The Functions Of Nonstructural P3 And P6 Encoded By Rice Yellow Stunt Virus,in Insect Vector Cell Cultures

Rice yellow stunt virus(RYSV),a member of Nucleorhabdovirus,persistently transmitted by Nephotettix cincticeps in a propagative way.Fang’s team had a systemically research on the functions of nonstructural proteins encoded by RYSV in plant hosts,and showed that P3 was a cell-to-cell movement protein and P6 was a gene silencing suppressor.However,the functions of these two nonstructural proteins during the infection process of RYSV in its insect vector remain unknown.Herein,we preliminarily explored the subcellular localization and function of P3 and P6 by using insect vector cultured cells.We constructed a transient expression vector system by cloning the actin promoter sequence of N.cincticeps.And then,we optimized this vector by adding an enhancer sequence and choosing the best transfection conditions.Fortunately,this vector can be used for protein expression in different insect cell lines,including N.cincticeps,Nephotettix virescens,Recilia dorsalis and Spodoptera frugiperda cultured cells.For the first time,we can express foreign protein in leafhopper cultured cells by transient expression vector system.We successfully expressed encoded proteins by RYSV,including nucleoprotein(N),phosphoprotein(P),matrix protein(M),glycoprotein(G),P3 and P6 in healthy N.cincticeps cultured cells and we found that RYSV-N,P,P6 and M were located in nuclei,P3 formed distinct particle structures in cytoplasm;and G had a dispersive distribution in the cytoplasm of cultured cells.Based on the results about the localization of P3,we further explored the localization and function of P3 in virus-infected leafhopper cultured cells.Firstly,we prepared the P3 specific antibodies and used immunofluorescence technology to observe the subcellular localization of P3 in RYSV-infected cultured cells at different hours post RYSV inoculation(hpi).Interestingly,we found that P3 localized in cytoplasm and formed particle structures at 18 hpi,and distributed in the periphery of the nucleus at 36 or 48 hpi,and P3 was mostly condensed in the nuclei of infected cells at 72 hpi.Thus,P3 may be mediated by other proteins and play an important role in the nucleus.What’s more,we get to know that P3 were co-localized with N and M in Sf9 cells,by baculovirus expression system.When expressed separately,P3 was localized in the cytoplasm,N was located in the nucleus,and M was positioned in the cell membrane.When P3 and N were co-expressed,P3 entered into the nucleus and co-localized with N.When P3 and M were co-expressed,M was moved from cell membrane to cytoplasm and co-localized with P3.Furthermore,a yeast hybrid-two assay was used to verify the potential interaction between P3 with N and M in vitro,respectively.Taken together,we get to know that the nucleus entry of P3 in nucleus mediated by N,and P3 participates in the cellular trafficking of M.It is speculated that P3 may be related to the replication and the assembly of virus particles in its insect vector cells.We found that P6 localized in cell nucleus whether cultured cells were infected or not.We guess that P6 may join with N and P to participate in the replication progress of RYSV in its insect vector cells.Furthermore,we found that P6 were co-localized with N and P in the nucleus in RYSV-infected N.cincticeps cultured cells.The interaction relationships between P6 with N,or P6 with P were confirmed by transient expression vector system and yeast two-hybrid assay.Finally,we used RNA interference by transfection of dsRNAs targeting to P6(dsP6)to knock down the expression of P6 gene.We found that the transcript levels of P6,N and P genes were decreased significantly compared to the control(dsGFP)by RT-qPCR assay.It is clear that P6 is involved in viral replication in it’s insect vector cells.To sum up,this study firstly constructed a leafhoppers cell transient expression system.By using this unique system and the stable leafhoppers cultured cell technology,baculovirus expression system,immunofluorescence technology,yeast two hybrid assay and RNAi technology,we investigated the subcellular localization and functions of RYSV P3 and P6 in cultured insect vector cells,and proved that those two nonstructural proteins play important roles during RYSV infection in insect vector cells.Our results will shed light on the interaction of plant rhabdoviruses with their insect vectors and provide theoretical basis for developing new disease resistance strategies.