Development of broad virus resistance in non-transgenic cucumber using CRISPR/Cas9 technology.

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.

[1]  M. Mahfouz,et al.  CRISPR/Cas9-mediated viral interference in plants , 2015, Genome Biology.

[2]  D. Voytas,et al.  Conferring resistance to geminiviruses with the CRISPR–Cas prokaryotic immune system , 2015, Nature Plants.

[3]  H. Sanfaçon.  Plant Translation Factors and Virus Resistance , 2015, Viruses.

[4]  Hao Li,et al.  Generation of inheritable and “transgene clean” targeted genome-modified rice in later generations using the CRISPR/Cas9 system , 2015, Scientific Reports.

[5]  A. Dombrovsky,et al.  Seed disinfection treatments do not sufficiently eliminate the infectivity of Cucumber green mottle mosaic virus (CGMMV) on cucurbit seeds , 2015 .

[6]  N. Patron,et al.  Editing plant genomes with CRISPR/Cas9. , 2015, Current opinion in biotechnology.

[7]  Robert J. Schmitz,et al.  Targeted genome modifications in soybean with CRISPR/Cas9 , 2015, BMC Biotechnology.

[8]  Huw D Jones,et al.  Regulatory uncertainty over genome editing , 2015, Nature Plants.

[9]  Z. Lippman,et al.  Efficient Gene Editing in Tomato in the First Generation Using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-Associated9 System1 , 2014, Plant Physiology.

[10]  Yang Lei,et al.  CRISPR-P: a web tool for synthetic single-guide RNA design of CRISPR-system in plants. , 2014, Molecular plant.

[11]  Jian‐Kang Zhu,et al.  The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation. , 2014, Plant biotechnology journal.

[12]  Liangyu Liu,et al.  CRISPR–Cas system: a powerful tool for genome engineering , 2014, Plant Molecular Biology.

[13]  Jennifer A. Doudna,et al.  DNA interrogation by the CRISPR RNA-guided endonuclease Cas9 , 2014, Nature.

[14]  Kabin Xie,et al.  Genome-wide prediction of highly specific guide RNA spacers for CRISPR-Cas9-mediated genome editing in model plants and major crops. , 2014, Molecular plant.

[15]  S. Kamoun,et al.  Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system , 2013, Plant Methods.

[16]  Detlef Weigel,et al.  Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease , 2013, Nature Biotechnology.

[17]  Botao Zhang,et al.  Efficient genome editing in plants using a CRISPR/Cas system , 2013, Cell Research.

[18]  G. Church,et al.  CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering , 2013, Nature Biotechnology.

[19]  George M. Church,et al.  Multiplex and homologous recombination–mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9 , 2013, Nature Biotechnology.

[20]  J. Keith Joung,et al.  High frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells , 2013, Nature Biotechnology.

[21]  N. Schlaich,et al.  Delivery of multiple transgenes to plant cells by an improved version of MultiRound Gateway technology , 2013, Transgenic Research.

[22]  Aiming Wang,et al.  Eukaryotic translation initiation factor 4E-mediated recessive resistance to plant viruses and its utility in crop improvement. , 2012, Molecular plant pathology.

[23]  M. Aranda,et al.  Melon RNA interference (RNAi) lines silenced for Cm-eIF4E show broad virus resistance. , 2012, Molecular plant pathology.

[24]  M. Nicolaï,et al.  Knock-Down of Both eIF4E1 and eIF4E2 Genes Confers Broad-Spectrum Resistance against Potyviruses in Tomato , 2011, PloS one.

[25]  J. Laliberté,et al.  The genome-linked protein VPg of plant viruses-a protein with many partners. , 2011, Current opinion in virology.

[26]  K. Nakahara,et al.  Involvement of the P1 cistron in overcoming eIF4E-mediated recessive resistance against Clover yellow vein virus in pea. , 2010, Molecular plant-microbe interactions : MPMI.

[27]  D. Zamir,et al.  An Induced Mutation in Tomato eIF4E Leads to Immunity to Two Potyviruses , 2010, PloS one.

[28]  R. Jackson,et al.  The mechanism of eukaryotic translation initiation and principles of its regulation , 2010, Nature Reviews Molecular Cell Biology.

[29]  A. Levi,et al.  Non-synonymous single nucleotide polymorphisms in the watermelon eIF4E gene are closely associated with resistance to Zucchini yellow mosaic virus , 2009, Theoretical and Applied Genetics.

[30]  M. Aranda,et al.  Genetic resistance for the sustainable control of plant virus diseases: breeding, mechanisms and durability , 2009, European Journal of Plant Pathology.

[31]  I. Yeam,et al.  Double mutations in eIF4E and eIFiso4E confer recessive resistance to Chilli veinal mottle virus in pepper , 2009, Molecules and cells.

[32]  Rachid Senoussi,et al.  Estimation of the number of virus particles transmitted by an insect vector , 2007, Proceedings of the National Academy of Sciences of the United States of America.

