Wilson, L. F. L.; Lim, C.; Torres, M. A.; Scheiner, S.; Wan, Y.; Purushotham, P.; Ho, R.; Zimmer, J.

Dictating cell growth and morphology, cellulose biosynthesis is intrinsic to plant cell biology. Accordingly, cellulose biosynthesis inhibitors (CBIs) are important herbicides, toxins, and experimental tools. We currently lack mechanistic understanding of CBI activity, preventing engineering of herbicide selectivity and disease immunity. Contrasting classical inhibitors, we unexpectedly identify the unusual Streptomyces phytotoxin thaxtomin A as the only in vitro active CBI, with unprecedentedly broad-spectrum activity against various cellulose synthase enzymes. High-resolution cryo-electron microscopy reveals that thaxtomin A leverages exotic nitroaromatic chemistry to target a strictly conserved site in cellulose synthase's polysaccharide secretion channel. Strikingly, in vitro biosynthesis and biophysical assays demonstrate thaxtomin A's near-picomolar efficacy. Plant and algal systems reveal that its global arrest of cellulose biosynthesis produces an osmotically driven crisis in expanding cells. Finally, site-directed mutagenesis generates the first toxin-resistant cellulose synthase. Our results underscore cellulose's critical function in plant lifeforms and inform efforts to inhibit related enzymes across kingdoms.