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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   

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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), Acidovorax avenae(Acidovorax), Purification(净化), Plant(植物)

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

  1. 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. 

  2. 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.

  3. 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.

  4. H2O2 detection 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

  1. Skimmed milk solution
    10% skim milk powder
    80 mM sodium hydrogen L(+)-glutamate monohydrate
    Sterilize the solution by autoclaving
  2. 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
  3. 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)
  4. 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)
  5. 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)
  6. 500x Fe liquid solution
    10.07 mM Na2·EDTA
    9.89 mM FeSO4·7H2O
    Sterilize the solution using a sterile filter (0.22 µm)
  7. 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)
  8. 2,4-dichlorophenoxyacetic acid solution
    181 mM 2, 4-dichlorophenoxyacetic acid
    Dissolve 2, 4-dichlorophenoxyacetic acid in1 ml of dimethyl sulfoxide
  9. 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.
  10. 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
  11. Electrophoresis buffer
    3.5 mM SDS
    24.8 mM Tris
    192 mM glycine
  12. CBB staining solution
    0.25% (w/v) CBB
    50% (v/v) methanol
    5% (v/v) acetic acid
  13. Destaining solution
    50% (v/v) methanol
    10% (v/v) acetic acid
  14. Transfer buffer
    25 mM Tris
    192 mM glycine
    1.7 mM SDS
    20% (v/v) methanol
  15. TBST buffer
    25 mM Tris
    13.7 mM NaCl
    0.1% (v/v) Tween-20
  16. Blocking buffer
    5% (w/v) skim milk powder
    Dissolve skim milk powder in TBST buffer
  17. 50 mM potassium phosphate buffer (pH 7.9)
    50 mM KH2PO4
    Adjust pH to 7.9 with KOH
  18. Luminol solution
    1.1 mM luminol
    Dissolve luminol in 50 mM potassium phosphate buffer (pH 7.9)
  19. 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

  1. 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. 
  2. 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. 
  3. 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. 
  4. 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.

简介

植物通过识别引起PAMP触发的免疫(PTI)的保守的病原体相关分子模式(PAMP)来感测潜在的病原体,包括活性氧的产生,胼lose质沉积和几个PTI相关基因的表达。 酸性弧菌是一种革兰氏阴性细菌,其导致幼苗疾病,其特征在于在感染的植物的鞘上沉积棕色条纹。 我们以前报告了从无毒力的大米中分离的鞭毛蛋白。 avenae N1141菌株诱导PTI,而从水稻毒力分离的鞭毛蛋白。 avenae K1株不诱导PTI。 为了检查N1141鞭毛蛋白的特异性PTI诱导的分子机制,需要来自N1141或K1菌株的高度纯化的鞭毛蛋白。 在这里,我们描述了一种高质量的纯化方法。 avenae鞭毛蛋白,并用于PTI诱导研究。

