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

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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Figure 1: Overview of the VIGS protocol in N. benthamiana.
Figure 2: Different methods of TRV inoculation in N. benthamiana plants.
Figure 3: Silencing the NbPDS gene in N. benthamiana plants by TRV-VIGS.

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References

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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.

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Authors and Affiliations

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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.

Corresponding author

Correspondence to Kirankumar S Mysore.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Details of TRV1 and TRV2 constructs.

Tobacco rattle virus (TRV) is a bipartite, positive-sense RNA virus. TRV1 (i.e., RNA1) encodes 134 and 194 kDa replicase proteins from the genomic RNA, 29 kDa movement protein, and 16 kDa cysteine-rich protein from subgenomic RNAs. TRV1 can replicate and move systemically without TRV2 (i.e., RNA2). TRV2 encodes coat protein from the genomic and two nonstructural proteins from subgenomic RNAs (A). To develop the TRV-based VIGS vector, a cDNA clone of TRV1 or TRV2 (Ppk20 strain) was placed between a duplicated CaMV 35S promoter (2X35S) and nopaline synthase terminator (NOSt) in an Agrobacterium T-DNA vector. In the TRV2 cDNA construct, the nonessential structural genes were replaced with MCS for cloning the target gene sequences. Further, a self-cleaving ribozyme site was included at the 3' end of TRV2. The TRV1 (AF406990)-based viral vector, pTRV1 (linear plasmid 6.791 kb), is used along with pTRV2 for silencing. The pTRV2 has three variants. The first one is suitable for conventional cloning (AF406991, linear plasmid 9663 nucleotide). The second variant is a gateway-cloning-compatible vector. The third variant is suitable for ligation-independent cloning. Rz, self-cleaving ribozyme; MCS, multiple cloning site; CP, coat protein; MP, movement protein. These vectors were created by Dr. S.P. Dinesh-Kumar, UC Davis, USA. Conventional cloning-compatible (YL156) and ligation-independent-compatible (YY13TRV2) vector plasmid DNA can be obtained from ABRC, Stock No. CD3-1040 vector name YL156. Ligation-independent-compatible TRV2 vector plasmid DNA is ABRC Stock No. CD3-1042, vector name YY13.

Supplementary Figure 2 Applications of VIGS to study gene function in various plant processes.

Some of the possible studies that can be performed on the silenced plants are given in this figure. VIGS has been used to dissect developmental processes like root and leaf development, meristem differentiation, reproductive organ development, the mechanism of symbiotic interaction with microbes, PAMP or effector-induced defense, nonhost disease resistance, nematode resistance, Agrobacterium-mediated T-DNA transfer, and abiotic stress tolerance in plants.

Supplementary information

Supplementary Figure 1

Details of TRV1 and TRV2 constructs. (PDF 390 kb)

Supplementary Figure 2

Applications of VIGS to study gene function in various plant processes. (PDF 434 kb)

Supplementary Table 1

Plant species having publicly available and popularly used mutant resources conducive for cloning the gene of interest. (PDF 108 kb)

Supplementary Table 2

Currently available popular VIGS vectors and their target plant species. (PDF 216 kb)

Supplementary Table 3

Plant species suitable for TRV-VIGS of target genes. (PDF 175 kb)

Supplementary Table 4

Different methods of TRV inoculation for achieving VIGS. (PDF 125 kb)

Supplementary Table 5

Details of some useful primers for performing TRV-VIGS in N. benthamiana. (PDF 178 kb)

Protocol for TRV-mediated VIGS in N. benthamiana. (MP4 124881 kb)

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Senthil-Kumar, M., Mysore, K. Tobacco rattle virus–based virus-induced gene silencing in Nicotiana benthamiana. Nat Protoc 9, 1549–1562 (2014). https://doi.org/10.1038/nprot.2014.092

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