RNA Interference Pathway Modulates The Infection Of Rice Viruses In Insect Vectors

Rice viruses,such as southern rice black-streaked dwarf virus(SRBSDV),rice stripe virus(RSV),rice ragged stunt virus(RRSV),rice gall dwarf virus(RGDV)and rice dwarf virus(RDV),are transmitted by leafhoppers or planthoppers in a persistent-propagative manner.After their ingestion by insects during feeding on diseased plants,these viruses must enter and replicate in the epithelial cells of the alimentary canal of insect vectors,then exit and move to the salivary glands to be transmitted to healthy plants.Thus,these rice viruses must overcome not only the tissue barriers but also the immunity barriers during the life cycle in respective insect vector,which affected the ability of insect vectors to transmit these rice viruses or the competence of insect vectors for these rice viruses.RNA interference(RNAi)is generally thought to be an evolutionarily mainly antiviral immunity pathway in insects.However,whether the RNAi pathway could modulate the ability of these insect vectors to transmit these rice viruses or the vector competence for these rice viruses,the establishment or maintenance of the persistent infection and efficient spread of these rice viruses has been the subject of debate.Here,we mainly focus on the relationship of the RNAi antiviral pathway with the insect vector competence for these rice viruses and the persistent infection of them.1.The small interfering RNA(siRNA)pathway modulates the ability of SBPH to transmit SRBSDV.The variation of an insect species competence for a virus or the ability of an insect species to transmit a virus is common in nature.SRBSDV is transmitted by the white-backed planthopper(WBPH;Sogatella furcifera Horvath)in a highly efficient manner.However,the inability of the small brown planthoppers(SBPH;Laodelphax striatellus)to transmit SRBSDV could be due to the restriction of viral dissemination from the midgut of SBPH,which led to the failure of viral spread to the salivary glands for virus transmission.The RNAi antiviral pathway in insects affected the infection rates,accumulation and dissemination of arboviruses.However,whether the RNAi antiviral pathway could control the persistent replication of SRBSDV,thereby modulating vector competence of WBPH and SBPH for SRBSDV is unknown.Firstly,to identify the barriers confronted by SRBSDV in SBPHs or WBPHs,second-instar nymphs of SBPHs and WBPHs were microinjected with SRBSDV inoculum through microinjection system.Immunofluorescence microscopy and RT-qPCR showed that the viral infection,proliferation,dissemination and transmission rates were lower in SBPHs when compared with that in WBPHs at 5,8,11 and 15 days post-first access to diseased plants(padp).Thus,three specific barriers to viral efficient transmission were confronted by SRBSDV in SBPHs.These barriers include the significant midgut barrier,the secondary salivary glands barrier and possible the immunity barrier.Secondly,to confirm whether the RNAi pathway could modulate SBPHs competence for SRBSDV or its ability to transmit the virus,we firstly used small-RNA deep sequencing to characterize abundant viral-derived small interference RNAs(vsiRNAs),22-nt in length,in the SBPH midguts and its continuous cultured cells,suggesting that the RNAi pathway was triggered by SRBSDV infection.In addition,immunofluorescence,RT-qPCR and small RNA deep sequencing showed that the knockdown of the expression of the core component Dicer2 of siRNA pathway due to RNA interference by transfecting and injecting double stand RNA of Dicer2 gene(dsDicer2)strongly increased the propagation ability of SRBSDV in the continuous cultured cells and in the midgut epithelium of SBPH,allowing copies of viral major outer capsid protein P10 gene of SRBSDV in the midgut epithelium to reach the certain threshold,1.99×109copies/?g midgut RNA,above which SRBSDV disseminated from midgut epithelium into midgut muscles,then into the salivary glands of SBPH,suggetting SBPH became a competent insect vector to transmit the virus to rice plants at last.Thus,the siRNA antiviral pathway modulates the SBPH competence for SRBSDV.Furthermore,to confirm whether the siRNA antiviral pathway could control the persistent infection of SRBSDV in competent insect vector WBPH,we used immunofluorescence,RT-qPCR and small RNA deep sequencing to show that the knockdown of Dicer2 expression promoted the viral infection in WBPH cultured cells.In addition,the knockdown of Dicer2 expression suppressed the accumulation of vsiRNAs,promoted the SRBSDV infection in the WBPH vector and reduced the survival rate and lifespan of the viruliferous insects.Taken together,these results indicate that the siRNA pathway effectively modulates the persistent infection of SRBSDV in the competent vector WBPH.This