Artificially Edited Alleles of the Eukaryotic Translation Initiation Factor 4E1 Gene Differentially Reduce Susceptibility to Cucumber Mosaic Virus and Potato Virus Y in Tomato

Abstract

Eukaryotic translation initiation factors, including eIF4E, are susceptibility factors for viral infection in host plants. Mutation and double-stranded RNA-mediated silencing of tomato eIF4E genes can confer resistance to viruses, particularly members of the Potyvirus genus. Here, we artificially mutated the eIF4E1 gene on chromosome 3 of a commercial cultivar of tomato (Solanum lycopersicum L.) by using CRISPR/Cas9. We obtained three alleles, comprising two deletions of three and nine nucleotides (3DEL and 9DEL) and a single nucleotide insertion (1INS), near regions that encode amino acid residues important for binding to the mRNA 5' cap structure and to eIF4G. Plants homozygous for these alleles were termed 3DEL, 9DEL, and 1INS plants, respectively. In accordance with previous studies, inoculation tests with potato virus Y (PVY; type member of the genus Potyvirus) yielded a significant reduction in susceptibility to the N strain (PVYN), but not to the ordinary strain (PVYO), in 1INS plants. 9DEL among three artificial alleles had a deleterious effect on infection by cucumber mosaic virus (CMV, type member of the genus Cucumovirus). When CMV was mechanically inoculated into tomato plants and viral coat accumulation was measured in the non-inoculated upper leaves, the level of viral coat protein was significantly lower in the 9DEL plants than in the parental cultivar. Tissue blotting of microperforated inoculated leaves of the 9DEL plants revealed significantly fewer infection foci compared with those of the parental cultivar, suggesting that 9DEL negatively affects the initial steps of infection with CMV in a mechanically inoculated leaf. In laboratory tests, viral aphid transmission from an infected susceptible plant to 9DEL plants was reduced compared with the parental control. Although many pathogen resistance genes have been discovered in tomato and its wild relatives, no CMV resistance genes have been used in practice. RNA silencing of eIF4E expression has previously been reported to not affect susceptibility to CMV in tomato. Our findings suggest that artificial gene editing can introduce additional resistance to that achieved with mutagenesis breeding, and that edited eIF4E alleles confer an alternative way to manage CMV in tomato fields.

Targeted Knockout of Eukaryotic Translation Initiation Factor 4E Confers Bymovirus Resistance in Winter Barley

“…Owing to the loss-of-function of the eIF4E protein, the plant lacks one of its most important interaction partners for bymoviruses (Wang and Krishnaswamy, 2012). That mechanism has been reported to confer broad resistance to several bymoviruses in a number of dicotyledonous plants as, for instance, cucumber, tomato and Arabidopsis (Chandrasekaran et al, 2016;Pyott et al, 2016;Atarashi et al, 2020;Moury et al, 2020). However, in a temperate cereal such as barley, the full knock-out of EIF4E was rather expected to be lethal; Yang et al (2016) screened over 2,900 wild and domesticated barley accessions and identified 65 haplotypes for HvEIF4E, of which 19 were associated with resistance to bymoviruses.…”

Resistance induction based on the understanding of molecular interactions between plant viruses and host plants

“…However, for functionally redundant factors, emerging resistance-breaking viruses represent another risk, potentially switching the factor in use from a null to a redundant allele. Moreover, in terms of conferring antiviral resistance, the functional alleles of eIF4E1 carrying non-synonymous base substitutions or a small in-frame deletion reportedly outstripped the null allele in tomato plants [ 101 , 102 ]. Tomato exhibits two eIF4Es , eIF4E1 and eIF4E2 , eIF(iso)4E, and nCBP .…”

“…Recently, we edited eIF4E1 by CRISPR/Cas9 and obtained three alleles, including a nucleotide insertion ( 1INS ) and nine nucleotide deletion ( 9DEL ) within the eIF4E1 protein coding region [ 101 ]. 1INS , containing a frameshift, is considered to be a null allele, and its homozygote showed resistance to the N strain of potato virus (PVY N ).…”

Integrating CRISPR-Cas and Next Generation Sequencing in Plant Virology

“…Consequently, these investigations validated the applicability of CRISPR/Cas9 to augment the development of high-quality tomato crops for higher yield and biomass. Atarashi et al (2020) demonstrated CRISPR/Cas9-mediated mutagenesis in the eIF4E1 gene of a commercial tomato cultivar. In addition to eIF4G, two deletions of three and nine nucleotides (3DEL and 9DEL) and a single nucleotide insertion (1INS) were found in close proximity to regions encoding amino acid residues essential for binding the 5՛ mRNA cap structure.…”