Vacuole Structure Analysis during Cell Death Subsequent to Application of Erwinia carotovora Culture Filtrates to Cell Cultures of Nicotiana tabacum

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Journal of Integrative Plant Biology
Jan 2015



We recently established an experimental model system for efficient defense-related cell death using tobacco BY-2 cultured cells treated with culture filtrates of the pathogenic bacterium Erwinia carotovora (E. carotovora) (Hirakawa et al., 2015). Applying this experimental system to transgenic BY-2 cells stably expressing the vacuolar membrane marker GFP-VAM3 (Kutsuna and Hasezawa, 2002) allowed us to monitor changes in vacuolar membrane structures including a decrease of transvacuolar strands during cell death (Hirakawa et al., 2015). Our model system can help to investigate organelle dynamics in defense-related cell death. Here, we show protocol for applying E. carotovora filtrates to BY-2 cells and confocal observation of vacuolar membrane dynamics and subsequent cell death. We used cell cycle synchronized BY-2 cells to effectively monitor invaginated vacuolar membranes such as transvacuolar strands in our recent report (Hirakawa et al., 2015); however, we do not describe the protocol for cell cycle synchronization in this article. For the step-by-step protocol for BY-2 cell synchronization, please refer to previous protocol papers (Nagata and Kumagai, 1999; Kumagai-Sano et al., 2006).

Keywords: Cell death (细胞死亡), Vacuole (液泡), Erwinia (欧文氏菌), Tobacco (烟草), Confocal microscopy (共聚焦显微镜)

Materials and Reagents

  1. Sterile filter with pore size 0.22 µm (Merck Millipore Corporation, Millex®–GV Filter unit, catalog number: SLGV033RS )
  2. 50 ml syringe (TERUMO CORPORATION, catalog number: SS-50ESZ )
  3. 12 well plate (Sumitomo Bakelite Co., catalog number: MS-80120 )
  4. Erwinia carotovora subsp. carotovora (National Institute of Technology and Evaluation, catalog number: 103133)
  5. Transgenic tobacco BY-2 cell culture (N. tabacum L. cv. Bright Yellow 2) stably expressing GFP-VAM3 (RIKEN Bioresource Center, catalog number: RPC00039 )
  6. Aphidicolin (Wako Pure Chemical Industries, Siyaku, catalog number: 015-09814 )
  7. Yeast extract (KANTO KAGAKU, KANTO Chemical, catalog number: 712021-5 )
  8. BactoTM Tryptone (BD bioscience, catalog number: 211705 )
  9. NaCl (KANTO KAGAKU, KANTO Chemical, catalog number: 37144-01 )
  10. Murashige and Skoog plant salt mixture (Wako Pure Chemical Industries, Siyaku, catalog number: 392-00591 )
  11. Sucrose (Wako Pure Chemical Industries, Siyaku, catalog number: 196-00015 )
  12. myo-Inositol solution (Wako Pure Chemical Industries, Siyaku, catalog number: 094-00281 )
  13. Thiamine hydrochloride (Wako Pure Chemical Industries, Siyaku, catalog number: 201-00852 )
  14. Sodium 2,4-Dichlorophenoxyacetate Monohydrate (Tokyo Chemical Industry, catalog number: D1319 )
  15. Lysogeny broth (LB) medium (see Recipes)
  16. Modified Linsmaier and Skoog (LS) medium (see Recipes)


  1. Rotary shaker for E. carotovora culture (TAITEC CORPORATION, model: BR-40LF )
  2. 300 ml flask for E. carotovora culture (Sansyo, Iwaki, catalog number: 82-0087 )
  3. Spectrophotometer (Beckman Coulter, model: DU® 640 )
  4. Centrifuge (TOMY SEIKO CO, model: MX-300 )
  5. Deep freezer (Nihon Freezer, catalog number: CLN-30U )
  6. 100 ml flask for BY-2 culture (Sansyo, Iwaki, catalog number: 82-0085 )
  7. Rotary shaker for the untreated BY-2 culture (TAITEC CORPORATION, catalog number: 300LF )
  8. Rotary shaker for the treated BY-2 culture (TAITEC CORPORATION, catalog number: NR-30 )
  9. Incubator for the treated BY-2 culture (Sanyo, catalog number: MIR-553 )
  10. Glass base dish (Sansyo, Iwaki, catalog number: 3911-035 )
  11. Confocal laser scanning microscope (OLYMPUS, model: FV300 )