关键字:鞭毛蛋白, Acidovorax, 净化, 植物

材料和试剂

  1. 0.22μm灭菌过滤器(Merck Millipore,目录号:SLGS033SB)
  2. 硝化纤维素膜(GE Healthcare,编号:10401196)
  3. 火棉涂覆的网格(Ted Pella,Inc。,目录号:12575-CU)
  4. 线OC(水稻C5928;获自RIKEN BioResource Center)的培养的稻细胞
  5. N1141(MAFF 301141)和K1(MAFF301755)(National Institute of Agrobaiological Sciences genebank,目录号:MAFF 301141; MAFF301755)。
  6. 脱脂奶粉(Wako,目录号:190-12865)
  7. 钠L(+) - 谷氨酸钠一水合物(Wako,目录号:198-02035)
  8. Luria-Bertani(LB)液体培养基(MO BIO实验室,目录号:12107-05)
  9. 2-氨基-2-羟甲基-1,3-丙二醇(Tris)(Wako,目录号:204-07885)
  10. NaCl(Nacalai Tesque,目录号:31320-05)
  11. KCl(Nacalai Tesque,目录号:28514-75)
  12. 1%(w/v)磷钨酸(pH 6.9)(Sigma-Aldrich,目录号:79690)
  13. 十二烷基硫酸钠(SDS)(Wako,目录号:191-07145)
  14. 分子量标记物低(APRO,目录号:SP-0110)
  15. 抗鞭毛蛋白兔抗体(Eurofins Genomics)
    注意:将从牛痘病毒N1141菌株纯化的鞭毛蛋白作为抗原注射到兔中。 使用从A. avenae N1141菌株纯化的鞭毛蛋白从兔抗血清(抗鞭毛蛋白)纯化抗鞭毛蛋白兔抗体。
  16. 山羊HRP缀合的抗兔IgG抗体(H + L链)[Medical& 生物实验室(MBL),目录号:458]
  17. ECL + Western印迹检测试剂(GE Healthcare,代码:RPN2132)
  18. Hybri-Bag(Cosmo Bio,目录号:S-1001)
  19. 铁氰化钾(Wako,目录号:169-03721)
  20. KNO <3> (Nacalai Tesque,目录号:28704-85)
  21. (NH 4)2 SO 4(Wako,目录号:019-03435)
  22. MgSO 4·7H 2 O(Nacalai Tesque,目录号:21003-75)
  23. CaCl 2 2·2H 2 O(Nacalai Tesque,目录号:06731-05)
  24. NaH 2 PO 4·2H 2 O(Wako,目录号:192-02815)
  25. MnSO 4·5H 2 O(Nacalai Tesque,目录号:21229-35)
  26. ZnSO 4·7H 2 O(Nacalai Tesque,目录号:37011-75)
  27. CuSO 4·5H 2 O(Nacalai Tesque,目录号:09605-04)

  28. (Wako,目录号:021-02195)。
  29. Na 2 MoO 4·2H 2 O(Nacalai Tesque,目录号:31621-52)
  30. EDTA·2Na(DOJINDO,目录号:345-01865)
  31. FeSO 4 7HH 2 O(Wako,目录号:098-01085)
  32. MS维生素粉1000x(Sigma-Aldrich,目录号:M7150-100ML)
  33. 2,4-二氯苯氧基乙酸(Wako,目录号:040-18532)
  34. 蔗糖(Wako,目录号:196-00015)
  35. 3-巯基-1,2-丙二醇(Wako,目录号:131-16451)
  36. 甘油(Nacalai Tesque,目录号:17018-25)
  37. 溴酚蓝(BPB)(Wako,目录号:021-02911)
  38. 甘氨酸(Wako,目录号:077-00735)
  39. 考马斯亮蓝R-250(CBB)(Wako,目录号:031-17922)
  40. 甲醇(Wako,目录号:137-01823)
  41. 乙酸(Wako,目录号:017-00251)
  42. 聚氧乙烯失水山梨醇单月桂酸酯(Tween 20)(Wako,目录号:167-11515)

  43. (Wako,目录号:169-04245)


  44. 脱脂牛奶溶液(见配方)
  45. N1141和K1菌株脱脂牛奶储备溶液(参见食谱)
  46. 25mM TBS缓冲液(pH 7.4)(参见配方)
  47. 20x R2主要解决方案(参见配方)
  48. 1,000x R2小型解决方案(请参阅配方)
  49. 500x铁液体溶液(参见配方)
  50. 1,000x MS维生素溶液(见配方)
  51. 2,4-二氯苯氧基乙酸溶液(见配方)
  52. R2S培养基(pH 5.6)(参见配方)
  53. 2x样品缓冲液(pH 6.8)(参见配方)
  54. 电泳缓冲液(参见配方)
  55. CBB染色溶液(见配方)
  56. 解决方案(参见配方)
  57. 传输缓冲区(请参阅配方)
  58. 具有Tween 20的Tris缓冲盐水(TBST)缓冲液(参见Recipes)
  59. 阻止缓冲区(参见配方)
  60. 50 mM磷酸钾缓冲液(pH 7.9)(参见配方)
  61. 鲁米诺溶液(见配方)
  62. 铁氰化钾溶液(参见配方)

