Identification And Functional Analysis Of Vector Proteins Interacted With Nucleocapsid Protein Of Barley Yellow Striate Mosaic Virus In Small Brown Planthopper

Small brown planthopper(Laodelphax striatellus Fallen)(SBPH)is an important migratory pest on crops,which has a extremely wide host range,can tranmit virus of multiple genera including Fijivirus,Tenuivirus and cytorhabdoviruses through feeding the sap on plants.Its outbreak pose a great threat to crop production and have caused severe economic losses.Blocking the transmission of the virus by SBPH is one of the most effective ways to control the spread of viral diseases.At present,some progress has been made in the study of the transmission mechanisms of Fijivirus and Tenuivirus in SBPH,but the interaction mechanism between SBPH and plant Rhabdovirus has rarely been studied.In this study,the BYSMV-planthopper system was employed as a model to investigate the transmission mechanism of rhabovirus in SBPH.The library screening was performed to identify the related proteins invovled in virus acquisition or/and transmission in SBPH and the candidate proteins was used to further functional analysis.It is of great scientific significance to provide new targets for blocking the spread of plant rhabdovirus and even various viruses transmitted by SBPH.In this study,the bait vector pDHB1-N containing the full-length BYSMV nucleocapsid protein(N)gene was first constructed.The functional verification results showed that pDHB1-N could normally express and was also functional in yeast.The library screening conditions were optimized to determine the presence of the competitive inhibitor of the HIS3 gene product,3-amino-1,2,4-triazole(3-AT),at a concentration of 12 mM to remove the possible slight screening background.The cDNA library of SBPH was screened by the bait pDHB1-N using the split-ubiquitin yeast membrane system.57 positive colonies was isolated and 20 of which was identified by sequence BLAST matching to species such as Nilaparvata lugens.The 20 putative proteins such as cuticular,ubiquitin and ribosomal membrane-associated protein were further analyzed based on Gene Ontology(GO)annotation,which were found to have activitives on transport,oxidoreductase and structural molecular,participating in many physiological processes such as cellular processes,metabolic processes and response to stimulus and present in various cellular components such as cell membranes,intracellular and extracellular regions.Analysis of signal pathways revealed that they are mainly involved in multiple pathway processes such as fatty acid biosynthesis,lipid metabolic and signaling pathway.At the same time,Yeast two-hybrid co-transformation assay and ?-galactosidase assay further confirmed that 17 of the 20 candidate proteins were interacted with BYSMV N.The interaction mechanism between SBPH and rice stripe virus(RSV)were performed on the previous study in our lab,suggesting that the combination of the CPR1 protein of SBPH with RSV could help the virus to evade the immunity of the host hemolymph and prevent the degradation of virus.In this study,the full-length cDNA of the same cuticular protein(CPR1)was also obtained from the library screening.After extensive expression and purification,the interaction between CPR1 and BYSMV N was confirmed in vitro by GST pull-down assay.To clarify the function of CPR1 in the transmission of BYSMV in SBPH,CPR1 was knocked down by RNA interference(RNAi)technology with microinjection of dsCPR1 or dsGFP(negative control)in the third-instar viruliferous nymphs of SBPH and the transcription levels of CPR1 and BYSMV N were detected by real-time qRT-PCR respectively.The results revealed that the transcription level of BYSMV decreased with the decrease of CPR1 transcription level in SBPH and most significantly on the sixth day after injection,decreasing by 71.84%.At the same time,the biological inoculation experiment also found that the transmission efficiency of SBPH decreased by 51.73% after knocking down CPR1.The results above shows that CPR1 plays an important role in the process of viral acquisition or transmission in the body of SBPH.In addition,the related studies suggested that a variety of cuticular proteins containing chitin-binding conserved domains were involved in the transmission of CYDV,CMV and other viruses in their vector insect.It is speculated that cuticular proteins may play a broad-spectrum role to help the virus overcome the transmission barrier or evade immunity and the key target site maybe lie in the chitin-binding conserved domain.This study will establish a foundation for further exploration of the molecular interaction mechanism between rhabdovirus and vector and provide a scientific basis for the new strategy through blocking the insect-mediated virus transmission pathway to control viral diseases.