Cloning The Sequence Of EIF4Es In Capsicum Chinense And Confirming Their Interaction With PVMV-VPg

Yellow Lantern chili peppers(Capsicum chinense)is an economically important specialty crop in Hainan island,while plant viruses had gradully became the most serious pathogen of Capsicum chinense.Pepper veinal mottle virus(PVMV)causes a major disease on the Capsicum chinense.PVMV is a member of Potyvirus,and it has a genome-linked protein(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 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.In this study,we cloned Four eIF4E genes and investigated the protein-protein interactions through the LexA yeast two hybrid system(Y2HS)and bimolecular fluorescence complementation technology(BiFC).This study lay the foundation for anti-PVMV study with eIF4E as a target.The research results are as follows:(1)Four eIF4E genes were identified from bioinformatic analysis of pepper genomes,cloned by RT-PCR,specific primers with Capsicum chinense leaves as template.Temporarily named as Cc-eIF4E,Cc-eIF(iso)4E,Cc-eIF4E-Xm and Cc-nCBP9090.Sequence analysis showed that Pepper eIF4E family consists of two eIF4E genes(Cc-eIF4E and Cc-eIF4E-Xm),one eIF(iso)4E gene(Cc-eIF(iso)4E),and one novel cap binding protein gene(Cc-nCBP9090).(2)PVMV-VPg coding region was obtained by PCR with PVMV isolated in Hainan,saved in our laboratory as template.The ORF consists of 573 nueleotides,encoding a protein of 191 amino acids.(3)To investigate the interaction between PVMV-VPg and eIF4E family of Capsicum chinense,construct bait vector containing PVMV-VPg gene,and activation domain vectors containing Cc-eIF4E,Cc-eIF(iso)4E,Cc-eIF4E-Xm and Cc-nCBP9090 gene respectively and these vectors are verified through self-activation and toxicity testing and nutritional deficiencies and X-galactosidase screening.The identified vectors were transferred into EGY48 yeast cell by lithium acetate method.The results of Y2HS showed that PVMV-VPg interacts with Cc-eIF4E and Cc-eIF(iso)4E,but can't interacts with Cc-eIF4E-Xm and Cc-nCBP9090.(4)To confirm the results of Y2HS we construct BiFC vectors as following:YC1-VPg vector and YNl-eIF4E vector and YNl-eIF(iso)4E vector and YNl-eIF4E-Xm vector and YN1-nCBP9090 vector,and transform them into Agrobacterium cells.Then co-infection YCl-VPg and YN1-eIF4E,YN1-eIF(iso)4E,YNl-eIF4E-Xm,YNl-nCBP9090 respectively into the tobacco leaf.The observation of confocal laser scanning microscope shows that Cc-eIF4E and Cc-eIF(iso)4E showed a strong and consistent interaction with PVMV-VPg.The result confirmed the interaction results of Y2HS.The main hypothesis for this interaction is that Cc-eIF4E and Cc-eIF(iso)4E proteins interact with PVMV-VPg.According to this results of the study lay a theoretical foundation in revealing the virus pathogenic mechanism and contribute to disruption of the VPg-eIF4E interaction by genome editing to engineer PVMV resistance.