设备

  1. 旋转振荡器(TAITEC,型号:NR-20)
  2. 高速冷藏微量离心机(TOMY,型号:MX-300)
  3. 高速冷冻离心机(Hitachi koki,型号:CR20G)
  4. 超速离心机(Hitachi koki,型号:CP70MX)
  5. 纤维混合机(Panasonic,型号:MX-X58-SW)
  6. 孵育器(TAITEC,型号:BR-42FL·MR)
  7. 透射电子显微镜(日立,型号:H-7100)
  8. 植物生长室(NK系统,型号:LH-411SP)
  9. 电泳槽(ATTO,型号:AE-6530M)
  10. 半干印迹(Bio-Rad,目录号:1703940JA)
  11. 发光图像分析仪(GE Healthcare,型号:ImageQuant LAS 4000)
  12. Lumi计数器(ATTO,型号:AB-2350)

程序

  1. 从酸性弧菌(Acidovorax avenae)中纯化鞭毛丝
    1. 对于酸性弧菌的预培养,加入2μl的A。 avenae将牛奶储备溶液倒入2ml LB液体培养基中,在30℃下摇动(200rpm)过夜。
    2. 将100μl预培养的悬浮液加入到1升新鲜的LB培养基中,并在30℃下摇动(200rpm)24小时。
    3. 收获。 avenae细胞,在室温下以6000xg离心20分钟。
    4. 向沉淀中加入300ml的25mM TBS缓冲液并重新悬浮。 在室温下以6000xg离心20分钟后,弃去上清液
    5. 用90ml TBS缓冲液重悬沉淀,转移到纤维混合器中
    6. 为了从细菌细胞中取出鞭毛,用纤维混合器在4℃剪切1分钟,并在冰上孵育5分钟。 重复此步骤7次。
    7. 为了除去完整的细菌细胞,在4℃下以6,000×g离心细菌悬浮液30分钟并保存上清液。
    8. 为了除去细菌细胞碎片,在4℃下以16,000×g离心上清液60分钟并保存上清液。
    9. 在4℃下以200,000×g离心上清液60分钟,并弃去上清液。
    10.  向颗粒中加入1.5 ml冰冷的蒸馏水,然后重新悬浮
    11. 在4℃下以20,000×g离心再悬浮的沉淀物20分钟,弃去上清液。 颗粒含有单体和聚合物鞭毛蛋白。
    12. 用0.5ml冰冷的蒸馏水重新悬浮沉淀,并储存在-80℃
  2. 使用透射电子显微镜观察纯化的鞭毛丝
    1. 使用50μl纯化的鞭毛细丝液滴(20,000×g颗粒)。
    2. 将纯化的鞭毛吸附到火棉胶涂覆的网格上,碳网支撑并使碳表面亲水,1分钟。
    3. 用1%(w/v)磷钨酸(pH 6.9)将网格染色1分钟,并用两滴蒸馏水洗涤。
    4. 使用在80kV下操作的透射电子显微镜将图像作为数字化图像。

  3. 通过Western印迹检测纯化的鞭毛细丝中鞭毛蛋白的纯度
    1. 鞭毛细丝。 avenae 仅由鞭毛蛋白组成。 通过SDS-PAGE和Western印迹分析鞭毛细丝中鞭毛蛋白的纯度
    2. 将半体积的2x样品缓冲液加入100ng纯化的鞭毛悬浮液中
    3. 将样品在95℃下加热5分钟,并在冰上冷却
    4. 在SDS-PAGE凝胶上加载20 ng样品和2μl低量程预染XL-Ladder Low标记。
    5. 在恒定100伏电泳,直到染料到达凝胶底部
    6. 使用转移缓冲液(恒定10伏3小时)电转移至硝酸纤维素膜
    7. 在室温下将膜在25ml封闭缓冲液中孵育1小时
    8. 用25ml TBST洗涤三次,每次5分钟
    9. 将膜与初级抗鞭毛蛋白兔抗体(1:2,500)在10ml TBST中在室温下孵育1小时。
    10. 用25ml TBST洗涤5次,每次5分钟
    11. 在第二山羊HRP缀合的抗兔IgG抗体(1:2,000)在10ml TBST中孵育膜30分钟室温。
    12. 用25ml TBST洗涤三次,每次5分钟。
    13. 孵育膜与1毫升的ECL +蛋白质印迹检测试剂在室温下3分钟,然后包裹在Hybri袋。
    14. 用LAS-4000测量每个条带的强度。

