Using Viral Vectors to Silence Endogenous Genes

Jeffrey Caplan1, S.P. Dinesh‐Kumar1

1 Yale University, New Haven, Connecticut
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 16I.6
DOI:  10.1002/9780471729259.mc16i06s01
Online Posting Date:  June, 2006
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Abstract

Virus‐induced gene silencing (VIGS) is a fast but transient method for knocking down expression of endogenous genes in plants. Replicating plant viruses activate a defense mechanism called post‐transcriptional gene silencing (PTGS), which protects the plant by silencing viral transcripts. VIGS of endogenous genes is accomplished by inserting a gene of interest into a viral vector. When the virus replicates in the plant, PTGS silences both the viral genome and the corresponding endogenous gene. The most robust and widely implemented VIGS system uses tobacco rattle virus (TRV) vectors and N. benthamiana as the plant host. This unit will explain how to introduce TRV‐based VIGS vectors into N. benthamiana plants by two methods: syringe infiltration or the Agrobacterium drench method. Furthermore, it will provide two alternate protocols optimized for VIGS in tomato plants: spray inoculation and vacuum infiltration.

Keywords: virus-induced gene silencing; VIGS; post-transcriptional gene silencing; PTGS; RNAi; Nicotiana benthamiana; tomato; Lycospericon esculentum; Agrobacterium tumefaciens

     
 
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Table of Contents

  • Basic Protocol 1: Virus‐Induced Gene Silencing by Syringe Infiltration
  • Basic Protocol 2: Virus‐Induced Gene Silencing by Agrodrench Inoculation
  • Alternate Protocol 1: Virus‐Induced Gene Silencing by Spray Inoculation
  • Alternate Protocol 2: Virus‐Induced Gene Silencing by Vacuum Infiltration
  • Support Protocol 1: Growth of Agrobacterium Containing Either TRV1 OR TRV2 Vector for Virus‐Induced Gene Silencing
  • Support Protocol 2: Construction of TRV Vector Containing Target Gene for Virus‐Induced Gene Silencing
  • Support Protocol 3: Growth of N. benthamiana Plants for Virus‐Induced Gene Silencing
  • Support Protocol 4: Growth of Tomato Plants for Virus‐Induced Gene Silencing
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Virus‐Induced Gene Silencing by Syringe Infiltration

  Materials
  • Agrobacterium GV2260 containing pTRV1 vector ( protocol 5)
  • Agrobacterium GV2260 containing pTRV2 with target gene (pTRV2‐TARGET; protocol 5)
  • VIGS agroinfiltration medium (see recipe)
  • N. benthamiana or tomato plants ( protocol 6 or protocol 73)
  • 1‐ml syringe without needle

Basic Protocol 2: Virus‐Induced Gene Silencing by Agrodrench Inoculation

  Materials
  • Agrobacterium GV2260 containing pTRV1 vector ( protocol 5)
  • Agrobacterium GV2260 containing pTRV2 with target gene (pTRV2‐TARGET; protocol 5)
  • VIGS agroinfiltration medium (see recipe)
  • Plant of Solanaceae family: N. benthamiana ( protocol 7), tomato ( protocol 8), pepper, potato, petunia, or eggplant
  • 10‐ml pipet

Alternate Protocol 1: Virus‐Induced Gene Silencing by Spray Inoculation

  Materials
  • Agrobacterium GV3101 containing pTRV1 vector ( protocol 8)
  • Agrobacterium GV3101 containing pTRV2‐TARGET vector ( protocol 8)
  • VIGS agroinfiltration medium (see recipe)
  • Carborundum
  • Tomato plants (see protocol 8)
  • Artist's airbrush (Model V180; Paasche; http://www.paascheairbrush.com)
  • Portable air compressor (Campbell Hausfeld; http://www.chpower.com/)
CAUTION: Carborundum may cause eye, skin, respiratory‐tract, or digestive‐tract irritation, as well as lung damage. It may cause cancer, based on the results of animal studies. Read the Material Safety Data Sheet (MSDS) before use.

