Transfecting Protoplasts by Electroporation to Study Viroid Replication

Xuehua Zhong1, Asuka Itaya1, Biao Ding1

1 Ohio State University, Columbus, Ohio
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 16D.4
DOI:  10.1002/9780471729259.mc16d04s00
Online Posting Date:  July, 2005
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Abstract

Protoplasts have been extensively used to study replication of plant viruses at the cellular level. However, their use in studying viroid replication has been very limited because of various technical barriers. As a result, mechanisms of viroid replication remain poorly understood. The electroporation protocol presented in this unit offers a simple, fast and reproducible method to inoculate protoplasts derived from cultured cells of tobacco (Nicotiana tabacum) and the related species Nicotiana benthamiana to study viroid replication. The protocol has proven to be useful in characterizing sequence/structural features of potato spindle tuber viroid (PSTVd) that are important for replication at the cellular level, and may be adapted to study the replication of other viroids.

Keywords: viroid; RNA; replication; protoplast; electroporation

     
 
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Table of Contents

  • Basic Protocol 1: Transfecting Protoplasts with Viroids by Electroporation
  • Support Protocol 1: Synthesis of RNA Inoculum for Transfecting Protoplasts with Potato Spindle Tuber Viroid
  • Support Protocol 2: Extraction of Total RNA From Transfected Protoplasts
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Transfecting Protoplasts with Viroids by Electroporation

  Materials
  • Tobacco BY‐2 (N. tabacum L. var Bright Yellow 2) cultured cells; available from many sources including authors' laboratory ( )
  • N. benthamiana cultured cells (Qi and Ding, ; Sunter and Bisaro, ); available from authors' laboratory ( )
  • Murashige & Skoog (MS) medium (see recipe)
  • Digestion and washing buffer (see recipe) containing 1.5% (w/v) cellulase (“Onozuka” RS; Yakult) and 0.2% (w/v) macerase (Calbiochem‐Novabiochem)
  • Digestion and washing buffer (see recipe), prechilled to 4°C
  • Electroporation buffer (see recipe), prechilled to 4°C
  • RNA prepared from plasmid containing PSTVd cDNA by in vitro transcription (see protocol 2)
  • Protoplast culturing solution (see recipe)
  • Protoplast culturing solid medium (see recipe) in 6‐well culture plates
  • RNeasy Plant Mini Kit (Qiagen; also see protocol 3)
  • 250‐ml flasks
  • 50‐ml sterile polypropylene conical tubes (e.g., Fisher)
  • Swinging‐bucket centrifuge (e.g., Marathon 21000R, Fisher)
  • Platform shaker (New Brunswick Scientific)
  • 100‐ and 40‐µm nylon mesh (Spectrum)
  • 400‐ml beakers
  • Electroporator Plus and 4‐mm‐gap electroporation cuvettes (all available from BTX; also see )
  • Disposable sterile 1.5‐ml microcentrifuge tubes (Fisher)
  • UV spectrophotometer (e.g., Thermo Spectronic)
  • Additional reagents and equipment for RNA quantitation (Gallagher, ) and northern blot analysis (Brown et al., )
NOTE: Autoclave all glassware and plasticware prior to beginning this protocol, unless they are purchased sterile.NOTE: Use large‐orifice pipet tips to handle protoplasts. Prepare large‐orifice 200‐ and 1000‐µl pipet tips by cutting tips and autoclave them. Large‐orifice tips are commercially available as well. The wider opening is necessary to avoid damaging the protoplasts.NOTE: When testing replication of PSTVd mutants or a different type of viroid, include wild‐type PSTVd as a positive control. In all experiments, use mock‐ (i.e., water‐) inoculated cells as a negative control.

