Materials and Reagents

1 mL needleless plastic syringe (TERUMO, catalog number: SS-01T)
20 mL needleless plastic syringe (TERUMO, catalog number: SS-20ESz)
50 mL centrifuge tube (FALCON, catalog number: 352070)
GD/X syringe filter (PES 0.45 μm) (GE healthcare, catalog number: 6876-2504)
500 mL Pyrex beaker
300 mL Pyrex beaker
Vacuum desiccator
15 mL centrifuge tube (FALCON, catalog number: 352097)
Soil-grown Nicotiana benthamiana
pEAQ-HT plasmid (Sainsbury et al., 2009)
Agrobacterium tumefaciens C58C1 carrying pCH32 (Hamilton et al., 1996; Hellens et al., 2000)
LB Broth (Lennox) (Sigma-Aldrich, catalog number: L7275-500TAB)
Kanamycin (FUJIFILM Wako Chemicals, catalog number: 113-00343)
Rifampicin (FUJIFILM Wako Chemicals, catalog number: 185-01003)
4'-Hydroxy-3',5'-dimethoxyacetophenone (acetosyringone) (Sigma-Aldrich, catalog number: D134406-25G)
Bis-Tris (Dojindo, catalog number: 6976-37-0)
Tween 20 (polyoxyethylene sorbitan monolaurate) (Nacalai Tesque, catalog number: 35624-15)
cOmplete^TM ULTRA Tablets, EDTA-free, Protease Inhibitor Cocktail (Merck, catalog number: 5892953001)
Sodium Chloride (FUJIFILM Wako Chemicals, catalog number: 195-01663)
Magnesium Chloride (FUJIFILM Wako Chemicals, catalog number: 136-03995)
Immobilized Ni²⁺-affinity column, HisTrap excel (Cytiva, catalog number: 17371205)
Note: Whilst any standard Ni²⁺-affinity column should be acceptable, we recommend using the HisTrap excel column by Cytiva. In this method, the His-tagged protein will be purified in buffer at pH 6.0, given that apoplast space is usually weakly acidic. According to the manufacturer’s instructions, HisTrap excel can capture His-tagged proteins even at pH 6.0.
Superloop, 1/16" fittings (ÄKTAdesign), 50 mL (Cytiva, catalog number: 18111382)
Imidazole (FUJIFILM Wako Chemicals, catalog number: 095-00015)
Vivaspin Turbo 15 (10 kDa molecular weight cut off) (Sartorius, catalog number: VST15T01)
Coomassie protein stain, such as InstantBlue (Expedeon, catalog number: ISB01L)
Agroinfiltration buffer (see Recipes)
Vacuum infiltration buffer (see Recipes)
Equilibration buffer (see Recipes)
Wash buffer (see Recipes)
Elution buffer (see Recipes)

Equipment

Vacuum pump (e.g., ULVAC DTC-41, or equivalent model)
Electroporation system (e.g., Bio-Rad Gene Pulser XCell^TM, or equivalent)
Centrifuge with a swing rotor for 50 mL centrifuge tubes (e.g., Hitachi CF16RXII with swing rotor T4SS31, or equivalent)
Centrifuge with a fixed angle rotor for 15 mL centrifuge tubes (e.g., Hitachi CR20GIII with fixed angle rotor R15A, or equivalent)
Fast Protein Liquid Chromatography machine (we used the equivalent of GE healthcare AKTA pure 25 M1 (Cytiva, catalog number 29018227), with optional accessories installed).
Note: Any fast protein liquid chromatography machine from companies such as Cytiva (https://www.cytivalifesciences.com/) or from Bio-Rad (https://www.bio-rad.com/) are fine.
Spectrophotometer (e.g.,Thermo Fisher Scientific NanodDrop One^C, or equivalent)
Standard SDS-PAGE equipment (such as Mini-PROTEAN^® Tetra Cell from Bio-Rad). For the SDS-PAGE protocol, please refer to Green and Sambrook (2014).

