Materials and Reagents

0.22 µm sterilization filter (Merck Millipore, catalog number: SLGS033SB )
Nitrocellulose membrane (GE Healthcare, code number: 10401196 )
Collodion-coated grid (Ted Pella, Inc., catalog number: 12575-CU )
Cultured rice cells of line OC (Oryza sativa C5928; obtained from RIKEN BioResource Center)
Acidovorax avenae N1141 (MAFF 301141) and K1 (MAFF301755) (National Institute of Agrobaiological Sciences genebank, catalog number: MAFF 301141 ; MAFF301755 )
Skim milk powder (Wako, catalog number: 190-12865 )
Sodium hydrogen L(+)-glutamate monohydrate (Wako, catalog number: 198-02035 )
Luria-Bertani (LB) liquid medium (MO BIO laboratories, catalog number: 12107-05 )
2-amino-2-hydroxymethyl-1,3-propanediol (Tris) (Wako, catalog number: 204-07885 )
NaCl (Nacalai Tesque, catalog number: 31320-05 )
KCl (Nacalai Tesque, catalog number: 28514-75 )
1% (w/v) phosphotungstic acid (pH 6.9) (Sigma-Aldrich, catalog number: 79690 )
Sodium dodecyl sulfate (SDS) (Wako, catalog number: 191-07145 )
Molecular-weight marker low (APRO, catalog number: SP-0110 )
Anti-flagellin rabbit antibody (Eurofins Genomics)
Note: Flagellin purified from A. avenae N1141 strain as antigen was injected in rabbit. Anti-flagellin rabbit antibody was purified from rabbit antiserum (anti-flagellin) using flagellin purified from A. avenae N1141 strain.
Goat HRP conjugated anti-rabbit IgG antibody (H + L chain) [Medical & Biological Laboratories (MBL), catalog number: 458 ]
ECL plus Western blotting detection reagents (GE Healthcare, code number: RPN2132 )
Hybri-Bag (Cosmo Bio, catalog number: S-1001 )
Potassium ferricyanide (Wako, catalog number: 169-03721 )
KNO₃ (Nacalai Tesque, catalog number: 28704-85 )
(NH₄)₂SO₄ (Wako, catalog number: 019-03435 )
MgSO₄·7H₂O (Nacalai Tesque, catalog number: 21003-75 )
CaCl₂·2H₂O (Nacalai Tesque, catalog number: 06731-05 )
NaH₂PO₄·2H₂O (Wako, catalog number: 192-02815 )
MnSO₄·5H₂O (Nacalai Tesque, catalog number: 21229-35 )
ZnSO₄·7H₂O (Nacalai Tesque, catalog number: 37011-75 )
CuSO₄·5H₂O (Nacalai Tesque, catalog number: 09605-04 )
H₃BO₃ (Wako, catalog number: 021-02195 )
Na₂MoO₄·2H₂O (Nacalai Tesque, catalog number: 31621-52 )
EDTA·2Na (DOJINDO, catalog number: 345-01865 )
FeSO₄·7H₂O (Wako, catalog number: 098-01085 )
MS vitamin powder 1,000x (Sigma-Aldrich, catalog number: M7150-100ML )
2, 4-dichlorophenoxyacetic acid (Wako, catalog number: 040-18532 )
Sucrose (Wako, catalog number: 196-00015 )
3-mercapto-1,2-propanediol (Wako, catalog number: 131-16451 )
Glycerol (Nacalai Tesque, catalog number: 17018-25 )
Bromophenol blue (BPB) (Wako, catalog number: 021-02911 )
Glycine (Wako, catalog number: 077-00735 )
Coomassie brilliant blue R-250 (CBB) (Wako, catalog number: 031-17922 )
Methanol (Wako, catalog number: 137-01823 )
Acetic acid (Wako, catalog number: 017-00251 )
Polyoxyethylene sorbitan monolaurate (Tween 20) (Wako, catalog number: 167-11515 )
KH₂PO₄ (Wako, catalog number: 169-04245 )
Skimmed milk solution (see Recipes)
Acidovorax avenae N1141 and K1 strain skimmed milk stock solution (see Recipes)
25 mM TBS buffer (pH 7.4) (see Recipes)
20x R2 Major solution (see Recipes)
1,000x R2 Minor solution (see Recipes)
500x Fe liquid solution (see Recipes)
1,000x MS vitamin solution (see Recipes)
2, 4-dichlorophenoxyacetic acid solution (see Recipes)
R2S medium (pH 5.6) (see Recipes)
2x sample buffer (pH 6.8) (see Recipes)
Electrophoresis buffer (see Recipes)
CBB staining solution (see Recipes)
Destaining solution (see Recipes)
Transfer buffer (see Recipes)
Tris buffered saline with Tween 20 (TBST) buffer (see Recipes)
Blocking buffer (see Recipes)
50 mM potassium phosphate buffer (pH 7.9) (see Recipes)
Luminol solution (see Recipes)
Potassium ferricyanide solution (see Recipes)

