An iterative gene-editing strategy broadens eIF4E1 genetic diversity in Solanum lycopersicum and generates resistance to multiple potyvirus isolates.

Resistance to potyviruses in plants has been largely provided by the selection of natural variant alleles of eukaryotic translation initiation factors (eIF) 4E in many crops. However, the sources of such variability for breeding can be limited for certain crop species, while new virus isolates continue to emerge. Different methods of mutagenesis have been applied to inactivate the eIF4E genes to generate virus resistance, but with limited success due to the physiological importance of translation factors and their redundancy. Here, we employed genome editing approaches at the base level to induce non-synonymous mutations in the eIF4E1 gene and create genetic diversity in cherry tomato (Solanum lycopersicum var. cerasiforme). We sequentially edited the genomic sequences coding for two regions of eIF4E1 protein, located around the cap-binding pocket and known to be important for susceptibility to potyviruses. We show that the editing of only one of the two regions, by gene knock-in and base editing, respectively, is not sufficient to provide resistance. However, combining amino acid mutations in both regions resulted in resistance to multiple potyviruses without affecting the functionality in translation initiation. Meanwhile, we report that extensive base editing in exonic region can alter RNA splicing pattern, resulting in gene knockout. Altogether our work demonstrates that precision editing allows to design plant factors based on the knowledge on evolutionarily selected alleles and enlarge the gene pool to potentially provide advantageous phenotypes such as pathogen resistance.

CRISPR-based knock-out of eIF4E2 in a cherry tomato background successfully recapitulates resistance to pepper veinal mottle virus.

The host susceptibility factors are important targets to develop genetic resistances in crops. Genome editing tools offer exciting prospects to develop resistances based on these susceptibility factors, directly in the cultivar of choice. Translation initiation factors 4E have long been known to be a susceptibility factor to the main genus of Potyviridae, potyviruses, but the inactivation of the eIF4E2 gene has only recently been shown to provide resistance to some isolates of pepper veinal mottle virus (PVMV) in big-fruit tomato plants. Here, using CRISPR-Cas9-NG, we show how eIF4E2 can be targeted and inactivated in cherry tomato plants. Three independent knockout alleles caused by indel in the first exon of eIF4E2, resulted in the complete absence of the eIF4E2 protein. All three lines displayed a narrow resistance spectrum to potyvirus, similar to the one described earlier for an eIF4E2 EMS mutant of M82, a big-fruit tomato cultivar; the plants were fully resistant to PVMV-Ca31, partially to PVMV-IC and were fully susceptible to two isolates of PVY assayed: N605 and LYE84. These results show how easily a resistance based on eIF4E2 can be transferred across tomato cultivar, but also confirm that gene redundancy can narrow the resistances based on eIF4E knockout.