  4. H 2 O 2检测和定量
    1. 在植物生长室中在30℃下在白荧光灯照射下培养水稻细胞的悬浮培养物(系OC)。将细胞每周在新鲜培养基中稀释,并在转移后4天进行H 2 O 2 O 2检测的实验。
    2. 将培养的水稻细胞放置在滤纸上以除去培养基。加入10毫克培养细胞到0.5毫升的新鲜培养基,并在30℃在植物生长室中培养细胞2小时。
    3. 将鞭毛蛋白(100nM)加入到培养的细胞中,并在30℃下在植物生长室中孵育。小于100μl的鞭毛蛋白悬浮液对于添加到培养基中是理想的。
    4. 孵育后,向含有160μl50mM磷酸钾缓冲液(pH7.9),10μl1.1mM发光氨和20μl14mM钾的化学发光反应缓冲液中加入10μl培养基 铁氰化物。使用lumi计数器量化H <2> O 2

代表数据



图1.从A中纯化的鞭毛细丝的透射电子显微照片。 avenae K1菌株。纯化的样品含有通过机械剪切形成的各种长度的鞭毛丝。右下角显示的比例尺为250 nm。


图2.从A中纯化的鞭毛细丝中鞭毛蛋白的纯度。通过SDS-PAGE(A)和蛋白质印迹分析(B)分析A119和A116和A116和A116菌株。通过考马斯亮蓝R-250(CBB)染色观察蛋白质。泳道1,N1141菌株的鞭毛蛋白(20,000×g/ml的沉淀);泳道2,K1菌株的鞭毛蛋白(20000×g的沉淀)。 N1141和K1鞭毛蛋白的分子量分别为50,820Da和51,254Da。 B.通过抗鞭毛蛋白抗体检测鞭毛蛋白。泳道1,N1141菌株的鞭毛蛋白(20,000×g/ml的沉淀);泳道2,K1菌株的鞭毛蛋白(20,000×g颗粒)

图3.通过从em纯化的鞭毛蛋白诱导培养的水稻细胞中H 2 O 2 O 2代的诱导。 avenae。 在用来自无毒Nll41菌株的100nM鞭毛蛋白处理的培养的水稻细胞中的H 2 O 2 O 2代的时间过程(实心正方形)或毒性K1株(空心正方形)。 Y轴表示相对于治疗前培养的细胞中H 2 O 2 O 2的量的倍数变化。条表示三个实验的平均值的标准偏差。

