Research On The Interaction And Molecular Modification Of SGS3-VISP1-ATG8a Antiviral Complex In Oilseed Rape

Viral disease is one of the major diseases affecting oilseed rape（Brassica napus）,seriously endangering its yield.Studying the mechanism of oilseed rape resistance to disease and finding solutions to this problem has become a hot research topic.Gene silencing is a natural plant defense mechanism against viruses.Suppressors of gene silencing 3（SGS3）is a core protein that mediates post-transcriptional gene silencing（PTGS）in plants.However,most viruses have evolved viral suppressors of RNA silencing（VSR）to counteract the anti-viral silencing activity of plants by blocking the PTGS pathway.Until now,all the VSRs target to SGS3 are viral source.Recently,a virus-induced small peptide from Arabidopsis thaliana named VISP1 was identified.This peptide mediates the interaction between SGS3 and autophagy-related protein 8（ATG8）,and subsequently degrades SGS3 in the cell’s autophagy pathway,thereby inhibiting the PTGS pathway and promoting viral infection.Knocking out the VISP1 gene in Arabidopsis plants resulted in stronger resistance to viruses than the wild type.And VISP1 is highly conserved in Cruciferae plants such as Brassica napus.Knocking out the VISP1 gene may mediate broad-spectrum resistance to oilseed rape viruses.Therefore,VISP1 is a promising gene editing target for breeding oilseed rape resistant to viruses.However,the molecular mechanism by which VISP1 mediates the interaction between SGS3 and ATG8 is still unclear.The main achievements are as follows:1.Optimization of the expression system and crystal screening of f SGS3ull-lengthAs a key component of plant gene silencing,SGS3 participates in the amplification of secondary si RNA with RDR6.However,it’s difficult to obtain SGS3 full-length protein due to the complexity of its structure.And the structural information of SGS3 has not been resolved,and there are no homologous protein structure resolutions.In this study,the full-length SGS3 protein,which is difficult to express in prokaryotic systems,was heterologously expressed using SF9 and High5 of insect cells.The expression system was optimized in terms of protein tags,cell lines,transfection ratios,and expression time.After multiple purification steps,10mg of SGS3 full-length protein can be obtained per liter of SF9.2.Identification of the interaction and key regions between VISP1,ATG8a,and SGS3ATG8a is an autophagy-related protein,which homologous family proteins have been resolved,such as ATG8f.But the mechanism of the virus hijacking the autophagy system to degrade key components involved in gene silencing is currently limited.In this study,we find there is no interaction between AtVISP1,AtATG8a and AtSGS3 by T2H assay.However,the fluorescence signal was reconstituted in tobacco leaves,indicating that three proteins have interaction in tobacco leaf and the interaction site was located in the cytoplasm by bimolecular fluorescence complementation（BiFC）experiments.In vitro pull-down experiments further indicated that AtVISP1 mediated the interaction of AtATG8a with AtSGS3.3.Analysis of the mechanism of VISP1-mediated interactions between ATG8a and SGS3In this study,we identified that VISP1 interacts with ATG8a and SGS3 through disulfide bonds by point mutation,sulfhydryl closure,and application of reducing or oxidizing agents.The mutation of VISP1 Cys to Ser,the use of NEM to close the sulfhydryl group and the reducing reagents such asβ-ME and DTT could prevent VISP1from interacting with ATG8a and SGS3.The oxidizing agents such as H₂O₂ could enhance the interaction.The Y2H experiment showed no interaction because its protein was expressed in the reduced condition intracellularly and the disulfide build could not be formed.In summary,this study verified that VISP1 interacts with autophagy-associated protein ATG8a and gene silencing key component SGS3 through disulfide bonds.It provides a theoretical basis for virus resistance breeding in oilseed rape and give a new idea to solve the virus-induced VSR hostage plant autophagy pathway to degrade key components of gene silencing.