Delivery and Expression of Functional Viral RNA Genomes In Planta by Agroinfiltration

Padmanaban Annamalai1, A. L. N. Rao1

1 University of California, Riverside, Riverside, California
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 16B.2
DOI:  10.1002/9780471729259.mc16b02s01
Online Posting Date:  June, 2006
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Abstract

Agroinfiltration is a simple, efficient, and powerful approach for transient expression of viral genes as well as DNA‐based expression of full‐length RNA genomes of plant viruses for studies leading to understanding of replication, movement, and assembly. Most importantly, it results in synchronous delivery of Agrobacterium transformants to a majority of cells encompassing the infiltrated area and is therefore ideal for examining the biological activities of viruses having multipartite genomes. Because of the high transformation rate and efficient accumulation of mRNAs, the method is also ideal for analyzing biological activities of viral genomes with defective replication and cell‐to‐cell movement characteristics.

Keywords: Agrobacterium tumefaciens; agroinfiltration; transient expression; expression of functional viral genes; Tiplasmid; viral genomes; RNA

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Agrobacterium Competent Cells for Transformation by Freeze‐thaw Method
  • Basic Protocol 2: Transformation of Competent Agrobacterium by Freeze‐thaw Method
  • Basic Protocol 3: Preparation of Agrobacterium Competent Cells for Transformation by Electroporation
  • Alternate Protocol 1: Glycerol Method for Preparation of Agrobacterium Electrocompetent Cells
  • Basic Protocol 4: Transformation of Competent Agrobacterium by Electroporation
  • Basic Protocol 5: Verification of Agrobacterium Transformation by Agarose GEL Electrophoresis
  • Alternate Protocol 2: Verification of Agrobacterium Transformation by PCR
  • Basic Protocol 6: Preparation and Infiltration of Transformed Agrobacterium Culture
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Agrobacterium Competent Cells for Transformation by Freeze‐thaw Method

  Materials
  • Agrobacterium strain (e.g., LBA 4404, C58C1, GV2260, or EHA 105; see Hellens et al., )
  • LB plates (see recipe)
  • LB medium (see recipe)
  • 150 mM NaCl: prepare from autoclaved 1 M NaCl with sterile H 2O and store up to 3 months at 4°C
  • 20 mM CaCl 2: prepare from filter‐sterilized (22‐µm filter) 1 M CaCl 2 and sterile H 2O; store up to 3 months at 4°C
  • Glycerol, sterile
  • 28°C incubator
  • 28°C orbital shaker
  • 250‐ml flask, sterile
  • 50‐ml polypropylene centrifuge tubes with tops, sterile (e.g., Fisher)
  • 1.5‐ml microcentrifuge tubes, sterile

Basic Protocol 2: Transformation of Competent Agrobacterium by Freeze‐thaw Method

  Materials
  • Competent Agrobacterium cells ( protocol 1), −80°C
  • Plasmid DNA: binary vector containing the gene of interest (e.g., see Sambrook and Russell, )
  • LB medium (see recipe)
  • LB plates with 50 µg/ml kanamycin and 50 µg/ml rifampicin (see recipe) or other appropriate antibiotic
  • 28°C orbital shaker
  • 28°C incubator

Basic Protocol 3: Preparation of Agrobacterium Competent Cells for Transformation by Electroporation

  Materials
  • Agrobacterium strain (e.g., LBA 4404, C58C1, GV2260, or EHA 105; see Hellens et al., )
  • LB plates (see recipe)
  • LB medium (see recipe)
  • 1 mM 4‐(2‐Hydroxyethyl)piperazine‐1‐ethane sulfonic acid (HEPES), pH 7: pass through a 0.45‐µm filter to sterilize; store up to 3 months at room temperature
  • Glycerol, sterile
  • 28°C incubator
  • 28°C orbital shaker
  • 250‐ml flask
  • 50‐ml polypropylene centrifuge tubes with tops, sterile (e.g., Fisher)
  • 1.5‐ml microcentrifuge tubes, sterile

Alternate Protocol 1: Glycerol Method for Preparation of Agrobacterium Electrocompetent Cells

  • SOB medium (see recipe)
  • 10% (v/v) glycerol, ice cold: prepare using autoclaved glycerol and sterile H 2O
  • 1.5‐ml microcentrifuge tubes, sterile