Equipment

Rotary shaker (TAITEC, model: NR-20 )
High-speed refrigerated micro centrifuge (TOMY, model: MX-300 )
High-speed refrigerated centrifuge (Hitachi koki, model: CR20G )
Ultracentrifuge (Hitachi koki, model: CP70MX )
Fiber blender (Panasonic, model: MX-X58-SW )
Incubator (TAITEC, model: BR-42FL· MR )
Transmission electron microscope (Hitachi, model: H-7100 )
Plant growth chamber (NK system, model: LH-411SP )
Electrophoresis tank (ATTO, model: AE-6530M )
Semi-dry blotter (Bio-Rad, catalog number: 1703940JA )
Luminescent image analyzer (GE Healthcare, model: ImageQuant LAS 4000 )
Lumi-counter (ATTO, model: AB-2350 )

Procedure

Purification of flagellar filaments from Acidovorax avenae
1. For pre-culture of Acidovorax avenae, add 2 µl of A. avenae skimmed milk stock solution to 2 ml of LB liquid medium, and shake (200 rpm) for overnight at 30 °C.
2. Add 100 µl of pre-cultured suspension to one liter fresh LB medium, and shake (200 rpm) for 24 h at 30 °C.
3. Harvest A. avenae cells by centrifugation at 6,000 x g for 20 min at room temperature.
4. Add 300 ml of 25 mM TBS buffer to the pellet and re-suspend. After centrifugation at 6,000 x g for 20 min at room temperature, discard the supernatant.
5. Re-suspend the pellet with 90 ml of TBS buffer, and transfer to fiber blender.
6. To take off flagellum from bacterial cells, shear with fiber blender for 1 min at 4 °C, and incubate for 5 min on ice. Repeat this step 7 times.
7. To remove intact bacterial cells, centrifuge the bacterial suspension at 6,000 x g for 30 min at 4 °C and save the supernatant.
8. To remove bacterial cellular debris, centrifuge the supernatant at 16,000 x g for 60 min at 4 °C and save the supernatant.
9. Ultracentrifuge the supernatant at 200,000 x g for 60 min at 4 °C and discard the supernatant.
10. Add 1.5 ml of ice-cold distilled water to the pellet and re-suspend.
11. Centrifuge the re-suspended pellet at 20,000 x g for 20 min at 4 °C, and discard the supernatant. The pellet contains the monomer and polymer flagellins.
12. Re-suspend the pellet with 0.5 ml of ice-cold distilled water and store at -80 °C.
Observation of purified flagellar filaments using transmission electron microscopy
1. 50 µl droplet of the purified flagellar filaments (pellet of 20,000 x g) are used.
2. Absorb the purified flagellum onto collodion-coated grids, which are supported with carbon and rendered the carbon surface hydrophilic, for 1 min.
3. Stain the grids with 1% (w/v) phosphotungstic acid (pH 6.9) for 1 min and wash with two drops of distilled water.
4. Images are taken as digitized pictures with transmission electron microscope operated at 80 kV.
Purity check of flagellin in the purified flagellar filaments by Western blotting
1. Flagellar filaments of A. avenae consists of only flagellin protein. Purity of flagellin in the flagellar filaments is analyzed by SDS-PAGE and Western blotting.
2. Add half volume of 2x sample buffer to 100 ng of purified flagellum suspension.
3. Heat the sample at 95 °C for 5 min, and cool on ice.
4. Load 20 ng of sample and 2 µl of Prestained XL-Ladder Low marker of low range onto SDS-PAGE gel.
5. Electrophoresis at constant 100 Volts until the dye reaches the bottom of the gel.
6. Electrotransfer to nitrocellulose membrane using transfer buffer (constant 10 Volts for 3 h).
7. Incubate membrane in 25 ml of blocking buffer for 1 h at room temperature.
8. Wash three times for 5 min each with 25 ml of TBST.
9. Incubate membrane with primary Anti-flagellin rabbit antibody (1:2,500) in 10 ml of TBST for 1 h at room temperature.
10. Wash five times for 5 min each with 25 ml of TBST.
11. Incubate membrane with secondary goat HRP conjugated anti-rabbit IgG antibody (1:2,000) in 10 ml of TBST for 30 min room temperature.
12. Wash three times for 5 min each with 25 ml of TBST.
13. Incubate membrane with 1 ml of ECL plus Western blotting detection reagent for 3 min at room temperature, and then wrap in Hybri-Bag.
14. Measure the intensity of each band with LAS-4000.
H₂O₂detection and quantification
1. Suspension cultures of rice cells (line OC) were grown at 30 °C under white fluorescent light irradiation in a plant growth chamber. The cells were diluted in fresh medium every week, and experiment for H₂O₂ detection was performed 4 days after transfer.
2. Place the cultured rice cells onto filter paper to remove the culture medium. Add 10 mg culture cells into 0.5 ml of fresh medium, and culture the cells in a plant growth chamber for 2 h at 30 °C.
3. Add the flagellin (100 nM) to the cultured cells, and incubate in a plant growth chamber at 30 °C. Less than 100 µl of the flagellin suspension is desirable for addition to a culture medium.
4. After incubation, add 10 µl of culture medium to chemiluminescence reaction buffer containing 160 µl of 50 mM potassium phosphate buffer (pH 7.9), 10 µl of 1.1 mM luminol and 20 µl of 14 mM potassium ferricyanide. Quantify H₂O₂ using a lumi-counter.

