Functional Analysis Of Host Protein Regulating Silencing Suppressor Of Tospovirus NSs

RNA interference（RNAi）is a Eukaryotic conserved pathway in defense mechanism against the invasion of viruses and other molecular parasites.At the same time,viruses have also evolved to express viral suppressors of RNA silencing（VSRs）to suppress host RNA-mediated silencing.More than 70%of plant viruses rely on insect for transmission and need to replicate in plants and insects.The encoded VSRs have silencing suppression activity not only in plant cells but also in insect cells.However,the mechanism of silencing suppression activity of such VSRs in arbovirus-vector has not been extensively explored.Tomato spotted wilt orthotospovirus（TSWV）is an ambisense RNA virus in the genus Orthotospovirus,family Tospoviridae.It is transmitted by the thrips Frankliniella occidentalis Pergande,in a persistent-propagative manner.The proliferation and transmission of TSWV in F.occidentalis requires its silencing supression NSs to response to insect RNAi defense.NSs has been reported to involve in the proliferation and spread of TSWV in F.occidentalis,but the mechanism of interaction between viruses and insect vectors is poorly understood.The FK506-binding protein 6（FKBP6）,which interacted with TSWV NSs,was preliminarily identified.In this study the interaction mechanism between TSWV NSs and FKBP6 was explored,and biological function of FKBP6 in the transmission of F.occidentalis was analyzed.The major results of this study are as follows:（1）FKBP6 was verified as an interacting protein of TSWV NSs in thrips cells.In addition,the expression level of FKBP6 was higher in adults and midgut.TSWV infection induced the expression of FKBP6 in F.occidentalis,and the gene expression changes with the virus replication.Knockdown of FKBP6 by RNAi reduced m RNA level,compared with those injected with ds GFP,while the expression level of the NSs gene was up-regulated,with the increase of TSWV amout,and the survival of F.occidentalis was not affected.In GFP-based transient silencing suppression assay in N.benthamiana,it was proved that the silencing activity of NSs was inhibited by FKBP6.In conclusion,FKBP6 targets TSWV NSs,and inhibits its silencing suppression activity,thereby regulates the proliferation of TSWV virus.（2）The TSWV NSs-interacting domain was mapped to the PPIase of FKBP6.It has been identified that NSs^1-434aa-FKBP6 interaction was required for silencing of RNA suppressor activity by NSs 1-434 aa,while NSs^1-433aa-FKBP6 interaction was disrupted because NSs 1-433aa lack of silencing suppression activity.It was confirmed that the interaction between FKBP6 and NSs were mainly affected the phosphorylation level in phosphorylated serine at 433 aa of NSs,further affected the silencing suppressor activity of NSs.In addition to study vector proteins regulated NSs to affect virus transmission in F.occidentalis,in this paper we also explored RNAi related proteins regulated silencing suppressor activity of NSs in virus-infected host plants.The main research object was tomato zone spot orthospovirus（TZSV）.The VSRs of othotospovirus have been widely publicized in plant cells,but little is known about the mechanism of targeted RNA silencing.In this paper we aimed to explore the structure and function of VSR and its relationship to the TSWV silencing suppressor and to explore the mechanism of the targeted RNAi pathway.The main results are as follows:（3）Using a GFP-based transient silencing suppression assay and a potato virus X（PVX）-based heterologous expression system,the NSs protein encoded by TZSV was identified by as a viral pathogenicity factor and VSR in Nicotiana benthamiana.The C-terminal integrity of TZSV NSs was identified to be responsible for silencing suppression activity and pathogenicity by agrobacterium-mediated transient expression of GFP and heterologous PVX virus expression system.In conclusion,NSs act as silencing suppressors and pathogenic factors of TZSV,and the C-terminal integrity of TZSV NSs is necessary for silencing suppressor activity and pathogenicity.（4）The results showed that the interaction between TZSV（TSWV）NSs and Nb SGS3 depended on the silencing suppression activity of NSs.TZSV NSs reduced the concentration of Nb SGS3 protein in plant cells,likely through the ubiquitination and autophagy pathways.Interestingly,TZSV infection or NSs overexpression significantly up-regulated the transcript levels of Nb SGS3.in response to host plant RNAi,and silencing of endogenous Nb SGS3 by VIGS can promote plant infection by TZSV.In conclusion,our data indicate that the interaction between TZSV NSs and Nb SGS3 depends on the silencing activity of NSs,and Nb SGS3 can attenuate the silencing inhibitory activity of TZSV to enhance the defense against viruses,while TZSV NSs degrade Nb SGS3 by hijacking the ubiquitination and autophagy pathways of host plants,to fight against host plants and to promote viral replication.The results of this research explain the mechanism of insects and hosts protein regulating silencing suppressor of tospovirus NSs,and provide a new target for finding new methods of virus control.Our results could promote the understanding of host defense virus immune pathway regulation network,and provide theoretical support for virus control.