Establishment Of The Reverse Genetics System For Two Representative Segmented Plant Negative-strand RNA Viruses

Negative-stranded RNA viruses(NSVs)include not only human pathogens but also many serious plant pathogens of agronomic production.Tomato spotted wilt virus(TSWV)and Rice stripe virus(RSV)are two representative members of the most important segmented plant NSVs,belonging to Bunyavidales,and pose major threats to global crop production and food security.The reverse genetics(RG)system is a core technology system in modern virology and also is an important molecular manipulation tool for studying the functions of virus genes and virus-host interactions.Although RG systems were first developed for animal-infecting viruses with segmented negative-stranded RNA genomes over 20 years ago,such a system for plant viruses with segmented negative-stranded/ambisense RNA genomes have not been developed until now.Many groups have tried to construct infectious clones for segmented plant NSVs using animal-infected NSV RG systems but all failed.In this study,TSWV and RSV were used for research model of segmented plant NSVs.A series of difficulties has hampered the construction of a successful RG system to rescue the TSWV entirely from infectious cDNA clone through technology innovation and system optimization.Our study makes breakthroughs in the study of segmented plant NSVs,representing establishment of the first segmented plant NSVs worldwide,which open a new era for the study of plant NSVs.Based on developing different new approaches,the RG systems of RSV were further constructed.The results are summarized as follows:1.Development of mini-replicon reverse genetics systems of tomato spotted wilt virusIn the last decade,Professor Tao's group has made no breakthrough in the RG system using the wild-type TSWV RNA-dependent RNA polymerase(RdRp)and the T7 promoter based on the strategy of animal negative-stranded RNA viruses.Recently,we found that TSWV RdRp sequence contains a large number of potential splicing site sequences.By optimizing the codon and intron sequences of TSWV RdRp gene,the expression of RdRp(?330 kDa)protein was successfully achived in plant system.Then,a TSWV S mini-genome reporter plasmid was generated based on 35S promoter-driven constructs of the S(-)-genomic(g)RNA template,flanked by the 5' hammerhead and 3' ribozyme sequence of hepatitis delta virus,NSs and N ORF were replaced by mCherry and eGFP reporter genes,respectively,and finally S mini-replicon system was successfully established.Based on TSWV S mini-replicon system,we found that N and RdRp were required for the replication and transcription of TSWV,and the four different viral suppressors of RNA silencing(VSRs:NSs from TSWV,P19 from Tomato bushy stunt virus[TBSV],HcPro from Tobacco etch virus[TEV]and yb from Barley stripe mosaic virus[BSMV])significantly enhanced the reporter genes(mCherry and eGFP)expression levels of SR(-)mCherry&eGFP-mini-replicon in plant cells.By using deletion mutation assays,we demonstrated that the roles of cis-acting elements 5'-UTR and 3'-UTR of TSWV S segment on SR(-)mcherry&eGFP-mini-replicon were required for the viral replication and transcription processes.Based on the TSWV S gRNA-derived mini-replicon system,we also developed the positive-stranded SR(+)eGFP-mini-replicon system based on the TSWV S(+)-antigenomic(ag)RNA,demonstrating that the N protein can also be expressed from the SR(+)eGFP replicon to support its transcription and replication.Next,a movement-competent mini-genome replication system was developed based on M(-)-gRNA,which directly expressed movement protein NSm that could promote cell-to-cell and systemic movement of TSWV.Finally,an L negative-sense mini-replicon system for low-replication was developed based on L(_)-gRNA.These RG systems now provide an important foundation for the further construction of TSWV full-length infectious clones,and also provide a powerful molecular tool to study the replication,transcription and cell-to-cell movement of tospoviruses.2.Systemic infection of the full-length infectious clone of tomato spotted wilt virusIn this part,TSWV S,M and L the full-length cDNA clones based on genomic and anti-genomic were constructed,similar to the ones made for S,M and L mini-replicons.However,all attempts to rescue the TSWV virus directly from full-length cDNA clones based on the wild-type viral genome sequences have failed.We further found that the ectopic co-expression of N and RdRp proteins could induce the superinfection exclusion effect of TSWV,leading to the S(-)-and M(-)-mini-replicon couldn't be expressed in the same plant cells and then seriously hindered the rescue of TSWV from cDNA clones based on RG systems.In order to solve this problem,we constructed a recombinant positive-sense L(+)opt anti-genome plasmid to express optimized RdRp that help us generate the functional RdRp protein in plant cells.The optimized full-length antigenomic L(+)opt was able to expressed RdRp required for viral RNA systhesis,S(+)-and M(-)-mini-replicons was also able to expressed the nucleocapsid N and movement protein NSm,respectively.Next,we successfully established the systemic infection of M(_)-and S(+)-mini-replicons by co-expression of full-length antigenomic L(+)in N.benthamiana.However,we found that wild-type negative-stranded M(-)was not able to complement and rescue the S(+)-mini-replicons into systemically infected leaves.Considering that primary M(-)transcripts were produced in the nucleus by the 35S promoter and putative splice sites were also predicted in the GP sequence,it was likely that the primary M(-)transcripts were prone to be spliced before sufficient replication of the mini-replicon and transcriptional-translational expression of the cell-to-cell movement protein gene.Therefore,codon optimization strategy was performed on the GP gene sequence in M(-),leading to a new construct designated as M(-)opt.A series of difficulties has been overcome for the construction of a successful RG system for TSWV.Finally,infectious TSWV was successfully rescued from full-length cDNA clones of L(+)opt,M(-)opt and S(+)in plant.3.Development of mini-replicon reverse genetics systems of rice stripe virusIn this study,we first developed an RSV RNA3-based mini-replicon system in plant.The RNA3(-)-gRNA MR3(-)eGFP-mini-replicon plasmid was generated based on 35S promoter-driven constructs,flanked by the 5' hammerhead and 3' ribozyme sequence of hepatitis delta virus.Our results showed that only optimized RSV RdRp was able to support the MR3(-)eGFp-mini-replicon system,and NP and RdRp were indispensable for the viral replication and transcription activity.Next,we found that four VSRs(NSs,P19,HcPro and yb)all significantly enhanced the reporter gene eGFP expression of MR3(-)eGFP-mini-replicon,but RSV NS3 seriously inhibited the eGFP expression of MR3(-)eGFp.Then,we demonstrated that the NS3 sequence and cis-acting elements 5'-UTR and 3'-UTR of RNA3 genome were required for replication and transcription processes of RSV;RSV-encoded movement protein NSvc4 was able to promote the cell-to-cell movement of reporter gene eGFP of MR3(-)eGFP-mini-replicon in plant cells.Based on the RNA3 mini-replicon system,RdRp of RNA1 genome,NSvc2 of RNA2 genome and NSvc4 of RNA4 genome were replaced by reporter gene eGFP,generating the MR1(-)eGFP,MR2(-)eGFP and NR4(-)eGFP from the RNA1,RNA2 and RNA4 genomic RNA segments,respectively.Finally,we also successfully established the mini-replicon systems based on RNA1,RNA2 and RNA4 segments using the same similar strategies in plant as shown above.The establishment of the RSV mini-replicon system is the first stage breakthrough on the RSV RG system,and the full-length infectious clone will be further studied and optimizied.To sum up,our research for the first time develops a tetradic-segmented plant NSV(RSV)mini-replicon RG system,providing a new and powerful technological platform for future studies of RSV infection,pathogenicity and effective control and promoting the rescue of RSV entirely from full-length infectious cDNA clones.