Reliable detection and quantification of plasmodesmal callose in Nicotiana benthamiana leaves during defense responses

Abstract

Callose, a beta-(1,3)-D-glucan polymer, is essential for regulating intercellular trafficking via plasmodesmata (PD). Pathogens manipulate PD-localized proteins to enable intercellular trafficking by removing callose at PD, or conversely by increasing callose accumulation at PD to limit intercellular trafficking during infection. Plant defense hormones like salicylic acid regulate PD-localized proteins to control PD and intercellular trafficking during innate immune defense responses such as systemic acquired resistance. Measuring callose deposition at PD in plants has therefore emerged as a popular parameter for assessing the intercellular trafficking activity during plant immunity. Despite the popularity of this metric there is no standard for how these measurements should be made. In this study, three commonly used methods for identifying and quantifying PD callose by aniline blue staining were evaluated to determine the most effective in the Nicotiana benthamiana leaf model. The results reveal that the most reliable method used aniline blue staining and fluorescent microscopy to measure callose deposition in fixed tissue. Manual or semi-automated workflows for image analysis were also compared and found to produce similar results although the semi-automated workflow produced a wider distribution of data points.

Plant phenotyping relevance

植物葉のPDカロース沈着という防御関連形質の画像測定法について、複数手法と画像解析ワークフローを比較・評価しており、測定法の検証が中心である。

Code and data availability

The preprint describes aniline blue staining methods and ImageJ/CalloseQuant image analysis for plasmodesmal callose quantification, but contains no data availability statement, no deposited phenotype datasets or confocal images, and no author code/scripts/workflows with a public URL. CalloseQuant is cited third-party,

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