  1. Preparation of E. carotovora culture filtrate
    1. Culture 100 ml of E. carotovora in liquid LB medium in a 300-ml flask at 37 ℃ overnight with rotary shaking at 150 rpm.
    2. Check OD600 with a spectrophotometer. The OD600 value should be in the range 0.8-1.0 for appreciable cell death induction.
    3. Centrifuge the bacterial cell culture for 10 min at 4,000 x g at room temperature.
    4. Collect the supernatant and sterile filter it with a syringe filter.
    5. The filtrate can be kept at -80 ℃ in a deep freezer for several months.

  2. Application of the filtrate to cell cycle synchronized tobacco culture cells
    1. Prepare the S-phase synchronized transgenic BY-2 cells expressing GFP-VAM3 by DNA polymerase inhibitor (aphidicolin) treatment and wash out with fresh modified LS medium (Nagata and Kumagai, 1999; Kumagai-Sano et al., 2006).
    2. Just after the washout, place 30 ml of the synchronized culture cells in a 100-ml flask on a rotary shaker culture at 27 ℃ for 30 min at 130 rpm.
    3. Add 1.6 ml of the cell cycle synchronized culture into a well of the 12-well plate.
    4. Add 0.4 ml of the E. carotovora culture filtrate into the cell culture well [final concentration: 20% (v/v)].
    5. Culture the treated cells on a rotary shaker in an incubator at 27 ℃ for two hours.
    6. Collect 150 μl of the cells into a microscope glass dish base for observation.
    7. Place the dish on the stage of the confocal laser scanning microscope.
    8. Acquire time-lapse images of GFP-VAM3 at 5-min intervals with 488 nm excitation lasers and 524-546 nm emission filters according to the microscope manufacturer’s instructions. See Figure 1 for representative results.

Representative data

The representative time-lapse images of GFP-VAM3 were shown in Figure 1. After the filtrate treatment, the invaginated vacuolar membrane structures including transvacuolar strands gradually decreased (Figure 1). The percentage of transvacuolar strand-less cells reached 12.8±3.00% 4 h after the filtrate treatment while 1.52±1.48% in the mock treatment (Hirakawa et al., 2015).

Figure 1. Time-lapse observation of tobacco BY-2 cells expressing GFP-VAM3 treated with culture filtrates of Erwinia carotovora. Note that the invaginated vacuolar membranes gradually decreased before cell death with cell shrinkage. The time-lapse images were obtained at 5-min intervals for 4.5 h. Scale bar indicates 50 μm.


You should wait around 10 min after the placement of a microscope glass dish base on the microscope stage to start a time-lapse imaging. The cells would be completely settled down to the bottom while waiting, resulting in appreciated results without the cell displacement.


  1. LB medium
    Note: No need to adjust pH.
    Yeast extract
    5 g/L
    BactoTM tryptone
    10 g/L
    5 g/L
  2. Modified LS medium
    Murashige and Skoog plant salt mixture
    4.6 g/L
    30 g/L
    myo-Inositol solution
    200 mg/L
    Thiamine hydrochloride
    1 mg/L
    2,4-Dichlorophenoxyacetic acid
    0.2 mg/L
    Adjust pH to 5.8 with KOH


The authors thank Dr. Toshihisa Nomura for preliminary experiments on E. carotovora culture filtrates. This work was supported by JSPS KAKENHI Grant Numbers 25711017 (T.H.), 25291056 (S.H.) and 24114007 (S.H.).


