Infection Mechanisms Of SRBSDV And RRSV In Vector Planthoppers

Southern rice black-streaked dwarf disease caused by southern rice black-streakeddwarf virus (SRBSDV) has recently prevailed in southern China and become one ofthe most damaging viral diseases of rice. SRBSDV is a newly proposed Fijivirusspecies in the family Reoviridae, which was transmitted mainly by white backedplanthopper (Sogatella furcifera, WBPH） in a persistent propagative manner.Meanwhile, Rice ragged stunt disease caused by rice ragged stunt virus (RRSV) hasrecently spread in southern China. RRSV is a well-established Oryzavirus species inthe family Reoviridae. It is transmitted by brown planthopper (Nilaparvata lugens,BPH), also in a persistent propagative manner. In recent years, transmissioncharacteristics of the two rice viruses by planthoppers have been studied by manyresearchers in China. However, few studies have been conducted on the mechanismsby which the two viruses infect their vector planthoppers. The infection route,replication and spread of viruses in insect vectors are still poorly understood.In this study, we investigated the infection route of SRBSDV in the digestivesystem of WBPH by immunofluorescence. SRBSDV particles initially traveledthrough the oesophagus into the midgut lumen of WBPH after insect acquisition ofthe virus from SRBSDV-infected rice plants. Then, only a few virus particles infectedand replicated in the epithelial cells of the midgut. At4days post-first access todiseased plants (padp), SRBSDV traversed the basal lamina and infected the visceralmuscle tissues encircling the midgut epithelium. From then on, SRBSDV spreadalone the circular and longitudinal muscles of the midgut to other organs. At6dayspadp, SRBSDV spread to salivary glands in some insects. At8days padp, SRBSDVinfected the whole alimentary canal and replicated in the salivary glands of WBPH.The compatibility between SRBSDV and WBPH is significantly different from thatbetween SRBSDV and SBPH. It may be related to the different infection route ofSRBSDV in different vector insects. The localization of SRBSDV in digestive systemof SBPHs at25days padp was detected by immunofluorescence microscopy. Wecould observe SRBSDV within the midgut, but not in the salivary glands of SBPHs.This result suggested that SRBSDV was restricted in midgut and failed to spread intothe salivary glands because of the midgut dissemination barrier in SBPHs, which ledto the SBPH failed to transmit SRBSDV to rice. During the infection of SRBSDV, viroplasms, the punctate inclusions for virusreplication and assembly of progeny virions, were formed in the alimentary canal andsalivary glands of BPHs. To study the infection mechanism of SRBSDV, we usedimmunofluorescence microscopy to observe the localization of P9-1in virus-infectedVCMs of WBPHs. P9-1formed punctate inclusions in VCMs, and progeny viralparticles and viral mRNA accumulated within the viroplasm matrix of P9-1. P9-1alsocould form viroplasm-like inclusions in the Sf9cells. All these results suggested thatP9-1is a constituent of the matrix of viroplasm and viral inclusions formed by P9-1were the sites for synthesising viral RNA and assembling viral particles. To furtherconfirm the function of P9-1in viral replication, the RNA interference induced bysynthesized dsRNA was used to investigate gene functions of P9-1in insect culturecells. By knocking down the expression of P9-1with synthesized dsRNA of P9-1, theformation of viroplasm and viral infection were significantly inhibited in the VCMs.Meanwhile, ingestion of dsRNAs from the P9-1gene via membrane feeding alsostrongly inhibited viral infection and replication in intact WBPHs. These resultssuggested that P9-1was essential for the infection and replication of SRBSDV inWBPHs.SRBSDV can only be effectively transmitted when it spread from the midgutepithelial cells, the primary site of virus entry into insect cells, to salivary glands. Thespread of SRBSDV in the insect vector plays a crucial role in the infection route. Inthis study, we observed that the tubular structures formed by non-structure proteinP7-1contained viral particles and scattered the surface of infected cells or extendedtoward neighboring cells. P7-1of SRBSDV also has the ability to form tubulesgrowing from the cell surface in the Sf9cells. Based on these results, we conjecturedthat the P7-1was involved in the cell-to-cell transmission of SRBSDV. To determinethe function of P7-1related to the spread of SRBSDV, we blocked the virus that hadbeen released into or was present in VCMs with virus-neutralizing antibodies. TheP7-1tubular structures were also extended from the primary cell toward neighboringhealthy cells. When the expression of P7-1was knocked down by RNAi induced bydsRNA specific for P7-1gene, the formation of tubules was inhibited and SRBSDVfailed to spread to the neighboring cells. All these results suggested that the P7-1participated in the cell-to-cell spread of SRBSDV in cells of WBPHs.Whether the P7-1participates in the spread of SRBSDV in WBPHs? We detectedthe localization of P7-1in WBPHs by confocal microscopy. The distribution oftubular structures formed by P7-1is correlated with the infection route of SRBSDV. P7-1firstly appeared in epithelial cells, then spread from the epithelial cells to circularmuscle of midgut and moved alone it. Tubules subsequently spread to the longitudinalmuscle that intersected with circular muscle and disseminated from the midgut toother organs. Virus started to replicate on the sites where tubules had arrived before.We confirmed that the P7-1specifically interacted with Actin of WBPH by yeast twohybrid experiments and co-immunoprecipitation, suggesting that the tubule formed byP7-1utilized the interaction with Actin of WBPH to spread on muscle of midgut.When the expression of P7-1was strongly inhibited by feeding synthesized dsRNA ofP7-1via membrane feeding, the formation of P7-1tubular structure was inhibited.This prevented the spread of SRBSDV in the digestive system of WBPHs, whichresulted in the fact that the tested WBPH failed to transmit virus to rice. All theseresults suggested that the P7-1was essential for the spread of SRBSDV in WBPH.We also determined the infection route and the replication mechanism of RRSV ininfected BPHs. We firstly analyzed the infection route of RRSV in BPHs byimmunofluorescence and found that it was roughly similar with that of SRBSDV.During the infection of RRSV, non-structure protein Pns10formed viroplasm inmidgut epithelial, muscle and salivary glands of RRSV-infected BPHs. To furtherconfirm the function of Pns10in viral infection, Second-instar nymphs of BPHs werefed synthesized dsRNA of Pns10by membrane feeding before acquisition ofSRBSDV. As a result, RNAi targeting Pns10significantly inhibited the formation ofviroplasm and prevented viral infection and replication in intact BPHs. These resultsindicate that Pns10is essential for infection of RRSV in BPHs.In all, the infection routes of SRBSDV and RRSV in different vector planthopperswere elucidated for the first time in this study. It was the first time to use RNAistrategy to investigate gene functions of plant viruses in insect culture cells. UsingRNAi induced by synthesized dsRNA, we found that P9-1of SRBSDV and Pns10ofRRSV were the constituent of the viroplasm matrix and were essential for viralreplication. Tubular structures formed by P7-1of SRBSDV contained viral particlesand participated in viral spread in insect. These studies are useful for understandingthe efficiently transmission mechanism of SRBSDV and RRSV in their vectors, andprovided theoretical basis and new targets to control viral diseases of rice by RNAistrategy.