Purification of Flagellin from Acidovorax avenae and Analysis of Plant Immune Responses Induced by the Purified Flagellin

Materials and Reagents

1. 0.22 µm sterilization filter (Merck Millipore, catalog number: SLGS033SB )
   
2. Nitrocellulose membrane (GE Healthcare, code number: 10401196 )
   
3. Collodion-coated grid (Ted Pella, Inc., catalog number: 12575-CU )
   
4. Cultured rice cells of line OC (Oryza sativa C5928; obtained from RIKEN BioResource Center)
   
5. Acidovorax avenae N1141 (MAFF 301141) and K1 (MAFF301755) (National Institute of Agrobaiological Sciences genebank, catalog number: MAFF 301141 ; MAFF301755 )
   
6. Skim milk powder (Wako, catalog number: 190-12865 )
   
7. Sodium hydrogen L(+)-glutamate monohydrate (Wako, catalog number: 198-02035 )
   
8. Luria-Bertani (LB) liquid medium (MO BIO laboratories, catalog number: 12107-05 )
   
9. 2-amino-2-hydroxymethyl-1,3-propanediol (Tris) (Wako, catalog number: 204-07885 )
   
10. NaCl (Nacalai Tesque, catalog number: 31320-05 )
    
11. KCl (Nacalai Tesque, catalog number: 28514-75 )
    
12. 1% (w/v) phosphotungstic acid (pH 6.9) (Sigma-Aldrich, catalog number: 79690 )
    
13. Sodium dodecyl sulfate (SDS) (Wako, catalog number: 191-07145 )
    
14. Molecular-weight marker low (APRO, catalog number: SP-0110 )
    
15. 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.
    
16. Goat HRP conjugated anti-rabbit IgG antibody (H + L chain) [Medical & Biological Laboratories (MBL), catalog number: 458 ]
    
17. ECL plus Western blotting detection reagents (GE Healthcare, code number: RPN2132 )
    
18. Hybri-Bag (Cosmo Bio, catalog number: S-1001 )
    
19. Potassium ferricyanide (Wako, catalog number: 169-03721 )
    
20. KNO₃ (Nacalai Tesque, catalog number: 28704-85 )
    
21. (NH₄)₂SO₄ (Wako, catalog number: 019-03435 )
    
22. MgSO₄·7H₂O (Nacalai Tesque, catalog number: 21003-75 )
    
23. CaCl₂·2H₂O (Nacalai Tesque, catalog number: 06731-05 )
    
24. NaH₂PO₄·2H₂O (Wako, catalog number: 192-02815 )
    
25. MnSO₄·5H₂O (Nacalai Tesque, catalog number: 21229-35 )
    
26. ZnSO₄·7H₂O (Nacalai Tesque, catalog number: 37011-75 )
    
27. CuSO₄·5H₂O (Nacalai Tesque, catalog number: 09605-04 )
    
28. H₃BO₃ (Wako, catalog number: 021-02195 )
    
29. Na₂MoO₄·2H₂O (Nacalai Tesque, catalog number: 31621-52 )
    
30. EDTA·2Na (DOJINDO, catalog number: 345-01865 )
    
31. FeSO₄·7H₂O (Wako, catalog number: 098-01085 )
    
32. MS vitamin powder 1,000x (Sigma-Aldrich, catalog number: M7150-100ML )
    
33. 2, 4-dichlorophenoxyacetic acid (Wako, catalog number: 040-18532 )
    
34. Sucrose (Wako, catalog number: 196-00015 )
    
35. 3-mercapto-1,2-propanediol (Wako, catalog number: 131-16451 )
    
36. Glycerol (Nacalai Tesque, catalog number: 17018-25 )
    
37. Bromophenol blue (BPB) (Wako, catalog number: 021-02911 )
    
38. Glycine (Wako, catalog number: 077-00735 )
    
39. Coomassie brilliant blue R-250 (CBB) (Wako, catalog number: 031-17922 )
    
40. Methanol (Wako, catalog number: 137-01823 )
    
41. Acetic acid (Wako, catalog number: 017-00251 )
    
42. Polyoxyethylene sorbitan monolaurate (Tween 20) (Wako, catalog number: 167-11515 )
    
43. KH₂PO₄ (Wako, catalog number: 169-04245 )
    
44. Skimmed milk solution (see Recipes)
    
45. Acidovorax avenae N1141 and K1 strain skimmed milk stock solution (see Recipes)
    
46. 25 mM TBS buffer (pH 7.4) (see Recipes)
    
47. 20x R2 Major solution (see Recipes)
    
48. 1,000x R2 Minor solution (see Recipes)
    
49. 500x Fe liquid solution (see Recipes)
    
50. 1,000x MS vitamin solution (see Recipes)
    
51. 2, 4-dichlorophenoxyacetic acid solution (see Recipes)
    
52. R2S medium (pH 5.6) (see Recipes)
    
53. 2x sample buffer (pH 6.8) (see Recipes)
    
54. Electrophoresis buffer (see Recipes)
    
55. CBB staining solution (see Recipes)
    
56. Destaining solution (see Recipes)
    
57. Transfer buffer (see Recipes)
    
58. Tris buffered saline with Tween 20 (TBST) buffer (see Recipes)
    
59. Blocking buffer (see Recipes)
    
60. 50 mM potassium phosphate buffer (pH 7.9) (see Recipes)
    
61. Luminol solution (see Recipes)
    
62. Potassium ferricyanide solution (see Recipes)
    

Equipment

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

Procedure