Abstract text: Plant cell wall plays a pivotal role in plant–pathogen interactions, where cell wall–derived oligosaccharides released during infection can trigger plant defence responses. The involvement of these oligosaccharides has largely been inferred indirectly through plant responses after exogenous treatments. However, the direct in planta analysis of their presence during infection remains technically challenging due to their low abundance and structural complexity. Here, we present an analytical workflow based on Hydrophilic Interaction Chromatography coupled to Mass Spectrometry (HILIC-MS), enabling sensitive and high-resolution profiling of cell wall–derived oligosaccharides during biotic interactions.

In this study, we focused on the maize–Colletotrichum graminicola interaction, where broad HILIC-MS profiling revealed a dynamic oligosaccharide profile during the interaction and confirmed their presence in planta during pathogen colonization. Notably, β-glucan oligosaccharides detected in infected tissues were shown to trigger immune responses in maize and enhance resistance to C. graminicola, supporting their biological relevance in this interaction. Our approach enables comprehensive profiling of the oligosaccharide and its temporal variation at low concentrations. The method has also been applied to additional plant–pathogen systems, plant–pest interactions and even nutritional studies. Altogether, this workflow provides a robust and transferable platform for investigating cell walls–derived oligosaccharides during biotic interactions.