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Tobacco rattle virus–based virus-induced gene silencing in Nicotiana benthamiana

Nature Protocols volume 9, pages 15491562 (2014) | Download Citation

Abstract

Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) is widely used in various plant species to downregulate the expression of a target plant gene. TRV is a bipartite, positive-strand RNA virus with the TRV1 and TRV2 genomes. To induce post-transcriptional gene silencing (PTGS), the TRV2 genome is genetically modified to carry a fragment of the target gene and delivered into the plant (along with the TRV1 genome) by agroinoculation. TRV1- and TRV2-carrying Agrobacterium strains are then co-inoculated into 3-week-old plant leaves by one of three methods: a needleless syringe, the agrodrench method or by pricking with a toothpick. Target gene silencing occurs in the newly developed noninoculated leaves within 2–3 weeks of TRV inoculation. The TRV-VIGS protocol described here takes only 4 weeks to implement, and it is faster and easier to perform than other gene silencing techniques that are currently available. Although we use Nicotiana benthamiana as an example, the protocol is adaptable to other plant species.

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Acknowledgements

This project was funded by The Samuel Roberts Noble Foundation. We thank J. Gallaway for excellent plant care. TRV constructs were obtained from S.P. Dinesh-Kumar, University of California Davis. We thank B. Stearns and S. McNeill for making and editing the video. We thank K. Brown for artwork in the figures and J. Kelley for editing the manuscript.

Author information

Affiliations

  1. Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, Oklahoma, USA.

    • Muthappa Senthil-Kumar
    •  & Kirankumar S Mysore
  2. National Institute of Plant Genome Research, New Delhi, India.

    • Muthappa Senthil-Kumar

Authors

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Contributions

M.S.-K. and K.S.M. designed the experiments; M.S.-K. performed the experiments; and M.S.-K. and K.S.M. wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Kirankumar S Mysore.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Figure 1

    Details of TRV1 and TRV2 constructs.

  2. 2.

    Supplementary Figure 2

    Applications of VIGS to study gene function in various plant processes.

  3. 3.

    Supplementary Table 1

    Plant species having publicly available and popularly used mutant resources conducive for cloning the gene of interest.

  4. 4.

    Supplementary Table 2

    Currently available popular VIGS vectors and their target plant species.

  5. 5.

    Supplementary Table 3

    Plant species suitable for TRV-VIGS of target genes.

  6. 6.

    Supplementary Table 4

    Different methods of TRV inoculation for achieving VIGS.

  7. 7.

    Supplementary Table 5

    Details of some useful primers for performing TRV-VIGS in N. benthamiana.

Videos

  1. 1.

    Protocol for TRV-mediated VIGS in N. benthamiana.

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DOI

https://doi.org/10.1038/nprot.2014.092

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