiting with the siRNA pathway gene AGO2 significantly increased the accumulation of viruses.Contrarily,DCL2 knockdown did not affect the replication of viruses.In addition,similar results were observed for the functions of AGO2 and DCL2 in white-backed planthopper(Sogatella furcifera)and brown planthopper(Nilaparvata lugens)compare to SBPHs through vsiRNA analysis,suggesting that AGO2,but not DCL2,plays essential roles in the antiviral immunity of rice planthoppers.This study found that RSV infection activates the JAK-STAT pathway,which regulates apoptosis and promotes viral accumulation in insect vectors.Research on the RNAi pathway of the SBPH demonstrated the crucial roles of the miRNA and piRNA pathways in its growth,development,and reproduction.AGO2 in the siRNA pathway plays a key role in antiviral defense,but DCL2 has no antiviral function.These findings enhance our understanding of the vector’s immune response to viruses.