Simultaneous visualization of callose deposition and plasma membrane for live-cell imaging in plants
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ORIGINAL ARTICLE
Simultaneous visualization of callose deposition and plasma membrane for live‑cell imaging in plants Masaki Kohari1 · Naoto Shibuya1 · Hanae Kaku1 Received: 31 March 2020 / Accepted: 14 August 2020 © Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract Key message The appropriate combination of fluorescent probes enabled the simultaneous visualization of callose deposition and plasma membrane in living Arabidopsis and can be useful for the cell biological study of papilla formation in plants. Abstract Localized callose deposition at the site of fungal infection is a central part of papilla formation, which creates a barrier between the host plasma membrane and the cell wall and plays an important role in preventing the penetration of fungal hyphae into the host cells. Using chitin-induced callose deposition as a model system, we examined suitable conditions for the simultaneous visualization of callose deposition and plasma membrane dynamics in living Arabidopsis cotyledons. We found that aniline blue fluorochrome (ABF) for callose staining selectively interferes with FM dyes for membrane visualization depending on the structure of the latter compounds and the proper combination of these fluorescent dyes and staining conditions is a key for successful live-cell imaging. The established conditions enabled the live-cell imaging of chitin-induced callose deposition and host membrane systems. The established system/conditions would also be useful for the cell biological studies on the localized callose deposition in other stress/development-associated processes. The finding that the slight difference in the structure of FM dyes affects the interaction with another fluorescent dye, ABF, would also give useful suggestions for the studies where multiple fluorescent dyes are utilized for live-cell imaging. Keywords Chitin oligosaccharide · Callose deposition · FM dyes · ABF · Arabidopsis · Live-cell imaging Abbreviations MAMP Microbe-associated molecular pattern (GlcNAc)7 N-Acetylchitoheptaose ABF Aniline blue fluorochrome
Introduction Callose, a β-1,3 glucan, is involved in various physiological processes in higher plants. Proper regulation of callose synthesis and its localized accumulation is important for both immune and stress responses (Chen and Kim 2009). Papilla formation at the site of fungal infection, for example, is a Communicated by Günther Hahne. * Hanae Kaku [email protected] 1
Department of Life Sciences, School of Agriculture, Meiji University, 1‑1‑1 Higashi‑Mita, Tama‑ku, Kawasaki 214‑8571, Japan
well-known example of the role of callose accumulation in plant immunity. Papilla, a complex structure mainly composed of callose and other compounds such as phenolics and proteins, is formed between the host cell wall and the plasma membrane at the infection site and functions as a barrier that interferes with the penetration of fungal hyphae (Voigt 2014). While the importance of papilla formation in plant immunity is well established, biochemical and cell biologica
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