Abstract
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, 119H/Y and 123S/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.
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This research work was supported by the Chief Scientist of the Israeli Ministry of Agriculture and Rural Development No. 20-02-0053.
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S.K.: virology, cross and self-pollination, mutant characterization, double mutant generation, genotyping, molecular analysis, western blotting, manuscript writing. B.A.: transformation and greenhouse establishment of T0 plants, mutant characterization, manuscript writing. M.K.: construct preparation, transformation, mutant characterization, pollination and genotyping, ToBRFV blotting. R.K.: virology, molecular and western blot analysis. M.B: growing experimental plants. D.L.: construct preparation. Y.S.: construct preparation, transformation, pollination. M.C.: genotyping and DNA extraction. S.K., M.K., R.K., B.A., D.L., V.G. and A.G. revised the manuscript. A.G.: conceived and designed the research. All authors contributed to the article and approved the submitted version.
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Supplementary Fig. 1
Amino acid alignment of SleIF4E1, SleIF4E2 and SleIF(iso)4E proteins. Amino acid residues colored in red are conserved in all proteins, whereas amino acids colored in green are conserved in SleIF4E1 and SleIF4E2, and not in SleIF(iso)4E (PNG 1546 kb)
Supplementary Table S1
Details of primers used for amplification of sgRNAs targeted regions, real time PCR and Vpg analysis (DOCX 13 kb)
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Kumar, S., Abebie, B., Kumari, R. et al. Development of PVY resistance in tomato by knockout of host eukaryotic initiation factors by CRISPR-Cas9. Phytoparasitica 50, 743–756 (2022). https://doi.org/10.1007/s12600-022-00991-7
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DOI: https://doi.org/10.1007/s12600-022-00991-7
Keywords
- eIF4E1
- eIF4E2
- PVY
- CRISPR/Cas9
- Susceptibility genes
- Recessive resistance