Objective To clone the papaya eukaryotic translation initiation factor 4E gene (CpeIF4E), and analyze its expression pattern in response to papaya leaf distortion mosaic virus (PLDMV) infection, the subcellular localization of CpeIF4E protein, as well as the interaction and key interaction sites between CpeIF4E and PLDMV genome-linked protein (PLDMV-VPg). This study will provide a theoretical foundation for the application of CpeIF4E in disease-resistant molecular breeding of papaya.

Method CpeIF4E and PLDMV-VPg genes were cloned from healthy and PLDMV-infected papaya leaves, respectively, and their coding sequences were identified. The subcellular localization vector CpeIF4E-GFP was constructed and transformed into rice protoplasts to determine the subcellular localization of CpeIF4E. The expression level of CpeIF4E under PLDMV infection was detected by realtime quantitative PCR (RT-qPCR). The interaction between CpeIF4E and PLDMV-VPg was verified using yeast twohybrid assay (Y2H), bimolecular fluorescence complementation (BiFC) assay, and luciferase complementation (LCI) assay. Based on natural variation sites of resistant eIF4E, site-directed mutagenesis of CpeIF4E at amino acid positions 73, 75, 82, 87, 114, and 117 was performed using overlap-extension PCR. Interaction assays were performed to identify the key residues for CpeIF4E and PLDMV-VPg interaction.

Result The full-length CDS of CpeIF4E was 711 bp, encoding 236 amino acids containing an IF4E domain. The coding sequence of PLDMV-VPg was 561 bp, encoding 186 amino acids. CpeIF4E was localized in the cytoplasm and nucleus. The expression level of CpeIF4E peaked at 7 days post PLDMV inoculation. CpeIF4E physically interacted with PLDMV-VPg. Mutations at positions 73, 75, 82, and 114 completely abolished the interaction between CpeIF4E and PLDMV-VPg, whereas mutations at positions 87 and 117 did not affect the interaction.

Conclusion CpeIF4E interacts with PLDMV-VPg, and the key interaction amino acid sites are positions 73, 75, 82, and 114. This study provides a clear theoretical basis and operable targets for creating novel PLDMV-resistant papaya germplasm through targeted modification of CpeIF4E via gene-editing technology.