RGDV Exploits Tubules To Facilitate Viral Transovariole Transmission By Leafhopper Recilia Dorsalis

In recent years,the epidemics of rice gall dwarf disease in Guangdong,Fujian,Hainan Province and causes a substantial yield loss in southern China.Rice gall dwarf virus(RGDV)is a member of the genus Phytoreovirus in the family Reoviridae and transmitted mainly by leafhopper Recilia dorsalis in a persistent propagative manner.At present,the researches on RGDV mainly focus on the genome structures and functions,infection of host or cultured insect vector cells.However,the vertical transmission of RGDV in Recilia dorsalis is still poorly understood.In this study,the different mating group trestments were performed to detect the viruliferous rate of offspring.We revealed a transmission efficiency of 18%when viruliferous females crossed to healthy males,compared with 70%infection in the offspring when viruliferous males crossed to healthy females.The result showed,both females and males were able to transmit RGDV to the next generation,and the males played an important role in the vertical transmission.1.The transovarial transmission of RGDV by female insects In our laboratory,electron microscopic observation showed that the non-structure protein Pns11 of RGDV can form virus-containing tubules,which possibly facilitate viral spread into the oocytes of R.dorsalis,but the mechanism was not clear.In this study,we determined that the Pns11 tubules may be associated with actin and propelled by actin-based tubule motility to overcome barriers of transovarial transmission in insect vectors.The effects of knockdown of Pns11 gene expression by RNA interference(RNAi)on viral transmission into offspring by female R.dorsalis,adults were detected by immunofluorescence microscopy,Western blot and RT-qPCR assay.The interference GFP protein expression was set as a control group.The female insects with obvious interference effect were selected for mating experiments.Experimental adults and their offspring were analyzed by RT-PCR or RT-qPCR assay to determine the prevalence of infection.The data showed that about 5.3%of the offspring from dsPns11-treated females were viruliferous,which significantly lower than 21%from the dsGFP-treated females.In addition,GST pull-down assay and yeast two-hybrid system confirmed that Pnsl 1 of RGDV specifically interacted with actin of R.dorsalis.Furthermore,we confirmed that RGDV could exploit Pnsll tubles to pass through actin-based junctions between follicular cells or through actin-based microvilli from follicular cells into oocyte of R.dorsalis.The ability of Pns11 tubules to passing through the actin-based machanies in insect ovaries was mediated by a specific interaction between Pns11 and actin,thus overcoming the barriers of transovarial transmission.2.Transmission of RGDV by sperms of male insectsWe found that the viruliferous males played an important role in the vertical transmission by the mating experiments.Immunofluorescence and electron microscopy showed that RGDV particles were mainly adhered to the plasma membrane of sperm nucleus.In addition,we incubated the purified expressed proteins of RGDV P2,P8 or Pns11 with the dissected sperms from male R.dorsalis adults in vitro,and then inculated these sperms with purified RGDV.We found that the adhesion of RGDV on the plasma membrane of sperm nucleus was mainly mediated by the association of outer capsid protein P8 with the sperm surface.In order to clarify the mechanism,proteins interacting with RGDV P8 from the sperms of R.dorsalis were screened by GST pull-down.Results showed that the vigilin and heparan sulfate proteoglycan(HSPG)and other ten candidate proteins possibly interacted with RGDV P8.The baculovirus expression vector system was used to verify the interactions of RGDV P8 and HSPG or Vigilin.We found that RGDV P8 and HSPG were co-localized in plasma membranes or nuclear membrane of Sf9 cells.When RGDV P8 was coexpressed with vigilin,they were co-localized in the cytoplasm of Sf9 cells.BIFC experiments further confirmed that RGDV P8 can interact with Vigilin or HSPG of R.dorsalis in the leaves of Nicotiana benthamiana.The RGDV P8 and Vigilin were mainly co-located on the plasma membrane and cytoskeleton,while RGDV P8 and HSPG were mainly co-located on the cytoplasmic membrane in the leaves of N.benthamiana.All together,we determined that both females and males were able to vertically transmit RGDV to the next generation of insect vectors,and the males play an important role in the vertical transmission.In the viruliferous famales,the ability of virus-containing Pns11 tubules to pass through actin-based microvilli from follicular cells into oocyte was mediated by a specific interaction of RGDV Pns11 and actin of R.dorsalis,thus overcoming transovarial transmission barriers.RGDV P8,the major outer capsid protein,interacted with the vigilin and HSPG on the plasma membrane of sperm nucleus in the viruliferous males.This attachment of viral particles on sperm surface led to the transfer of RGDV to the ovaries during fertilization process,which finally causing the vertical transmission of RGDV into offspring.Therefore,the preliminary researches revealed mechanism for RGDV to use insect male sperm a vehicle to vertical transmission,which provided new ideas and theoretical basis for blocking the vertical transmission of RGDV.