Simultaneous knockout of multiple eukaryotic translation initiation factor 4E genes confers durable and broad-spectrum resistance to potyviruses in tobacco.

Recessive resistance mediated by mutations in the eukaryotic translation initiation factor 4E (eIF4E), has proven effective against diverse potyviruses and is extensively utilized in breeding programs. However, the rise of resistance-breaking (RB) strains and emerging potyviral species necessitates the development of more durable and broad-spectrum resistance strategies. In this study, our field survey in Yunnan, China, identified potato virus Y (PVY) RB isolates, as well as the prevalence of tobacco vein banding mosaic virus (TVBMV) and chilli veinal mottle virus (ChiVMV), in tobacco carrying the recessive va locus, which lacks the eIF4E1-S susceptibility gene, due to a chromosomal deletion. Protein interaction and viral infection assays demonstrated that both eIF4E1-S and eIFiso4E-T are used by PVY RB as susceptibility factors for infection, with the combined inactivation of these genes confering durable resistance. Similarly, the knockout of eIFiso4E-S, in the va genetic background, provided effective resistance to TVBMV and reduced susceptibility to ChiVMV. Notably, pyramiding mutations in eIFiso4E-S and eIFiso4E-T, in va tobacco, generated plants exhibiting robust, broad-spectrum resistance, to all three viruses, without compromising plant development. These findings underscore the potential of stacking eIF4E mutations to engineer durable, broad-spectrum resistance to potyviruses in tobacco, offering a promising strategy for crop improvement. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42994-025-00216-5.