CRISPR/Cas9-mediated gene editing to confer turnip mosaic virus (TuMV) resistance in Chinese cabbage (Brassica rapa)

Abstract

Genome editing approaches, particularly the CRISPR/Cas9 technology, are becoming state-of-the-art for trait development in numerous breeding programs. Significant advances in improving plant traits are enabled by this influential tool, especially for disease resistance, compared to traditional breeding. One of the potyviruses, the turnip mosaic virus (TuMV), is the most widespread and damaging virus that infects Brassica spp. worldwide. We generated the targeted mutation at the eIF(iso)4E gene in the TuMV-susceptible cultivar “Seoul” using CRISPR/Cas9 to develop TuMV-resistant Chinese cabbage. We detected several heritable indel mutations in the edited T₀ plants and developed T₁ through generational progression. It was indicated in the sequence analysis of the eIF(iso)4E-edited T₁ plants that the mutations were transferred to succeeding generations. These edited T₁ plants conferred resistance to TuMV. It was shown with ELISA analysis the lack of accumulation of viral particles. Furthermore, we found a strong negative correlation ( r = − 0.938) between TuMV resistance and the genome editing frequency of eIF(iso)4E. Consequently, it was revealed in this study that CRISPR/Cas9 technique can expedite the breeding process to improve traits in Chinese cabbage plants.

Acknowledgements

This work was carried out with the support of “Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01652201)” Rural Development Administration, Republic of Korea.

Credit authorship contribution

Ye-Rin Lee: Conceptualization, Investigation, Formal analysis, Methodology, Software, Writing - original draft, Writing – review & editing. Muhammad Irfan Siddique: Investigation, Writing - original draft, Writing – review & editing Do-Sun Kim: Data curation, Formal analysis, Writing – review & editing. Eun Su Lee: Data curation, Formal analysis. Koeun Han: Data curation, Formal analysis. Sang-Gyu Kim: Formal analysis, Writing – review & editing. Hye-Eun Lee: Funding acquisition, Project Administration, Supervision, Validation, Writing – review & editing.

Data availability

The data and materials used to support the findings of this study are available from the corresponding author upon request.

Conflict of interest statement

None delcared.

Supplementary data

Supplementary data is available at Horticulture Research online.

References

[1] Chandrasekaran J, Brumin M, Wolf D et al. Development of broad virus resistance in non-transgenic cucumber using CRISPR/Cas 9 technology. Mol Plant Pathol. 2016; 17: 1140-53.

[2] Gill U, Scott JW, Shekasteband R et al. Ty-6, a major begomovirus resistance gene on chromosome 10, is effective against tomato yellow leaf curl virus and tomato mottle virus. Theor Appl Genet. 2019; 132: 1543-54.

[3] Kang BC, Yeam I, Jahn MM . Genetics of plant virus resistance. Annu Rev Phytopathol. 2005; 43: 581-621.

[4] Maule AJ, Caranta C, Boulton MI . Sources of natural resistance to plant viruses: status and prospects: review. Mol Plant Pathol. 2007; 8: 223-31.

[5] Yoon YJ, Venkatesh J, Lee JH et al. Genome editing of eIF4E1 in tomato confers resistance to pepper mottle virus. Front Plant Sci. 2020; 11: 1-11.

[6] Xu RF, Li H, Qin RY et al. Generation of inheritable and “transgene clean” targeted genome-modified rice in later generations using the CRISPR/Cas9 system. Sci Rep. 2015; 5: 1-10.

[7] Jones HD . Regulatory uncertainty over genome editing. Nat Plants. 2015; 1: 1-3.

[8] Jinek M, Chylinski K, Fonfara I et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012; 337: 816-21.

[9] Cong L, Zhang F . Genome engineering using CRISPR-Cas9 system. In: Chromosomal Mutagenesis. Springer, 2015, 197-217.

