RNAi in Transgenic Plants

Yanhai Yin1, Joanne Chory2, David Baulcombe3

1 Iowa State University, Ames, Iowa, 2 The Salk Institute for Biological Studies, La Jolla, California, 3 John Innes Centre, Norwich, United Kingdom
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 26.6
DOI:  10.1002/0471142727.mb2606s72
Online Posting Date:  November, 2005
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Abstract

RNA silencing in plants is a rapid and facile approach for assignment of gene function and virus resistance. It allows selective RNA degradation through a mechanism that is given specificity by short interfering RNAs. The RNA silencer sequences are normally delivered as transgenes or a part of virusā€vectors. This unit provides Basic Protocols for transgene and virus induced silencing. It also includes a Basic Protocol for protein overexpression using viral proteins that suppress silencing.

Keywords: double stranded RNA; posttranscriptional control; antisense RN

     
 
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Table of Contents

  • Basic Protocol 1: Constructing Stable Transgenic Plants that Express dsRNA
  • Basic Protocol 2: Virus‐Induced Gene Silencing
  • Basic Protocol 3: Suppression of Silencing for Protein Overexpression
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Constructing Stable Transgenic Plants that Express dsRNA

  Materials
  • Template DNA: cDNA clone of target gene
  • Restriction enzymes: e.g., KpnI, EcoRI, XhoI, XbaI, BamHI, HindIII, ClaI, or NotI (see Fig. )
  • Intermediate vector pHANNIBAL (Wesley et al., ) and binary vector pART27 (Gleave, )
  • Agrobacterium tumefaciens strain GV3101 with pSa‐rep (Hellens et al., )
  • YEP medium (see recipe): with no supplements, with 50 mg/liter gentamicin, and with 50 mg/liter gentamicin plus 50 mg/liter spectinomycin
  • 0.15 M NaCl (autoclave and store at room temperature)
  • 20 mM CaCl 2 (filter sterilize; store at 4°C) supplemented with 15% (v/v) glycerol (autoclave and store at room temperature)
  • Liquid nitrogen
  • YEP plates (see recipe) supplemented with 50 mg/liter gentamicin and 50 mg/liter spectinomycin
  • Arabidopsis thaliana ecotype Columbia (Col‐0) seeds (Lehle Seeds)
  • 0.1% (w/v) phytoagar (Caisson Laboratories; autoclave and store at room temperature)
  • Arabidopsis infiltration medium (see recipe)
  • Silwet L‐77 (Lehle Seeds)
  • 70% ethanol containing 0.1% (v/v) Triton X‐100
  • 95% or 100% ethanol
  • Arabidopsis transgenic plant selection plates (see recipe) and wild‐type control plates (omit kanamycin)
  • 100‐ml and 2‐liter Erlenmeyer flasks
  • 28°C shaking incubator
  • High‐speed refrigerated centrifuge and rotors
  • Equipment for growing Arabidopsis thaliana (Lehle Seeds):
    • Potting soil
    • 3 × 5–in. plastic pots
    • Nylon screens
    • Clear covers
    • Environmentally controlled 22°C growth chambers and growth rooms for Arabidopsis
    • 22°C incubator allowing control over light/dark cycles
  • Rubber bands or tape
  • Covers of pipet‐tip box or deep petri dishes as containers for Agrobacterium solution
  • Plastic wrap (e.g., Saran wrap)
  • Coin envelopes for seed drying and storage
  • 15‐ml tubes
  • 100‐mm diameter filter papers, sterilized by autoclaving
  • Additional reagents and equipment for PCR (unit 15.1), restriction enzyme digestion (unit 3.1), agarose gel electrophoresis (unit 2.5), DNA purification from agarose gels (unit 2.6), use of T4 DNA ligase (unit 3.14), transformation of E. coli (unit 1.8), plasmid DNA minipreps (unit 1.6), dephosphorylation of DNA with alkaline phosphatase (unit 3.10), culture of bacteria (unit 1.2), plant RNA preparation (unit 4.3), RT‐PCR (units 15.5& 15.8), northern blotting (unit 4.9), and immunoblotting (unit 10.8)

