Development of PVY resistance in tomato by knockout of host eukaryotic initiation factors by CRISPR-Cas9

Tomato (Solanum lycopersicum) is a valuable crop worldwide and is widely infected with the potyvirus potato virus Y (PVY), which causes serious yield loss. Viral infection depends on host elements and the eukaryotic translation initiation factor 4E (eIF4E) is essential in the potyvirus life cycle. To generate potyvirus resistance, the tomato SleIF4E1 and SleIF4E2 genes were disrupted and knockout mutants (sleif4e1, sleif4e2 and double mutant sleif4e1/e2) were generated using CRISPR/Cas9 technology. Mutant plants were evaluated for resistance to PVY following mechanical inoculation. The sleif4e1 and sleif4e1/e2 mutants did not display the PVY related symptoms as observed in wild type and sleif4e2 plants. During the first 21 days post infection, PVY coat protein accumulation was significantly lower in the sleif4e1, sleif4e1/e2 mutants than in the wild type and sleif4e2 plants and was undetectable 32 days post infection. However, PVY RNA accumulation was observed in sleif4e1 and sleif4e1/e2 virus resistant plants, reflecting that resistance is associated with impaired translation and not viral RNA accumulation. Interestingly, two amino acid changes, 119^H/Y and 123^S/N, were observed in the viral-encoded VPg gene in sleif4e1/e2 double mutant plants infected with PVY, indicating selection pressure on viral genes during replication. None of the mutant plants showed resistance to any virus but PVY when challenged with eggplant mild leaf mottle virus, cucumber mosaic virus, pepino mosaic virus and tomato brown rugose fruit virus. Thus, it was demonstrated that SleIF4E-mediated resistance is specific to PVY.