pathway thus plays a crucial role by preventing excessive plant virus accumulation in insect vectors,thereby protecting them from the negative impact of these viruses.Although the core component Dicer2 of siRNA pathway could modulate the persistent propagation of SRBSDV in SBPH or WBPH,the huge difference still exists in the competence of SBPH and WBPH for SRBSDV.The deduced amino acid sequence identity of DCR2 genes between SBPH and WBPH is about 84%,suggesting that the competence difference of SBPH or WBPH for SRBSDV may relate with the natural variations of Dicer2.Due to the lack of transgenic expression system for Hemipteran insects,the direct evidence to confirm the specific role of WBPH DCR2 in SBPH is not available.Thus,what roles the siRNA antiviral response played in insects depends on the specific pathogen systems.It seems that the siRNA pathway modulates the competence for a virus in incompetent insect vectors,but it plays a crucial role in protecting them from the negative impact of the virus in competent insect vectors.2.The siRNA pathway modulates the persistent infection and efficient transmission of RGDV in insect vector,Recilia dorsalis.Persistent-propagative viruses are more suitable to replicate highly efficiently in insect cells.But such viruses do not cause any disease to insect vectors,suggesting that persistent-propagative viruses could coexist peacefully with insect vectors under the controlling of the siRNA antiviral pathway in insects.RGDV is highly efficiently transmitted mainly by the leafhopper R.dorsalis in a persistent-propagative manner.To determine how the siRNA pathway modulates persistent infection of RGDV in its insect vector,R.dorsalis,we showed that a conserved siRNA antiviral response was triggered by the persistent replication of RGDV in cultured leafhopper cells and in intact insects,by appearance of vsiRNAs,primarily 21-nt long,and the increased expression of siRNA pathway core components Dicer2 and Argonaute2 by small RNA deep sequencing and RT-qPCR,respectively.Silencing of Dicer2 due to RNA silencing strongly suppressed production of vsiRNAs,promoted viral accumulation,and caused severe cytopathological changes such as vacuolization in the cytoplasm,reduction of cytoplasmic density,the loss of microvilli brush border integrity,and the degeneration of mitochondria and rough endoplasmic reticulum in vitro and in vivo,at last the high mortality of insects.When the viral accumulation level rose above a certain threshold of genome copy of viral major outer capsid protein P8 gene of RGDV(1.32×1014 copies/?g insect RNA),the viral infection in 55%of leafhoppers was lethal rather than persistent.Taken together,for the first time,we revealed that,from an evolutionary standpoint,a plant virus has developed a mechanism,by triggering a siRNA antiviral response,to optimize the delicate balance between robust replication and limited pathology in its insect vector,allowing for persistent infection and highly efficient vectoring in nature.3.The siRNA pathway modulates the persistent infection of other rice viruses in insect vector.To confirm whether the siRNA antiviral pathway could modulate the persistent infection of rice viruses in other insect vector,we used immunofluorescence,RT-qPCR and small RNA deep sequencing to show that silencing of Dicer2 and Argonaute2,the core components of siRNA pathway,in Nephotettix cincticeps and its cultured cells also suppressed the production of vsiRNAs,primarily 21-nt long,but promoted the infection,proliferation and transmission rates of RDV in insects,therefore increasing competence of N.cincticeps for RDV.Furthermore,silencing of Dicer2,the core component of the siRNA pathway also promoted the persistent infection of RSV and HiPV in SBPH.Thus,this study further confirmed that the siRNA pathway has general or conversed function in modulating the persistent replication of viruses in insect vectors such as leafhoppers and planthoppers.In summary,using insect cultured cells,RNAi silencing,immunofluorescence,small RNA deep sequencing and RT-qPCR,we confirms and reveals that(1)SRBSDV confronted with the midgut,the salivary gland and immunity barriers in SBPH;(2)there is a general siRNA antiviral response in insect vectors leafhopper and planthopper and the core components of the pathway are Dicer2 and Argonaute2;(3),the siRNA antiviral pathway modulates the competence of the insect vector SBPH and WBPH for SRBSDV;(4),the siRNA antiviral pathway modulates viral persistent infection,pathogenicity,efficient spread of RGDV in R.dorsalis and coexistence peacefully between them.The results of this study are not only important for exploring new efficient strategies to block the viral spread by insect vector,but also important for providing theoretic base for administering disease caused by rice viruses.