Engineered glycoside hydrolases as fluorescent probes reveal the spatial distribution of the pectic polysaccharide rhamnogalacturonan II in plant cell walls

Plant cell walls are dynamic composites whose architecture determines growth, mechanics, and environmental resilience. Efforts to link pectin structure to function have been limited by the lack of molecular probes with sufficient specificity, a gap that becomes even more pronounced for the intricately branched rhamnogalacturonon-II (RG-II) subclass. Here we report the first fluorescent probes with defined specificity to RG-II, engineered from catalytic site mutants of Bacteroides thetaiotaomicron glycoside hydrolases BT1010 and BT0996. These enzyme-derived probes bind RG-II monomer with high affinity, discriminate against dimeric forms, and localize to cell corners and junctions in Arabidopsis thaliana stems, consistent with RG-II’s unique ability among wall polysaccharides to form borate-mediated, covalent crosslinkages between molecules. Application of these probes revealed spatial partitioning distinct from the homogalacturonan (HG)- and rhamnogalacturonan I (RG-I)-enriched middle lamella, highlighting functional specialization among pectic domains, with RG-II reinforcing cell junctions while HG and RG-I mediate wall flexibility. Our work establishes a generalizable framework for transforming CAZymes into high-precision imaging reagents, enabling molecular-level visualization of structurally complex polysaccharides in the cell wall.