Characterization And Functional Analysis Of Interaction Proteins Involved In Rice Stripe Virus Transmission On Small Brown Planthopper

Rice stripe virus (RSV), a typical member of the genus Tenuivirus, causes rice stripe disease,which is one of the most serious rice diseases in subtropical and temperate regions of Asia. RSV ismainly transmitted by the small brown planthopper (SBPH)(Laodelphax striatellus) in apersisent-propagative manner. Transmission is a critical step in the infection cycle of every virusbecause it controls dispersal in space and time, thus directly influences its epidemiology. SBPH,especially in high density, can cause damage to rice plants when sucking the sap. Even at a much lowerdensity, it can lead to more significant disease epidemics and yield losses because of virus transmission.Therefore, it is important to study the transmission mechanism of RSV by SBPH. The viral componentsinvolved in these interactions are relatively well established. Virus coat protein (CP) plays a major rolefor the invasion of various insect vector tissues and hence for the successful infection of the salivaryglands and subsequent introduction of viruses into plants.In this study, two cDNA library of SBPH were constructed using yeast two hybrid GAL4systemand split-ubiquitin yeast membrane system. These two systems are used to studying the proteininteractions occured in the nucleus and cytoplasm respectively. Firstly, a SBPH cDNA library of yeasttwo hybrid GAL4system was screened by RSV CP and SP as bait proteins. Three proteins of SBPH,actin, one hypothetical protein of unknown function and a cuticular protein which belongs to RR2family, were obtained. Out of three proteins, actin interacted with RSV CP, hypothetical proteininteracted with RSV SP and a cuticular protein interacted with both RSV CP and SP respectively.Seondly, a SBPH cDNA library of split-ubiquitin yeast membrane system was screened by RSV CP as abait protein. One hundred and fourteen proteins were obtained as putatives interactors and17proteinswere chosen to confirm the interaction with RSV CP using yeast membrane system. Twelve proteins ofSPBH were confirmed and they are ribosome associated membrane protein, protein with NAC domain(NAC), Calcium-transporting ATPase, Synaptic vesicle protein, Selenoprotein T, Jagunal, G-proteincoupled receptor, Ribosomal protein L13, Vitellogenin (Vg),40S ribosomal protein S28, Atlastin andNovel cuticular protein (NCuP) which belong to RR1family respectively. Finally, five proteins ofSBPH were identified the interactions with RSV CP by CO-IP assay as an independent confirmationanalysis. Different efficiency of protein interactions from strong to weak were NCuP，Atlastin，Jagunal，Vg and NAC.Based on the screening sequences from yeast two hybrid，the full-length cDNA of NCuP and Vgwere acquired by Rapid Amplification of cDNA Ends (RACE). Two cDNA sequences of NCuP and Vghad been deposited in GenBank under accession no. KC485263and no. KC469581. NCuP couldcolocalized not only with RSV CP in Sf9cells, but also with ribonucleoprotein particles (RNPs) of RSVin the cell line and hemocytes of SBPH by fluorescent confocal microscopy. This demonstrated thatNCuP was involved in specific interactions that formed virus-vector combinations in the insect vectors.To learn more about NCuP, we investigated its expression levels among different organs of L. striatellusby ELISA. The results showed that the quantity of NCuP expression was higher in the hemolymph. To reveal the function of NCuP in virus transmission in the vector, we utilized a powerful strategy offunctional study, insect RNAi. The introduction of NCuP dsRNA into L. striatellus by microinjectionwas performed. The quantity of RSV in the SBPH silenced the expression of NCuP by RNAitechnology was65%lower than that of control groups and the virus transmission efficiency wasreduced40%. The declining quantity of virus demonstrated that NCuP could protect the virus fromdegradation in the hemolymph. Some persistent propagative virus could move from the intestine tohemolymph and be transported to the salivary glands. Colocalization of Vg and RNPs of RSV wasobserved in the germarium of SBPH ovarioles. Thus, VgR mediated endocytosis might haveoccasionally incorporated RSV encapsulated in Vg into the germarium of SBPH ovarioles and the RSVparticles then spread into the oocytes through nutritive cord.