Materials and Reagents

Materials
1. Soil mix or substrate such as Compo Sana^® Universal Ligera (COMPO, TSUSTPROF25L)
2. Plant pots such as Desch vol 11 (Desch Plantpak, catalog number: 1055278 )
3. Seed tray–40 cavities
4. Plant trays
5. Cling film
6. Syringes 1 ml or 2 ml
7. Tissue paper
8. Petri dishes
9. Falcon tubes
10. Eppendorf tubes
11. Pipette tips
12. Sterile toothpicks
13. Syringe filter 0.22 µm
Biological materials
1. Arabidopsis thaliana NahG seeds (Lawton et al., 1995)
2. Agrobacterium tumefaciens strain (LBA4404, GV3101 or C58C1) carrying a binary vector with the gene of interest
Reagents
1. Sterile deionized water
2. Glycerol (CARLO ERBA Reagents, catalog number: 453752 )
3. NaCl (AppliChem, catalog number: 121659.1210 )
4. Tryptone (Biolife, catalog number: 412290 )
5. Yeast Extract (AppliChem, catalog number: 403687.1210 )
6. Bacteriological agar (MICROKIT, catalog number: BCB006+ )
7. MES (2-(N-morpholino) ethanesulfonic acid) (Sigma-Aldrich, catalog number: M2933 )
8. MgCl₂ (AppliChem, catalog number: 131396.1210 )
9. DMSO (Sigma-Aldrich, catalog number: M81802 )
10. Acetosyringone (Sigma-Aldrich, catalog number: D134406 )
11. Rifampicin (Duchefa Biochemie, catalog number: R0146.0005 )
12. Tetracycline (Sigma-Aldrich, catalog number: T3383 )
13. Gentamycin (Sigma-Aldrich, catalog number: G3632 )
14. Spectinomycin (Duchefa Biochemie, catalog number: S0188.0005 )
15. Kanamycin (Sigma-Aldrich, catalog number: K4378 )
16. LB medium (see Recipes)
17. 1 M MES (see Recipes)
18. 1 M MgCl₂ (see Recipes)
19. 0.1 M Acetosyringone (see Recipes)
20. Infiltration solution (see Recipes)
21. Antibiotics solution (depending on construct and Agrobacterium strain, see Recipes)

Equipment

Sterile Erlenmeyer flasks
Plant growth chamber capable of sustaining 21 °C under short-day conditions (8 h light/16 h dark) with 140-150 µmol m^-2 sec^-1 light intensity (Radiber SA, catalog number: AGP-1400 )
Spectrophotometer capable of OD₆₀₀ measurements such as Shimadzu UV-1601 (Shimadzu, catalog number: 206-67001-34 )
Automatic P1000, P200 and P20 micropipettes
Incubator set at 28 °C such as Incubator D-6450 Hanau (Heraeus Instruments, model: D-6450 )
Incubator shaker capable of sustaining 28 °C and 180 rpm such as New Brunswick^TM I26 (Eppendorf, New Brunswick scientific, model: I26, catalog number: M1324-0000 )
Centrifuge for 50 ml tubes such as Rotofix 32A (Hettich, catalog number: 1206-01 )
Autoclave

Procedure

Plant growth conditions
Note: The preparation of Arabidopsis NahG plants is a key step in obtaining a satisfactory transient gene expression/protein expression. In this protocol, ~4 weeks old Arabidopsis NahG plants are used for Agrobacterium infiltration.
1. Sow seeds in water-soaked soil mix in a plant pot. Cover the pot with cling film and place it in a growth chamber with 8 h light/16 h dark cycle at 21 °C.
2. Grow until the seedlings have two true leaves (around 7-10 days).
3. Carefully transplant seedlings to the final destination in seed trays with 40 cavities.
4. Grow plants for approximately 3 more weeks inside the growth chamber with 8 h light/16 h dark cycle at 21 °C.
5. Plants are ready for infiltration when they have a robust rosette of 10-12 leaves and have not started flowering.
Agrobacterium cultures
Note: This protocol can be used with, at least, three different commonly used strains of Agrobacterium: LBA4404, GV3101 and C58C1.
1. Using a glycerol stock and a sterile toothpick, streak the Agrobacterium clone(s) to be used in LB solid plates supplemented with the appropriate antibiotics. Place the plates inside a 28 °C incubator for 48 h to obtain fresh and single colonies.
2. The day before starting the infiltration, start liquid Agrobacterium cultures in LB liquid medium using the fresh colonies on the plates. Pick Agrobacterium biomass from a single colony, using a sterile toothpick, place it inside a sterile Erlenmeyer flask with 20 ml LB liquid media supplemented with the appropriate antibiotics, and culture them at 28 °C and 180 rpm overnight.
Infiltration
1. Agrobacterium preparation
  1. Pour saturated cultures into 50 ml Falcon tubes. Spin down cells at 4,000 x g for 10 min.
  2. Discard LB medium supernatant by decanting. Eliminate as much supernatant as possible.
  3. Resuspend with vortex the cell pellets using 1 volume of freshly prepared infiltration buffer.
  4. After resuspension, leave cultures for 2-4 h in darkness at room temperature.
  5. Prepare a 1/20 dilution of the saturated culture, measure OD₆₀₀ and calculate necessary volume to have a final OD₆₀₀ of 0.05. Dilute using infiltration buffer.
2. Infiltration procedure
  1. Fill a 1 or 2 ml needleless syringe with the resuspended culture at a final OD₆₀₀ of 0.05. Perform the infiltration by pressing the syringe (without needle) on the abaxial side of the leaf while exerting counter-pressure with a fingertip on the adaxial side. Observe how the liquid spreads within the leaf if the infiltration is successful. Infiltrate whole leaves (ca. 100 µl of bacterial suspension/leave).
  2. Dry the excess of culture from the leaf surface using tissue paper (Figure 1, Video 1).
  3. Two to four days after infiltration, observe fluorescence of infiltrated proteins or harvest infiltrated leaves to do a protein extraction.
    
