Invasion Of Midgut Epithelial Cells By Rice Stripe Virus Is Mediated By Sugar Transporter 6 In Laodelphax Striatellus

Rice stripe disease(RSD is one of the most serious rice diseases in subtropical and temperate regions.Losses of 20–30% were observed commonly in regions where Oryza sativa var.japonica is grown in China during the early tears of 21 century.RSD was caused by rice stripe virus(RSV),a member of the genus Tenuivirus.RSV is mainly transmitted in a persistent-progative and transovarial manner by the small brown planthopper(SBPH,Laodelphax striatellus).SBPH is an important agricultural pest,not only infestingcereal plants,but also as a vectorof several viruses.The transmission process is a critical step for every virus because it controls dispersal in space and time,thus directly influencingvirus ecology and disease epidemiology.Evidences have suggested that particle replication,movement and dissemination in different tissues are mediated by proteins interaction between vector insectand viruses,and virus coat protein or nucleocapsid protein plays a major role for the recognization of receptors when viruses attempt to penetrate into the vector or host cells.Hence,screening and then confirmingproteins interacted with RSV NP are necessary for revealing the mechanism of RSV transmission by SBPH.In this study,a protein,screened from SPHB cDNA library was speculated to play an important role in RSV infecting the midgut epithelial cells of SBPH and named sugar transporter 6(LsST6)based on the high homologous with NlST6 of brown planthopper,at nucleotide and amino acid sequence levels.LsST6 predicted to contain 12 transmembrane(TM)domain by online software THMM and a GRK conserved domain between TM2 and TM3 was identified to belong the major facilitator superfamily(MFS).We confirmed the interaction between LsST6 and RSV NP by yeast two hybrid and co-immunoprecipitation.The results showed that LsST6 could interact with RSV NP in vivo and in vitro.LsST6 mainly localized on the cell membrane of Spodoptera frugiperda 9 cell(Sf9)while RSV NP distributed on the cytoplasm.Interestingly,when viral coat proteins respectly co-infected the sf9 cells with LsST6,the viral coat protein distributed on the membrane and colocalized with LsST6.These results suggested that LsST6 was able to change the localization of the viral coat protein.The data of RT-qPCR revealed that LsST6 highly expressed in the gut of SBPH and localized on both membrane and cytoplasm of midgut epithelium by laser scanning confocal microscopy(LSCM).To estimate the function of LsST6 for RSV particle infection or dissemination in the gut of SBPH,we used LSCM to observe the location after a 2-day acquisitionaccess period(AAP).LsST6 was observed to colocalized with RSV on the cell membrane of midgut epitheliums at different time after a 2-day APP,which suggested LsST6 might be a crucial factor for RSV entry the midgut epithelial cells.To confirm the assumption,purified RSV particles were added into the sf9 cells that could express the exogenous LsST6.We found RSV particle colocalized with LsST6 on the cell membrane after 7 h.By 15 h,only a few particles had entered into the cytoplasm of sf9 cells and abundant RSV particles appeared on the cytoplasm by 20 h.Meanwhile,the date of RT-qPCR and northern blot also confirmed that RSV particle entered into the cytoplasm of sf9 cells.All these results strongly indicated that LsST6 played a key role in RSV entry the cells.Finally,the results of RNA interference mediated by dsRNA injection on the SBPH LsST6 expression indicated that the non-viruliferous SBPH capacity of acquisition on RSV was significantly decline,while the titer of RSV in the viruliferous SBPH had no effection.This work uncovered a strategy by which LsST6 mediates viral entry into midgut epithelial cells and leads to successful transmission by the insect vector.