Representative data

Figure 1. Transmission electron micrograph of flagellar filaments purified from A. avenae K1 strain. The purified sample contains the flagellar filaments of various lengths which are formed by mechanical shearing. Scale bar shown at the bottom right corner is 250 nm.

Figure 2. Purity of flagellins in flagellar filaments purified from A. avenae N1141 and K1 strains by SDS-PAGE (A) and Western blot analysis (B). A. The proteins were visualized by Coomassie brilliant blue R-250 (CBB) staining. Lane 1, flagellin of N1141 strain (pellet of 20,000 x g); lane 2, flagellin of K1 strain (pellet of 20,000 x g ). Molecular masses of the N1141 and K1 flagellins are 50,820 Da and 51,254 Da, respectively. B. The flagellins were detected by anti-flagellin antibody. Lane 1, flagellin of N1141 strain (pellet of 20,000 x g); lane 2, flagellin of K1 strain (pellet of 20,000 x g).

Figure 3. Induction of H₂O₂ generation in cultured rice cells by flagellins purified from A. avenae. Time course of H₂O₂ generation in cultured rice cells treated with 100 nM flagellins from the avirulent N1141 strain (solid squares) or virulent K1 strain (open squares). The Y axis represents fold change relative to amount of H₂O₂ in cultured cells before treatment. Bars indicate the standard deviation of the mean of three experiments.

