Identification Of WBPH Proteins Interacted With SRBSDV P5-1

Southern rice black-streaked dwarf virus(SRBSDV)is transmitted by the White-backed planthopper(WBPH,Sogatella furcifera)in a propagative,persistent manner.After invading the insect vector WBPH,the virus requires associated proteins in the insec t to form viroplasma matrix,and then is able to replicate and proliferate.Previous studies have shown that,SRBSDV encoding nonstructural proteins P5-1 is an important component of the viral matrix.In order to explore which proteins in WBPH interact wit h SRBSDV P5-1 and their roles,this study using SRBSDV P5-1 as a bait to screen WBPH cDNA library by yeast two-hybrid assay.Two proteins,a ribosomal protein component L12 and an enolase,were found showing strong interaction with P5-1,confirmed by bimolecular fluorescence complement(BiFC)assay in plant cells.Detection of these two genes in the insect by RT-qPCR consequently,our results suggest these two proteins may involve in SRBSDV transmission in its vector WBPH.Main results were showed as follows:1.Analysis of the structure and function of virus nonstructural protein P5-1.Using the bioinformatics method,P5-1 is more stable,the primary structure contains the nuclear functional domain and multiple protein interaction sites,the interaction sites are mainly concentrated in the 338-499 aa region;SWISS MODEL built The three-dimensional structure model shows that the protein contains more helical structures and folded structures,which contain multiple holes that may provide access for nucleic acid templates and energy materialtransport.2.Screening and validation of two WBPH proteins interacted with viral P5-1.Two proteins interacting with SRBSDV P5-1 were identified by yeast two-hybrid technique.The ribosomal protein L12 and Enolase we re cloned from the cDNA library of white-backed planthopper.The results of bioinformatics showed that the ribosomal protein L12 had five protein binding sites and two DNA binding sites,suggesting that this protein might be recruited by viral P5-1 and involved in viral RNA replication,protein synthesis,and regulation of host cell development and other physiological activities;enolase contains 17 protein binding sites and a DNA binding site,suggesting that this protein might be associated with viral P5-1 and involved in host immune response to help the virus break the insect immune line and replication.3.Expression level detection of the genes encoding for the two WBPH proteins interacted with viral P5-1.The results of RT-qPCR showed that the expression of ribosomal protein L12 was significantly increased after infection with SRBSDV,but the expression of enolase did not change much.4.The functional model of P5-1 protein in virus replication.According to the results of this study and previous studies,it is speculated that P5-1 may recruiting virus P6 and P9-1 in the synthesis of SRBSDV matrix,and then recruiting host ribosomal protein L12 and enolase to realize viral replication function.Protein L12 might be involved in viral replication,transcription and protein synthesis,and enolase involved in the overcoming of host immune response,to help the virus replication.