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

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Original research article

A brief version of this protocol appeared in:
Journal of Integrative Plant Biology
Jan 2015

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, Vacuole, Erwinia, Tobacco, Confocal microscopy

Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: 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.
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