Using Satellite Tobacco Mosaic Virus Vectors for Gene Silencing

Véronique Gosselé1, Michael Metzlaff1

1 Bayer BioScience N.V., Gent
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 16I.5
DOI:  10.1002/9780471729259.mc16i05s00
Online Posting Date:  October, 2005
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Abstract

This unit describes the use of satellite tobacco mosaic virus (STMV) vectors in combination with native TMV particles for inducing transient gene silencing in tobacco plants. Target gene fragment selection and insertion, virus delivery procedures, and phenotype screening of silenced plants are described in detail. All critical parameters for tobacco plant cultivation, virus infection, and RNA silencing efficiency are discussed.

Keywords: RNA silencing; viral vectors; satellite virus–induced silencing system; Nicotiana tabacum; tobacco mosaic virus; plant gene function discovery

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Satellite Tobacco Mosaic Virus Constructs and Helper Virus for RNA Silencing
  • Basic Protocol 2: Co‐Inoculating Satellite Tobacco Mosaic Virus in Vitro Transcripts and TMV‐U2‐Infected Tobacco Leaf Sap into Tobacco Leaves
  • Support Protocol 1: Cultivation of Tobacco Plants
  • Support Protocol 2: Isolating Helper Virus Leaf Sap from TMV‐U2‐Infected Tobacco Plants
  • Alternate Protocol 1: Supplying Satellite Tobacco Mosaic Virus Constructs to Tobacco Via Agroinfiltration
  • Alternate Protocol 2: Supplying Chimeric Satellite Tobacco Mosaic Virus as Sap or as Total RNA from Previously Infected Plants
  • Basic Protocol 3: Screening for Silencing Phenotypes
  • Basic Protocol 4: Analyzing Virus RNA and Target Gene Messenger RNA Levels in Silenced Versus Nonsilenced Tissue
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Satellite Tobacco Mosaic Virus Constructs and Helper Virus for RNA Silencing

  Materials
  • SbfI and NotI and BsaI or HindIII restriction enzymes and appropriate buffers
  • Satellite tobacco mosaic virus (STMV) vectors pVE349 or pVE350 (Figs. and )
  • Buffered phenol ( appendix 2A)
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol ( appendix 2A)
  • 24:1 (v/v) chloroform/isoamyl alcohol
  • 5 M sodium perchlorate (NaClO 4)
  • Isopropanol
  • RNase‐free H 2O
  • MEGAscript high‐yield T7 transcription kit (Ambion)
  • Additional reagents and equipment for PCR, subcloning DNA fragments, DNA sequencing (optional), agarose gel electrophoresis, measuring DNA and RNA concentration by UV spectrophotometry, denaturing formaldehyde agarose gel electrophoresis (unit 16.3)

Basic Protocol 2: Co‐Inoculating Satellite Tobacco Mosaic Virus in Vitro Transcripts and TMV‐U2‐Infected Tobacco Leaf Sap into Tobacco Leaves

  Materials
  • STMV in vitro transcript of interest (see protocol 1)
  • 0.2 M sodium phosphate buffer, pH 7.0 ( appendix 2A), autoclaved
  • Carborundum powder (e.g., silicon carbide, 400 grinding compound; Alfa Aesar cat. no. 39800)
  • Nicotiana tabacumSR1 plants (see protocol 3), each with leaf chosen for inoculation
  • TMV‐U2‐containing sap (see protocol 4), store on ice
  • Plastic squirt bottle containing water
  • ∼0.5‐cm (diameter) brush, does not need to be sterilized

Support Protocol 1: Cultivation of Tobacco Plants

  Materials
  • Nicotiana tabacumSR1 seeds (available from many seed suppliers)
  • 5% (v/v) bleach solution
  • Tween 20
  • H 2O, sterile
  • 14‐cm sterile polystyrene plates (e.g., Falcon 3025) containing 100 ml N. tabacum germination medium (see recipe)
  • 12‐cm flowerpots containing soil
  • Plant growth room (unit 16.3), 24°C, equipped with timer‐controlled grow lights and controlled relative humidity

