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Infiltration of Nicotiana benthamiana Protocol for Transient Expression via Agrobacterium   

Abstract

Materials and Reagents

1. Agrobacterium strain hosting a plant expression construct (usually driven by Cauliflower mosaic virus 35S promoter)
2. Healthy Nicotiana benthamiana (N. benthamiana) plants 2-4 weeks
3. MES
4. MgCl₂ stock
5. Antibiotics
6. Acetosyringone
7. LB media with appropriate antibiotics (see Recipes)
8. Acetosyringone stock (see Recipes)
9. MES-K (see Recipes)
10. Resuspension solution (see Recipes)
11. Acetosyringone datasheet (Sigma-Aldrich) (see Recipes)
    

Equipment

1. Centrifuge for 50 ml tubes
2. Spectrometer
3. Syringe
4. UV lamp (optional)
5. Fluorescence microscope (optional)
6. Confocal laser scanning microscope (optional)
   

Procedure

1. Inoculate one single colony of Agrobacterium in 5 ml LB with appropriate antibiotics. Grow overnight at 28-30 °C.
   Note: I usually use 100 μg/ml gentamicin (maintain the virulence of Agrobacterium strain GV3101) and 50 μg/ml spectinomycin (selective marker for shuttle vector) for most of the shuttle vectors.
   
2. Use 1 ml of the overnight culture to inoculate 25 ml LB (with same antibiotics, plus 20 μM acetosyringone added after autoclaving and immediately before use) and grow overnight.
3. Measure the A₆₀₀ of overnight culture. 
4. Precipitate the bacteria (5,000 x g, 15 min), resuspend the pellet in Resuspension Solution. The final A₆₀₀ should be adjusted to 0.4. 
5. Leave on the bench (room temperature) for 2-3 h (or overnight) before infiltration.
6. Perform the infiltration with 5 ml syringe. Simple press the syringe (no needle) on the underside of the leaf (Note: Avoid cotyledons), and exert a counter-pressure with finger on the other side. Successful infiltration is often observed as a spreading “wetting” area in the leaf.
7. (Optional) Check the GFP fluorescence by a portable long-wavelength UV lamp 2-5 days after infiltration. This only applies to strong expression of GFP signal (as green from red background).
8. Observe the fluorescence labeled protein under a fluorescent microscope or confocal laser scanning microscope. Or harvest leaves for protein purification.
   

Recipes

1. LB media with appropriate antibiotics
   Usually two antibiotics used: one to maintain Agrobacteria virulence, one for the shuttle vector
   
2. Acetosyringone stock
   100 mM in ethanol, stored at -20 °C
   
3. MES-K (0.5 M) (pH 5.6)
   First make 0.5 M MES, adjust pH with KOH to 5.6
   
4. Resuspension solution
   10 mM MgCl₂
   10 mM MES-K (pH 5.6)
   Autocloave 15 min
   100 μM acetosyringone (note: Added after autoclaving and immediately before using)
5. Acetosyringone datasheet
   Synonyms     3’, 5’-Dimethoxy-4’-hydroxyacetophenone
   Synonyms     Acetosyringone
   4’-Hydroxy-3’, 5’-dimethoxyacetophenone
   Molecular Formula              C₁₀H₁₂O₄
   Molecular Weight                196.20
   CAS Number                       2478-38-8
   Beilstein Registry Number   1966119
   EG/EC Number                   2196105
   MDL number                      MFCD00008748
   

References

1. Li, X., Chanroj, S., Wu, Z., Romanowsky, S. M., Harper, J. F. and Sze, H. (2008). A distinct endosomal Ca²⁺/Mn²⁺ pump affects root growth through the secretory process. Plant Physiol 147(4): 1675-1689.