食谱

  1. 脱脂牛奶溶液
    10%脱脂奶粉
    80mM氢氧化钠L(+) - 谷氨酸单水合物 通过高压灭菌消毒溶液
  2. A。 avenae 脱脂牛奶储备液
    文化A。 avenae 在LB固体培养基上在30℃培养24小时 加入1ml脱脂牛奶溶液到培养固体培养基,收获
    立即在液氮中冷冻,并储存在-80℃
  3. 25mM TBS缓冲液(pH7.4) 25mM 2-氨基-2-羟甲基-1,3-丙二醇
    137 mM NaCl 2.68mM KCl
    使用无菌过滤器(0.22μm)消毒溶液
  4. 20x R2主要解决方案
    800mM KNO 3
    50.7mM(NH 4)2 SO 4 4
    20.3mM MgSO 4 7H 2 O 2·h/v 20.4mM CaCl 2·2H 2 O·h/v 35mM NaH 2 PO 4·4H 2·2H 2 O·dm / 使用无菌过滤器(0.22μm)消毒溶液
  5. 1,000x R2小型解决方案
    7.17mM MnSO 4·5H 2 O x/v 7.65mM ZnSO 4·7H 2 O·h/v 0.5mM CuSO 4·5H 2 O 2·h/v 48.52mM H 3 BO 3 sub。 0.52mM Na 2 MoO 4 H 2 2H 2 O
    使用无菌过滤器(0.22μm)消毒溶液
  6. 500x Fe液体溶液
    10.07mM Na 2 EDTA·
    9.89mM FeSO 4·7H 2 O·h/v 使用无菌过滤器(0.22μm)消毒溶液
  7. 1,000x MS维生素溶液(10ml)
    1.03克MS维生素粉(1,000x)
    使用无菌过滤器(0.22μm)消毒溶液
  8. 2,4-二氯苯氧基乙酸溶液 181mM 2,4-二氯苯氧基乙酸 将2,4-二氯苯氧基乙酸溶于1ml二甲基亚砜中
  9. R2S培养基(pH 5.6)(1000ml) 50 ml 20x R2主溶液
    1 ml 1,000x R2次要溶液
    2ml 500×Fe液体溶液 1 ml 1,000x维生素溶液
    0.1ml 2,4-二氯苯氧基乙酸溶液 30克蔗糖 用KOH调节pH至5.6。 用蒸馏水填充至1000ml,并通过高压灭菌消毒。
  10. 2x样品缓冲液(pH 6.8)
    125 mM Tris 139 mM SDS
    12%(v/v)3-巯基-1,2-丙二醇 20%(v/v)甘油 用HCl调节pH至6.8,加入1%BPB
  11. 电泳缓冲液
    3.5mM SDS
    24.8mM Tris 192mM甘氨酸
  12. CBB染色溶液
    0.25%(w/v)CBB
    50%(v/v)甲醇
    5%(v/v)乙酸
  13. 脱色解决方案
    50%(v/v)甲醇
    10%(v/v)乙酸
  14. 传输缓冲区
    25 mM Tris
    192 mM甘氨酸 1.7mM SDS
    20%(v/v)甲醇
  15. TBST缓冲区
    25 mM Tris
    13.7mM NaCl 0.1%(v/v)Tween-20
  16. 阻塞缓冲区
    5%(w/v)脱脂奶粉
    将脱脂奶粉溶于TBST缓冲液中
  17. 50mM磷酸钾缓冲液(pH7.9) 50mM KH 2 PO 4 4/v/v 用KOH调节pH至7.9
  18. 鲁米诺溶液
    1.1 mM luminol
    将鲁米诺溶解在50mM磷酸钾缓冲液(pH7.9)
  19. 铁氰化钾溶液
    14mM铁氰化钾 将铁氰化钾溶于50mM磷酸钾缓冲液(pH7.9)中

致谢

该方案改编自以前公开的研究(Che等人,2000; Hirai等人,2011; Katsuragi等人, 2015; Schwacke和Hager,1992)。这项工作得到日本技术和促进生物产业创新基础和应用研究计划和科学研究资助(B)(25292067)的支持,来自教育,文化,体育,科学。

参考文献

  1. Che,F.S.,Nakajima,Y.,Tanaka,N.,Iwano,M.,Yoshida,T.,Takayama,S.,Kadota,I.and Isogai,A。(2000)。 来自不相容的 Pseudomonas avenae菌株的鞭毛蛋白诱导培养的水稻细胞中的抗性反应。 J Biol Chem 275(41):32347-32356。
  2. Hirai,H.,Takai,R.,Iwano,M.,Nakai,M.,Kondo,M.,Takayama,S.,Isogai,A.and Che,F.S.(2011)。 糖基化调节由氨基酸菌(Acidovorax avenae)对水稻免疫应答的特异性诱导 flagellin。 J Biol Chem 286(29):25519-25530。
  3. Katsuragi,Y.,Takai,R.,Furukawa,T.,Hirai,H.,Morimoto,T.,Katayama,T.,Murakami,T.and Che,F.S.(2015)。 CD2-1,C- 末端区域,调节水稻中免疫应答的诱导。 Mol Plant Microbe Interact 28(6):648-658。
  4. Schwacke,R。和Hager,A。(1992)。 真菌诱导剂诱导活性氧从培养的云杉细胞中瞬时释放 这取决于Ca( 2 + )和蛋白激酶活性。植物 187(1):136-141。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
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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