Basic Protocol 4: Transformation of Competent Agrobacterium by Electroporation

  • Electrocompetent Agrobacterium cells ( protocol 3 or protocol 4)
  • Plasmid DNA: binary vector containing gene of interest (e.g., see Sambrook and Russell, )
  • YEP or SOC medium (see reciperecipes)
  • YEP plates supplemented with 50 µg/ml kanamycin and 50 µg/ml rifampicin (see recipe) or other appropriate antibiotic
  • Electroporation cuvettes with 0.1‐ or 0.2‐cm path length (e.g., BioRad), ice cold
  • Electroporator (e.g., BioRad)
  • 1.5‐ml microcentrifuge tubes or 5‐ml plastic tubes, sterile
  • 28°C orbital shaker
  • 28°C incubator
  • Microcentrifuge

Basic Protocol 5: Verification of Agrobacterium Transformation by Agarose GEL Electrophoresis

  Materials
  • Transformed Agrobacterium cultures ( protocol 2 or protocol 5)
  • LB medium with appropriate antibiotics (see recipe)
  • Plasmid DNA extraction kit (e.g., QIAprep Spin miniprep kit; Qiagen; optional)
  • 28°C orbital shaker
  • Additional reagents and equipment for isolation (Engebrecht et al., ) and digestion (Sambrook and Russell, ) of plasmid DNA, and agarose gel electrophoresis (Voytas, )

Alternate Protocol 2: Verification of Agrobacterium Transformation by PCR

  Materials
  • Transformed Agrobacterium cultures ( protocol 2 or protocol 5)
  • H 2O, sterile
  • 10× reaction buffer (NEB or Promega)
  • 20 µM primer 1 (5′ forward primer of gene of interest)
  • 20 µM primer 2 (3′ reverse primer of gene of interest)
  • 2.0 mM dNTPs (Amersham or Promega)
  • 100 mM MgCl 2 (Promega)
  • Taq polymerase (Promega)
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, )

Basic Protocol 6: Preparation and Infiltration of Transformed Agrobacterium Culture

  Materials
  • Positively identified, transformed Agrobacterium cultures ( protocol 6 or protocol 7)
  • LB plates supplemented with 50 µg/ml kanamycin and 50 µg/ml rifampicin (see recipe) or other appropriate antibiotic
  • LB medium supplemented with 50 µg/ml kanamycin and 50 µg/ml rifampicin (see recipe) or other appropriate antibiotic
  • 1 M 2‐(N‐morpholino)ethanesulfonic acid (MES) pH 5.6: pass through a 0.45‐µm filter to sterilize and store up to 3 months at room temperature in a brown bottle
  • 100 mM 5′dimethoxy‐4′‐hydroxyacetophenone (acetosyringone) in DMSO
  • 10 mM MgCl 2: prepare from filter‐sterilized (0.45‐µm filter) 1 M MgCl 2 and sterile H 2O
  • Nicotiana benthamiana seedlings: five‐leaf stage, i.e., three‐ to four‐week‐old plants having two to three well expanded leaves, or other appropriate seedling
  • 28°C incubator
  • 28°C orbital shaker
  • 50‐ml plastic tubes, sterile (e.g., Fisher or Oak Ridge)
  • 1‐ml syringe without needle
  • Greenhouse, growth room, or 20°C to 23°C growth chamber with adjustable lighting conditions
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Figures

  •   FigureFigure 16.B0.1 (A) Events leading to Agrobacterium‐mediated transient gene expression in plant cells. Cauliflower mosaic virus 35S promoter (35S) initiates transcription of the gene of interest (GOI) which terminates at the Nos terminator (T). (B) Representative example of a typical binary vector suitable for agroinfiltration. The left (L) and right (R) borders indicate the sequences of 25‐bp imperfect repeats that flank the T‐DNA and are required for its transfer.

