Horizontal And Vertical Transmission Mechanisms Of Rice Gall Dwarf Virus By Its Insect Vector

The rice gall dwarf disease caused by rice gall dwarf virus(RGDV)is one of the important rice viral diseases and leads to normal epidemic pattern throughout southern provinces of China in recent years.RGDV is transmitted by leafhopper vector Recilia dorsalis,in a persistent-propagative manner and can be vertically transmitted to insect offspring.Viral transmission through insect vectors is often associated with epidemics of plant diseases.Thus mechanisms for viral horizontal transmittion to plant hosts from salivary glands,and for viral vertical transmission to insect offspring from reproductive system were studied here.We also discussed the effects of different transmission pathways on disease epidemics in field.The persistent viruses circulate in the vector body,enter the salivary gland,and then are released into the apical plasmalemma-lined cavities where saliva is stored.The cavity plasmalemma of vector salivary glands thus represents the last membrane barrier for viral transmission.Here,we studied the mechanism used by RGDV to overcome this essential barrier.We observed that the infection of RGDV induced the formation of virus-associated filaments constructed by viral nonstructural protein Pns11 within the salivary glands of its leafhopper vector by electron and immunofluorescence microscopy.Such filaments attached to actin-based apical plasmalemma and induced an exocytosis-like process for viral release into vector salivary gland cavities,through a direct interaction of Pns11 of RGDV and actin of R.dorsalis.Failure of virus-induced filaments assembly by RNA interference with synthesized double-stranded RNA targeting the Pns11 gene inhibited the dissemination of RGDV into salivary gland cavities,preventing viral transmission by R.dorsalis.For the first time,we show that a virus can exploit virus-induced inclusion as a vehicle to pass through the apical plasmalemma into vector salivary gland cavities,thus overcoming the last membrane barrier for viral transmission by insect vectors.Virions suspended in saliva infected plants while vector insects sucking plant saps.The saliva plays a critical role in tripartite relationships among insect,host plant and virus.A comparative proteomic analysis of secreted saliva from viruliferous and nonviruliferous leafhoppers identified 955 individual proteins.Out of 209 proteins were identified from viruliferous leafhopper saliva particularly while 237 proteins belonged to nonviruliferous ones specifically.Most of the differential expression proteins were categorized into oxidoreductase family,which took part in detoxification during insect feeding and were related to feeding behavior.Thus,RGDV infection caused proteomic changes on saliva secretion of R.dorsalis and may effects the feeding efficiency.Viruses can be transmitted from parent to offspring,called vertical transmission.It is an essential maintenance mechanism during adverse conditions for horizontally transmission.Previous studies showed that RGDV can be transmitted to more than 80%of offspring,while only about a quarter of ovaries of viruliferous females were infected by virus.There was a contradiction betweenhigh viruliferous rate of offspring and low efficiency of transovarial transmission.To test the transmission mode of vertical transmission of RGDV,we first carried out the crosses including possible mating combinations within and between viruliferous and nonviruliferous leafhoppers.Statistical analysis showed that RGDV was vertically transmitted from both viruliferous female and males to their offspring.The cross within viruliferous insects(V♀×V♂)transmitted the virus to 81%of their offspring,and the nonviruliferous females mated with viruliferous males(N♀×V♂)produced offspring with an average of 73%infection.In contrast,when viruliferous females mated with nonviruliferous males(V♀×N♂),virus was transmitted to only 22%of their offspring.The result indicated that paternal transmission was the dominant mode for vertical transmission,while the maternal transmission was a minor mode for vertical transmission.Electron microscopy and immunofluorescence were used to study how RGDV transmitted from male parent to offspring.The results revealed that virus particles can bind to insect sperm head and then were transported to female adults through mating.Virions invaded eggs together with sperm during fertilization.We then conducted a virus-sperm incubation system in vitro to test the interaction between virus and sperm and found that the major outer capsid protein P8 of RGDV can bind to live sperms in vitro.A comprehensive GST pull-down screening for protein-protein interactions between P8 of RGDV and sperm proteins were performed to explore the possible receptors during the binding of virus particles to sperms and the candidate receptor heparan sulfate proteoglycan(HSPG)was obtained.The yeast two-hybrid assay and GST pull-down conformed that the Domain III of HSPG interacted with P8 of RGDV.HSPG located at the plasma membrane of the sperm head and virus attached to HSPG.The pretreatment of sperms with HSPG-specific antibody blocked viral binding in vitro.Thus,this novel vertical transmission was possibly mediated by the interaction of major outer capsid protein P8 of RGDV with the surface receptor HSPG of insect sperm head.The direct effects of RGDV on male and female leafhoppers were tested and compared.RGDV infection reduced the longevity of female adults significantly,whereas the longevity and mating ability of male adults were no significant difference compared with the nonviruliferous ones.Eggs produced by cross between nonviruliferous female and viruliferous male(N♀×V♂)exhibited no significant difference from those produced by cross within nonviruliferous insects(N♀×N♂)in egg development rate and morphology parameters.However,the two aspects of eggs produced by viruliferous females mating with nonviruliferous males(V♀×N♂)degraded significantly.The results suggested that viral infection affected female more than male adults and the offspring acquired virus through paternal transmission showed higher performance than maternal transmission.The viruliferous rate of males was higher than that of females in the field during both overwintering season and planting season.Disease severity of RGDV was positively correlated with viruliferous rate of the main insect vector R.dorsalis based on systematic monitoring between 2013 and 2016.It was supposed that the viruliferous rate of overwintering genetation was a good indicator of RGDV disease epidemics.In the overwintering season,virus was maintained through vertical transmission since the lack of host plants and caused normal epidemic pattern.In summary,we conformed and revealed that(i)RGDV can exploit the virus-associated Pnsll filament to perform an exocytosis-like process for viral dissemination via the apical plasmalemma into salivary cavities of its insect vectors;(ii)the component variation of the watery saliva of R.dorsalis induced by RGDV infection was proteomic analyzed;(iii)the vertical transmission of RGDV by R.dorsalis was dominantly mediated by males mating with a low frequency of transovarial transmission by females,and such unique vertical transmission pathway was mediated by the interaction of major outer protein P8 of RGDV with the membrane receptor of sperm,HSPG;(iv)the association of this vertical transmission of viruses with normal epidemic of viral diseases in the field was investigated and discussed.The results will promote the development of the study field of vector-transmitted viral pathogens,and provide the theoretical bases for control exploration of the viruses.