[33]  A. Maule,et al.  Sources of Natural Resistance to Plant Viruses: Status and Prospects , 2022 .

[34]  A. Palloix,et al.  Simultaneous mutations in translation initiation factors eIF4E and eIF(iso)4E are required to prevent pepper veinal mottle virus infection of pepper. , 2006, The Journal of general virology.

[35]  A. Palloix,et al.  Different mutations in the genome-linked protein VPg of potato virus Y confer virulence on the pvr2(3) resistance in pepper. , 2006, Molecular plant-microbe interactions : MPMI.

[36]  E. Hébrard,et al.  Emergence of a resistance-breaking isolate of Rice yellow mottle virus during serial inoculations is due to a single substitution in the genome-linked viral protein VPg. , 2006, The Journal of general virology.

[37]  M. Jahn,et al.  Genetics of plant virus resistance. , 2005, Annual review of phytopathology.

[38]  M. Ishikawa,et al.  Selective involvement of members of the eukaryotic initiation factor 4E family in the infection of Arabidopsis thaliana by potyviruses , 2005, FEBS letters.

[39]  O. Lachman,et al.  Transgenic cucumbers harboring the 54-kDa putative gene of Cucumber fruit mottle mosaic tobamovirus are highly resistant to viral infection and protect non-transgenic scions from soil infection , 2005, Transgenic Research.

[40]  M. Aranda,et al.  Development and use of detection methods specific for Cucumber vein yellowing virus (CVYV) , 2004, European Journal of Plant Pathology.

[41]  M. Aranda,et al.  Advances in understanding recessive resistance to plant viruses. , 2004, Molecular plant pathology.

[42]  A. Palloix,et al.  Mutations in potato virus Y genome-linked protein determine virulence toward recessive resistances in Capsicum annuum and Lycopersicon hirsutum. , 2004, Molecular plant-microbe interactions : MPMI.

[43]  S. Sacristán,et al.  Estimation of Population Bottlenecks during Systemic Movement of Tobacco Mosaic Virus in Tobacco Plants , 2003, Journal of Virology.

[44]  S. German-Retana,et al.  The Eukaryotic Translation Initiation Factor 4E Controls Lettuce Susceptibility to the Potyvirus Lettuce mosaic virus1 , 2003, Plant Physiology.

[45]  K. Browning,et al.  The Arabidopsis eukaryotic initiation factor (iso)4E is dispensable for plant growth but required for susceptibility to potyviruses. , 2002, The Plant journal : for cell and molecular biology.

[46]  A. Palloix,et al.  A natural recessive resistance gene against potato virus Y in pepper corresponds to the eukaryotic initiation factor 4E (eIF4E). , 2002, The Plant journal : for cell and molecular biology.

[47]  K. Kasschau,et al.  Loss-of-Susceptibility Mutants of Arabidopsis thaliana Reveal an Essential Role for eIF(iso)4E during Potyvirus Infection , 2002, Current Biology.

[48]  J. Laliberté,et al.  Complex Formation between Potyvirus VPg and Translation Eukaryotic Initiation Factor 4E Correlates with Virus Infectivity , 2000, Journal of Virology.

[49]  A. Gal‐On.  A Point Mutation in the FRNK Motif of the Potyvirus Helper Component-Protease Gene Alters Symptom Expression in Cucurbits and Elicits Protection Against the Severe Homologous Virus. , 2000, Phytopathology.

[50]  J. Laliberté,et al.  Interaction of the viral protein genome linked of turnip mosaic potyvirus with the translational eukaryotic initiation factor (iso) 4E of Arabidopsis thaliana using the yeast two-hybrid system. , 1997, Virology.

[51]  B. Raccah,et al.  A zucchini yellow mosaic virus coat protein gene mutation restores aphid transmissibility but has no effect on multiplication. , 1992, The Journal of general virology.

[52]  P. Palukaitis,et al.  Construction of full-length cDNA clones of cucumber mosaic virus RNAs 1, 2 and 3: Generation of infectious RNA transcripts , 1990, Molecular and General Genetics MGG.

[53]  James B. Hicks,et al.  A plant DNA minipreparation: Version II , 1983, Plant Molecular Biology Reporter.

[54]  Rainer Fischer,et al.  The CRISPR/Cas9 system for plant genome editing and beyond. , 2015, Biotechnology advances.

[55]  P. Palukaitis,et al.  Transgenic resistance. , 2014, Advances in virus research.

[56]  S. German-Retana,et al.  Involvement of the cylindrical inclusion (CI) protein in the overcoming of an eIF4E-mediated resistance against Lettuce mosaic potyvirus. , 2009, Molecular plant pathology.

[57]  M. Aranda,et al.  Recessive resistance to plant viruses. , 2009, Advances in virus research.

[58]  Annett Baier,et al.  Plant resistance to viruses. , 1987, Ciba Foundation symposium.