Videos

Literature Cited

Literature Cited
   Annamalai, P. and Rao, A.L.N. 2005. Agrobacterium‐mediated DNA expression of functional genome components of brome mosaic virus in planta: Transient expression of viral capsid protein and RNA packaging independent of replication. Virology 338:96‐111.
   Balucombe, D.C. 1999. Fast forward genetics based on virus induced gene silencing. Curr. Opin. Plant Biol. 2:109‐113.
   Been, M.D. 1994. Cis‐and trans‐acting ribozymes from human pathogen, hepatitis delta virus. Trends Biochem. Sci. 19:251‐256.
   Bendahmane, A., Querci, M., Kanyuka, K., and Baulcombe, D.C. 2000. Agrobacterium transient expression system as a tool for the isolation of disease resistance genes: Application to the R×2 locus in potato. Plant J. 21:73‐81.
   Boyer, J.C. and Haenni, A.L. 1994. Infectious transcripts and cDNA clones of RNA viruses. Virology 198:415‐426.
   Brigneti, G., Voinnet, O., Li, W.X., Ji, L.H., Ding, S.W., and Baulcombe, D.C. 1998. Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana. EMBO J. 17:6739‐6746.
   Bruening, G., Buzayan, J., Gerlach, W., and Hampel, A. 1988. Small RNA autolytic cleavage and ligation. In From Proteins to Ribosomes, Vol. 1 (R.H. Sarma and M.H. Sarma, eds.) pp. 239‐248. Adenine Press, Schenectady, New York.
   Daros, J.A. and Flores, R. 2004. Arabidopsis thaliana has the enzymatic machinery for replicating representative viroid species of the family pospiviroidae. Proc. Natl. Acad. Sci. U.S.A. 101:6792‐6797.
   Engebrecht, J., Brent, R., and Kaderbhai, M.A. 1991. Minipreps of plasmid DNA. 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. 1.6.1‐1.6.10. John Wiley & Sons, Hoboken, N.J.
   Grimsley, N., Hohn, T., Davies, J.W., and Hohn, B. 1987. Agrobacterium mediated delivery of infectious maize streak virus into maize plants. Nature 325:177‐179.
   Hellens, R., Mullineaux, P., and Klee, H. 2000. A guide to Agrobacterium binary Ti vectors. Trends Plant Sci. 5:446‐451.
   Ishikawa, M., Janda, M., Krol, M.A., and Ahlquist, P. 1997. In vivo DNA expression of functional brome mosaic virus RNA replicons in Sacchromyces cerevisiae. J. Virol. 71:7781‐7790.
   Johansen, L.K. and Carrington, J.C. 2001. Silencing on the spot. Induction and suppression of RNA silencing in Agrobacterium‐mediated transient expression system. Plant Physiol. 126:930‐938.
   Kapila, J., De Rycke, R., van Montagu, M., and Angenon, G. 1997. An Agrobacterium–mediated transient gene expression system for intact leaves. Plant Sci. 122:101‐108.
   Leiser, R.M., Ziegler‐Graff, V., Reutenauer, A., Herrbach, E., Lemaire, O., Guilley, H., Richards, K., and Jonard, G. 1992. Agroinfection as an alternative to insects for infecting with beet western yellows luteovirus. Proc. Natl. Acad. Sci. U.S.A. 89:9136‐9140.
   Li, H.W., Lucy, A.P., Guo, H.S., Li, W.X., Ji, L.H., Wong, S.M., and Ding, S.W. 1999. Strong host resistance targeted against a viral suppressor of the plant gene silencing defense mechanism. EMBO J. 18:2683‐2691.
   Liu, L. and Lomonossoff, G.P. 2002. Agroinfection as a rapid method for propagating cowpea mosaic virus based constructs. J. Virol. Methods 105:343‐348.
   Lu, R., Malcuit, I., Moffett, P., Ruiz, M.T., Peart, J., Wu, A.J., Rathjen, J.P., Bendahmane, A., Day, L., and Baulcombe, D.C. 2003. High‐throughput virus induced gene silencing implicates heat shock protein 90 in plant disease resistance. EMBO J. 22:5690‐5699.
   Palanichelvam, K., Cole, A.B., Shababi, M., and Schoelz, J.E. 2000. Agroinfiltration of cauliflower mosaic virus gene VI elicits hypersensitive response in Nicotiana species. Mol. Plant. Microbe Interact. 13:1275‐1279.
   Sambrook, J. and Russell, D.W. 2001. Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Vlot, C.A., Neelman, L., Linthorst, H.J.M., and Bol, J.F. 2001. Role of the 3′‐untranslated regions of alfalfa mosaic virus RNAs in the formation of a transiently expressed replicase in plants and in the assembly of virions. J. Virol. 75:6440‐6449.
   Voinnet, O., Rivas, S., Mestre, P., and Baulcombe, D.C. 2003. An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J. 33:949‐956.
   Voytas, D. 2000. Agarose gel electrophoresis. 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. 2.5A.1‐2.5A.9. John Wiley & Sons, Hoboken, N.J.
   Weigel, D. and Glazebrook, J. 2002. Arabidopsis: A laboratory manual, pp. 119‐141. Cold Spring Harbor Laboratory Press, New York.
   Yang, Y., Li, R., and Qi, M. 2000. In vivo analysis of plant promoters and transcription factors by agroinfiltration of tobacco leaves. Plant J. 22:543‐551.
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