Blaukopf, M. | bioRxiv Science

bioRxiv2026

Towards a comprehensive chemical and genetic tool library for rhamnogalacturonan-II oligosaccharides and exploitation

Rhamnogalacturonan-II (RG-II) is considered the most complex glycan in nature. It forms part of an intricate network of complex glycans in the plant cell wall where it plays a critical role in plant growth, development and defence. It has been identified as an important nutrient source for the human gut microbiota (HGM), a key modulator of human health and disease status. Increasing evidence also suggests that RG-II can modulate plant-microbe interactions. Given its importance and potential, detailed studies of RG-IIs structure-function relationships and metabolism are required to underpin future crop-improvement strategies and to harness its benefits for plant and human health. Progress in this field is however hampered by RG-IIs structural complexity and limited access to enabling tools, in particular chemically defined RG-II-derived oligosaccharide (CDRO) substructures. Achieving targeted, efficient, and scalable production of CDROs remains a significant challenge and is indeed one of the major reasons why RG-II and glycomic research in general, significantly lag behind genomic and proteomic research. Here, we have genetically engineered as well as screened a diverse set of genetic strains, including transposon (Tn) mutants of the prominent model human gut microbe Bacteroides thetaiotaomicron (B. theta) and its gut and plant-associated relatives for new CDRO-generating and/or RG-II-utilising strains. Several CDROs, some of which had never been produced before by any other means (including chemical synthesis), where generated and characterised by a combination of high-resolution mass spectrometry (MS), enzymatic profiling and 2D-NMR. In addition to expanding the CDRO toolbox, we identified key genetic strains that will serve as a base or platform for the production of an unprecedented amount of CDROs covering the complexity and diversity of chemical modifications in RG-II. CDROs were later exploited to gain new insights into the microbial metabolism of RG-II in the human gut, revealing key aspects of its chemical structure that drive or limit its metabolism in B. theta. Notably, we generated new evidence in support of an alternative operational paradigm for polysaccharide utilisation systems that are widespread in the Bacteroidota phylum. We confirmed the presence of pathways for the metabolism of RG-II and/or RG-II core sugars D-apiose (D-Apif), and 3-deoxy-D-manno-2-octulosonic acid (D-Kdo) in aerobic plant-associated microbes including fungi and Flavobacterium spp., highlighting their potential to be exploited as cost-effective alternatives to B. theta for the generation of CDROs.

biochemistry↗

bioRxiv2025

High-Throughput Glycan Array Screening Reveals Rhamnogalacturonan-I as a Ligand for Arabidopsis Leucine-Rich Repeat Receptor Kinases

The plant cell wall not only serves as a physical barrier against pathogens but, when damaged, also functions as a source of cell wall-derived molecules that play crucial roles in plant immunity as damage-associated molecular patterns (DAMPs). While oligogalacturonides from homogalacturonan are well-studied DAMPs, the immune-signaling potential of other cell wall components remains largely unexplored. Conventional genetic and biochemical approaches aimed at identifying ligand-receptor pairs in plant immunity have been limited by the vast diversity of potential ligand molecules and functional redundancy of putative receptors. Here, we developed a high-throughput screening pipeline that simultaneously examines multiple interactions between plant cell wall-derived glycans and >350 extracellular domains (ECDs) of receptor kinases and receptor like proteins in Arabidopsis, resulting in the screening of >40,000 interactions. We discovered a group of leucine-rich repeat receptor kinases named ARMs (AWARENESS of RG-I MAINTENANCES) that interact with rhamnogalacturonan-I (RG-I), a major component of pectin. RG-I treatment induced pattern-triggered immunity responses, with distinct kinetics compared to oligogalacturonide responses. We identified RG-I oligosaccharide structures required for interaction with ARM receptors and immune activation, and found that ARM receptors are redundantly involved in plant immunity. Collectively, our approach provides a powerful platform for discovering glycan-receptor pairs in plants, facilitating a more comprehensive understanding of cell wall surveillance mechanisms in plant immunity.

plant biology↗