    Figure 1. Experimental procedure of transient expression in NahG Arabidopsis thaliana plants using Agrobacterium tumefaciens. A. Eppendorf tube with Agrobacterium carrying the desired construct, 1 ml syringe, 4-week-old NahG Arabidopsis plants, and piece of paper. B. Plant infiltration (see details in the procedure section).
    
    Video 1. Experimental procedure of transient expression in NahG Arabidopsis thaliana plants using Agrobacterium tumefaciens

Notes

Some leaves might suffer necrosis after infiltration with the bacterial suspension.
Growing NahG Arabidopsis under long day condition (16 h light/8 h dark cycle), might not affect the transient expression if the plants have not started flowering. However, plant leaves are bigger and easier to infiltrate when plants are grown in short day conditions.
It is also possible to adjust the OD₆₀₀ first and then incubate the cultures for 2-4 h in darkness.
Infiltrate no more than 4-5 leaves per plant. Survival rate of the plants diminishes considerably when more leaves are infiltrated.
Using this protocol the level of protein accumulation is optimal at 3 days after infiltration.
Practically any leaf of the rosette can be infiltrated with positive results but the eldest leaves tend to show a lower signal and the youngest leaves tend to show a stronger response to the bacterial inoculation, with the concomitant necrosis.
Drying the excess of culture is not determinant for the success of the infiltration but highly recommendable due to leaves sticking together when wet, causing higher levels of necrosis.

Recipes

LB medium (1 L)
NaCl 5 g
Tryptone 10 g
Yeast extract 5 g
(Only for solid medium) Bacteriological agar 16 g
1 M MES (100 ml)
17.5 g MES
Complete with sterile deionized water
Adjust the pH to 5.6
Sterilize by filtration
Store at room temperature
1 M MgCl₂ (100 ml)
20.3 g MgCl₂
Complete with sterile deionized water
Sterilize by autoclaving
Store at room temperature
0.1 M Acetosyringone (10 ml)
0.196 g acetosyringone
10 ml DMSO
Prepare 1 ml aliquots and store them at -20 °C
Infiltration solution (100 ml)

Top up with sterile deionized water
Note: Prepare infiltration solution just before use.
Antibiotics solution

Acknowledgments

The authors thank Huang Tan for his technical help during the imaging. This work was supported by the Spanish Ministerio de Ciencia y Tecnología (AGL2016-75819-C2) and the Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences. This protocol is derived from a previous publication (Rosas-Díaz et al., 2017).

Competing interests

The authors declare that they have no conflict of interest.

References

Krenek, P., Samajova, O., Luptovciak, I., Doskocilova, A., Komis, G. and Samaj, J. (2015). Transient plant transformation mediated by Agrobacterium tumefaciens: Principles, methods and applications. Biotechnol Adv 33(6 Pt 2): 1024-1042.
Lawton, K., Weymann, K., Friedrich, L., Vernooij, B., Uknes, S. and Ryals, J. (1995). Systemic acquired resistance in Arabidopsis requires salicylic acid but not ethylene. Mol Plant Microbe Interact 8(6): 863-870.
Rosas-Díaz, T., Cana-Quijada, P., Amorim-Silva, V., Botella, M. A., Lozano-Duran, R. and Bejarano, E. R. (2017). Arabidopsis NahG plants as a suitable and efficient system for transient expression using Agrobacterium tumefaciens. Mol Plant 10(2): 353-356.