Support Protocol 1: Synthesis of RNA Inoculum for Transfecting Protoplasts with Potato Spindle Tuber Viroid

  Materials
  • Plasmid containing PSTVd cDNA with T7, T3, or SP6 promotor, suitable for in vitro transcription (e.g., pRZ6‐2; available from the authors; )
  • Restriction endonuclease (e.g., HindIII for pRZ6‐2) and buffer (e.g., Invitrogen)
  • MEGAscript kit (Ambion)
  • MEGAclear kit (Ambion)
  • UV spectrophotometer (e.g., Thermo Spectronic)
  • Additional reagents and equipment for restriction digestion (Bloch and Grossmann, ), in vitro transcription (Tabor, ), and RNA quantitation (Gallagher, )

Support Protocol 2: Extraction of Total RNA From Transfected Protoplasts

  Materials
  • TRIzol Reagent (Invitrogen)
  • Transfected protoplasts, harvested (see protocol 1, steps to )
  • 25:24:1 phenol:chloroform:isoamyl alcohol ( appendix 2A)
  • 100% and 75% (v/v) ethanol
  • 5 M ammonium acetate (see appendix 2A for 10 M)
  • DEPC‐treated H 2O ( appendix 2A)
  • UV spectrophotometer (e.g., Thermo Spectronic)
  • Additional reagents and equipment for RNA quantitation (Gallagher, ) and northern blot analysis (Brown et al., )
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Figures