  1. Hirakawa, Y., Nomura, T., Hasezawa, S. and Higaki, T. (2015). Simplification of vacuole structure during plant cell death triggered by culture filtrates of Erwinia carotovora. J Integr Plant Biol 57(1): 127-135.
  2. Kumagai-Sano, F., Hayashi, T., Sano, T. and Hasezawa, S. (2006). Cell cycle synchronization of tobacco BY-2 cells. Nat Protoc 1(6): 2621-2627.
  3. Kutsuna, N. and Hasezawa, S. (2002). Dynamic organization of vacuolar and microtubule structures during cell cycle progression in synchronized tobacco BY-2 cells. Plant Cell Physiol 43(9): 965-973.
  4. Nagata, T. and Kumagai, F. (1999). Plant cell biology through the window of the highly synchronized tobacco BY-2 cell line. Methods Cell Sci 21(2-3): 123-127.


我们最近建立了使用烟草BY-2培养细胞的有效防御相关细胞死亡的实验模型系统,所述培养细胞用致病性细菌欧文氏菌(Erwinia carotovora)( E。carotovora (Hirakawa ,,2015)。将该实验系统应用于稳定表达液泡膜标记物GFP-VAM3的转基因BY-2细胞(Kutsuna和Hasezawa,2002),使我们能够监测液泡膜结构的变化,包括细胞死亡期间跨血管链的减少(Hirakawa et al。 al。,2015)。我们的模型系统可以帮助调查细胞器动力学在国防相关的细胞死亡。在这里,我们显示应用 E的协议。 carotovora过滤到BY-2细胞和共聚焦观察液泡膜动力学和随后的细胞死亡。我们使用细胞周期同步的BY-2细胞来有效地监测内陷的液泡膜,例如在我们最近的报告中的跨血管链(Hirakawa等人,2015);然而,我们不描述本文中的细胞周期同步的协议。对于BY-2细胞同步的逐步方案,请参考先前的方案论文(Nagata和Kumagai,1999; Kumagai-Sano等人,2006)。

关键字:细胞死亡, 液泡, 欧文氏菌, 烟草, 共聚焦显微镜


  1. 孔径为0.22μm的无菌过滤器(Merck Millipore Corporation,Millex -GV Filter unit,目录号:SLGV033RS)
  2. 50ml注射器(TERUMO CORPORATION,目录号:SS-50ESZ)
  3. 12孔板(Sumitomo Bakelite Co.,目录号:MS-80120)
  4. Erwinia carotovora subsp。 carotovora(国立技术与评估研究所,目录号:103133)
  5. 稳定表达GFP-VAM3(RIKEN Bioresource Center,目录号:RPC00039)的转基因烟草BY-2细胞培养物(紫um um
  6. Aphidicolin(Wako Pure Chemical Industries,Siyaku,目录号:015-09814)
  7. 酵母提取物(KANTO KAGAKU,KANTO Chemical,目录号:712021-5)
  8. 胰蛋白胨(BD bioscience,目录号:211705)
  9. NaCl(KANTO KAGAKU,KANTO Chemical,目录号:37144-01)
  10. Murashige和Skoog植物盐混合物(Wako Pure Chemical Industries,Siyaku,目录号:392-00591)
  11. 蔗糖(Wako Pure Chemical Industries,Siyaku,目录号:196-00015)
  12. 肌醇溶液(Wako Pure Chemical Industries,Siyaku,目录号:094-00281)</em>
  13. 盐酸硫胺素(Wako Pure Chemical Industries,Siyaku,目录号:201-00852)
  14. 2,4-二氯苯氧基乙酸钠一水合物(Tokyo Chemical Industry,目录号:D1319)
  15. 溶菌酶培养基(LB)培养基(参见配方)
  16. 修改的Linsmaier和Skoog(LS)介质(参见配方)


