Abstract
The plant cell wall is a physical barrier, which fulfills a plethora of functions, for example it can efficiently prevent pathogen’s entry into the cell. In addition, its changing composition contributes to plants inducible defense mechanisms. This layer of defense includes pathogen perception and is followed by the activation of defense responses resulting, among others, in a modification and remodeling of the cell wall. This relatively late defense response (hours or days after contact with pathogen) comprises the accumulation of polysaccharides, such as the 1,3-ß-glucan callose, phenolic compounds and reactive oxygen species. Callose depositions occur during normal plant growth (e.g. in the phloem), they can be also a response to different stress stimuli. During the response to pathogen attack, callose depositions are essential part of cell wall reinforcement and are important for successful plant defense. Here, we describe a method to stain callose apposition spots, which can be used to quantify this defense response.
Materials and Reagents
Equipment
Procedure
Representative data
Notes
Callose depositions can be easily quantified by counting all stained spots in the defined leaf area(s). In addition, the amount of deposits can be categorized in groups, ranked from the lowest to the highest density of spots per leaf or leaf area. Quantification of the intensity of callose with image analysis software is also possible, however focusing of the image to visualize all callose depositions in a particular leaf area may be difficult. To achieve high reproducibility of results, it is important to perform the experiments with leaves at equal age and to prevent injuries during the transfer of plants or leaves to the different solutions during sample preparation.
Recipes
Acknowledgments
The protocol was modified after Clay et al. (2009). This work was supported by the Federal Office for Agriculture and Food (BLE), Germany.
References
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