Development of broad virus resistance in non-transgenic cucumber using CRISPR/Cas9 technology
Summary Genome editing in plants has been boosted tremendously by the development of CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) technology. This powerful tool allows substantial improvement in plant traits in addition to those provided by classical breeding. Here, we dem...
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| Published in | Molecular plant pathology Vol. 17; no. 7; pp. 1140 - 1153 |
|---|---|
| Main Authors | , , , , , , , , |
| Format | Journal Article |
| Language | English |
| Published |
England
Blackwell Publishing Ltd
01.09.2016
John Wiley & Sons, Inc John Wiley and Sons Inc |
| Subjects | |
| Online Access | Get full text |
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| Abstract | Summary
Genome editing in plants has been boosted tremendously by the development of CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) technology. This powerful tool allows substantial improvement in plant traits in addition to those provided by classical breeding. Here, we demonstrate the development of virus resistance in cucumber (Cucumis sativus L.) using Cas9/subgenomic RNA (sgRNA) technology to disrupt the function of the recessive eIF4E (eukaryotic translation initiation factor 4E) gene. Cas9/sgRNA constructs were targeted to the N′ and C′ termini of the eIF4E gene. Small deletions and single nucleotide polymorphisms (SNPs) were observed in the eIF4E gene targeted sites of transformed T1 generation cucumber plants, but not in putative off‐target sites. Non‐transgenic heterozygous eif4e mutant plants were selected for the production of non‐transgenic homozygous T3 generation plants. Homozygous T3 progeny following Cas9/sgRNA that had been targeted to both eif4e sites exhibited immunity to Cucumber vein yellowing virus (Ipomovirus) infection and resistance to the potyviruses Zucchini yellow mosaic virus and Papaya ring spot mosaic virus‐W. In contrast, heterozygous mutant and non‐mutant plants were highly susceptible to these viruses. For the first time, virus resistance has been developed in cucumber, non‐transgenically, not visibly affecting plant development and without long‐term backcrossing, via a new technology that can be expected to be applicable to a wide range of crop plants. |
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| AbstractList | Summary Genome editing in plants has been boosted tremendously by the development of CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) technology. This powerful tool allows substantial improvement in plant traits in addition to those provided by classical breeding. Here, we demonstrate the development of virus resistance in cucumber (Cucumis sativus L.) using Cas9/subgenomic RNA (sgRNA) technology to disrupt the function of the recessive eIF4E (eukaryotic translation initiation factor 4E) gene. Cas9/sgRNA constructs were targeted to the N' and C' termini of the eIF4E gene. Small deletions and single nucleotide polymorphisms (SNPs) were observed in the eIF4E gene targeted sites of transformed T1 generation cucumber plants, but not in putative off-target sites. Non-transgenic heterozygous eif4e mutant plants were selected for the production of non-transgenic homozygous T3 generation plants. Homozygous T3 progeny following Cas9/sgRNA that had been targeted to both eif4e sites exhibited immunity to Cucumber vein yellowing virus (Ipomovirus) infection and resistance to the potyviruses Zucchini yellow mosaic virus and Papaya ring spot mosaic virus-W. In contrast, heterozygous mutant and non-mutant plants were highly susceptible to these viruses. For the first time, virus resistance has been developed in cucumber, non-transgenically, not visibly affecting plant development and without long-term backcrossing, via a new technology that can be expected to be applicable to a wide range of crop plants. Genome editing in plants has been boosted tremendously by the development of CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) technology. This powerful tool allows substantial improvement in plant traits in addition to those provided by classical breeding. Here, we demonstrate the development of virus resistance in cucumber ( Cucumis sativus L.) using Cas9/subgenomic RNA (sgRNA) technology to disrupt the function of the recessive eIF4E ( eukaryotic translation initiation factor 4E ) gene. Cas9/sgRNA constructs were targeted to the N′ and C′ termini of the eIF4E gene. Small deletions and single nucleotide polymorphisms (SNPs) were observed in the eIF4E gene targeted sites of transformed T1 generation cucumber plants, but not in putative off‐target sites. Non‐transgenic heterozygous eif4e mutant plants were selected for the production of non‐transgenic homozygous T3 generation