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

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. Bacto^TM 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)
    

Equipment

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 )
    

Procedure