Engineering of CRISPR/Cas9-mediated potyvirus resistance in transgene-free Arabidopsis plants

Journal Article / 2016

Pyott, Douglas E.; Sheehan, Emma; Molnar, Attila

Members of the eukaryotic translation initiation factor (eIF) gene family, including eIF4E and its paralogue eIF(iso)4E, have previously been identified as recessive resistance alleles against various potyviruses in a range of different hosts. However, the identification and introgression of these alleles into important crop species is often limited. In this study, we utilise CRISPR/Cas9 technology to introduce sequence-specific deleterious point mutations at the eIF(iso)4E locus in Arabidopsis thaliana to successfully engineer complete resistance to Turnip mosaic virus (TuMV), a major pathogen in field-grown vegetable crops. By segregating the induced mutation from the CRISPR/Cas9 transgene, we outline a framework for the production of heritable, homozygous mutations in the transgene-free T2 generation in self-pollinating species. Analysis of dry weights and flowering times for four independent T3 lines revealed no differences from wild-type plants under standard growth conditions, suggesting that homozygous mutations in eIF(iso)4E do not affect plant vigour. Thus, the established CRISPR/Cas9 technology provides a new approach for the generation of Potyvirus resistance alleles in important crops without the use of persistent transgenes.

Amino Acid Sequence; Arabidopsis/genetics/growth & development/virology; Base Sequence; Biomass; Chromosome Segregation/genetics; CRISPR-Cas Systems/genetics; Crosses, Genetic; Disease Resistance/genetics; Electrophoresis, Agar Gel; Eukaryotic Initiation Factor-4E/genetics; Flowers/physiology; gene editing; Genetic Engineering/methods; Green Fluorescent Proteins/metabolism; Mutagenesis, Site-Directed; Mutation/genetics; Plants, Genetically Modified; polymerase chain reaction; Potyvirus/physiology; RNA, Guide/metabolism; Transgenes

Molecular plant pathology

8

1276–1288

17

10.1111/mpp.12417

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