Screening,Identification And Expression Analysis Of Proteins Interacting With Mulberry Vein Banding Associated Virus N Protein In Mulberry

Mulberry vein banding associated virus(MVBa V),the main pathogenic virus of mulberry virus disease,belongs to the genus Orthotospovirus in the family Tospoviridae.MVBa V S RNA encodes nucleocapsid(N)protein.Screening and identification of MVBa V N protein interacting proteins and further study of their interaction mechanism will play an important role in revealing the pathogenesis of MVBa V.In the previous study,we prepared MVBa V N protein polyclonal antibody.Based on the detection of its sensitivity and specificity,this study used co-immunoprecipitation to screen the interacting proteins of N protein,and used Biomolecular fluorescence complementation and Yeast-two-hybrid system to candidate interacting proteins to verify,on the part of genes encoding the interacting protein expression analysis.The main research results are as follows:1.Verification by Western blot showed that the prepared antibody could detect the MVBa V N fusion protein as low as 0.50 ng,and can specifically recognized the fusion N protein expressed by bacteria and the N protein in infected mulberry leaves at the dilution ratio of 1:1000,without cross-reaction with the host protein.It showed good specificity and high sensitivity.2.The total proteins were extracted from mulberry leaves with typical symptoms of mulberry vein disease,and 106 proteins interacting with MVBa V N were screened by co-immunoprecipitation combined with mass spectrometry(COIP-MS),including the non-structural movement protein(NSm),nonstructural suppressor protein(NSs)and nucleocapsid protein(N protein)encoded by MVBa V.3.Select MLP-like protein 423(MLP-LP423),Nodulin-related protein 1(NRP1),Pectinesterase(PECT),Enolase(ENO),14-3-3-like protein B(14-3-3LPB),Fructose-bisphosphate aldolase(FBA),Ribosome-recycling factor(RRF),Serine hydroxymethyltransferase(SHMT),Adenosylhomocysteinase(SAHH)and Allene oxide synthase(AOS),a total of 10 candidate interacting proteins were subjected to Biomolecular fluorescence complementation and Yeast-two-hybrid verification.The results showed that there are interactions between MLP-LP423,NRP1,PECT,14-3-3LPB,FBA,RRF and SAHH proteins and virus N proteins.4.The expression patterns of 14-3-3LPB,PECT,RRF and SAHH genes in healthy mulberry trees,virus infected mulberry trees and mulberry seedlings under different abiotic stresses and hormone treatments were detected by real-time fluorescent quantitative PCR.After mulberry was infected by MVBa V,the expression of 14-3-3LPB was up-regulated in root and stem,PECT was up-regulated in leaf and stem,and down-regulated in root RRF was up-regulated in upper leaves and stems,while SAHH was up-regulated in roots and leaves and down-regulated in stems.The expression of PECT under different abiotic stresses and hormone treatments showed a downward trend,while the14-3-3LPB,RRF and SAHH showed different trends.These results lay a foundation for further elucidating the pathogenesis of MVBa V and the interaction mechanism between MVBa V and host protein.