Optimization Of The Mini-Replicon-Based Reverse Genetics System Of Rice Stripe Virus And Function Analysis Of Cis-Acting Elements And Trans-Acting Factors

Rice stripe virus（RSV）is one of the most serious pathogens that harms rice,mainly infects rice and other gramineous plants.Through mechanical inoculation,RSV can also infect dicotyledonous model plants such as Nicotiana benthamiana and Arabidopsis thaliana.RSV is a segmental plant negative strand RNA virus,a member of genus Tenuivirus,family Phenuiviridae,order Bunyavirales.Reverse genetics system is a key technical system to reveal the function of virus genes and the infection mechanism.However,it is quite difficult to construct reverse genetics system of RSV because of its four segmented genomes.The RSV minireplicon system was initially established in our laboratory at the early stage.Based on this,the present study optimized the RSV minireporter system,and prepared mouse polyclonal antibody of nucleocapsid NP protein,which was used to detect the NP protein expressed by the RSV MR3_{（-）e GFP} system in N.benthamiana.The efficiency of MR3_{（-）e GFP} microreplicon system was improved and the interaction relationships among RSV NS2,NS3,SP protein and Jasmonate ZIM-domain（JAZ）protein of Jasmonic acid（JA）signaling was screened by yeast two hybrid technique,which lay the foundation to elucidate the pathogenesis of RSV.The results are summarized as follows:1.The preparation of mouse polyclonal antibody of nucleocapsid NP protein and its application on MR3（-）e GFP system in N.benthamiana.The RSV RNA3 minireporter system had been established by our laboratory,but the NP protein expressed by the minireporter system could not be detected with the original monoclonal antibody.In this study,purified RSV virions were used as antigens to make the new RSV NP mouse polyclonal antiserum which is sensitive and could successfully detect the NP protein expressed in the MR3_{（-）e GFP} system in N.benthamiana.Thus,this study laid a foundation for the optimization of MR3_{（-）e GFP} system and the establishment of reverse genetic system of RSV full-length infectious clone.2.To explore the regulation effect of NS2、NSvc2 and SP encoded by RSV on the RSV MR3（-）e GFP and the optimization study.In this study,the RSV MR3_{（-）e GFP} system was optimized by exploring the effects of NS2,NSvc2 and SP encoded by RSV on the minireplicon system.Finally,it was found that NS2 and SP can promote the MR3_{（-）e GFP} system to a certain degree,and the optimal concentration of NS2 is OD₆₀₀ 0.1 and the optimal concentration of SP is OD₆₀₀ 0.3.Consequently,the efficiency of RSV MR3_{（-）e GFP} system can be significantly improved,which will create favorable conditions for studying the pathogenic mechanism of RSV.3.To explore the positive regulatory effects of RSV NS3,as a cis-acting element,on the replication and transcription of RSVOn the basis of MR3_{（-）e GFP},we completely deleted the NS3 sequence and constructed MR3_（-）e GFPΔNS3 and there was no green fluorescence appeared.Then,two mutants were constructed:RSV MR3（-）e GFP&NS3stop1 and MR3（-）e GFP&NS3stop2,to make the NS3 protein unable to be expressed.As a result,green fluorescence could still be observed,indicating that there is no significant effect of NS3 on the MR3_{（-）e GFP} system.We continued to build another four mutants:MR3_{（-）e GFPMut1-4}.Each mutant has a quarter of NS3 sequence,and only MR3_{（-）e GFPMut1}can not express e GFP,the other three mutants can work normally,which showed that the first quarter of NS3 is not enough to express the e GFP.Besides,the g RNA and ag RNA of MR3_{（-）e GFPMut1-4}can still be detected by Northern blot.Therefore,we conclude that the NS3 ORF sequence is not necessary for viral replication but is required for the expression of e GFP from the MR3_{（-）e GFP} in N.benthamiana cells.4.The interaction among RSV NS3,SP and resistance factor JAZs proteins of JA signaling pathway was screened preliminarily.NS3 is a VSR of RSV,SP is a disease-related protein,these two proteins both are essential for RSV infection.In this study,the weak interaction relationships between NS3and JAZ8,JAZ11 of Arabidopsis thaliana,SP and JAZ2 of Arabidopsis thaliana were screened via yeast two-hybrid system.What’s more,SP could interact strongly with JAZ7and JAZ12 of Oryza sativa,and the interactions were also verified by Bi FC technology,which showed that,the interaction between SP and JAZ7 occurred in the cytoplasm,while the interaction between SP and JAZ12 occurred in the nucleus.Revealing these interactions is helpful to further explore the defense and anti-defense relationships between host plants and RSV.