[10] Wang A . Dissecting the molecular network of virus-plant interactions: the complex roles of host factors. Annu Rev Phytopathol. 2015; 53: 45-66.

[11] Gomez MA, Lin ZD, Moll T et al. Simultaneous CRISPR/Cas9-mediated editing of cassava eIF 4E isoforms nCBP-1 and nCBP-2 reduces cassava brown streak disease symptom severity and incidence. Plant Biotechnol J. 2019; 17: 421-34.

[12] Macovei A, Sevilla NR, Cantos C et al. Novel alleles of rice eIF4G generated by CRISPR/Cas9-targeted mutagenesis confer resistance to rice tungro spherical virus. Plant Biotechnol J. 2018; 16: 1918-27.

[13] Peng A, Chen S, Lei T et al. Engineering canker-resistant plants through CRISPR/Cas9-targeted editing of the susceptibility gene Cs LOB 1 promoter in citrus. Plant Biotechnol J. 2017; 15: 1509-19.

[14] Pyott DE, Sheehan E, Molnar A . Engineering of CRISPR/Cas9-mediated Potyvirus resistance in transgene-free Arabidopsis plants. Mol Plant Pathol. 2016; 17: 1276-88.

[15] Wang F, Wang C, Liu P et al. Enhanced rice blast resistance by CRISPR/Cas9-targeted mutagenesis of the ERF transcription factor gene OsERF922. PLoS One. 2016; 11: 1-18.

[16] Cui H, Wang A . The biological impact of the hypervariable N-terminal region of potyviral genomes. Annu Rev Virol. 2019; 6: 255-74.

[17] Ward CW, Shukla DD . Taxonomy of potyviruses: current problems and some solutions. Intervirology. 2004; 32: 269-96.

[18] Kim J, Kang WH, Hwang J et al. Transgenic Brassica rapa plants over-expressing eIF (iso) 4E variants show broad-spectrum turnip mosaic virus (TuMV) resistance. Mol Plant Pathol. 2014; 15: 615-26.

[19] Revers F, García J . Molecular biology of potyviruses. Adv Virus Research. 2015; 92: 101-99.

[20] Sanfaçon H. Plant translation factors and virus resistance. Viruses. 2015; 7: 3392-419.

[21] Truniger V, Aranda MA . Recessive resistance to plant viruses. Adv Virus Res. 2009; 75: 119-59.

[22] Beauchemin C, Boutet N, Laliberté JF . Visualization of the interaction between the precursors of VPg, the viral protein linked to the genome of turnip mosaic virus and the translation eukaryotic initiation factor iso 4E in planta. J Virol. 2007; 81: 775-82.

[23] Duprat A, Caranta C, Revers F et al. The Arabidopsis eukaryotic initiation factor (iso)4E is dispensable for plant growth but required for susceptibility to potyviruses . Plant J. 2002; 32: 927-34.

[24] Lellis AD, Kasschau KD, Whitham SA et al. Loss-of-susceptibility mutants of Arabidopsis thaliana reveal an essential role for elF(iso)4E during potyvirus infection . Curr Biol. 2002; 12: 1046-51.

[25] Léonard S, Plante D, Wittmann S et al. Complex formation between Potyvirus VPg and translation eukaryotic initiation factor 4E correlates with virus infectivity. J Virol. 2000; 74: 7730-7.

[26] Miyoshi H, Okade H, Muto S et al. Turnip mosaic virus VPg interacts with Arabidopsis thaliana eIF (iso) 4E and inhibits in vitro translation . Biochimie. 2008; 90: 1427-34.

[27] Kim J, Kang WH, Yang HB et al. Identification of a broad-spectrum recessive gene in Brassica rapa and molecular analysis of the eIF4E gene family to develop molecular markers . Mol Breed. 2013; 32: 385-98.

[28] Qian W, Zhang S, Zhang S et al. Mapping and candidate-gene screening of the novel turnip mosaic virus resistance gene retr02 in Chinese cabbage (Brassica rapa L.) . Theor Appl Genet. 2013; 126: 179-88.