  1. 旋转振荡器。 carotovora 文化(TAITEC CORPORATION,型号:BR-40LF)
  2. 300ml烧瓶中。 carotovora 文化(Sansyo,Iwaki,目录号:82-0087)
  3. 分光光度计(Beckman Coulter,型号:DU 640)
  4. 离心机(TOMY SEIKO CO,型号:MX-300)
  5. 深冷冻器(Nihon Freezer,目录号:CLN-30U)
  6. 100ml培养瓶(Sansyo,Iwaki,目录号:82-0085)用于BY-2培养
  7. 用于未处理的BY-2培养物(TAITEC CORPORATION,目录号:300LF)的旋转振荡器
  8. 用于处理的BY-2培养物(TAITEC CORPORATION,目录号:NR-30)的旋转振荡器
  9. 用于处理的BY-2培养物(Sanyo,目录号:MIR-553)的培养箱
  10. 玻璃底皿(Sansyo,Iwaki,目录号:3911-035)
  11. 共聚焦激光扫描显微镜(OLYMPUS,型号:FV300)


  1. E的制备。 carotovora 培养滤液
    1. 培养100毫升E。 carotovora)在液体LB培养基中在37℃下在旋转振荡下在150rpm下培养过夜。
    2. 用分光光度计检查OD <600>。对于明显的细胞死亡诱导,OD <600>值应该在0.8-1.0的范围内
    3. 在室温下以4,000×g离心细菌细胞培养物10分钟
    4. 收集上清液,用注射器过滤器无菌过滤
    5. 滤液可在-80℃下在深冷冻箱中保存几个月。

  2. 滤液对细胞周期同步的烟草培养细胞的应用
    1. 准备S期同步转基因BY-2细胞表达 GFP-VAM3通过DNA聚合酶抑制剂(aphidicolin)处理和洗涤 用新鲜的改性LS培养基(Nagata和Kumagai,1999; Kumagai-Sano et al。,2006)。
    2. 刚洗完后,放30 ml 的同步培养细胞在旋转振荡器上的100-ml烧瓶中 在27℃下以130rpm培养30分钟
    3. 将1.6ml细胞周期同步培养物加入12孔板的孔中
    4. 加入0.4ml的E。 carotovora 培养物滤液进入细胞培养孔[最终浓度:20%(v/v)]。
    5. 将处理的细胞在27℃的培养箱中的旋转振荡器上培养2小时
    6. 收集150微升的细胞进入显微镜玻璃皿基地观察。
    7. 将盘放在共焦激光扫描显微镜的平台上。
    8. 获得GFP-VAM3的延时图像与488 nm 5分钟间隔 ?激发激光器和524-546nm发射滤光器 显微镜制造商的说明。代表性见图1 结果。







  1. LB培养基
    5 g/L
    Bacto TM 胰蛋白酶
    10 g/L
    5 g/L
  2. 修改的LS培养基
    4.6 g/L
    30 g/L
    myo - 肌醇溶液
    200 mg/L
    盐酸硫胺素 1 mg/L
    0.2 mg/L


作者感谢Toshihisa Nomura博士进行初步实验。 carotovora 培养物滤液。该工作由JSPS KAKENHI批准号25711017(T.H。),25291056(S.H。)和24114007(S.H。)支持。


  1. Hirakawa,Y.,Nomura,T.,Hasezawa,S.and Higaki,T。(2015)。 在植物细胞死亡过程中,通过 Erwinia carotovora的培养物滤液引发的液泡结构的简化 >。
  2. Kumagai-Sano,F.,Hayashi,T.,Sano,T.and Hasezawa,S。(2006)。 烟草BY-2细胞的细胞周期同步。 Nat Protoc < (6):2621-2627。
  3. Kutsuna,N。和Hasezawa,S。(2002)。 在同步烟草BY-2细胞的细胞周期进程中,液泡和微管结构的动态组织。 a> 植物细胞生理 l 43(9):965-973。
  4. Nagata,T.and Kumagai,F。(1999)。 通过高度同步的烟草BY-2细胞系窗口的植物细胞生物学。 Methods Cell Sci 21(2-3):123-127。
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引用:Hirakawa, Y., Hasezawa, S. and Higaki, T. (2015). Vacuole Structure Analysis during Cell Death Subsequent to Application of Erwinia carotovora Culture Filtrates to Cell Cultures of Nicotiana tabacum. Bio-protocol 5(20): e1629. DOI: 10.21769/BioProtoc.1629.