Construction Of Papaya EIF4E Mutant Genes And Study Of The Interaction Between Papaya EIF4E And PRSV-VPg

The papaya ringspot virus (PRSV) is the main pathogen that restricts papaya production, belonging to the genus Potyvirus family, Potyviridae. The PRSV genome consists of a single-stranded positive sense RNA with a5'terminal genome-linked protein (VPg) and3'terminal poly(A) tail. VPg has an essential role in the virus infection cycle. The eukaryotic translation initiation factor4E (eIF4E) which can bind with cap structure of mRNA or cap homologue plays a key role in the initiation step of protein synthesis. In many plant/RNA virus interaction, eIF4E is an indispensable factor for infection of RNA virus with plant. The mutation or loss of eIF4E may results in resistance to the virus. It has found the interaction between Cp-eIF4E and PRSV-VPg. Conventional breeding of papaya resistant varieties is difficult, but there is the great value in the study of resistance against PRSV by genetic engineering strategy. However, this method that results in resistance against PRSV is by genes of virus, and its application is limited by narrow spectrum of resistance and biosecurity. Therefore, this study decided to make Cp-eIF4E as the target, studying the effect of point mutations on the PRSV-VPg/Cp-eIF4Einteraction. The major research work include:(1) aligned susceptible Cp-eIF4E sequence with other species of eIF4E sequence and analyzed the three-dimensional structure of Cp-eIF4E, then, three types of mutant sites were determined:W83and W129(locating in the cap binding domain edge), W100and G162((locating in the eIF4G binding region), G115and A229[the active sites that pepper eIF4E resistance gene (pvrl) and melon eIF4E resistance gene (nsv) resistance site lie in the Cp-eIF4E, being near the cap binding domain]. Point mutations were produced in Cp-eIF4E byoverlap extension PCR in this paper, and six Cp-eIF4E mutant genes were successfully constructed. Interactions were tested by yeast two-hybrid and bimolecular fluorescence complementation assays. The results suggest that all these sites can affect the PRSV-VPg/Cp-eIF4E interaction. Therefore, this paper gains mutant Cp-eIF4Es that do not interact with PRSV-VPg and lays the foundation for cultivating eIF4E mediated resistance of new papaya varieties.