Effect Of Satellite RNA On Cross-protection Of Attenuated Vaccine Based On Cucumber Mosaic Virus

Plant virus diseases are serious,which will lead to the decline of crop yield and quality,so the control is difficult.In contrast,cross-protection is an effective strategy to control plant virus diseases.Pre-inoculation of attenuated vaccine can stimulate the host plants to produce immune ability and achieve the purpose of reducing or preventing the disease.Cucumber mosaic virus(CMV)is one of the most widely distributed and economically important plant viruses with the largest host range.Some CMV isolates contain satellite RNAs,which is a kind of noncoding RNA.Some satellite RNAs can affect the pathogenicity of helper virus in varying degrees.In this paper,we studied the regulatory effect of satellite RNAs on attenuated vaccine of CMV and explored a new feasible way for the application of satellite RNAs in cross-protection.Two kinds of CMV satellite RNAs,sat CMV-TA-ca and sat CMV-TA-tb,were cloned in our laboratory to study the effects of two kinds of satellite RNAs on CMV virulence(CMV_Fny),CMV natural attenuated isolate(CMV_TA-pe)and CMV artificial attenuated mutant(CMV_{Fny2-2b PT})and CMV heterologous virus fragment insertion mutant(CMV_{Fny2-2b PT-TM5088}),and the cross-protection of satellite RNAs and helper virus in equal or unequal proportion.Firstly,two binary plasmids PCB-sat CMV-TA-ca and PCB-sat CMV-TA-tb were constructed,which were suitable for agrobacterium tumefaciens infiltration inoculation.The binary plasmids PCB-sat CMV-TA-ca and PCB-sat CMV-TA-tb were co-inoculated in N.benthamiana with the plasmids containing CMV_Fny RNA1,RNA2 and RNA3 respectively.The results showed that satellite RNAs could replicate in plants and reduce the pathogenicity of CMV_Fny.The quantitative inoculation system of satellite RNA and helper virus was established.The results that mixed inoculation of different types of CMV and satellite RNAs in equal proportion(equal concentration)were as follows:(1)When CMV_Fny was co-inoculated with satellite RNAs,it showed a certain degree of cross-protection to CMV_Fny,and sat CMV-TA-tb was more effective than sat CMV-TA-ca.(2)CMV_TA-pe,as a natural attenuated isolate,only caused slight dwarfing when infected with N.benthamiana.Pre-inoculation of CMV_TA-pe has cross-protection on CMV_Fny,and the cross-protection effect of satellite RNAs and CMV_TA-pemixed inoculation is more obvious.These results indicated that the cross-protection of CMV natural attenuated isolate by satellite RNAs was enhanced.(3)CMV_{Fny2-2b PT} is an artificially attenuated mutant of CMV_Fny RNA2.Pre-inoculation of it has cross-protection on CMV_Fny,and the best protection effect can be achieved by injection of CMV_Fny on the fifth day.The co-inoculation of satellite RNAs and CMV_{Fny2-2b PT} enhanced the pathogenicity,and the cross-protection of CMV_{Fny2-2b PT} was weakened due to the presence of satellite RNAs.(4)CMV_{Fny2-2b PT-TM5088} is an insertional weak mutant of CMV_Fny RNA2.The cross-protection effect of CMV_{Fny2-2b PT-TM5088} on CMV_Fny was demonstrated by pre-inoculation.The presence of satellite RNAs did not significantly change the cross-protection effect of CMV_{Fny2-2b PT-TM5088} on CMV_Fny,but it can improve the cross-protection of CMV and TMV mixed infection.The results that the attenuated mutants were inoculated with satellite RNAs in different proportion(unequal concentration)showed:(1)When CMV_{Fny2-2b PT} and sat CMV-TA-ca were inoculated at 2:1 and 1:1,the cross-protection effect was better.(2)When CMV_{Fny2-2b PT} and sat CMV-TA-tb were inoculated at 1:1 and 1:2,the cross-protection effect was obvious.The results showed that the satellite RNAs had regulatory effect on cross-protection of CMV artificial attenuated mutants,and the occurrence of positive and negative regulation depended on the inoculation ratio of the satellite RNAs to the attenuated vaccine.This study analyzed the regulatory effect of satellite RNAs on different types of CMV attenuated vaccines and explored a new feasible way for the application of satellite RNA in cross-protection.