Videos

Literature Cited

   Bloch, K.D. and Grossmann, B. 1995. Digestion of DNA with restriction endonucleases. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 3.1.1‐3.1.21. John Wiley & Sons, Hoboken, N.J.
   Branch, A.D. and Robertson, H.D. 1984. A replication cycle for viroids and other small infectious RNAs. Science 223:450‐454.
   Brown, T. Mackey, K., and Du, T. 2004. Analysis of RNA by northern and slot blot hybridization. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 4.9.1‐4.9.19. John Wiley & Sons, Hoboken, N.J.
   Faustmann, O., Kern, R., Sänger, H.L., and Mühlbach, H.‐P. 1986. Potato spindle tuber viroid (PSTV) RNA oligomers of (+) and (−) polarity are synthesized in potato protoplasts after liposome‐mediated infection with PSTV. Virus Res. 4:213‐227.
   Feldstein, P.A., Hu, Y., and Owens, R.A. 1998. Precisely full length, circularizable, complementary RNA: An infectious form of potato spindle tuber viroid. Proc. Natl. Acad. Sci. U.S.A. 95:6560‐6565.
   Flores, R., Randles, J.W., Bar‐Josef, M., and Diener, T.O. 2000. Subviral agents: Viroids. In Virus Taxonomy, Seventh Report of the International Committee on Taxonomy of Viruses (M.H.V. van Regenmortel, C.M. Fauquet, D.H.L. Bishop, E.B. Carstens, M.K. Estes, S.M. Lemon, J. Manilof, M.A. Mayo, D.J. McGeoch, C.R. Pringle, and R.B. Wickner, eds.) pp. 1009‐1024. Academic Press, San Diego.
   Gallagher, S. 2004. Quantitation of DNA and RNA with absorption and fluorescence spectroscopy. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. A.3D.1‐A.3D.12. John Wiley & Sons, Hoboken, N.J.
   Hammond, R.W. and Owens, R.A. 1987. Mutational analysis of potato spindle tuber viroid reveals complex relationships between structure and infectivity. Proc. Natl. Acad. Sci. U.S.A. 84:3967‐3971.
   Hu, Y., Feldstein, P.A., Bottino, P.J., and Owens, R.A. 1996. Role of the variable domain in modulating potato spindle tuber viroid replication. Virology 219:45‐56.
   Hu, Y, Feldstein, P.A., Hammond, J., Hammond, R.W., Bottino, P.J., and Owens, R.A. 1997. Destabilization of potato spindle tuber viroid by mutations in the left terminal loop. J. Gen. Virol. 78:1199‐1206.
   Loss, P., Schmitz, M., Steger, G., and Riesner, D. 1991. Formation of a thermodynamically metastable structure containing hairpin II is critical for infectivity of potato spindle tuber viroid RNA. EMBO J. 10:719‐727.
   Mühlbach, H.‐P. and Sänger, H.L. 1977. Multiplication of cucumber pale fruit viroid in inoculated tomato leaf protoplasts. J. Gen. Virol. 35:377‐386.
   Owens, R.A., Hammond, R.W., Gardner, R.C., Kiefer, M.C., Thompsom, S.M., and Cress, D.E. 1986. Site‐specific mutagenesis of potato spindle tuber viroid cDNA. Plant Mol. Biol. 6:179‐192.
   Owens, R.A., Thompson, S.M., and Steger, G. 1991. Effects of random mutagenesis upon potato spindle tuber viroid replication and symptom expression. Virology 185:18‐31.
   Owens, R.A., Chen, W., Hu, Y., and Hsu, Y.‐H. 1995. Suppression of potato spindle tuber viroid replication and symptom expression by mutations which stabilize the pathogenicity domain. Virology 208:554‐564.
   Owens, R.A., Steger, G., Hu., Y., Fels, A., Hammond, R.W., and Riesner, D. 1996. RNA structural features responsible for potato spindle tuber viroid pathogenicity. Virology 222:144‐158.
   Qi, Y. and Ding, B. 2002. Replication of potato spindle tuber viroid in cultured cells of tobacco and Nicotiana benthamiana: The role of specific nucleotides in determining replication levels for host adaptation. Virology 302:445‐456.
   Qi, Y. and Ding, B. 2003a. Inhibition of cell growth and shoot development by a specific nucleotide sequence in a noncoding viroid RNA. Plant Cell 15:1360‐1374.
   Qi, Y. and Ding, B. 2003b. Differential subnuclear localization of RNA strands of opposite polarity derived from an autonomously‐replicating viroid. Plant Cell 15:2566‐2577.
   Qi, Y., Pélissier, T., Itaya, A., Hunt, E., Wassenegger, M., and Ding, B. 2004. Direct role of a viroid RNA motif in mediating directional RNA trafficking across a specific cellular boundary. Plant Cell 16:1741‐1752.
   Qu, F., Heinrich, C., Loss, P., Steger, G., Tien, P., and Riesner, D. 1993. Multiple pathways of reversion in viroids for conservation of structural elements. EMBO J. 12:2129‐2139.
   Semancik, J.S. and Conejero‐Tomas, V. 1987. Viroid pathogenesis and expression of biological activity. In Viroids and Viroid‐Like Pathogens (J. S. Semancik, ed.) pp. 71‐126. CRC Press, Boca Raton, Fla.
   Sinha, A., Wetten, A.C., and Caligari, P.D.S. 2003. Effect of biotic factors on the isolation of Lupinus albus protoplasts. Aust. J. Bot. 51:103‐109.
   Sunter, G. and Bisaro, D.M. 2003. Identification of a minimal sequence required for activation of the tomato golden mosaic virus coat protein promoter in protoplasts. Virology 305:452‐462.
   Tabler, M. and Sänger, H.L. 1984. Cloned single‐ and double‐stranded DNA copies of potato spindle tuber viroid (PSTV) RNA and co‐inoculated subgenomic DNA fragments are infectious. EMBO J. 3:3055‐3062.
   Tabler, M. and Tsagris, M. 2004. Viroids: Petite RNA pathogens with distinguished talents. Trends Plant Sci. 9:339‐348.
   Tabor, S. 1987. DNA‐dependent RNA polymerases. In Current Protocols in Microbiology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 3.8.1‐3.8.4. John Wiley & Sons, Hoboken, N.J.
Key References
   Qi and Ding, 2002. See above.
  Establishes electroporation protocol to inoculate tobacco and N. benthamiana protoplasts for studying PSTVd replication.
Internet Resources
   http://www.btxonline.com/products/
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