Alternate Protocol 2: Virus‐Induced Gene Silencing by Vacuum Infiltration

  Materials
  • Agrobacterium GV3101 containing pTRV1 vector ( protocol 5)
  • Agrobacterium GV3101 containing pTRV2‐TARGET vector ( protocol 5)
  • VIGS agroinfiltration medium (see recipe)
  • Tomato plants (see protocol 8)
  • Silwet L‐77
  • 500‐ml plastic beaker
  • Desiccator (240 mm diameter; Bel‐Art Co.)
  • Vacuum pump (Model FB DVP0352; Fisher)

Support Protocol 1: Growth of Agrobacterium Containing Either TRV1 OR TRV2 Vector for Virus‐Induced Gene Silencing

  Materials
  • TRV1 or TRV2 vector (available by request from Dr. S. Dinesh‐Kumar; )
  • Kit for BP Gateway or LR Gateway reactions (Invitrogen)
  • Agrobacterium strain GV2260 or GV3101
  • LB plates ( appendix 4A) containing 50 mg/liter kanamycin, 25 mg/liter rifampicin, 50 mg/liter streptomycin, and 50 mg/liter carbenicillin (for strain GV2260) or 50 mg/liter kanamycin and 15 mg/liter gentamicin (for strain GV3101)
  • LB medium ( appendix 4A) containing 50 mg/liter kanamycin, 25 mg/liter rifampicin, 50 mg/liter streptomycin, and 50 mg/liter carbenicillin (for strain GV2260) or 50 mg/liter kanamycin and 15 mg/liter gentamicin (for strain GV3101)
  • 200 mM acetosyringone (see recipe)
  • 1 M MES (see recipe)
  • Additional reagents and equipment for transformation of Agrobacterium (unit 16.2)

Support Protocol 2: Construction of TRV Vector Containing Target Gene for Virus‐Induced Gene Silencing

  Materials
  • Target gene of interest
  • TRV2 vector pYL156 or pYL279 (available by request from Dr. S. Dinesh‐Kumar; )
  • pDONR221 (Invitrogen)
  • Competent Agrobacterium strain GV2260 or GV3101 (available by request from Dr. S. Dinesh‐Kumar; )
  • LB plates ( appendix 4A) containing 50 mg/liter kanamycin, 25 mg/liter rifampicin, 50 mg/liter streptomycin, and 50 mg/liter carbenicillin (for strain GV2260) or 50 mg/liter kanamycin and 15 mg/liter gentamicin (for strain GV3101)
  • Additional reagents and equipment for PCR (Kramer and Coen, ), restriction digestion (Bloch and Grossman, ) and ligation (Tabor, ), and transformation of Agrobacterium (unit 16.2)

Support Protocol 3: Growth of N. benthamiana Plants for Virus‐Induced Gene Silencing

  Materials
  • N. benthamiana seeds
  • Super Fine Germinating Mix (Conrad Fafard, Inc.; http://www.fafard.com)
  • Professional Growth Medium No. 2 (Conrad Fafard, Inc.; http://www.fafard.com)
  • 1‐pint (∼500‐ml) pots, 4 in. (∼10 cm) square
  • Pot trays
  • Clear plastic domes
  • Light source (40‐W Gro‐Lux fluorescent light bulb, Sylvania)

Support Protocol 4: Growth of Tomato Plants for Virus‐Induced Gene Silencing

  Materials
  • VF36 or MicroTom tomato seeds (Tomato Genetics Resource Center, TGRC; http://tgrc.ucdavis.edu)
  • Professional Growth Medium No. 2 (Conrad Fafard, Inc.; http://www.fafard.com)
  • Vermiculite
  • 1‐pint (∼500‐ml) pots, 4 in. (∼10 cm) square
  • Pot trays
  • Clear plastic domes
  • Light source (40‐W Gro‐Lux fluorescent light bulb, Sylvania)
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Figures

Videos

Literature Cited

Literature Cited
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   Burch‐Smith, T.M., Anderson, J.C., Martin, G.B., and Dinesh‐Kumar, S.P. 2004. Applications and advantages of virus‐induced gene silencing for gene function studies in plants. Plant J. 39:734‐746.
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