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

Summary 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...

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Published inMolecular plant pathology Vol. 17; no. 8; pp. 1276 - 1288
Main Authors Pyott, Douglas E., Sheehan, Emma, Molnar, Attila
Format Journal Article
LanguageEnglish
Published England Blackwell Publishing Ltd 01.10.2016
John Wiley & Sons, Inc
John Wiley and Sons Inc
Subjects
alleles
Amino Acid Sequence
Arabidopsis
Arabidopsis - genetics
Arabidopsis - growth & development
Arabidopsis - virology
Arabidopsis thaliana
Base Sequence
Biomass
Chromosome Segregation - genetics
CRISPR-Cas Systems - genetics
CRISPR/Cas9
Crops
Crosses, Genetic
Disease Resistance - genetics
eIF(iso)4E
Electrophoresis, Agar Gel
engineering
Eukaryotic Initiation Factor-4E - genetics
flowering
Flowers - physiology
Gene Editing
Genetic Engineering - methods
Green Fluorescent Proteins - metabolism
Growth conditions
homozygosity
hosts
introgression
loci
mutagenesis
Mutagenesis, Site-Directed
Mutation
Mutation - genetics
Original
pathogens
Plants, Genetically Modified
point mutation
Polymerase Chain Reaction
Potyvirus
Potyvirus - physiology
RNA, Guide, CRISPR-Cas Systems - metabolism
self-pollination
transgene-free
Transgenes
translation (genetics)
TuMV
Turnip mosaic virus
vegetable crops
vigor
virus resistance
potyvirus
CRISPR/Cas9
TuMV
eIF(iso)4E
transgene-free
virus resistance
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Abstract Summary 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.
AbstractList 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.
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 T sub(2) generation in self-pollinating species. Analysis of dry weights and flowering times for four independent T sub(3) 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.
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 T 2 generation in self‐pollinating species. Analysis of dry weights and flowering times for four independent T 3 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.
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.
Summary 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.
Summary 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.
Author Sheehan, Emma
Pyott, Douglas E.
Molnar, Attila
AuthorAffiliation 1 Institute of Molecular Plant Sciences, University of Edinburgh Edinburgh EH9 3JR UK
AuthorAffiliation_xml – name: 1 Institute of Molecular Plant Sciences, University of Edinburgh Edinburgh EH9 3JR UK
Author_xml – sequence: 1
  givenname: Douglas E.
  surname: Pyott
  fullname: Pyott, Douglas E.
  organization: Institute of Molecular Plant Sciences, University of Edinburgh, EH9 3JR, Edinburgh, UK
– sequence: 2
  givenname: Emma
  surname: Sheehan
  fullname: Sheehan, Emma
  organization: Institute of Molecular Plant Sciences, University of Edinburgh, EH9 3JR, Edinburgh, UK
– sequence: 3
  givenname: Attila
  surname: Molnar
  fullname: Molnar, Attila
  email: attila.molnar@ed.ac.uk, attila.molnar@ed.ac.uk
  organization: Institute of Molecular Plant Sciences, University of Edinburgh, EH9 3JR, Edinburgh, UK
BackLink https://www.ncbi.nlm.nih.gov/pubmed/27103354$$D View this record in MEDLINE/PubMed
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Issue 8
Keywords potyvirus
CRISPR/Cas9
TuMV
eIF(iso)4E
transgene-free
virus resistance
Language English
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2016 The Authors. Molecular Plant Pathology Published by British Society for Plant Pathology and John Wiley & Sons Ltd.
This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
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Snippet Summary Members of the eukaryotic translation initiation factor (eIF) gene family, including eIF4E and its paralogue eIF(iso)4E, have previously been...
Members of the eukaryotic translation initiation factor (eIF) gene family, including eIF4E and its paralogue eIF(iso)4E , have previously been identified as...
Members of the eukaryotic translation initiation factor (eIF) gene family, including eIF4E and its paralogue eIF(iso)4E, have previously been identified as...
Summary Members of the eukaryotic translation initiation factor (eIF) gene family, including eIF4E and its paralogue eIF(iso)4E, have previously been...
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StartPage 1276
SubjectTerms alleles
Amino Acid Sequence
Arabidopsis
Arabidopsis - genetics
Arabidopsis - growth & development
Arabidopsis - virology
Arabidopsis thaliana
Base Sequence
Biomass
Chromosome Segregation - genetics
CRISPR-Cas Systems - genetics
CRISPR/Cas9
Crops
Crosses, Genetic
Disease Resistance - genetics
eIF(iso)4E
Electrophoresis, Agar Gel
engineering
Eukaryotic Initiation Factor-4E - genetics
flowering
Flowers - physiology
Gene Editing
Genetic Engineering - methods
Green Fluorescent Proteins - metabolism
Growth conditions
homozygosity
hosts
introgression
loci
mutagenesis
Mutagenesis, Site-Directed
Mutation
Mutation - genetics
Original
pathogens
Plants, Genetically Modified
point mutation
Polymerase Chain Reaction
Potyvirus
Potyvirus - physiology
RNA, Guide, CRISPR-Cas Systems - metabolism
self-pollination
transgene-free
Transgenes
translation (genetics)
TuMV
Turnip mosaic virus
vegetable crops
vigor
virus resistance
Title Engineering of CRISPR/Cas9-mediated potyvirus resistance in transgene-free Arabidopsis plants
URI https://api.istex.fr/ark:/67375/WNG-N4S6ZT4X-7/fulltext.pdf
https://onlinelibrary.wiley.com/doi/abs/10.1111%2Fmpp.12417
https://www.ncbi.nlm.nih.gov/pubmed/27103354
https://www.proquest.com/docview/1817414591
https://www.proquest.com/docview/1827900078
https://www.proquest.com/docview/1846324838
https://pubmed.ncbi.nlm.nih.gov/PMC5026172
Volume 17
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