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

燕麦食酸菌鞭毛蛋白的纯化及其诱导植物免疫反应的分析

HH Hiroyuki Hirai
TF Takehito Furukawa
YK Yuya Katsuragi
FC Fang-Sik Che

发布: 2016年08月20日第6卷第16期 DOI: 10.21769/BioProtoc.1898 浏览次数: 9386

评审: Zhaohui LiuChong HeArsalan Daudi

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The authors used this protocol in:

Cover of Molecular Plant Microbe Interactions, featuring study using the protocol.

Jun 2015

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Abstract

Plants sense potential pathogens by recognizing conserved pathogen-associated molecular patterns (PAMPs) that cause PAMP-triggered immunity (PTI) including the generation of reactive oxygen species, callose deposition, and expression of several PTI-related genes. Acidovorax avenae is a Gam-negative bacterium that causes a seedling disease characterized by the deposition of brown stripes on the sheaths of infected plants. We previously reported that flagellin isolated from the rice avirulent A. avenae N1141 strain induces PTI, while flagellin isolated from the rice virulent A. avenae K1 strain does not induce PTI. To examine the molecular mechanism of specific PTI induction by N1141 flagellin, highly purified flagellin from N1141 or K1 strains is required. Here, we describe a high quality purification method for the A. avenae flagellins and for using it in PTI induction study.

Keywords: Flagellin (鞭毛蛋白) search Acidovorax avenae (Acidovorax) search Purification (净化) search Plant (植物) search

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

English
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文章信息

版权信息

© 2016 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Hirai, H., Furukawa, T., Katsuragi, Y. and Che, F. (2016). Purification of Flagellin from Acidovorax avenae and Analysis of Plant Immune Responses Induced by the Purified Flagellin. Bio-protocol 6(16): e1898. DOI: 10.21769/BioProtoc.1898.

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分类

生物化学 > 蛋白质 > 免疫检测

植物科学 > 植物免疫 > 病害生物测定

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I have a couple of questions about this protocol1) You centrifuge...
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