Establishment Of Mini-replicon Based Reverse-Genetics Systems Of Orthotospovirus And Functional Analysis Of N Interacting Host Factors

Orthotospovirus has a wide range of hosts and has caused serious damage to many agricultural and horticultural crops.The typical species Tomato spotted wilt virus（TSWV）is listed as one of the top ten most serious plant viruses.There are more than thirty species of viruses in Orthotospovirus.The classification of the genus mainly relies on the amino acid sequence homology of nucleocapsid（N）and geographical distribution,which can be further classified as American or Euro-Asian clade.At present,the most serious viruses of American clade in this genus have been reported in China,including TSWV,Impatiens necrotic spot virus（INSV）,while viruses of Euro-Asian clade including Watermelon silver mottle virus（WSMo V）,Calla lily chlorotic spot virus（CCSV）,Capiscum chlorosis virus（Ca CV）and Tomato zonate spot virus（TZSV）.Due to the lack of reverse genetic systems,the research on the mechanisms of replication,transcription,movement of Orthotospovirus have been severely limited.The reverse genetic system of TSWV has been established in the laboratory,while the reverse genetic systems of many other viruses in this genus need to be further established.Whether N,RNA dependent RNA polymerase（RdRp）and NSm of viruses from American and Euro-Asian clades can support genomic replication,transcription and movement of different viral groups is unclear.Which host factors can interact with nucleocapsid and regulate the infection of viruses remain to be studied.Aiming at the above scientific problems,this paper has carried out researchs from the following four aspects:1.Establishment of four mini-replicon reverse genetics systems of OrthotospovirusAccording to the distribution of geographical types,TSWV and INSV belong to the American clade,while WSMo V,Ca CV,TZSV and CCSV belong to the Euro-Asian clade.In order to establish mini-replicon systems of different clades,TSWV and INSV were selected as the representatives of American clade and Ca CV,TZSV,WSMo V and CCSV as the representatives of Euro-Asian clade in this study.The mini-replicon system of INSV was further established based on the successfully established mini-replicon system of TSWV in laboratory.The codon and intron sequences of RdRp^INSV was first optimized to obtain RdRp_opt^INSV,which was successfully expressed in plants.INSV S genome was inserted into the plant binary expression vector p CB301 and the reporter gene e GFP replaced N^INSV to form a mini-replicon SR^INSV_{（-）e GFP}.SR^INSV_{（-）e GFP} was co-infiltrated with RdRp_opt^INSV,N^INSV and viral suppressor of RNA silencing（VSR）into N.benthamiana,which formed functional mini-replicon system.The mini-replicon systems of Ca CV,TZSV and WSMo V could also be established by using the RdRp_opt^CCSV of laboratory following the construction strategy of INSV mini-replicon.2.Study on the interacted recognition mechanism of genomic mini-replicon with RdRp and N encoded by American and Euro-Asian clades OrthotospovirusBased on the mini-replicon system of Orthotospovirus has been established,this study further investigated the compatibility of N and RdRp in supporting different clades of Orthotospovirus.The mini-replicons of TSWV,INSV,Ca CV,TZSV and CCSV were infiltrated into the N.benthamiana in the following combinations,results indicated that:when RdRp and N belonged to both American or Euro-Asian clade,the co-expression of RdRp and N could support the work of the mini-replicon systems of the same clade efficiently;when both RdRp and N belonged to TSWV or CCSV,the co-expression of RdRp and N could support the work of the mini-replicon systems of other clade in an inefficient way.It is speculated that the Orthotospovirus of different geographical types can also cause the reassortment by exploring the mini-replicon systems of different clades,which provides an important theoretical basis for the prevention and control of this virus disease.3.Study on the interacted recognition and cell to cell movement mechanisms of genomic mini-replicon with NSm encoded by American and Euro-Asian clades OrthotospovirusThe regulation role of NSm on the cell to cell movement of genomic mini-replicons in the two geographic clades was further investigated based on the existing reverse genetic systems of Orthotospovirus.Results were as follows:the movement-deficient reporter of positive strand RNA virus represented by Cucumber mosaic virus（CMV）was successfully complemented by movement proteins（MP）of TSWV,INSV,Ca CV,TZSV,CCSV and Rice stripe virus（RSV）that were members of the segmented negative strand RNA virus.It indicated that all the above MPs could support the cell to cell movement of CMV;NSm encoded by Orthotospovirus could also promote the cell to cell movement of reporter gene e GFP in the mini-replicon system of TSWV or CCSV.Moreover,NSm encoded by Orthotospovirus complemented the cell to cell movement of viruses within the genus,which was positively correlated with the genetic relationship;MPs encoded by heterologous virus CMV or RSV could promote the cell to cell movement of reporter gene e GFP in the mini-replicon system of TSWV or CCSV,while the 3a^CMV and NSvc4^RSVpromoted the cell to cell movement of reporter gene e GFP more strongly in the mini-replicon system of TSWV.These results suggest that the cell to cell movement of Orthotospovirus is specific to some extent,and the NSm encoded by more closely related virus can better support the cell to cell movement of mini-replicon system.4.A preliminary study on the TSWV N interacting host factors Nb B3 or Nb Bobber1TSWV N was used as bait to screen N.benthamiana c DNA libaray by yeast-two hybird（Y2H）.In this study,we choosed the B3 domain containing protein and small heat shock protein Bobber1 to explore their preliminary functions.The interaction between TSWV N and Nb B3 or Nb Bobber1 was confirmed by Y2H system and bimolecular fluorescence complementation（Bi FC）.Tobacco rattle virus（TRV）-mediated gene silencing and transient expression of Nb B3 or Nb Bobber1 indicated that Nb B3 negatively regulated TSWV infection,while Nb Bobber1 positively regulated TSWV infection.These results lay a good foundation for further revealing the role of host interaction proteins in virus infection.