Fuctional Analysis Of EEF1A In Tomato Spotted Wilt Virus Replication

Tomato spotted wilt virus(TSWV)is a devastating plant virus,which has been nominated one of the top 10 plant viruses.TSWV has world-wide distribution and wide host range,it can cause severe economic losses not only for many grain crops,but also for horticultural crops.The eukaryotic elongation factor 1A(e EF1A)is an important factor in protein synthesis.It has been shown to be part of replicase complex in several positive-strand RNA virus and stimulate viral RNA synthesis.In recent reports,eEF1A is also engaged in negative-strand RNA viruses and plays similar roles as in positive-strand RNA virus,such as promoting the viral RNA translation,regulating viral RNA replication,accelerating the process of virus infection,etc.However,the mechanism of negative-strand RNA virus replication facilitated by eEF1A is not known.This work concentrated on the hotspots of plant virology and the urgent demand of agriculture,intended to analyze the interaction mode between eEF1A and protein of TSWV in virus and TSWV in virus replication,and revealed the molecular mechanism of eEF1A in virus replicase complex,which presents most important theoretical significance and practical application value.Briefly results are as follows:(1)N.benthamiana eEF1A genes and their 3'-UTR sequences were cloned by amplification;(2)Phylogenetictree analysis of the homologous sequences of eEF1A were used to classify all cloned sequences into group A and B.There are some differences between the two groups;(3)Virus-induced gene silencing(VIGS)was performed to down-regulate eEF1A gene in N.benthamiana leaves,while virus accumulation were decreased in TSWV infected plants.Repeat experiments showed that the minimum amount of virus accumulation occurred in 28 days;(4)The phenotype of plants showed lower effect in virus-induced gene silencing(VIGS)by using selected parts of the 3'-UTR sequence respectively;(5)Directly interacts between eEF1A and TSWV-NSm was identified and defined by yeast two-hybrid technique;(6)The interaction between eEF1A and TSWV-NSm were also verifiedby bilamolecular fluorescence complementation technique,which excluded the possibility of self-activation.