Abstract
Virus-Induced Gene Silencing (VIGS) is an excellent reverse genetic tool for the study of gene function in plants, based on virus infection. In this chapter, we describe a high-throughput approach based on VIGS for the study of tomato fruit biochemistry. It comprises the selection of the sequence for silencing using bioinformatics tools, the cloning of the fragment in the Tobacco Rattle Virus (TRV), and the agroinfiltration of tomato fruits mediated by Agrobacterium tumefaciens.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Baulcombe DC (1999) Fast forward genetics based on virus-induced gene silencing. Curr Opin Plant Biol 2(2):109–113
Robertson D (2004) VIGS vectors for gene silencing: many targets, many tools. Annu Rev Plant Biol 55:495–519
Burch‐Smith TM, Anderson JC, Martin GB et al (2004) Applications and advantages of virus‐induced gene silencing for gene function studies in plants. Plant J 39(5):734–746
Senthil-Kumar M, Mysore KS (2011) New dimensions for VIGS in plant functional genomics. Trends Plant Sci 16(12):656–665
Sahu PP, Puranik S, Khan M et al (2012) Recent advances in tomato functional genomics: utilization of VIGS. Protoplasma 249(4):1017–1027
Lacomme C (2014) Milestones in the development and applications of plant virus vector as gene silencing platforms. In: Palmer K, Gleba Y (eds) Plant viral vectors. Springer, Berlin, pp 89–105
Scofield SR, Nelson RS (2009) Resources for virus-induced gene silencing in the grasses. Plant Physiol 149(1):152–157
Jiang Y, Ye S, Wang L et al (2014) Heterologous gene silencing induced by tobacco rattle virus (TRV) is efficient for pursuing functional genomics studies in woody plants. Plant Cell Tissue Organ Cult 116(2):163–174
Orzaez D, Medina A, Torre S et al (2009) A visual reporter system for virus-induced gene silencing in tomato fruit based on anthocyanin accumulation. Plant Physiol 150(3):1122–1134
Quadrana L, Rodriguez MC, López M et al (2011) Coupling virus-induced gene silencing to exogenous green fluorescence protein expression provides a highly efficient system for functional genomics in Arabidopsis and across all stages of tomato fruit development. Plant Physiol 156(3):1278–1291
Ratcliff F, Martin‐Hernandez AM, Baulcombe DC (2001) Technical advance: tobacco rattle virus as a vector for analysis of gene function by silencing. Plant J 25(2):237–245
Senthil‐Kumar M, Mysore KS (2011) Virus‐induced gene silencing can persist for more than 2 years and also be transmitted to progeny seedlings in Nicotiana benthamiana and tomato. Plant Biotech J 9(7):797–806
Liu Y, Schiff M, Marathe R et al (2002) Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N‐mediated resistance to tobacco mosaic virus. Plant J 30(4):415–429
Liu Y, Schiff M, Dinesh‐Kumar SP (2002) Virus‐induced gene silencing in tomato. Plant J 31(6):777–786
Tomato Genome Consortium (2012) The tomato genome sequence provides insights into fleshy fruit evolution. Nature 485(7400):635–641
Fantini E, Falcone G, Frusciante S et al (2013) Dissection of tomato lycopene biosynthesis through Virus-Induced Gene Silencing. Plant Physiol 163(2):986–998
Fu DQ, Zhu BZ, Zhu HL et al (2005) Virus-induced gene silencing in tomato fruit. Plant J 43(2):299–308
Liu E, Page JE (2008) Optimized cDNA libraries for virus-induced gene silencing (VIGS) using tobacco rattle virus. Plant Methods 4(5):1–13
Xu P, Zhang Y, Kang L et al (2006) Computational estimation and experimental verification of off-target silencing during posttranscriptional gene silencing in plants. Plant Physiol 142(2):429–440
Acknowledgments
This work was supported by the Italian Ministry of Research (Project “Integrated Knowledge for the Sustainability and Innovation of Italian Agri-Food”), by the Italian Ministry of Agriculture (Projects “Nutrisol” and “Biomassval”), and by the European Commission (FP7 Project “From discovery to products: A next generation pipeline for the sustainable generation of high-value plant products,” contract no. 613513; Horizon 2020 project “Traditional tomato varieties and cultural practices: a case for agricultural diversification with impact on food security and health of European population,” contract no. 634561) and benefited from the networking activities of COST Action FA1106 “QualityFruit.” We thank Alessandro Nicolia for critical reading of the manuscript.
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Fantini, E., Giuliano, G. (2016). Virus-Induced Gene Silencing as a Tool to Study Tomato Fruit Biochemistry. In: Botella, J., Botella, M. (eds) Plant Signal Transduction. Methods in Molecular Biology, vol 1363. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3115-6_7
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3115-6_7
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3114-9
Online ISBN: 978-1-4939-3115-6
eBook Packages: Springer Protocols