Imprecise Cas12a/ <scp>ssODN</scp> ‐Mediated Editing of <i>eIF4E1</i> Confers Dominant‐Negative Resistance to Potato Virus Y in <i>Solanum tuberosum</i>

ABSTRACT Mutants in the eukaryotic initiation factor 4E ( eIF4E ) gene family provide recessive resistance to plant viruses. Previously, we demonstrated that knocking out SteIF4E1 potato alleles ( A, B, B, B1 ) resulted in limited resistance to a necrogenic PVY isolate (NTN). Expanding on this, we used a non‐transgenic method employing Cas12a and a single‐stranded oligodeoxynucleotide (ssODN) as a repair template to introduce amino acid changes in SteIF4E1, attempting to mimic the Capsicum PVY resistance allele pvr2 1 . However, no plants that had been regenerated from transfected protoplast cells displayed correct SteIF4E1 editing. Instead, truncated sequence duplications (pvr2 1 SD) appeared at some mutated alleles, suggesting that ssODN‐mediated repair predominantly occurs via microhomology‐mediated end joining. Importantly, out of the 39 edited lines tested, the dwarf line Bb29 exhibited resistance to PVY‐NTN and showed reduced systemic infection sustained by PVY‐O. Notably, PVY‐NTN resistance‐breaking isolates occasionally emerged in Bb29, confirming genuine eIF4E‐mediated resistance. In Bb29, three of four eIF4E1 copies were mutated ( AΔ12, BΔ6, B, B1pvr2 1 SD ), with SteIF4E1_B1pvr2 1 SD featuring a premature stop codon at the 5′ end of the mutated sequence, and the unique SteIF4E1_AΔ12 allele in our gene‐edited lines. We found that SteIF4E1_BΔ6 , but not SteIF4E1_AΔ12 , partially complemented an eIF4E‐deficient yeast strain. In addition, introducing a wild‐type copy of SteIF4E1 into Bb29 restored virus susceptibility and partially reversed dwarfism. Conversely, transforming wild‐type potatoes with SteIF4E1_AΔ12 mimicked the resistance profile of Bb29, conferring stronger and broader resistance against PVY than the SteIF4E1 knockout approach. These findings reveal a dominant‐negative resistance mechanism, opening new avenues for engineering potyvirus resistance.