Abstract
Virus-induced gene silencing (VIGS) is a post-transcriptional gene silencing method used for unraveling gene functions. As an attractive alternative to mutant collections or stable transgenic plants, it has been widely used in reverse-genetics studies owing to its ease use and quick turnaround time. Turnip yellow mosaic virus (TYMV) has the ability to induce VIGS in Arabidopsis thaliana. However, the conventional vector construction is difficult and the efficiencies of the infection methods are low. Here, we improved the vector construction and viral infection methods, inserted an inverted-repeat fragment of the phytoene desaturase gene into a TYMV-derived vector by homologous recombination and transformed Brassica rapa with plasmid DNA harboring a cDNA copy of the TYMV genome through particle bombardment. An apparent photobleaching phenotype was detected and efficient VIGS was induced. An 80-bp fragment was sufficient to produce VIGS in leaves, stems, roots, flowers, siliques, and stalks of B. rapa. Because TYMV has a wide host range in Brassica, the VIGS system described here will contribute to the improvement of high-throughput technology and efficient functional research in B. rapa and other Brassicaceae crops.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- CaLCuV:
-
cabbage leaf curl virus
- CP:
-
coat protein
- FPKM:
-
reads per kilobase of exon model per million mapped reads
- GADPH:
-
glyceraldehyde-3-phosphate dehydrogenase
- PDS:
-
phytoene desaturase
- PTGS:
-
post-transcriptional gene silencing
- TGMV:
-
tomato golden mosaic virus
- TMV:
-
tobacco mosaic virus
- TRV:
-
tobacco rattle virus
- TYMV:
-
turnip yellow mosaic virus
- VIGS:
-
virus-induced gene silencing
References
Bai, C., Wang, P.Q., Fan, W.D., Fu, L., Wang, Z.N., Zhang, Z., Song, G.L., Zhang, J., Wu, H.: Analysis of the role of the drought-induced gene DRI15 and salinity-induced gene SI1 in aternanthera philoxeroides plasticity using a virus-based gene silencing tool. - Front. Plant Sci. 8: 262, 2017.
Baulcombe, D.: Viruses and gene silencing in plants. - Arch. Virol. 7: 189–201, 1999.
Benedito, V.A., Visser, P.B., Angenent, G.C., Krens, F.A.: The potential of virus-induced gene silencing for speeding up functional characterization of plant genes. - Genet. mol. Res. 3: 323–341, 2004.
Burch-Smith, T.M., Anderson, J.C., Martin,G.B., Dinesh-Kumar, S.P.: Applications and advantages of virus-induced gene silencing for gene function studies in plants. - Plant J. 39: 734–746, 2004.
Campisi, L., Fambrini, M., Michelotti, V., Salvini, M., Giuntini,D., Pugliesi, C.: Phytoene accumulation in sunflower decreases the transcript levels of the phytoene synthase gene. - Plant Growth Regul. 48: 79–87, 2006.
Cheng, L., Li, H.P., Qu, B., Huang, T., Tu, J.X., Fu, T.D., Liao, Y.C.: Chloroplast transformation of rapeseed (Brassica napus) by particle bombardment of cotyledons. - Plant Cell Rep. 29: 371–381, 2010.
Dheda, K.., Huggett, J.F., Bustin, S.A., Johnson, M.A., Rook, G., Zumla, A.: Validation of housekeeping genes for normalizing RNA expression in real-time PCR. - Biotechniques 37: 112, 2004.
Dinesh-Kumar, S.P., Anandalakshmi, R., Marathe, R., Schiff, M., Liu, Y.: Virus-induced gene silencing. - Methods mol. Biol. 236: 287–94, 2003.
Fantini, E., Giuliano, G.: Virus-induced gene silencing as a tool to study tomato fruit biochemistry. - Plant Signal Transduction 1363: 65–78, 2016.
Faivre, R.O., Gilroy, E.M., Hrubikova, K., Hein, I., Millam, S., Loake, G.J., Birch, P., Taylor, M., Lacomme, C.: Potato virus X-induced gene silencing in leaves and tubers of potato. - Plant Physiol. 134: 1308–1316, 2004.
Gao, X., Wheeler, T., Li, Z., Kenerley, C.M., He, P., Shan, L.: Silencing GhNDR1 and GhMKK2 compromises cotton resistance to Verticillium wilt. - Plant J. 66: 293–305, 2011.
Jia, H., Shen, Y.: Virus-induced gene silencing in strawberry fruit. - Methods mol. Biol. 975: 211–218, 2013.
Juvale, P.S., Hewezi, T., Zhang, C.,. Kandoth, P.K., Mitchum, M.G., Hill, J.H., Whitham, S.A., Baum, T.J.: Temporal and spatial bean pod mottle virus-induced gene silencing in soybean. - Mol. Plant Pathol. 13: 1140–1148, 2012.
Kay, R., Chan, A., Daly, M., McPherson, J.: Duplication of CAMV-35S promoter sequences creates a strong enhancer for plant genes. - Sci. 236: 1299–1302, 1987.
Lacomme, C., Hrubikova, K., Hein, I.: Enhancement of virusinduced gene silencing through viral-based production of inverted-repeats. - Plant J. 34: 543–553, 2003.
Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R.J., Thompson, D., Gibson, T.J., Higgins, D.G.: Clustal W and Clustal X version 2.0. - Bioinformatics 23: 2947–2948, 2007.