[29] Tilman D, Balzer C, Hill J et al. Global food demand and the sustainable intensification of agriculture. Proc Natl Acad Sci U S A. 2011; 108: 20260-4.

[30] van Regenmortel MH, Mahy BW . Desk Encyclopedia of Plant and Fungal Virology . Oxford: Academic Press; 2009: 426-30.

[31] An JW, Lee JH, Choi S et al. Identification of the determinant of tomato yellow leaf curl Kanchanaburi virus infectivity in tomato. Virus Res. 2021; 291: 198192.

[32] Siddique MI, Lee JH, Ahn JH et al. Genotyping-by-sequencing-based QTL mapping reveals novel loci for pepper yellow leaf curl virus (PepYLCV) resistance in Capsicum annuum. PLoS One. 2022; 17: 1-17.

[33] Cillo F, Palukaitis P . Transgenic resistance. Adv Virus Res Elsevier. 2014; 90: 35-146.

[34] Murovec J, Guček K, Bohanec B et al. DNA-free genome editing of brassica oleracea and B. rapa protoplasts using CRISPR-cas9 ribonucleoprotein complexes . Front Plant Sci. 2018; 9: 1-9.

[35] Jenner CE, Nellist CF, Barker GC et al. Turnip mosaic virus (TuMV) is able to use alleles of both eIF4E and eIF(iso)4E from multiple loci of the diploid Brassica rapa. Mol Plant-Microbe Interact. 2010; 23: 1498-505.

[36] Brooks C, Nekrasov V, Lipppman ZB et al. Efficient gene editing in tomato in the first generation using the clustered regularly interspaced short palindromic repeats/CRISPR-associated9 system. Plant Physiol. 2014; 166: 1292-7.

[37] Feng Z, Mao Y, Xu N et al. Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis . Proc Natl Acad Sci U S A. 2014; 111: 4632-4637.

[38] Liu H, Wang K, Jia Z et al. Efficient induction of haploid plants in wheat by editing of TaMTL using an optimized agrobacterium-mediated CRISPR system . J Exp Bot. 2020; 71: 1337-49.

[39] Ma X, Zhang Q, Zhu Q et al. A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant. 2015; 8: 1274-84.

[40] Pan C, Ye L, Qin L et al. CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations. Sci Rep. 2016; 6: 1-10.

[41] Hong JK, Suh EJ, Park SR et al. Multiplex CRISPR/Cas9 mutagenesis of BrVRN1 delays flowering time in Chinese cabbage (Brassica rapa L. ssp. pekinensis) . Agric. 2021; 11: 1-15.

[42] Jeong SY, Ahn H, Ryu J et al. Generation of early-flowering Chinese cabbage (Brassica rapa spp. pekinensis) through CRISPR/Cas9-mediated genome editing . Plant Biotechnology Rep. 2019; 13: 491-9.

[43] Shin N, Shin Y, Kim H et al. Function analysis of the PR55/B gene related to self-incompatibility in Chinese cabbage using CRISPR/Cas9. Int J Mol Sci. 2022; 23: 5062.

[44] Murashige T, Skoog F . A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol Plant. 1962; 15: 473-97.

[45] Oh Y, Lee B, Kim H et al. A multiplex guide RNA expression system and its efficacy for plant genome engineering. Plant Methods. 2020; 16: 1-11.

[46] Doyle J. DNA protocols for plants. In: Molecular Techniques in Taxonomy . Springer: Berlin, Heidelberg, 1991, 283-93.

[47] Shokralla S, Porter TM, Gibson JF et al. Massively parallel multiplex DNA sequencing for specimen identification using an Illumina MiSeq platform. Sci Rep. 2015; 9687.

[48] Hull R. Mechanical inoculation of plant viruses. Curr Protoc Microbiol. 2009; 13: 16B-6.