plants. Homozygous T3 progeny following Cas9/sgRNA that had been targeted to both eif4e sites exhibited immunity to Cucumber vein yellowing virus ( Ipomovirus ) infection and resistance to the potyviruses Zucchini yellow mosaic virus and Papaya ring spot mosaic virus‐W . In contrast, heterozygous mutant and non‐mutant plants were highly susceptible to these viruses. For the first time, virus resistance has been developed in cucumber, non‐transgenically, not visibly affecting plant development and without long‐term backcrossing, via a new technology that can be expected to be applicable to a wide range of crop plants. Summary Genome editing in plants has been boosted tremendously by the development of CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) technology. This powerful tool allows substantial improvement in plant traits in addition to those provided by classical breeding. Here, we demonstrate the development of virus resistance in cucumber (Cucumis sativus L.) using Cas9/subgenomic RNA (sgRNA) technology to disrupt the function of the recessive eIF4E (eukaryotic translation initiation factor 4E) gene. Cas9/sgRNA constructs were targeted to the N′ and C′ termini of the eIF4E gene. Small deletions and single nucleotide polymorphisms (SNPs) were observed in the eIF4E gene targeted sites of transformed T1 generation cucumber plants, but not in putative off‐target sites. Non‐transgenic heterozygous eif4e mutant plants were selected for the production of non‐transgenic homozygous T3 generation plants. Homozygous T3 progeny following Cas9/sgRNA that had been targeted to both eif4e sites exhibited immunity to Cucumber vein yellowing virus (Ipomovirus) infection and resistance to the potyviruses Zucchini yellow mosaic virus and Papaya ring spot mosaic virus‐W. In contrast, heterozygous mutant and non‐mutant plants were highly susceptible to these viruses. For the first time, virus resistance has been developed in cucumber, non‐transgenically, not visibly affecting plant development and without long‐term backcrossing, via a new technology that can be expected to be applicable to a wide range of crop plants. Genome editing in plants has been boosted tremendously by the development of CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) technology. This powerful tool allows substantial improvement in plant traits in addition to those provided by classical breeding. Here, we demonstrate the development of virus resistance in cucumber (Cucumis sativus L.) using Cas9/subgenomic RNA (sgRNA) technology to disrupt the function of the recessive eIF4E (eukaryotic translation initiation factor 4E) gene. Cas9/sgRNA constructs were targeted to the N′ and C′ termini of the eIF4E gene. Small deletions and single nucleotide polymorphisms (SNPs) were observed in the eIF4E gene targeted sites of transformed T1 generation cucumber plants, but not in putative off‐target sites. Non‐transgenic heterozygous eif4e mutant plants were selected for the production of non‐transgenic homozygous T3 generation plants. Homozygous T3 progeny following Cas9/sgRNA that had been targeted to both eif4e sites exhibited immunity to Cucumber vein yellowing virus (Ipomovirus) infection and resistance to the potyviruses Zucchini yellow mosaic virus and Papaya ring spot mosaic virus‐W. In contrast, heterozygous mutant and non‐mutant plants were highly susceptible to these viruses. For the first time, virus resistance has been developed in cucumber, non‐transgenically, not visibly affecting plant development and without long‐term backcrossing, via a new technology that can be expected to be applicable to a wide range of crop plants. |
| Author | Chandrasekaran, Jeyabharathy Leibman, Diana Wolf, Dalia Brumin, Marina Gal-On, Amit Klap, Chen Pearlsman, Mali Arazi, Tzahi Sherman, Amir |
| AuthorAffiliation | 1 Department of Plant Pathology and Weed Research ARO, Volcani Center Bet‐Dagan 50250 Israel 4 Department of Ornamental Plants and Agricultural Biotechnology ARO, Volcani Center Bet‐Dagan 50250 Israel 3 Department of Fruit Tree Sciences ARO, Volcani Center Bet‐Dagan 50250 Israel 2 Department of Vegetable Research ARO, Volcani Center Bet‐Dagan 50250 Israel |
| AuthorAffiliation_xml | – name: 3 Department of Fruit Tree Sciences ARO, Volcani Center Bet‐Dagan 50250 Israel – name: 1 Department of Plant Pathology and Weed Research ARO, Volcani Center Bet‐Dagan 50250 Israel – name: 2 Department of Vegetable Research ARO, Volcani Center Bet‐Dagan 50250 Israel – name: 4 Department of Ornamental Plants and Agricultural Biotechnology ARO, Volcani Center Bet‐Dagan 50250 Israel |
