Analysis of β-Glucans in the Cell Walls of Plants and Fungi Using Thin-Layer Chromatography

Plants are colonized by several fungal pathogens in their lifecycle. Apoplast is an important component in the plant cells where communication between the host and the invading pathogen is initiated. During infections, the extracellular polysaccharide components in the host and pathogens play a crucial role in determining the outcome of plant-pathogen interactions. β-glucans are abundant and structurally diverse polysaccharides dominating the extracellular cell wall surface of plants and fungal pathogens. The variation in glucosyl linkage patterns (e.g., β-1,3-, β-1,4-, and β-1,6-) provides structural diversity in β-glucans, and these patterns dictate their biological functions. β-glucans in the cell walls of plants and fungi are prone to modification during infections, leading to structural change or release of bioactive oligosaccharides that modulate plant immune responses. Therefore, it is crucial to analyze the β-glucan oligosaccharides and their glucosyl linkage patterns in plant and fungal cell walls. A detailed protocol to analyse β-glucan oligosaccharides released from fungal and plant cell walls using thin-layer chromatography (TLC) and linkage-specific glucanases is provided. TLC is a cost-effective and less time-consuming approach to analyze β-glucan oligosaccharides when compared to other chromatographic and mass spectrometry approaches. The provided protocol can be easily established in any lab and can also be applied to characterize novel biologically relevant carbohydrate-active enzymes (CAZymes) that display β-glucanases activity. The information obtained from TLC analysis using linkage-specific glucanases will be also useful for structural elucidation and validation when combined with approaches such as Nuclear Magnetic Resonance.