Cloning The Full Sequence Of EIF4E And EIFiso4E In Papaya And Verifing Their Interaction With PRSV-VPg

The potyvirus papaya ringspot virus (PRSV) is an important pathogen that restricts papaya cultivation and cucurbits production. At the 5'end of the potyvirus genome, there is a viral protein linked to the genome (VPg) which may intervene at several steps in the virus infection cycle, including viral RNA replication, and viral cell-to-cell and long-distance movement.The eukaryotic translation initiation factor 4E which can bind with m7GpppN cap structure of mRNA or cap homologue specifically play important roles in protein translation. Usually, mRNA binds with 4E through 5'cap structures, but there is no cap structure in 5'end of the potyvirus genome, the virus just binds with 4E by VPg protein. The study found that the interaction between virus genome-linked protein VPg and eukaryotic translation initiation factor 4E plays a key role in virus infection, it can affect the virus-plant interaction and mediate resistance of plants to viruses by changing the key sites of 4E.eIF4E belongs to a small multigenic family, and in plants, it has an isoform eIFiso4E. The two factors are mechanistically equivalent during the process of translation,but exhibit differences in their ability to bind m7GpppN and to other cap analogues It is different in the use of eIF4E and its isomer for potyvirus in one plant, The binding capacity of papaya eIF4E and eIFiso4E with PRSV-VPg has not yet reported.So papaya eIF4E and eIFiso4E genes were clond by RT-PCR in this study and then the interaction between PRSV-VPg with Cp-eIF4E and Cp-eIFiso4E were verified by the GAL4 yeast system and Bimolecular fluorescence compleme-ntation technique (BiFc). The results lay the foundation for anti-PRSV study with eIF4E as a taget.In this study, full length of papaya eIF4E and eIFiso4E genes were obtained by RT-PCR,5'-Full RACE and 3'-Full RACE with papaya leaves as template. The full length of Cp-eIF4E is 1185bp with an open reading frame of 708 nucleotides encoding a protein of 236 amino acids and Cp-eIFiso4E is 995bp with an open reading frame of 618 nucleotides encoding a protein of 206 amino acids. PRSV-VPg coding region was obtained by PCR with PRSV isolate in Hainan saved in our laboratory as template. The open reading frame consists of 561 nucleotides, encoding a protein of 187 amino acids.To investigate the interaction between PRSV-VPg and Cp-eIF4E/Cp-eIFiso4E yeast-two-hybrid-GAL4 system was employed to construct bait vectors containing PRSV-VPg gene, and activation vectors containing Cp-eIF4E and Cp-eIFiso4E gene respectively and these vectors are verified through self-activation and toxicity testing. The identified vectors were transferred into YRG-2 by lithium acetate method. It showed that PRSV-VPg interacts with both Cp-eIF4E and Cp-eIFiso4E after nutritional deficiencies andβ-galactosidase filter screening.To further verify the above results, Bimolecular fluorescence complementation technique was used to construct BiFc-pSATN-nEYFP vetors containing PRSV-VPg gene and BiFc-pSATN-cEYFP vectors containing Cp-eIF4E and Cp-eIFiso4E gene respectively which were bombarded into the onion epidermis for cultivating 12-16 hours. The interactions between PRSV-VPg with Cp-eIF4E and Cp-eIFiso4E were obsvered in onion cells under fluorescence microscope. The result confirmed the interaction results of GALA. The main hypothesis for this interaction is that eIF4E/ eIFiso4E proteins interact with VPg of potyvirus promoting viral protein translation and accumulation.The results of this study lay a theoretical foundation in revealing the virus pathogenic mechanism and pave the way for anti-PRSV papaya research as 4E target.