The Interaction Between Nonstructural Protein P7-1 Of Southern Rice Black-streaked Dwarf Virus And Its Vector Sogatella Furcifera

Recent outbreaks of Southern rice black-streaked dwarf disease in southern China caused by southern rice black-streaked dwarf virus(SRBSDV),have seriously threaten stable crop production.SRBSDV is transmitted by white backed planthopper(Sogatella furcifera,Horvath)in a persistent propagative manner with higher affinity.The virus-containing tubules composed of SRBSDV encoded nonstructural protein P7-1 is important channel for virions to overcome various barriers and rapid spread.Since the spread of virions need the participation of vector proteins,identification of WBPH factors interacted with SRBSDV P7-1 will contribute to studying affinity mechanism between SRBSDV and WBPH.In this study,we constructed the WBPH cDNA library using total RNA of viruliferous WBPHs.After screening WBPH Library with SRBSDV P7-1 as bait protein in the yeast two-hybrid system,63 putative proteins were successfully identified.We selected 6 candidate prey proteins after sequence and analysis for the following various tests.The BiFC results showed that 5 WBPH candidate prey proteins as B-cell receptor-associated protein 31-like(BAP31),Ly-6/neurotoxin superfamily member 1(Neurotoxin),DNA-J/hsp40 protein(DNA J),ubiquitin-protein E3 ligase(E3)and myosin RLC2(Myosin)interacted with P7-1.But,the protein of skeletal muscle growth protein 5(Skeletal)couldn't interact with P7-1.Moreover,P7-1 and BAP31 formed some granular and vesicular structures in the cytoplasm and plasma membrane.DNA J or Myosin with P7-1 was observed in some small granular structures around the plasma membrane.To further prove the interactions of P7-1 with candidate prey proteins,subcellular co-localization assay was conducted.The results indicated that BAP31,Neurotoxin,DNA J and E3 could co-localize with P7-1 in epidermal cells of N.benthamiana plants,and Skeletal couldn't co-localize with P7-1.To further prove the interactions of P7-1 with WBPH proteins,Baculovirus Expression Vector System was used.P7-1 could interact with candidate prey proteins in different ways in Sf9 cells.When these proteins were expressed in Sf9 cells individually,P7-1 formed tubular structures which could extend out the surface of cells.BAP31 could form vesicular structures in cytoplasm.Neurotoxin formed granular inclusions dispersed in cytoplasm closed with nuclear membrane.DNA J and E3 formed granular structures in cytoplasm.Myosin dispersed in cytoplasm and could form pseudopodia.When co-expressed P7-1 and candidate prey proteins in Sf9 cells respectively,BAP31 and P7-1 were co-localized in cytoplasm.But BAP31 failed to form vesicular structure.The tubular structures formed by P7-1 were restricted in cytoplasm and couldn't extend out of cell.Neurotoxin formed more granular inclusions and tubular structures distributing around it.DNA J was recruited apparently by P7-1 and co-localized with tubular structures.The granular structures formed by E3 localization with P7-1 partially,and E3 were distributed around tubular structures.Moreover,when co-expressed,BAP31 and Neurotoxin could co-localize completely in cytoplasm,but BAP31 could not form vesicular structures too.These results indicated that tubular structures formed by P7-1 could interact with multiple candidate prey proteins in Sf9 cells,BAP31 and Neurotoxin could also interact with each other.Finally,to further confirm the influence of the 5 candidate prey proteins on the infection of SRBSDV in WBPHs,RNA interference was used to inhibit the expression of the candidate proteins.The results showed that when the gene of BAP31 was knocked down,the expression of P7-1 and P10 significantly increased compared with the control.While the knockdown of the Neurotoxin,DNA J or Myosin,significantly reduced the expression of P7-1 and P10 in constrast to the control.The Real-time PCR assay demonstrated that the relative expression of J,E3 and Myosin were increased,whereas that of the BAP31 was reduced in viruliferous WBPHs compared to the nonviruliferous ones.It was assumed that the proteins of Neurotoxin,DNA J,E3 and Myosin may participate in the formation of tubular structures and be advantageous for virus infection,but the BAP31 may inhibit the infection of SRBSDV in WBPHs.All together,in present study,63 WBPH proteins interacted with SRBSDV encoded nonstructural protein P7-1 tubules were obtained by yeast two-hybrid system,and the interaction between 5 candidate prey proteins and P7-1 were proved in other N.benthamiana plants or Sf9 cells.We identified the functions of candidate prey proteins in virus infection process by RNA interference,however,the mechanism of interaction needs to further study.This research will provide evidences for studying the interaction mechanism between virus and vector.