| Author_xml | – sequence: 1 givenname: Jeyabharathy surname: Chandrasekaran fullname: Chandrasekaran, Jeyabharathy organization: Department of Plant Pathology and Weed Research, ARO, Volcani Center, 50250, Bet-Dagan, Israel – sequence: 2 givenname: Marina surname: Brumin fullname: Brumin, Marina organization: Department of Plant Pathology and Weed Research, ARO, Volcani Center, 50250, Bet-Dagan, Israel – sequence: 3 givenname: Dalia surname: Wolf fullname: Wolf, Dalia organization: Department of Vegetable Research, ARO, Volcani Center, 50250, Bet-Dagan, Israel – sequence: 4 givenname: Diana surname: Leibman fullname: Leibman, Diana organization: Department of Plant Pathology and Weed Research, ARO, Volcani Center, 50250, Bet-Dagan, Israel – sequence: 5 givenname: Chen surname: Klap fullname: Klap, Chen organization: Department of Plant Pathology and Weed Research, ARO, Volcani Center, 50250, Bet-Dagan, Israel – sequence: 6 givenname: Mali surname: Pearlsman fullname: Pearlsman, Mali organization: Department of Plant Pathology and Weed Research, ARO, Volcani Center, 50250, Bet-Dagan, Israel – sequence: 7 givenname: Amir surname: Sherman fullname: Sherman, Amir organization: Department of Fruit Tree Sciences, ARO, Volcani Center, 50250, Bet-Dagan, Israel – sequence: 8 givenname: Tzahi surname: Arazi fullname: Arazi, Tzahi organization: Department of Ornamental Plants and Agricultural Biotechnology, ARO, Volcani Center, 50250, Bet-Dagan, Israel – sequence: 9 givenname: Amit surname: Gal-On fullname: Gal-On, Amit email: amitg@volcani.agri.gov.il organization: Department of Plant Pathology and Weed Research, ARO, Volcani Center, 50250, Bet-Dagan, Israel |
| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/26808139$$D View this record in MEDLINE/PubMed |
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(2003) Estimation of population bottlenecks during systemic movement of tobacco mosaic virus in tobacco plants. J. Virol. 77, 9906-9911. Maule, A.J., Caranta, C. and Boulton, M.I. (2007) Sources of natural resistance to plant viruses: status and prospects. Mol. Plant Pathol. 8, 223-231. Sternberg, S.H., Redding, S., Jinek, M., Greene, E.C. and Doudna, J.A. (2014) DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature, 507, 62-67. Sanfaçon, H. (2015) Plant translation factors and virus resistance. Viruses, 7, 3392-3419. Lei, Y., Lu, L., Liu, H.-Y., Li, S., Xing, F. and Chen, L 1997; 234 2010; 11 2007; 104 2007; 145 1983; 1 2013; 23 2002; 12 2015; 33 2015; 32 2005; 579 2004; 5 2000; 90 1990; 222 2012; 13 2013; 9 2010; 23 2009; 10 2007; 8 2009; 120 2014; 166 2014; 7 2010; 5 2009; 125 2014; 12 2015; 1 2015; 15 2015; 16 2015; 5 2014; 90 2011; 1 2011 2002; 32 2009 2006; 19 2005; 43 2014; 85 2011; 6 2015; 7 2009; 27 2003; 132 1992; 73 2003; 77 2004; 110 2014; 507 2006; 87 2004; 17 2015; 64 2013; 31 2000; 74 2015 2005; 14 e_1_2_7_5_1 e_1_2_7_3_1 e_1_2_7_9_1 e_1_2_7_7_1 e_1_2_7_19_1 e_1_2_7_60_1 e_1_2_7_17_1 e_1_2_7_15_1 e_1_2_7_41_1 e_1_2_7_13_1 e_1_2_7_43_1 e_1_2_7_11_1 e_1_2_7_45_1 e_1_2_7_47_1 e_1_2_7_26_1 e_1_2_7_49_1 e_1_2_7_28_1 e_1_2_7_50_1 e_1_2_7_25_1 e_1_2_7_31_1 e_1_2_7_52_1 e_1_2_7_23_1 e_1_2_7_33_1 e_1_2_7_54_1 Truniger V. (e_1_2_7_55_1) 2009 e_1_2_7_21_1 e_1_2_7_35_1 e_1_2_7_56_1 e_1_2_7_37_1 e_1_2_7_58_1 e_1_2_7_39_1 e_1_2_7_6_1 e_1_2_7_4_1 e_1_2_7_8_1 e_1_2_7_18_1 e_1_2_7_16_1 e_1_2_7_40_1 e_1_2_7_2_1 e_1_2_7_14_1 e_1_2_7_42_1 e_1_2_7_12_1 e_1_2_7_44_1 e_1_2_7_10_1 e_1_2_7_46_1 e_1_2_7_48_1 e_1_2_7_27_1 e_1_2_7_29_1 e_1_2_7_51_1 e_1_2_7_30_1 e_1_2_7_53_1 e_1_2_7_24_1 e_1_2_7_32_1 e_1_2_7_22_1 e_1_2_7_34_1 e_1_2_7_57_1 e_1_2_7_20_1 e_1_2_7_36_1 e_1_2_7_59_1 e_1_2_7_38_1 |
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Genome editing in plants has been boosted tremendously by the development of CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats)... Genome editing in plants has been boosted tremendously by the development of CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats)... Summary Genome editing in plants has been boosted tremendously by the development of CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats)... |
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| SubjectTerms | backcrossing Base Sequence Chromosome Segregation - genetics CRISPR-Cas Systems - genetics CRISPR/Cas9 crops cucumber Cucumber vein yellowing virus cucumbers Cucumis sativus Cucumis sativus - genetics Cucumis sativus - virology Disease Resistance - genetics eIF4E Eukaryotic Initiation Factor-4E - genetics Gene Editing Genes Genome editing Genotype heterozygosity homozygosity Homozygote immunity mutants Mutation - genetics Original Papaya mosaic virus Papaya ringspot virus plant development Plant Diseases - virology Plant Viruses - physiology Plants, Genetically Modified Potyviridae progeny RNA single nucleotide polymorphism technology translation (genetics) Vegetables virus resistance Viruses Zucchini yellow mosaic virus |
| Title | Development of broad virus resistance in non-transgenic cucumber using CRISPR/Cas9 technology |
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