Recipes

Skimmed milk solution
10% skim milk powder
80 mM sodium hydrogen L(+)-glutamate monohydrate
Sterilize the solution by autoclaving
A. avenae skimmed milk stock solution
Culture A. avenae on LB solid medium for 24 h at 30 °C
Add 1 ml of skimmed milk solution to the culture solid medium, and harvest
Freeze immediately in liquid nitrogen, and store at -80 °C
25 mM TBS buffer (pH 7.4)
25 mM 2-amino-2-hydroxymethyl-1,3-propanediol
137 mM NaCl
2.68 mM KCl
Sterilize the solution using a sterile filter (0.22 µm)
20x R2 Major solution
800 mM KNO₃
50.7 mM (NH₄)₂SO₄
20.3 mM MgSO₄·7H₂O
20.4 mM CaCl₂·2H₂O
35 mM NaH₂PO₄·2H₂O
Sterilize the solution using a sterile filter (0.22 µm)
1,000x R2 Minor solution
7.17 mM MnSO₄·5H₂O
7.65 mM ZnSO₄·7H₂O
0.5 mM CuSO₄·5H₂O
48.52 mM H₃BO₃
0.52 mM Na₂MoO₄·2H₂O
Sterilize the solution using a sterile filter (0.22 µm)
500x Fe liquid solution
10.07 mM Na₂·EDTA
9.89 mM FeSO₄·7H₂O
Sterilize the solution using a sterile filter (0.22 µm)
1,000x MS vitamin solution (10 ml)
1.03 g MS vitamin powder (1,000x)
Sterilize the solution using a sterile filter (0.22 µm)
2,4-dichlorophenoxyacetic acid solution
181 mM 2, 4-dichlorophenoxyacetic acid
Dissolve 2, 4-dichlorophenoxyacetic acid in1 ml of dimethyl sulfoxide
R2S medium (pH 5.6) (1,000 ml)
50 ml 20x R2 major solution
1 ml 1,000x R2 minor solution
2 ml 500x Fe liquid solution
1 ml 1,000x vitamin solution
0.1 ml 2, 4-dichlorophenoxyacetic acid solution
30 g sucrose
Adjust pH to 5.6 with KOH. Fill up to 1,000 ml with distilled water, and sterilize by autoclaving.
2x sample buffer (pH 6.8)
125 mM Tris
139 mM SDS
12% (v/v) 3-mercapto-1,2-propanediol
20% (v/v) glycerol
Adjust pH to 6.8 with HCl, and add to 1% BPB
Electrophoresis buffer
3.5 mM SDS
24.8 mM Tris
192 mM glycine
CBB staining solution
0.25% (w/v) CBB
50% (v/v) methanol
5% (v/v) acetic acid
Destaining solution
50% (v/v) methanol
10% (v/v) acetic acid
Transfer buffer
25 mM Tris
192 mM glycine
1.7 mM SDS
20% (v/v) methanol
TBST buffer
25 mM Tris
13.7 mM NaCl
0.1% (v/v) Tween-20
Blocking buffer
5% (w/v) skim milk powder
Dissolve skim milk powder in TBST buffer
50 mM potassium phosphate buffer (pH 7.9)
50 mM KH₂PO₄
Adjust pH to 7.9 with KOH
Luminol solution
1.1 mM luminol
Dissolve luminol in 50 mM potassium phosphate buffer (pH 7.9)
Potassium ferricyanide solution
14 mM potassium ferricyanide
Dissolve potassium ferricyanide in 50 mM potassium phosphate buffer (pH 7.9)

Acknowledgments

This protocol was adapted from the previously published studies (Che et al., 2000; Hirai et al., 2011; Katsuragi et al., 2015; Schwacke and Hager, 1992). This work was supported by Technology of Japan and the Program for Promotion of Basic and Applied Research for Innovations in Bio-Oriented Industry and Grant-in-Aid for Scientific Research (B) (25292067) from the Ministry of Education, Culture, Sports, Science.

References

Che, F. S., Nakajima, Y., Tanaka, N., Iwano, M., Yoshida, T., Takayama, S., Kadota, I. and Isogai, A. (2000). Flagellin from an incompatible strain of Pseudomonas avenae induces a resistance response in cultured rice cells. J Biol Chem 275(41): 32347-32356.
Hirai, H., Takai, R., Iwano, M., Nakai, M., Kondo, M., Takayama, S., Isogai, A. and Che, F. S. (2011). Glycosylation regulates specific induction of rice immune responses by Acidovorax avenae flagellin. J Biol Chem 286(29): 25519-25530.
Katsuragi, Y., Takai, R., Furukawa, T., Hirai, H., Morimoto, T., Katayama, T., Murakami, T. and Che, F. S. (2015). CD2-1, the C-terminal region of flagellin, modulates the induction of immune responses in rice. Mol Plant Microbe Interact 28(6): 648-658.
Schwacke, R. and Hager, A. (1992). Fungal elicitors induce a transient release of active oxygen species from cultured spruce cells that is dependent on Ca(²⁺) and protein-kinase activity. Planta 187(1): 136-141.