Abstract text: Carbohydrate-binding modules (CBMs) can be used as probes to image plant cell wall polysaccharides, offering advantages over antibodies in size, production cost, and batch-to-batch consistency. However, many CBMs remain incompletely characterized, and fluorescent labeling approaches suffer from notable drawbacks: fluorescent protein fusions restrict fluorophore choice, while dye conjugation is constrained by protein sequence and produces heterogeneous labeling stoichiometry.

We first address these limitations by expanding the CBM repertoire, introducing CBM probes for unique marine algal polysaccharides. Furthermore, we harness SnapTag, a self-labeling protein tag that reacts covalently with benzylguanine-derivatized fluorophores, so that each CBM only needs to be produced once for labeling post-purification. This enables stoichiometric, single-site labeling, and is compatible with a broad palette of fluorophores, including dyes suited for super-resolution microscopy, making the same CBM stock readily adaptable for multiplexed imaging.

Here, we present examples of how the new CBM repertoire can be applied, including binding validation, bioimaging of developing cell walls, super-resolution microscopy to deduce polymer distribution, and electron microscopy imaging. Taken together, our platform provides an expanded and versatile toolkit for the cell wall research community.