Support Protocol 2: Isolating Helper Virus Leaf Sap from TMV‐U2‐Infected Tobacco Plants

  Materials
  • Leaf material from TMV‐U2‐infected plants, frozen at −70°C or freeze‐dried
  • 0.2 M sodium phosphate buffer, pH 7.0 ( appendix 2A), autoclaved
  • Mortar and pestle, prechilled
  • Additional reagents and equipment for preparing total RNA from leaves (unit 16.1), measuring RNA concentration by UV spectrophotometry, and denaturing agarose gel electrophoresis

Alternate Protocol 1: Supplying Satellite Tobacco Mosaic Virus Constructs to Tobacco Via Agroinfiltration

  • T‐DNA vector (e.g., pTVE334; available from the authors: )
  • Chimeric STMV of interest (see protocol 1, steps to )
  • Tobacco‐infecting Agrobacterium tumefaciens strain
  • LB medium ( appendix 4A)
  • Agroinfiltration medium (see recipe)
  • Incubator, 28°C, with shaking
  • Spectrophotometer, 600 nm
  • Razor blades
  • 1‐ or 2‐ml syringes
  • Additional reagents and equipment for subcloning DNA fragments and transforming Agrobacterium (unit 16.2),

Alternate Protocol 2: Supplying Chimeric Satellite Tobacco Mosaic Virus as Sap or as Total RNA from Previously Infected Plants

  Materials
  • Frozen leaf samples, harvested at appropriate time points (see protocol 7), including noninoculated leaf samples for transcript quantification only
  • STMV in vitro transcript (see protocol 1), for transcript quantification only
  • Hybond N+ filter (e.g., Amersham Biosciences)
  • STMV or target gene probe, either subcloned in an appropriate vector or as PCR fragment
  • Additional reagents and equipment for preparing RNA from leaf samples (unit 16.1) and northern analysis of plant RNA (unit 16.3)
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Figures

Videos

Literature Cited

   Baulcombe, D. 2004. RNA silencing in plants. Nature 431:356‐363.
   Bernstein, E., Caudy, A.A., Hammond, S.M., and Hannon, G.J. 2001. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409:363‐366.
   Gosselé, V., Faché, I., Meulewaeter, F., Cornelissen, M., and Metzlaff, M. 2002. SVISS—a novel transient gene silencing system for gene function discovery and validation in tobacco plants. Plant J. 32:859‐866.
   Muangsan, N. and Robertson, D. 2004. Geminivirus vectors for transient gene silencing in plants. Methods Mol. Biol. 265:101‐115.
   Routh, G., Dodds, J.A., Fitzmaurice, L., and Mirkov, T.E. 1995. Characterization of deletion and frameshift mutants of satellite tobacco mosaic virus. Virology 212:121‐127.
   Van Larebeke, N., Engler, G., Holsters, M., Van den Elsacker, S., Zaenen, I., Schilperoort, R.A., and Schell, J. 1974. Large plasmid in Agrobacterium tumefaciens essential for crown gall‐inducing ability. Nature 252:169‐170.
   Waterhouse, P.M., Graham, M.W., and Wang, M.B. 1998. Virus resistance and gene silencing can be induced by simultaneous expression of sense and antisense RNA. Proc. Natl. Acad. Sci. U.S.A. 95:13959‐13964.
Key References
   Baulcombe, D.C. 1999. Fast forward genetics based on virus‐induced gene silencing. Curr. Opin. Plant Biol. 2:109‐113.
  Describes how the technology of virus‐induced gene silencing is being refined and adapted as a high‐throughput procedure for functional genomics in plants.
   Routh et al., 1995. See above.
  Describes the first conversion of a satellite RNA virus into an infectious vector.
Internet Resources
   http://www.ambion.com/catalog
  Provides much information on RNA, RNAi, and enzymes for in vitro RNA procedures.
   http://www.esi‐topics.com/genesil
  Summarizes publication and citation data from ISI Essential Science Indicators for the analysis of research trends and performance in gene silencing.
   http://tgi.ncsu.edu
  Provides information on the progress of the Tobacco Genome Initiative, launched in 2003, with the aim to sequence the whole genome of Nicotiana tabacum.
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