Virus-induced gene silencing in plants. Methods

The Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK.
Methods (Impact Factor: 3.65). 09/2003; 30(4):296-303. DOI: 10.1016/S1046-2023(03)00037-9
Source: PubMed


Virus-induced gene silencing (VIGS) is a technology that exploits an RNA-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying inserts derived from host genes the process can be additionally targeted against the corresponding mRNAs. VIGS has been used widely in plants for analysis of gene function and has been adapted for high-throughput functional genomics. Until now most applications of VIGS have been in Nicotiana benthamiana. However, new vector systems and methods are being developed that could be used in other plants, including Arabidopsis. Here we discuss practical and theoretical issues that are specific to VIGS rather than other gene "knock down" or "knockout" approaches to gene function. We also describe currently used protocols that have allowed us to apply VIGS to the identification of genes required for disease resistance in plants. These methods and the underlying general principles also apply when VIGS is used in the analysis of other aspects of plant biology.

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Available from: Rui Lu, Nov 06, 2014
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    • "In this article, we report the use of a virus-induced gene-silencing (VIGS)-based, fast-forward genetics approach (Baulcombe, 1999; Lu et al., 2003b; del Pozo et al., 2004) to identify plant genes that play a role in nonhost disease resistance. One of the genes identified through the screen encodes the photorespiratory enzyme , GOX. "
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    • "Virus-induced gene silencing (VIGS), a technique developed for gene transcript suppression, has been used for the characterization of the function of plant genes and avoids the disadvantages of regenerating plants from difficult species ( Burch-Smith et al. 2004, Hileman et al. 2005, Wege et al. 2007, Senthil-Kumar and Mysore 2011, Ramegowda et al. 2014). Virus-induced gene silencing has been widely used for functional gene analysis in species like Nicotiana benthamiana (Solanaceae) ( Ratcliff et al. 2001, Lu et al. 2002), A. thaliana (Brassicaceae) ( Burch-Smith et al. 2006, Wang et al. 2006), tomato (Solanaceae) ( Liu et al. 2002a, Ekengren et al. 2003), barley (Gramineae) ( Holzberg et al. 2002), Papaver somniferum (Papaveraceae) ( Hileman et al. 2005), Phalaenopsis amabilis (Orchidaceae) ( Lu et al. 2007), Aquilegia vulgaris and Thalictrum sp. (Ranunculaceae) ( Gould and Kramer 2007, Kramer et al. 2007, Di Stilio et al. 2010), Spinacea oleracea (Amaranthaceae ) ( Golenberg et al. 2009), Jatropha curcas (Euphorbiaceae ) ( Ye et al. 2009), Zingiber officinale (Zingiberaceae) ( Renner et al. 2009), and Malus domestica and Pyrus pyrifolia (Rosaceae) ( Sasaki et al. 2011). "
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    ABSTRACT: Virus-induced gene silencing (VIGS) has been shown to be an effective tool for investigating gene functions in herbaceous plant species, but has rarely been tested in trees. The establishment of a fast and reliable transformation system is especially important for woody plants, many of which are recalcitrant to transformation. In this study, we established a tobacco rattle virus (TRV)-based VIGS system for two Populus species, Populus euphratica and P. × canescens. Here, TRV constructs carrying a 266 bp or a 558 bp fragment of the phytoene desaturase (PDS) gene were Agrobacterium-infiltrated into leaves of the two poplar species. Agrobacterium-mediated delivery of the shorter insert, TRV2-PePDS266, into the host poplars resulted in expected photobleaching in both tree species, but not the longer insert, PePDS558. The efficiency of VIGS was temperature-dependent, increasing by raising the temperature from 18 to 28 °C. The optimized TRV-VIGS system at 28 °C resulted in a high silencing frequency and efficiency up to 65-73 and 83-94%, respectively, in the two tested poplars. Moreover, syringe inoculation of Agrobacterium in 100 mM acetosyringone induced a more efficient silencing in the two poplar species, compared with other agroinfiltration methods, e.g., direct injection, misting and agrodrench. There were plant species-related differences in the response to VIGS because the photobleaching symptoms were more severe in P. × canescens than in P. euphratica. Furthermore, VIGS-treated P. euphratica exhibited a higher recovery rate (50%) after several weeks of the virus infection, compared with TRV-infected P. × canescens plants (20%). Expression stability of reference genes was screened to assess the relative abundance of PePDS mRNA in VIGS-treated P. euphratica and P. × canescens. PeACT7 was stably expressed in P. euphratica and UBQ-L was selected as the most suitable reference gene for P. × canescens using three different statistical approaches, geNorm, NormFinder and BestKeeper. Quantitative real-time PCR showed significant reductions in PDS transcripts (55-64%) in the photobleached leaves of both VIGS-treated poplar species. Our results demonstrate that the TRV-based VIGS provides a practical tool for gene functional analysis in Populus sp., especially in those poplar species which are otherwise recalcitrant to transformation. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email:
    Tree Physiology 07/2015; 35(9). DOI:10.1093/treephys/tpv064 · 3.66 Impact Factor
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    • "These are detected by the plant host and trigger PTGS, which involves the generation of small interfering RNAs (siRNAs) that are then loaded into the RNA-induced silencing complex containing at least one Argonaute endonuclease to guide the cleavage of complementary viral RNA (Baulcombe, 2004). In VIGS, a fragment of a plant gene is inserted into a virus vector to form a recombinant virus that, upon infection of a plant host, induces PTGS targeting both the virus RNA and homologous endogenous plant RNA sequences for degradation (Lu et al., 2003). Only one plant virus, Barley stripe mosaic virus (BSMV), which has a tri-partite RNA genome comprising RNAa, RNAb and RNAc, has so far been developed into a VIGS vector for wheat (Scofield et al., 2005). "
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    ABSTRACT: Virus-induced gene silencing (VIGS) has emerged as a powerful reverse genetic technology in plants supplementary to stable transgenic RNAi and, in certain species, as a viable alternative approach for gene functional analysis. The RNA virus Barley stripe mosaic virus (BSMV) was developed as a VIGS vector in the early 2000s and since then it has been used to study the function of wheat genes. Several variants of BSMV vectors are available, with some requiring in vitro transcription of infectious viral RNA, while others rely on in planta production of viral RNA from DNA-based vectors delivered to plant cells either by particle bombardment or Agrobacterium tumefaciens. We adapted the latest generation of binary BSMV VIGS vectors for the identification and study of wheat genes of interest involved in interactions with Zymoseptoria tritici and here present detailed and the most up-to-date protocols. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Fungal Genetics and Biology 06/2015; 79. DOI:10.1016/j.fgb.2015.04.006 · 2.59 Impact Factor
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