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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Abstract

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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Materials

Basic Protocol 1: Cloning into the Expression Plasmids

  Materials
  • TBSV gene vector (pHST2) DNA (can be obtained from the authors at http://scholthoflab.tamu.edu)
  • 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; http://www.conviron.com)
  • 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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Figures

  •   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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Literature Cited

   Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. (eds.). 2007. Current Protocols in Molecular Biology. John Wiley & Sons, Hoboken, N.J.
   Blanc, S., Schmidt, I., Vantard, M., Scholthof, H.B., Kuhl, G., Esperandieu, P., Cerutti, M., and Louis, C. 1996. The aphid transmission factor of cauliflower mosaic virus forms a stable complex with microtubules in both insect and plant cells. Proc. Natl. Acad. Sci. U.S.A. 93:15158‐15163.
   Chu, M., Desvoyes, B., Turina, M., Noad, R., and Scholthof, H.B. 2000. Genetic dissection of tomato bushy stunt virus p19‐protein‐mediated host‐dependent symptom induction and systemic invasion. Virology 266:79‐87.
   Desvoyes, B., Faure‐Rabasse, S., Chen, M.‐H., Park, J.‐W., and Scholthof, H.B. 2002. A novel plant homeodomain protein interacts in a functionally relevant manner with a virus movement protein. Plant Physiology 129:1521‐1532.
   Desvoyes, B. and Scholthof, H.B. 2002. Host‐dependent recombination of a Tomato bushy stunt virus coat protein mutant yields truncated capsid subunits that form virus‐like complexes which benefit systemic spread. Virology 304:434‐442.
   Engebrecht, J., Brent, R., and Kaderbhai, M.A. 1991. Minipreps of plasmid DNA. Curr. Protoc. Mol. Biol. 15:1.6.1‐1.6.10.
   Gallagher, S.R. 2006. One‐dimensional SDS gel electrophoresis of proteins. Curr. Protoc. Mol. Biol. 75:10.2A.1‐10.2A.37.
   Gallagher, S.J. and Desjardins, P.R. 2006. Quantitation of DNA and RNA with absorption and fluorescence spectroscopy. Curr. Protoc. Mol. Biol. 76:A.3D.1‐A.3D.21.
   Gallagher, S.R., Winston, S.E., Fuller, S.A., and Hurrell, J.G.R. 2004. Immunoblotting and immunodetection. Curr. Protoc. Mol. Biol. 66:10.8.1‐10.8.24.
   Hearne, P.Q., Knorr, D.A., Hillman, B.I., and Morris, T.J. 1990. The complete genome structure and synthesis of infectious RNA from clones of tomato bushy stunt virus. Virology 177:141‐151.
   Hou, H.S. and Qiu, W.P. 2003. A novel co‐delivery system consisting of a Tomato bushy stunt virus and a defective interfering RNA for studying gene silencing. J. Virol. Meth. 111:37‐42.
   Hull, R. and Matthews, R.E. 2001. Matthews' Plant Virology. Academic Press, New York.
   Joelson, T., Akerblom, L., Oxelfelt, P., Strandberg, B., Tomenius, K., and Morris, T.J. 1997. Presentation of a foreign peptide on the surface of tomato bushy stunt virus. J. Gen. Virol. 78:1213‐1217.
   Kimple, M.E. and Sondek, J. 2004. Overview of affinity tags for protein purification. Curr. Protoc. Protein Sci. 36:9.9.1‐9.9.19.
   Knorr, D.A., Mullin, R.H., Hearne, P.Q., and Morris, T.J. 1991. De novo generation of defective interfering RNAs of tomato bushy stunt virus by high multiplicity passage. Virology 181:193‐202.
   Kramer, M.F. and Coen, D.M. 2001. The polymerase chain reaction. Curr. Protoc. Mol. Biol. 56:15.1.1‐15.1.14.
   Moore, D. and Dowhan, D. 2002. Purification and concentration of DNA from aqueous solutions. Curr. Protoc. Mol. Biol. 59:2.1.1‐2.1.10.
