CRISPR/Cas9-mediated editing of eukaryotic elongation factor 1B gamma (eEF1Bγ) reduces Tobacco etch virus accumulation in Nicotiana benthamiana

Tobacco etch virus accumulation declined in Nicotiana benthamiana eEF1Bγ gene-edited lines, suggesting that eEF1Bγ may be a host factor for this virus. Viruses use host factors to replicate and move from cell to cell. Therefore, the editing of genes encoding viral host factors that are not essential for plant survival enables the rapid development of plants with durable virus resistance. Eukaryotic initiation factors, such as eIF4E and eIF4G, function as host factors for viral infection, and loss-of-function mutations of these factors lead to virus resistance. Broadening the spectrum of host factor targets would help expand resources for engineering virus resistance. In this study, we tested whether editing the eukaryotic translation elongation factor gene eEF1Bγ would produce virus-resistant plants. Accordingly, we targeted the four eEF1Bγ genes in Nicotiana benthamiana for editing using virus-induced gene editing (VIGE) with Tobacco rattle virus (TRV). Although we attempted to obtain plants edited for all four eEF1Bγ homologs, we failed to identify such plants. Instead, we obtained plants with three of the four homologs knocked out, harboring 1-bp insertion/deletions resulting in premature stop codons. These eEF1Bγ-edited plants did not exhibit resistance to Potato virus X (PVX), Tobacco mosaic virus (TMV), or Tomato bushy stunt virus (TBSV) but showed reduced accumulation of Tobacco etch virus (TEV) compared to wild-type plants. These findings demonstrate the feasibility of conferring resistance in plants through gene editing of eEF1Bγ, underscoring the importance of exploring diverse host factor targets for comprehensive virus resistance.