Abstract
The outwardly directed cell wall and associated plasma membrane of epidermal cells represent the first layers of plant defense against intruding pathogens. Cell wall modifications and the formation of defense structures at sites of attempted pathogen penetration are decisive for plant defense. A precise isolation of these stress-induced structures would allow a specific analysis of regulatory mechanism and cell wall adaption. However, methods for large-scale epidermal tissue preparation from the model plant Arabidopsis thaliana, which would allow proteome and cell wall analysis of complete, laser-microdissected epidermal defense structures, have not been provided. We developed the adhesive tape – liquid cover glass technique (ACT) for simple leaf epidermis preparation from A. thaliana, which is also applicable on grass leaves. This method is compatible with subsequent staining techniques to visualize stress-related cell wall structures, which were precisely isolated from the epidermal tissue layer by laser microdissection (LM) coupled to laser pressure catapulting. We successfully demonstrated that these specific epidermal tissue samples could be used for quantitative downstream proteome and cell wall analysis. The development of the ACT for simple leaf epidermis preparation and the compatibility to LM and downstream quantitative analysis opens new possibilities in the precise examination of stress- and pathogen-related cell wall structures in epidermal cells. Because the developed tissue processing is also applicable on A. thaliana, well-established, model pathosystems that include the interaction with powdery mildews can be studied to determine principal regulatory mechanisms in plant–microbe interaction with their potential outreach into crop breeding.
Highlights
The plant cell wall and its underlying plasma membrane are required to perceive environmental changes like biotic and mechanic stresses and represent the first layers of defense to invadingEpidermal tissue for quantitative analysis pathogens
In case of a non-uniform drying of the liquid cover glass coat, which might impair epidermis preparation quality, a short heating of the coated slide at 30◦C promoted the formation of an optimal adhesive surface
The ACT method can be seen as further development of the perforated-tape epidermal detachment method, which was applicable on A. thaliana adaxial epidermal tissue (Ibata et al, 2013), but not compatible with downstream laser microdissection (LM) applications
Summary
The plant cell wall and its underlying plasma membrane are required to perceive environmental changes like biotic and mechanic stresses and represent the first layers of defense to invadingEpidermal tissue for quantitative analysis pathogens. A specific analysis of separated epidermal tissue can provide new insights especially into the early regulation and organization of stress-related changes and adaption This is of special interest in the field of plant–microbe interactions where cell wall thickenings, socalled papillae, are one of the most prominent and long-studied responses to invading pathogens in epidermal cells (de Bary, 1863). Conventional sample preparation from infected tissues, e.g., complete leaves after fungal infection, results in a relative strong dilution of the targeted stress-related defense structures because they only affect a relatively small part of the whole tissue This is a major restriction in a time-resolved and plant line- or mutant-specific quantification of altered cell wall components, papillae constituents, and associated proteins
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