Prolifecation And Apoptosis Induction Of Rice Gall Dwarf Virus In Its Insect Vector

Rice gall dwarf virus(RGDV)is a member of the genus Phytoreovirus in the family Reoviridae.RGDV is transmitted mainly by the leafhopper Recilia dorsalis in a persistent propagative manner.To complete the infection in insect body,virus need to overcome the multiple tissue barriers,including midgut infection barrier,midgut escape barrier,salivary gland infection barrier and salivary gland escape barrier etc..The mechanisms by which RGDV infects and moves in R.dorsalis and the effects of RGDV infection on R.dorsalis remain elusive.In this study,the infection route of RGDV in R.dorsali was investigated.RGDV proteins involved in virus replication and spread in R.dorsalis were identified and their modes of action were characterized.Gelsolin of R.dorsalis was found to interact with Pnsllof RGDV and negatively affects RGDV infection.Besides,RGDV was found to induce apoptosis,which promotes RGDV replication in R.dorsalis.1.The infection route of RGDV in the body of R.dorsalisImmunofluorescence techniques were used to track the route of RGDV infection in leafhopper R.dorsalis.RGDV was first detected in the filter chamber epithelium at 2 days post-first access to diseased plants(padp).At 5 padp,RGDV accumulated in the filter chamber visceral muscle tissue,the midgut and the hindgut visceral muscle tissues.At the same time,the virus was detected in the hemolymph.At 9 padp,RGDV was found in the salivary glands and could be detected in the entire alimentary canal.The results suggested that RGDV first infects the filter chamber.Then RGDV crosses the basal lamina into the visceral muscles,from where it spreads into the hemolymph and into the salivary glands of its leafhopper vector.This is the first study to describe the infection route RGDV in R.dorsalis.2.Involvement of Pns7,Pns9 and Pns12 in replication of RGDV in its insect vectorPhytoreoviruses can induce the formation of electron dense inclusions named viroplasm,which is the site for viral replication and assembly.Normally,viroplasm is composed of six non-structural proteins.In this study,to investigate the roles of Pns7,Pns9 and Pns12 of RGDV in the formation of viroplasm,the polyclonal antibodies against Pns7,Pns9 and Pnsl2 were prepared,purified and conjugated to fluorescein isothiocyanate,respectively.Immunofluorescence microscopy and transmission electron microscopy demonstrated that Pns7,Pns9 and Pnsl2 were present in the viroplasm formed during RGDV infection in the continuous cell cultures(VCMs).Double-strand RNAs(dsPns7,dsPns9 and dsPnsl2)were synthesized in vitro and used to silence Pns7,Pns9 and Pns12 genes,respectively,in VCMs.In all treatments,viroplasm formation and viral replicatioin were significantly inhibited.However,treatment with dsGFP had no obvious effects on viroplasm formation and viral replication.In order to confirm the role of Pns7,Pns9 and Pnsl2 in the replication of RGDV in leafhopper R.dorsalis,dsPns7,dsPns9 and dsPnsl2,respectively,were injected into the thorax at 2 padp of R.dorsalis.Then,RGDV was detected using immunofluorescence at 4,10 and 15 padp.In all treatments,RGDV could be detected only in the filter chamber but did not accumulate in visceral muscle tissue and the salivary glands.The insect vector could not transmit the virus.These results indicated that Pns7,Pns9 and Pns12 of RGDV played important roles in viroplasm formation and viral replication in its insect vector.Genes involved in viroplasm formation are promising targets for interventing RGDV transmission by RNAi.3.Involvement of Pns11 in cell to cell spread of RGDV in VCMs.The localization of RGDV Pns11 in Spodoptera frugiperda(Sf9)and VCMs of R.dorsalis was studied by immunefluorescence techniques and electron microscopy.Filamentous and tubular structures composed of Pns11 were observed in Sf9 cells expressing Pns11 alone and in VCMs infected with RGDV.Virus particles were arranged around the filamentous structures in the cytoplasm of the VCMs.Tubular structures containing virus particles were able to protrude from infected cell surface to adjacent cells.We speculated that the tubular structure formed by Pnsll may be involved in the spread of the virus in VCMs.In order to verify this,virus antibodis were used to neutralize virus particles released to the medium in order to inhibit virus secondary infection in VCMs.The results showed that RGDV could spread from infected cells to adjacent cells when Pnsll was expressed normally.However,knockdown of Pnsll expression by RNAi abolished the formation of tubules and prevented cell-to-cell spread of RGDV,although it did not significantly affect viral multiplication.All these results showed that RGDV exploits virus-containing tubules to facilitate viral spread among its insect vector cells.4.Interaction of Gelsolin with Pns11 and involvement of Gelsolin in RGDV infection in VCMsYeast two hybrid experiments showed that RGDV Pns11 interacted with Gelsolin,an actin-binding protein,of R.dorsalis.The interaction was confirmed by co-localization of Pns11 and Gelsolin in Sf9 cells and in VCMs.In Sf9 cells,Gelsolin showed a diffuse localization when expressed alone.However,it formed filamentous-like structures when it was co-expressed with RGDV Pns11.Knockdown of Gelsolin by RNAi significantly increased the accumulation of RGDV in VCMs,suggesting that Gelsolin plays a negative role in RGDV infection.5.Induction of apoptosis by RGDV and its role in RGDV infection of R.dorsalisDecreasing in cell size,cell shrinkage and cell death were observed in VCMs infected with RGDV.Moreover,filamentous structures formed by Pns11 were found to target the mitochondrion and induce mitochondrial degradation.The leafhopper R.dorsalis infected by RGDV have a shorter life-span than healthy controls,which imply that RGDV infection may disturb normal cell physiology of its insect vector.Consistent with this,TUNEL and flow cytometry showed that apoptosis was induced in RGDV-infected VCMs.Apoptosis induced by RGDV is dependent on the Caspase pathway.The RT-qPCR results showed that the Caspase family genes and apoptosis inhibiting IAP1 expression level increased in response to RGDV infection.Knockdown of Caspase 2 and Caspase 8 expression by RNA interference significantly decreased virus multiplication in VCMs and insect bodies.In contrast,knockdown of IPA1 expression promoted virus multiplication.In summary,the present study clarified the infection route of RGDV in its insect vector.Pns7,Pns9 and Pns12 of RGDV were found to play an important role in viroplasm formation and viral replication in VCMs and bodies of R.dorsali.RGDV exploits virus-containing tubules to facilitate viral spread among its insect vector cells.Gelsolin interacted with RGDV Pnsll and negatively regulated virus infection in VCMs.Moreover,apoptosis induced by RGDV infection promoted virus infection.These results provide a basic foundation for further studies on the interaction between viruses and their insect vectors.