Using Vectors Derived from Tomato Bushy Stunt Virus (TBSV) and TBSV Defective Interfering RNAs (DIs)

Wenping Qiu1, Herman B. Scholthof2

1 Department of Agriculture, Missouri State University at Mountain Grove, Mountain Grove, null, 2 Department of Plant Pathology and Microbiology, Texas A&M University, College Station, null
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 16I.4
DOI:  10.1002/9780471729259.mc16i04s7
Online Posting Date:  November, 2007
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This unit describes principles and protocols for expressing a gene of interest in plant cells using gene vectors that are derived from an infectious full‐length cDNA plasmid of the tomato bushy stunt virus (TBSV) genomic RNA, and from defective interfering RNAs (DIs). The TBSV gene vector system permits convenient cloning, allows modification and abundant expression of the gene of interest, and facilitates biosecure containment of the gene vectors. These vectors can be employed for functional genomics studies and for analyzing the biochemical properties and subcellular distribution of expressed RNAs and/or their cognate proteins. As with other plant virus gene vectors, recombination and deletion of the gene of interest during virus multiplication limits the application of the TBSV gene vectors to the inoculated cells or leaves. Curr. Protoc. Microbiol. 7:16I.4.1‐16I.4.16. © 2007 by John Wiley & Sons, Inc.

Keywords: tomato bushy stunt virus; defective interfering RNAs; gene vectors; gene of interest

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Cloning into the Expression Plasmids
  • Alternate Protocol 1: DNA‐Based TBSV Gene Vectors
  • Alternate Protocol 2: DI as a Gene Vector for RNAi
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Cloning into the Expression Plasmids

  • TBSV gene vector (pHST2) DNA (can be obtained from the authors at
  • E. coli DH5α competent cells
  • LB medium containing 50 to 100 µg/ml ampicillin ( appendix 4A)
  • Column‐based plasmid purification kit (e.g., QIAprep Spin Miniprep kit; Qiagen)
  • Gene‐of‐interest DNA fragment
  • Restriction endonucleases XhoI, SacI, and SnaBI (and other restriction enzymes as needed) and corresponding buffers
  • Forward and reverse PCR primers for gene of interest (see Kramer and Coen, )
  • PfuUltraII Fusion HS DNA polymerase (Stratagene, cat. no. 600670; optional)
  • Klenow fragment of DNA polymerase I
  • Plasmid harboring Kanr gene (e.g., pKAN‐2 or Gateway pENTR)
  • 1% agarose gel prepared in TBE buffer (Voytas, )
  • TE buffer ( appendix 2A)
  • 50 U/µl T7 RNA polymerase (Invitrogen, cat. no. 18033‐19) and 5× T7 buffer
  • 100 mM dithiothreitol (DTT)
  • 10 mM NTP mix (Invitrogen, cat. no. 18109‐017)
  • RNAsin ribonuclease inhibitor (Promega)
  • RNA inoculation buffer (see recipe)
  • Nicotiana benthamiana plants
  • Climate‐controlled growth chambers (Conviron;
  • Additional reagents and equipment for transformation of E. coli (Seidman et al., ), plasmid DNA isolation (Engebrecht et al., ), polymerase chain reaction (PCR; Kramer and Coen, ), insertion of DNA fragments into plasmids (Struhl, ), use of Klenow fragment of DNA polymerase I to generate blunt ends (Tabor et al., ), phenol/chloroform extraction and ethanol precipitation of DNA (Moore and Dowhan, ), agarose gel electrophoresis (Voytas, ), spectrophotometric quantitation of nucleic acids (Gallagher and Desjardins, ), and inoculation of RNA transcripts into whole plants (unit 16.1 in this manual)
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  •   FigureFigure 16.I0.1 TBSV‐based gene vectors. Schematic diagram of TBSV RNA‐ and DNA‐based vectors to illustrate the viral genome and the composition of defective interfering RNAs (DIs). The function of each protein (numbers represent molecular mass in kDa) is listed on top of the diagram. Rectangles denote coding regions of the TBSV genome, and lines represent untranslated sequences. The four regions (I, II, III, and IV) of DIs are presented on top of the diagram for the plasmid pHS21. Unique restriction sites for cloning purposes are listed below each diagram. Abbreviations: MSC, multiple cloning sites; MP, movement protein; sGFP, synthetic green fluorescent protein; HDVagrz, hepatitis delta virus antigenomic ribozyme; CaMV 35S, the cauliflower mosaic virus 35S promoter; T7, the DNA‐dependent RNA polymerase promoter of the T7 bacteriophage.
  •   FigureFigure 16.I0.2 Levels of TBSV RNA accumulation. (A) An agarose gel image is shown to illustrate that TBSV genomic RNA accumulates abundantly and can be readily visualized upon ethidium bromide staining. (B) The identity of the single‐stranded genomic (gRNA) and subgenomic RNAs (sgRNA1 and sgRNA2) can be confirmed by hybridization with TBSV‐specific probes. These particular results were obtained with wild‐type TBSV viral RNAs that accumulate in infected Nicotiana benthamiana protoplasts. Abbreviations: M, mock‐inoculated; T, TBSV‐infected. The double‐stranded genomic RNA is visible above the gRNA in the lanes labeled T on the ethidium bromide‐stained gel.
  •   FigureFigure 16.I0.3 TBSV‐mediated expression of green fluorescent protein (GFP) from a TBSV vector reminiscent of pHST2 (construct kindly provided by Teresa Rubio and Andy Jackson). (A) Protoplasts prepared from leaf tissues of Nicotiana benthamiana transfected with a TBSV vector expressing GFP. The protoplasts shown in the image were illuminated with blue light. The red‐colored cells (appearing as dark in the black and white reproduction) represent uninfected protoplasts containing autofluorescent chloroplasts. The green and yellow color (green superimposed on red; gray in the black and white version) that is due to GFP expression can be observed in some of the protoplasts. The image serves as an example to illustrate that protoplasts can be infected with RNA transcripts of TBSV gene vectors for transient expression of a foreign gene and for inspecting infectivity of a recombinant TBSV virus. (B) N. benthamiana leaves 5 days post‐inoculation with TBSV expressing GFP (right), compared to mock‐inoculated leaves (left). The bright‐field images (top) show the slight chlorotic symptoms typical upon inoculation of this host with TBSV vectors; the UV‐illuminated panels (bottom) show the GFP fluorescence. Images courtesy of Yi‐Cheng Hsieh.


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Key References
   Scholthof, 1999. See above.
  Describes features and methodology of DNA‐based TBSV gene vectors pHST12 and pHST34.
   Scholthof, et al. 1996. See above.
  A detailed review on background, principles, and application of virus gene vectors.
   Qiu, et al. 2002. See above.
  Presents an example of using TBSV gene vectors to induce silencing, allowing for functional studies of coexpressed suppressor protein.
   Hou and Qiu, 2003. See above.
  A case study of applying both TBSV and defective interfering RNA (DI) as gene vectors in gene‐silencing research.
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