| The rice dwarf disease caused by rice dwarf virus(RDV)is one of the major rice viral pathogens in south China.RDV is transmitted by the leafhopper Nephotettix cincticeps in a persistent propagative and transovarian manner.Vertical virus transmission through transovarial passage plays an important role in maintenance of viral population in cold seasons,and viral propagation across vector generations,which facilitates viral epidemics in the field.The mechanism for vertical virus transmission is always a focus and difficulty.In this study,I investigated the mechanism for transovarial transmission of RDV by N.cincticeps,and the interaction of leafhopper vitellogenin(Vg),endosymbionts and RDV during their transovarial transmission,aiming to decipher the multiple cooperations among insects,endosymbionts and plant viruses during their vertical transmissionFirst,we determined the distribution and transovarial transmission of the two obligate symbionts,Sulcia and Nasuia in N.cincticeps.Sulcia and Nasuia propagate abundantly in adult female insects.Electron and immunofluorescent microscopy indicated that Sulcia and Nasuia initially gathered around the epithelial plug in ovary,and then invaded into the cytoplasm of follicular cells surrounding epithelial plug,possibly via membrane fusion,and Sulcia and Nasuia were able to infect the same follicular cell.They further accumulated within epithelial plug and moved into the oocyte,forming a characteristic'symbiont ball' in the oocyte.These results suggested that Sulcia and Nasuia simultaneously migrate intothe oocyte following the same path.We have demonstrated previously that RDV can hitchhike Sulcia for transovarial transmission,since Sulcia and Nasuia moved together into the oocyte and frequently occurred in the same follicular cell,we deduced that Nasuia might also be involved in the oocyte entry of RDVElectron and immunofluorescent microscopy revealed that RDV hitchhiked both Nasuia and Sulcia to enter the oocyte,however,the associations of RDV with Sulcia and Nasuia were different.RDV distributed on the outer membrane of Sulcia but within the periplasmic space between the outer and inner membranes of Nasuia,suggesting that distinct interaction strategies of RDV with Nasuia and Sulcia may occur.The yeast two hybrid and GST pull down assay showed that RDV minor capsid protein P2 interacted with Sulcia outer membrane protein(OMP),and the major capsid protein interacted with Nasuia porin,indicating that RDV virions may directly pass through the protein channels on the outer membrane of Nasuia and accumulate in the periplasmic space.In particular,the association of RDV and Nasuia was abolished by pretreatment with Nasuia specific antibody,which also inhibited the transovarial transmission rate of RDV but did not affect the oocyte entry of Nasuia.The results demonstrated that RDV has evolved different strategies to employ Sulcia and Nasuia for transovarial transmission,and they have formed complex tripartite interactions during their joint transovarial transmission to the next insect generation.The N.cincticeps vitellogenin(NcVg)specifically accumulated with adult female insects,and was sequestered into the developing oocytes from the nurse cell of the germarimm via the nutritive cord.We observed that NcVg was absorbed into the oocyte via the epithelial plug at the vitellogenic stage,suggesting that a new oocyte entry route for NcVg existed.Since Sulcia and Nasuia always entered the oocyte via the epithelial plug at the vitellogenic stage,we hypothesized that NcVg might be involved in the oocyte entry of the symbionts.To test the hypothesis,we observed the distribution pattern of NcVg,Nasuia and Sulcia within the hemolymph,epithelial plug and symbiont ball.we found that NcVg colocalized with Nasuia but not Sulcia,indicating Nasuia and NcVg moved jointly from hemolymph into epithelial plug,and finally into oocytes.The yeast two hybrid and GST pull down assay revealed that NcVg interacted specifically with Nasuia porin,but not with Sulcia OMP.Nasuia porin,Sulcia OMP or NcVg antibody treatment significantly reduced the binding efficiency between Nasuia and NcVg.Microimjecting dsRNA targeting insect proline-rich protein(Ncprp)which is critical for Nasuia propagation significantly reduced the accumulation of NcVg and Nasuia im ovary,but had no effect on Sulcia accumulation,indicating Nasuia is critical in the process of oocyte entry of NcVg.To further determine whether Nasuia exploited NcVg system for entering the oocyte,the vitellogenin receptor(VgR),we knocked down the in vivo expression of VgR,and found that the amount of NcVg decreased in female ovary,whereas the oocyte entry of Nasuia and Sulcia was not affected,indicating that Nasuia is employed by NcVg for the joint entry into oocytes,which is independent of NcVgR systemSince Nasuia was hitchhiked by RDV and NcVg,we then observed their distribution pattern within insect ovary.The result showed that they colocalized in both the hemocytes and the epithelial plug,indicating RDV and the leafhopper N.cincticeps have evolved to exploit Nasuia to enter the oocyteIn summary,we have revealed the cooperative interactions of RDV,NcVg and Nasuia during their maternal transfer,and RDV has evolved different strategies to exploit Sulcia and Nasuia for transovarial transmission.Moreover,we has discovered a new oocyte entry path for NcVg which hitchhikes Nasuia for entering the oocyte.The study has characterized the mechanism for the triple interactions among Nasuia,NcVg and RDV during their joingt movement into the oocyte,and provided new knowledge for further deciphering the cooperative mechanisms among insect,plant viruses and insect symbiont. |
PDF Full Text Request