Lindgren, L.O., Stalberg, K.G., Hoglund, A.S.: Seed-specific overexpression of an endogenous Arabidopsis phytoene synthase gene results in delayed germination and increased levels of carotenoids, chlorophyll, and abscisic acid. - Plant Physiol. 132: 779–785, 2003.
Liu, Y.L., Schiff, M., Dinesh-Kumar, S. P.: Virus-induced gene silencing in tomato. - Plant J. 31: 777–786, 2002.
Liu, Y.L., Schiff, M., Marathe, R., Dinesh-Kumar S.P.: Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus. - Plant J. 30: 415–429, 2002.
Liu, D., Hu, R., Palla, K.J., Tuskan, G.A., Yang, X.: Advances and perspectives on the use of CRISPR/Cas9 systems in plant genomics research. - Curr. Opin. Plant Biol. 30: 70–77, 2016.
MacFarlane, S.A.: Molecular biology of the tobraviruses. - J. gen. Virol. 80: 2799–2807, 1999.
Molnar, A., Csorba, T., Lakatos, L., Varallyay, E., Lacomme, C., Burgyan, J.: Plant virus-derived small interfering RNAs originate predominantly from highly structured singlestranded viral RNAs. - J. Virol. 79: 7812–7818, 2005.
Peele, C., Jordan, C.V., Muangsan, N., Turnage, M., Egelkrout, E., Eagle, P., Hanley-Bowdoin, L., Robertson, D.: Silencing of a meristematic gene using geminivirus-derived vectors. - Plant J. 27: 357–366, 2001.
Pflieger, S., Blanchet, S., Camborde, L., Drugeon, G., Rousseau, A., Noizet, M., Planchais, S., Jupin, I.: Efficient virus-induced gene silencing in Arabidopsis using a 'onestep' TYMV-derived vector. - Plant J. 56: 678–690, 2008.
Pflieger, S., Richard, M.M.S., Blanchet, S., Meziadi, C., Geffroy, V.: VIGS technology: an attractive tool for functional genomics studies in legumes. - Funct. Plant Biol. 40: 1234–1248, 2013.
Ratcliff, F., Martin-Hernandez, A.M., Baulcombe, D.C.: Tobacco rattle virus as a vector for analysis of gene function by silencing. - Plant J. 25: 237–245, 2001.
Robertson, D.: VIGS vectors for gene silencing: many targets, many tools. - Annu. Rev. Plant Biol. 55: 495–519, 2004.
Schmittgen, T.D., Livak, K.J.: Analyzing real-time PCR data by the comparative C-T method. - Nat. Protocols 3: 1101–1108, 2008.
Schuck, J., Gursinsky, T., Pantaleo, V., Burgyan, J., Behrens, S.E.: AGO/RISC-mediated antiviral RNA silencing in a plant in vitro system. - Nucl. Acids Res. 41: 5090–5103, 2013.
Senthil-Kumar, M., Mysore, K.S.: New dimensions for VIGS in plant functional genomics. - Trends Plant Sci. 16: 656–665, 2011.
Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S.: MEGA6: molecular evolutionary genetic analysis version 6.0. - Mol. Biol. EVol. 30: 2725–2729, 2013.
Tang, J., Wang, F., Wang, Z., Huang, Z., Xiong, A., Hou, X.: Characterization and co-expression analysis of WRKY orthologs involved in responses to multiple abiotic stresses in Pak-choi (Brassica campestris ssp. chinensis). - BMC Plant Biol. 13: 188, 2013.
Tijsterman, M., Plasterk, R.H.A.: Dicers at RISC: the mechanism of RNAi. - Cell 117: 1–3, 2004.
Turnage, M.A., Muangsan, N., Peele, C.G., Robertson, D.: Geminivirus-based vectors for gene silencing in Arabidopsis. - Plant J. 30: 107–114, 2002.
Valentine, T., Shaw, J., Blok, V.C., Phillips, M.S., Oparka, K.J., Lacomme, C.: Efficient virus-induced gene silencing in roots using a modified tobacco rattle virus vector. - Plant Physiol. 136: 3999–4009, 2004.
Venegas, A.R., Zhang, Y.P., Konrad, K., Lomas, T.: Flowering without vernalization in winter canola (Brassica napus): use of virus-induced gene silencing (VIGS) to accelerate genetic gain. - Nova Scientia 3: 29–50, 2010.
Voinnet, O.: Induction and suppression of RNA silencing: insights from viral infections. - Nat. Rev. Genet. 6: 206–210, 2005.
Author information
Authors and Affiliations
Corresponding author
Additional information
Acknowledgment: We thank A. Bouteilly (Centre National de la Recherche Scientifique) for providing the plasmid pTY-S. This work was supported by the grants from the key program of the National Natural Science Foundation of China (31330067), the National Natural Science Foundation of Jiangsu (BK20130093), the Fundamental Research Funds for the Central Universities (KYTZ201401), the Jiangsu Province Agriculture Research System (SXGC[2017]273), and the China Agriculture Research System (CARS-25-A-12). The first two authors contributed equally to this work.
Electronic supplementary material
Rights and permissions
About this article
Cite this article
Yu, J., Yang, XD., Wang, Q. et al. Efficient virus-induced gene silencing in Brassica rapa using a turnip yellow mosaic virus vector. Biol Plant 62, 826–834 (2018). https://doi.org/10.1007/s10535-018-0803-6
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10535-018-0803-6