Basic Protocol 2: Virus‐Induced Gene Silencing

  Materials
  • A. tumefaciens binary vector pTRV: TRV RNA2 vector construct (e.g., pTV00 based on TRV strain PPK20; Ratcliff et al., ); freely available to academic scientists from the Baulcombe laboratory (http://www.sainsbury‐laboratory.ac.uk/dcb/)
  • SOB medium (see recipe)
  • 10% (v/v) glycerol, ice cold
  • L medium and plates (see recipe) supplemented with 50 µg/ml kanamycin, 5 µg/ml tetracycline, and 50 µg/ml rifampicin
  • A. tumefaciens strain C58C1 carrying pBINTRA6 (GeneBank accession no. AF314165; Ratcliff et al., ); freely available to academic scientists from the Baulcombe laboratory (http://www.sainsbury‐laboratory.ac.uk/dcb/)
  • SOB medium (see recipe) supplemented with 50 µg/ml kanamycin, 5 µg/ml tetracycline, and 50 µg/ml rifampicin
  • 10 mM MgCl 2 with 100 µM acetosyringone (3′,5′‐dimethoxy‐4′‐hydroxyacetophenone; Acros Organics) and 1 mM MES, pH 5.6
  • Nicotiana benthamiana seedlings (5 cm high, 2 weeks old)
  • 2‐liter Erlenmeyer flasks
  • 250‐m1 centrifuge bottles
  • Electroporator (Cell‐Porator and Voltage Booster; Invitrogen) and 0.15‐cm cuvettes, prechilled
  • Culture tubes
  • 1‐ml syringes
  • 20° to 24°C greenhouse or growth chamber
  • Additional reagents and equipment for constructing RNAi vector and transforming A. tumefaciens (see protocol 1), electroporation (optional; unit 1.8), alkaline lysis minipreps (unit 1.6), PCR (unit 15.1), and restriction enzyme digestion (unit 3.1)

Basic Protocol 3: Suppression of Silencing for Protein Overexpression

  Materials
  • pBin61 binary plasmid vector with XbaI and SmaI restriction sites in the CaMV 35S expression cassette (Bendahmane et al., ); freely available to academic scientists from the Baulcombe laboratory (http://www.sainsbury‐laboratory.ac.uk/dcb/)
  • Restriction enzymes: SmaI and XbaI
  • cDNA of full open reading frame to be overexpressed
  • E. coli strain DH5α
  • L plates (see recipe) supplemented with 50 µg/ml kanamycin
  • A. tumefaciens strain C58C1:pCH32
  • L plates (see recipe) supplemented with 50 µg/ml kanamycin, 5 µg/ml tetracycline, and 50 µg/ml rifampicin
  • A. tumefaciens strain C58C1:pCH32:p35S‐p19 (or C58C1:pCH32:p35S‐pHC‐Pro) harboring expression plasmids of the p19 (or HC‐pro) viral suppressors of silencing (Voinnet et al., ); freely available to academic scientists from the Baulcombe laboratory (http://www.sainsbury‐laboratory.ac.uk/dcb/)
  • L medium (see recipe) supplemented with 50 µg/ml kanamycin and 5 µg/ml tetracycline
  • 10 mM MgCl 2 with 100 µM acetosyringone (3′,5′‐dimethoxy‐4′‐hydroxyacetophenone; Acros Organics) and 1 mM MES, pH 5.6
  • Nicotiana benthamiana seedlings (5 cm high, 2 weeks old)
  • 28°C shaking incubator
  • Culture tubes
  • 1‐ml syringes
  • 20°C to 25°C greenhouse or growth chamber allowing control of light/dark cycles
  • Additional reagents and equipment for restriction digestion (unit 3.1), use of T4 DNA ligase (unit 3.14), transformation of E. coli (unit 1.8), transformation of A. tumefaciens (see protocol 1 or protocol 22), and growth of bacteria (unit 1.2)
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Figures

Videos

Literature Cited

Literature Cited
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