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Editing of TOM1 gene in tobacco using CRISPR/Cas9 confers resistance to Tobacco mosaic virus

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Abstract

Background

Genome editing technology has become one of the excellent tools for precise plant breeding to develop novel plant germplasm. The Tobacco mosaic virus (TMV) is the most prominent pathogen that infects several Solanaceae plants, such as tobacco, tomato, and capsicum, which requires critical host factors for infection and replication of its genomic RNA in the host. The Tobamovirus multiplication (TOM) genes, such as TOM1, TOM2A, TOM2B, and TOM3, are involved in the multiplication of Tobamoviruses. TOM1 is a transmembrane protein necessary for efficient TMV multiplication in several plant species. The TOM genes are crucial recessive resistance genes that act against the tobamoviruses in various plant species.

Methods and results

The single guided RNA (sgRNA) was designed to target the first exon of the NtTOM1 gene and cloned into the pHSE401 vector. The pHSE401-NtTOM1 vector was introduced into Agrobacterium tumefaciens strain LBA4404 and then transformed into tobacco plants. The analysis on T0 transgenic plants showed the presence of the hptII and Cas9 transgenes. The sequence analysis of the NtTOM1 from T0 plants showed the indels. Genotypic evaluation of the NtTOM1 mutant lines displayed the stable inheritance of the mutations in the subsequent generations of tobacco plants. The NtTOM1 mutant lines successfully conferred resistance to TMV.

Conclusions

CRISPR/Cas genome editing is a reliable tool for investigating gene function and precision breeding across different plant species, especially the species in the Solanaceae family.

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Data Availability

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Acknowledgements

PJ and VRA are grateful to the University Grant Commission, New Delhi, for the financial assistance under the SAP-DRS-II program (No. F-5-24/2015/DRS-II). DS is thankful to the Department of Science and Technology, Government of India, for providing Inspire Fellowship (No. IF160264). SA is grateful to the Council of Scientific & Industrial Research, Government of India, for giving CSIR-Emeritus Scientist (No. 21(1067)/19/EMR-II).

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This research received no external funding.

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Authors and Affiliations

  1. Department of Biotechnology, Kakatiya University, Warangal, 506009, Telangana, India

    Phanikanth Jogam, Dulam Sandhya, Sadanandam Abbagani & Venkateswar Rao Allini

  2. Department of Plant Pathology, University of Minnesota, Saint Paul, MN, 55108, USA

    Anshu Alok

  3. Department of Microbiology, Kakatiya University, Warangal, 506009, Telangana, India

    Venkataiah Peddaboina

  4. Center of Innovative and Applied Bioprocessing (DBT-CIAB), Mohali, 140306, Punjab, India

    Sudhir P. Singh

  5. Department of Biology, East Carolina University, Greenville, NC, 27858, USA

    Baohong Zhang

Authors
  1. Phanikanth Jogam
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  2. Dulam Sandhya
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  3. Anshu Alok
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  4. Venkataiah Peddaboina
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  5. Sudhir P. Singh
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  8. Venkateswar Rao Allini
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Contributions

PJ performed experiments and wrote the manuscript. DS helped while conducting experiments and data analysis. AA helped in bioinformatics, construct, and manuscript preparation. VP and SPS data curation and revision of the manuscript. SA experimental suggestions and critical revision of the manuscript, BZ and VRA conceived the idea, designed the experiments, and finalized the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Baohong Zhang or Venkateswar Rao Allini.

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Jogam, P., Sandhya, D., Alok, A. et al. Editing of TOM1 gene in tobacco using CRISPR/Cas9 confers resistance to Tobacco mosaic virus. Mol Biol Rep 50, 5165–5176 (2023). https://doi.org/10.1007/s11033-023-08440-2

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