   Palmer, I. and Wingfield, P.T. 2004. Preparation and extraction of insoluble (inclusion‐body) proteins from Escherichia coli. Curr. Protoc. Protein Sci. 38:6.3.1‐6.3.18.
   Pogue, G.P., Lindbo, J.A., Garger, S.J., and Fitzmaurice, W.P. 2002. Making an ally from an enemy: Plant virology and the new agriculture. Annu. Rev. Phytopathol. 40:45‐74.
   Qiu, W.P., Park, J.‐W., Jackson, A.O., and Scholthof, H.B. 2001. Retention of a small replicase gene segment in Tomato bushy stunt virus defective RNAs inhibits their helper‐mediated trans‐accumulation. Virology 281:51‐60.
   Qiu, W.P., Park, J.‐W., and Scholthof, H.B. 2002. Tombusvirus P19‐mediated suppression of virus induced gene silencing is controlled by genetic and dosage features that influence pathogenicity. Mol. Plant‐Microbe Interact. 15:269‐280.
   Qiu, W.P. and Scholthof, K.‐B.G. 2004. Satellite panicum mosaic virus capsid protein elicits symptoms on a nonhost plant and interferes with a suppressor of virus‐induced gene silencing. Mol. Plant‐Microbe Interact. 17:263‐271.
   Qu, F. and Morris, T.J. 2002. Efficient infection of Nicotiana benthamiana by Tomato bushy stunt virus is facilitated by the coat protein and maintained by p19 through suppression of gene silencing. Mol. Plant‐Microbe Interact. 15:193‐202.
   Rubio, T., Borja, M., Scholthof, H.B., Feldstein, P.A., Morris, T.J., and Jackson, A.O. 1999. Broad‐spectrum protection against Tombusviruses elicited by defective interfering RNAs in transgenic plants. J. Virol. 73:5070‐5078.
   Russo, M., Burgyan, J., and Martelli, G.P. 1994. Molecular biology of Tombusviridae. Adv. Virus Res. 44:381‐428.
   Scholthof, H.B. 1999. Rapid delivery of foreign genes into plants by direct rub‐inoculation with intact plasmid DNA of a tomato bushy stunt virus gene vector. J. Virol. 73:7823‐7829.
   Scholthof, H.B., Morris, T.J., and Jackson, A.O. 1993. The capsid protein gene of tomato bushy stunt virus is dispensable for systemic movement and can be replaced for localized expression of foreign genes. Mol. Plant‐Microbe Interact. 6:309‐322.
   Scholthof, H.B., Scholthof, K.‐B.G., and Jackson, A.O. 1996. Plant virus gene vectors for transient expression of foreign proteins in plants. Annu. Rev. Phytopathol. 34:299‐323.
   Scholthof, K.‐B.G., Mirkov, T.E., and Scholthof, H.B. 2002. Plant virus gene vectors: Biotechnology applications in agriculture and medicine. Genet. Eng (NY) 24:67‐85.
   Seidman, C.E., Struhl, K., Sheen, J., and Jessen, T. 1997. Introduction of plasmid DNA into cells. Curr. Protoc. Mol. Biol. 37:1.8.1‐1.8.10.
   Sit, T.L., Vaewhongs, A.A., and Lommel, S.A. 1998. RNA‐mediated trans‐activation of transcription from a viral RNA. Science 281:829‐832.
   Struhl, K. 1991. Subcloning of DNA fragments. Curr. Protoc. Mol. Biol. 13:3.16.1‐3.16.2.
   Tabor, S., Struhl, K., Scharf, S.J., and Gelfand, D.H. 1997. DNA‐dependent DNA polymerases. Curr. Protoc. Mol. Biol. 37:3.5.1‐3.5.15.
   Turina, M., Omarov, R.T., Murphy, J.F., Bazaldua‐Hernandez, C., Desvoyes, B., and Scholthof, H.B. 2003. A newly identified role for Tomato bushy stunt virus P19 in short distance spread. Mol. Plant Pathology 4:67‐72.
   Voytas, D. 2000. Agarose gel electrophoresis. Curr. Protoc. Mol. Biol. 51:2.5A.1‐2.5A.9.
   White, K.A., and Morris, T.J. 1999. Defective and defective interfering RNAs of monopartite plus‐strand RNA plant viruses. In Current Topics in Microbiology and Immunology: Satellites and Defective RNAs (P.K. Vogt and A.O. Jackson eds.) pp. 1‐17. Springer, New York.
   Zhang, G., Leung, C., Murdin, L., Rovinski, B., and White, K.A. 2000. In planta expression of HIV‐1 p24 protein using an RNA plant virus‐based expression vector. Mol. Biotechnology 14:99‐107.
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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