Procedure

Expression of His-tagged CARD1 ectodomain in apoplastic space of N. benthamiana
Note: We expressed non-tagged CARD1ecto, and purified it by conventional column chromatographies in our previous study (Laohavisit et al., 2020). Although it is possible to purify non-tagged proteins, the purification procedure is simpler in His-tagged proteins.
1. Clone the nucleotide sequence encoding CARD1 from Arabidopsis genomic DNA (amino acids 1–546) into the pEAQ-HT vector, in frame with a His tag at C-terminus (CARD1ecto-His), using standard molecular biology techniques (Figure 1).
  Notes:
  1. The native signal peptide of CARD1 should be intact, so that CARD1ecto-His is secreted into the apoplastic space.
  2. In the pEAQ-HT vector system, a gene of interest is inserted between a modified 5’-untranslated region (UTR) and the 3’-UTR from Cowpea mosaic virus RNA-2, and co-expressed together with silencing suppressor p19 (Sainsbury et al., 2009; Sainsbury and Lomonossoff, 2008). This permits an extremely high-level and rapid production of proteins of interest (see Figure 1).
  3. We used genomic DNA to clone the construct, but cDNA should also work.
  4. For His-tag at C-terminus, pEAQ-HT should be digested with AgeI and SmaI restriction enzymes (Figure 1, see Sainsbury et al., 2009 for details).
  Figure 1. The pEAQ-HT vector map. Top, a simplified pEAQ-HT vector map. Bottom, a close-up of the restriction enzyme map.
2. After sequence confirmation, transform electrocompetent cells of Agrobacterium tumefaciens C58C1 with pEAQ-CARD1ecto-His, using an electroporation system (machine default settings for Agrobacterium tumefaciens are capacitor = 25 μF, pulse controller = 200 Ω, and Voltage = 2.4 kV). Plate out the transformed cells onto LB agar plates supplemented with 100 μg/mL rifampicin and 50 μg/mL kanamycin.
3. Confirm the Agrobacterium colonies with correct transformation using PCR, and make glycerol stocks for long term storage.
4. Pick a fresh colony or take a small portion of the glycerol stocks of Agrobacterium into 5 mL of LB liquid media supplemented with 100 μg/mL rifampicin and 50 μg/mL kanamycin and incubate it with shaking at 120 rpm at 28°C overnight.
5. Dilute 2 mL of overnight culture with 18 mL of fresh LB liquid media supplemented with 100 μg/mL rifampicin and 50 μg/mL kanamycin, and incubate with shaking at 120 rpm and 28°C for 4 h.
6. Centrifuge the Agrobacterium at 3,000 × g for 5 min, and discard the supernatant.
7. Add 10 mL of Agroinfiltration buffer, and resuspend the pellet.
8. Repeat steps 6–7.
9. Measure OD₆₀₀ of the suspension and adjust it to 0.3.
10. Infiltrate the Agrobacterium suspension into whole leaves of 5-weeks-old N. benthamiana from the abaxial side, using 1 mL needleless syringes. The infiltrated area should turn darker in color compared to the unfiltrated area (Yin et al., 2017).
  Note: In our experiment, we infiltrated approximately 30 leaves to obtain approximately 1 mg of highly purified CARD1ecto-His protein. For other proteins of interest, it is recommended that the experimenter perform small-scale pilot experiments (for instance, 8–10 leaves per construct) to ascertain how much protein can be obtained, since different proteins will show different expression levels.
Extraction of apoplastic fluid from N. benthamiana leaves
1. After 4–7 days post inoculation, harvest the infiltrated N. benthamiana leaves and pile them together, such that the abaxial side of the leaves points upward.
2. Carefully place the pile of leaves into a 500-mL Pyrex beaker containing 150 mL of vacuum-infiltration buffer. Afterward, place a 300-mL Pyrex beaker on top of the pile, such that leave samples are submerged in buffer (Figure 2).
  
  Figure 2. Set-up for vacuum infiltration.
3. Place the assembled beaker (with leave samples) into a vacuum desiccator, and apply a pressure of 60 hPa for 10 min using a pump (or other appropriate equipment). Bubbles should be released from the leaves.
4. Slowly release the pressure inside the desiccator. We usually allow at least 10 min for the pressure to return to normal.
  Note: In this step, expanded air bubbles in the apoplastic space shrink significantly and vacuum-infiltration buffer will replace this space. The color of the leaves should turn darker as the apoplastic space has been filled with buffer. If you do not notice any differences, this suggests that the vacuum infiltration process has not been successful, and step 3 should be carefully repeated.
5. Carefully take out individual leaves from the beaker and gently remove excess buffer using paper towels. Set these aside.
6. Meanwhile, assemble the apoplastic fluid collection unit. This consists of the following materials:
  1. 20-mL single-use needleless plastic syringe with the plunger removed (i.e., only the barrel part is needed)
  2. 50-mL centrifuge tubes, non-skirted
  To assemble the apoplastic fluid collection unit, simply place the barrel part of a 20-mL sized syringe into a 50-mL centrifuge tube (Figure 3A).
  
