Enhancing papaya resistance to ringspot virus through CRISPR/Cas9-mediated gene editing of eIF4E

Papaya is a tropical fruit crop with high nutritional and medicinal properties. Its production worldwide is significantly hindered by papaya ringspot disease, caused by a potyvirus known as papaya ringspot virus (PRSV). While the eukaryotic translation initiation factor 4E gene (eIF4E) and its paralogue eIF(iso)4E have been linked to potyvirus resistance in various model and crop species, their role in PRSV susceptibility has not been systematically investigated in papaya. We employed the CRISPR/Cas9 gene editing technology to create gene editing reagents that induce two independent double-strand DNA breaks at the eIF4E and eIF(iso)4E target genes. We then regenerated stable transgenic plants by Agrobacterium-mediated transformation using somatic embryos. Heteroduplex and Sanger sequencing analyses revealed chimeric and heterozygous mutations in eIF4E and eIF(iso)4E genes in the regenerated transgenic papaya T0 lines, respectively. Notably, only eIF4E-edited plants demonstrated resistance to PRSV across all time points tested. Both DAS-ELISA and qPCR analyses confirmed undetectable viral accumulation in these mutant lines. In contrast, PRSV accumulation was observed in the systemic leaves of both wild-type (WT) and eIF(iso)4E-edited lines, which developed characteristic symptoms over time. These results identify eIF4E as a critical host factor for PRSV and show that targeted mutations can confer robust resistance. These highly resistant lines will serve as foundational germplasm for further selection and breeding. Despite the limitations of using chimeric T0 mutant lines, this study establishes a framework for developing improved local papaya cultivars through advanced gene-editing technologies.