Construction Of The Attenuated Vaccine Vector From Cucumber Mosaic Virus And Research On The Mechanism Of Mutation Repair

Plant virus disease was the second largest type of disease in agricultural production,causing severe damage to most crops worldwide.The management of plant virus disease was the difficult and hot spot.The cross-protection strategy based on attenuated vaccine was an effective way to prevent plant virus disease,but there were some limiting factors such as narrow host range,narrow target virus spectrum and genetic unstability of vaccine.The unstability of the vaccine was related to mutation repair due to the low fidelity of Rd Rp,which could be achieved through RNA recombination and other mechanisms.However,the factors triggerring these repair mechanisms were not identified deeply.Cucumber mosaic virus(CMV)was one of plant viruses with the broadest host range and the widest distribution.The attenuated vaccine using CMV as the basic viral vector had the advantage of broad host range.The CMV genome contained three single-stranded RNAs(RNA1,RNA2 and RNA3)totally encoding five proteins(1a,2a,2b,MP,CP).The multi-segment genome provided multiple choices for inserting heterologous fragment to construct the multivalent vaccines.In this study,we constructed the basic vectors for wide-host and multivalent attenuated vaccine based on CMV and analyzed the triggering factors and occurrence occasion of mutation repair based on the mutation repair of mild mutants,which could provide the revelation on virus evolution and the development and application of vaccines.Firstly,two types of 2b premature termination mutants R2-2bPTI and R2-2bPT?were constructed in CMV RNA2.R2-2bPTI still showed severe pathogenicity,while R2-2bPTII was an attenuated mutant.The heterologous fragment insertion mutant R2-2bPTII-P200 based on R2-2bPTII was also an attenuated mutant.Both R2-2bPT?and R2-2bPT?-P200 had a cross-protection effect on CMV,if they pre-inoculated plants.Inoculation assay of continuous 10generations presented the stability of these two kinds of attenuated mutants.Therefore,R2-2bPT?could be used not only as a vaccine to prevent CMV,but also as a basic vector for constructing multivalent vaccine by inserting fragments of heterologous virus.Different heterologous fragments were inserted into R2-2bPT?to test the stability and the capacity triggering gene silencing.100 nt or longer fragments of PDS gene could trigger gene silencing,shown on leaves of plant.Insertion of fragments more than 200 nt was inclined to be repaired showing unstability.Therefore,R2-2bPT?could be the basic vector to develop a potential trivalent attenuated vaccine with 200 nt heterologous fragments.To enhance the ability to contain heterologous fragments,attenuated mutant R2-2bPT?was constructed by deleting the sequence from original 2661G to 2751A from R2-2bPT?.Pre-inoculation of R2-2bPT?or its fragment insertion mutant R2-bPT?-P200 had the cross-protection effect on wild type of CMV.Insertion of 350 nt heterologous fragments into R2-2bPT?still kept the genetic stability.Therefore,R2-2bPT?could be the basic vector to develop a potential quadrivalent attenuated vaccine with 300 nt heterologous fragments.To further explore the weakening sites and insertion sites of heterologous fragments through the CMV genome,mutation was made on other viral proteins and cis-acting elements.Four deletion mutants(R3-m1,R3-m2,R3-m3 and R3-m4),and replacement mutants(R3-m1-P300,R3-m2-P400,R3-m3-P400and R3-m4-P400)were made on 100 nt at the 3'end of the MP,100 nt at the 5'end of the CP and the intergenic region(IGR)in CMV RNA3.However,all these mutants failed to infect plants systematically,which did not meet the basic requirements of systemic infection of the vaccine.The mutants R1-3Um and R3-3Um on the3'UTR of RNA1 and RNA3 showed the stability,while their insertion mutants R1-3Um-P200and R3-3Um-P200 occurred mutation repair,which did not meet the basic requirements of genetic stability of the vaccine.The 18 nt replacement mutant T_m1(R1-T_m1,R2-T_m1,R3-T_m1)and the 18 nt insertion mutant T_m2(R1-T_m2,R2-T_m2,R3-T_m2)were made in TLS of different RNAs of CMV.However,some of these TLS mutants showed severe pathogenicity,and further insertion mutation T_m3(R1-T_m3,R2-T_m3,R3-T_m3)and T_m4(R1-T_m4,R2-T_m4,R3-T_m4)based on them occurred mutation repair.It was suggested that mutants of TLS region was not suitable candidate for the vaccine.Although TLS mutants was not the candidate of the vaccine,they can be used to clarify the mechanism of mutation repair mediated by RNA recombination due to the conservation of TLS among different RNAs of CMV.TLS mutants of RNA1,RNA2 and RNA3 had different rates of mutation repair when they were inoculated with the conventional concentration(OD₆₀₀=1.2).TLS mutations R2-T_m1,R2-T_m2,R3-T_m1 and R3-T_m2 in RNA2 and RNA3 were respectively 50.0%,37.5%,80.0%and 62.5%,while TLS mutations in RNA1 failed to be repaired.Same type of TLS mutation in RNA3 had higher mutation repair frequency than that in RNA2.In addition,replacement type of TLS mutation in RNA2 or RNA3 had higher mutation repair than insertion type of TLS mutation.However,co-inoculation of R1-T_m1+R2-T_m1+R3-T_m1 or R1-T_m2+R2-T_m2+R3-T_m2failed to be repaired,which implied that the repair of TLS mutation was mediated by template-selective type of RNA recombination.Northern blot identified that TLS mutation caused the relative quantity defect of RNA2 or the quality defect of RNA3 showing larger size of RNA3.These results indicated that the quantitative defect of decrease in relative RNA2 or the qualitative defect of larger RNA3 size was factor related with mutation repair.Further studies shown that the TLS mutation in RNA1 could also be repaired under specific conditions.The repair rate of TLS mutation in RNA1 was 100%when the TLS mutants of RNA1 and the RNA2 attenuated mutant R2-2bPT?co-infected plants,and the repair rate of TLS mutation was 100%.Similarly,co-infection of the TLS mutant of RNA3 and R2-2bPT?also caused 100%mutation repair of TLS.These results suggested that mutation repair of TLS mutation in RNA1 was related with the low accumulation rate of the genome caused by the mutation of gene silencing inhibitor 2b.The gradient dilution assay also identified the mutation repair of TLS mutantion in RNA1 around dilution end-point.In summary,multiple levels of triggered factors in mutation repair included mutation characteristic of TLS,quantity defect or quality defect of genome segments,or quantity defect of whole genome,especially around dilution end-point.In addition,the co-infection of TLS mutants in RNA2 and the intercellular movement-deficient mutant M5 did not present the mutation repair on TLS mutation,which suggested the requirement of cell-to-cell movement on mutation repair.In this study,three type of basic vector from CMV were constructed and showed potential to develop multi-targets attenuated vaccine.In addition,we also identified the multiple triggering factors of mutation repair and its requirement on cell-to-cell movement,which provided important revelations on evolution of RNA viruses as well as application of viral mild vaccine.