Genome Editing of eIF4E1 in Tomato Confers Resistance to Pepper Mottle Virus

Many of the recessive virus-resistance genes in plants encode eukaryotic translation initiation factors (eIFs), including eIF4E, eIF4G, and related proteins. Notably,eIF4Eand its isoformeIF(iso)4Eare pivotal for viral infection and act as recessive resistance genes against various potyviruses in a wide range of plants. In this study, we used Clustered Regularly Interspaced Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated targeted mutagenesis to test whether novel sequence-specific mutations ateIF4E1inSolanum lycopersicum(tomato) cv. Micro-Tom could confer enhanced resistance to potyviruses. This approach produced heritable homozygous mutations in the transgene-free E(1)generation. Sequence analysis ofeIF4E1from E(0)transgenic plants expressingCas9andeIF4E-sgRNAtranscripts identified chimeric deletions ranging from 11 to 43 bp. Genotype analysis of theeIF4E1-edited lines in E-0, E-1, and E(2)transgenic tomato plants showed that the mutations were transmitted to subsequent generations. When homozygous mutant lines were tested for resistance to potyviruses, they exhibited no resistance to tobacco etch virus (TEV). Notably, however, several mutant lines showed no accumulation of viral particles upon infection with pepper mottle virus (PepMoV). These results indicate that site-specific mutation of tomatoeIF4E1successfully conferred enhanced resistance to PepMoV. Thus, this study demonstrates the feasibility of the use of CRISPR/Cas9 approach to accelerate breeding for trait improvement in tomato plants.