Screening And Function Analysis Of Interaction Factors Of Insect Vectors With Nonstructural Protein Pns10 Of Rice Dwarf Virus

Rice dwarf virus(RDV),a persistent-propagative virus,can be transmitted by Nephotettix cincticeps.Nonstructural viral protein Pns10 of RDV assembles into tubular structure to contain viral particles and spread among cultured leafhopper cells and epitheilla cells of N.cincticeps.RDV spread successfully by this "safe way" with the help of insect peoteins.Previous yesat two-hybrid assay showed candidate proteins interacting with Pns10,such as actin,mysion,etc.The interaction of Pns10 with cytoplasmic actin of leafhoppers was crucial for insect vector specificity.In present study,the Matchmake Gold yeast two-hybrid system was applied.The pGBKT7-Pns10 as a bait vector was used to screen a cDNA library derived from adults and nymphs of N.cincticeps.The screening proteins were related to the development and growth of insect,and celluar components,etc,based on the analysis of Blastx in Genbank.Then we selected 6 candidate prey proteins which possibly participated in viral infection,including trpomodulin(Tmod),mitochondrial porin(Mito P),lipophorin precursor(LP),Vigilin,apoptosis inducing factor(AIF),vitellogenin(Vg).Bimolecular fluorescence complementation(BiFC)assay demonstrated that Pns10 could interact with Tmod,Vg and LP.Nevertheless,Mito P,AIF and vigilin could not interact with Pns10.Subcellular co-localization assay in Nicotians benthamiana showed that Tmod,Vg,LP and vigilin co-localized with Pns10 respectively,but Mito P,AIF still showed absence of interaction with Pns10.To further understand the role of 6 candidate proteins playing in RDV spread,we detected relative expression of them in RDV-infected N.cincticeps and cultured cells preliminarily.The results indicated that relative expression of Tmod,Vg,LP,vigilin or AIF was higher than the control mock-infected treatment.Tmod,the specific actin-binding peotein in slow-growing end,is an associated protein with actin.For the reason that virus-containing Pns10 tubules transport virions based on actin,GST-Pull down assay were performed.We confirmed that Tmod could interact with Pns10 specifically in vitro.In RDV-infected N.cincticeps and cultured cells,Pns10 tubular could pass through the Tmod-labled microvilli,which was consistent with previous study.We then analyzed the expression of Tmod and Pns10,RDV P8 by RT-qPCR assay during viral infection in insect vector and cultured cells.The results indicated that relative expression of Tmod displayed similar trend with Pns10,P8 with time.Inhibiting Tmod in RDV-infected N.cincticeps and cultured cells,relative expression of Tmod decreased significantly than mock-infected treatment,and relative expression of Pns10 or P8 also reduced as Tmod.When dstropomodulin(dsTmod)treatment was up to 14th day,viruliferous rate was decreased about 50%.It was suggested that Tmod regulated Pns10 positively.Therefore,it was assumed that PnslO made full use of Tmod to manipulate actin-based tubule motility(ABTM)in insect vectors,so that actin could extend safely to help Pns10 cross into adjacent cell,even overcome the immune attacking to facilitate viral spread.Vg,the precursor of vitellin,the nutrition of ovarian development,is synthesized by fat body and secreted into hemolymph to oocytes,but the function of Vg in virus infection is unknown.Here,we confirmed the interaction of Vg and Pns10 by the GST-Pull down assay.And we found Vg could co-localize with Pns10 in cultured cells by immunofluorescent labeling.Besides,Pns10 could also co-localize with Vg in follicular cells,mycetocyte and pedicle.But Pns10 cound not be detected in ovary inside.Therefore,RDV possibly infected pedicle firstly,and interaction of Vg with Pns10 may assist virus to spread.