  Figure 3. Set-up of the apoplastic fluid collection unit.
7. Per apoplastic fluid collection unit, take 6–8 leaves from step 5 and separate them into two piles (3–4 leaves per pile). Next, carefully roll the leaves from one pile (from the base to the apex of the leaf) and place this into the barrel part of the apoplastic fluid collection unit. The abaxial or adaxial sides can be faced in any direction. Repeat for the second pile (Figure 3B).
  Note: For our experiment, we used approximately 30 leaves to purify CARD1ecto-His protein, which corresponded to 6 apoplastic fluid collection units.
8. Place the apoplastic fluid collection units (with leaves) in a swing rotor, and centrifuge at 1,500 × g and 4°C for 10 min.
  Note: Centrifugation should be performed using a swing rotor, to maximize recovery of the apoplast fluid.
9. Apoplastic fluid should appear at the bottom of the 50-mL centrifuged tubes. Transfer the fluid to a separate tube and centrifuge again at 1,500 × g and 4°C for 3 min, to ensure all fluid has been extracted. Finally, pool all the apoplastic fluid into a single tube.
10. Centrifuge the collected apoplastic fluid from step 9 at 10,000 × g and 4°C for 10 min. Collect the supernatant, and centrifuge it again at 10,000 × g and 4°C for 10 min.
11. Filter the supernatant with a GD/X syringe filter. The resulting filtrate is now ready for the next step of purification.
  Note: The filter unit should have a 0.45 µm pore size.
CARD1ecto-His purification by Ni²⁺-affinity chromatography
Note: Monitor the absorbance at 280 nm to detect proteins throughout the purification process.
1. Connect a HisTrap excel column (capacity of 5 mL) to an FPLC system (such as the AKTA pure), and wash this column with 25 mL of ultrapure water (5 column volumes, CV) at 5 mL/min (1 CV/min).
  Note: Wash volume and the flow rate should be adjusted according to the column size used.
2. Equilibrate the column with 25 mL (5 CV) of equilibration buffer at 5 mL/min (1 CV/min).
3. Load the apoplastic fluid sample onto the column with a superloop at 2.5 mL/min (0.5 CV/min).
  Note: Depending on your FPLC machine configuration and your sample volume, you can also load your sample manually.
4. Wash the column with 25 mL (5 CV) of wash buffer at 5 mL/min (1 CV/min).
5. Elute the His-tagged protein with 25 mL (5 CV) of elution buffer at 5 mL/min (1 CV/min).
6. Pool the peak fractions in one sampling tube and check the purity of CARD1ecto-His by SDS-PAGE, followed by Coomassie brilliant blue staining (InstantBlue; Expedeon) (Figure 4).
  
  Figure 4. SDS-PAGE analysis of purified CARD1ecto-His. Lane 1: Apoplastic fluid from intact N. benthamiana leaves. Lane 2: Apoplastic fluids from the CARD1ecto-His expressing N. benthamiana leaves. Lane 3: Purified CARD1ecto-His by Ni²⁺-affinity chromatography. The gel was stained with Coomassie brilliant blue (InstantBlue).
7. Measure protein concentration by absorbance at 280 nm, using a spectrophotometer (such as NanoDrop One^C).
8. (Optional) Apply the pooled fractions in an ultrafiltration unit with a 10-kDa cut-off membrane (Vivaspin turbo 15) for buffer exchange and/or concentration.
  Note: When buffer exchanging or sample concentration are necessary for downstream processes.

Recipes

Agroinfiltration buffer
10 mM MES/NaOH (pH 5.6)
10 mM MgCl₂
150 μM acetosyringone
Vacuum infiltration buffer
20 mM Bis-Tris/HCl (pH 6.0)
0.01% Tween20
1× cOmplete^TM Protease Inhibitor Cocktail
Equilibration buffer
20 mM Bis-Tris/HCl (pH 6.0)
300 mM NaCl
Wash buffer
20 mM Bis-Tris/HCl (pH 6.0)
300 mM NaCl
5 mM Imidazole
Elution buffer
20 mM Bis-Tris/HCl (pH 6.0)
300 mM NaCl
300 mM Imidazole

Acknowledgments

This protocol was adapted from our published work (Laohavisit et al., 2020). This work was supported by JSPS Grant-in-Aid for 17H06172 and 20H05909 to K.S.

Competing interests

The authors declare no competing interests.

References

Green, M.R. and Sambrook, J. (2014). Molecular Cloning: A Laboratory Manual (4^th Edition). Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press.
Hamilton, C.M., Frary, A., Lewis, C. and Tanksley, S.D. (1996). Stable transfer of intact high molecular weight DNA into plant chromosomes. Proc Natl Acad Sci U S A 93(18): 9975-9979.
Hellens, R., Mullineaux, P. and Klee, H. (2000). Technical Focus: a guide to Agrobacterium binary Ti vectors. Trends Plant Science 5(10): 446-451.
Laohavisit, A., Wakatake, T., Ishihama, N., Mulvey, H., Takizawa, K., Suzuki, T. and Shirasu, K. (2020). Quinone perception in plants via leucine-rich-repeat receptor-like kinases. Nature 587(7832): 92-97.
Meyer, A.J., Riemer, J. and Rouhier, N. (2019). Oxidative protein folding: state-of-the-art and current avenues of research in plants. New Phytol 221(3): 1230-1246.
Sainsbury, F. and Lomonossoff, G. P. (2008). Extremely high-level and rapid transient protein production in plants without the use of viral replication. Plant Physiol 148(3): 1212-1218.
Sainsbury, F., Thuenemann, E. C. and Lomonossoff, G. P. (2009). pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants. Plant Biotechnol J 7(7): 682-693.
Souza, A. D. (2015). Expression and partial purification of His-tagged proteins in a plant system. Bio-protocol 5(17). e1572.
Yin, K., Han, T. and Liu, Y. (2017). Use of Geminivirus for delivery of CRISPR/Cas9 components to tobacco by Agro-infiltration. Bio-protocol 7(7): e2209.