欧文氏杆菌培养滤液处理烟草细胞培养物后细胞死亡过程中的液泡结构分析

Yumi Hirakawa; Seiichiro Hasezawa; Takumi Higaki

doi:10.21769/BioProtoc.1629

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Peer-reviewed

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

欧文氏杆菌培养滤液处理烟草细胞培养物后细胞死亡过程中的液泡结构分析

Yumi Hirakawa

Seiichiro Hasezawa

Takumi Higaki email

发布: 2015年10月20日第5卷第20期 DOI: 10.21769/BioProtoc.1629 浏览次数: 7726

评审: Samik BhattacharyaDaniel SavatinAnonymous reviewer(s)

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Cover of Journal of Integrative Plant Biology, featuring study using the protocol.

Jan 2015

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Abstract

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

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

Equipment

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

Procedure

English

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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.