Ligand-Baited Proteomics Reveals Vesicle Trafficking Proteins as Putative Site of Action of Indaziflam

Abstract Indaziflam is a potent herbicide that has become an important tool for pre-emergence weed control. Although it is classified as a cellulose biosynthesis inhibitor, its precise site of action remains unresolved. Here, we show that indaziflam preferentially affects the root elongation zone, where cells exhibit swelling and frequent rupture. We performed ligand-baited coprecipitation assays using root tissue lysates from Arabidopsis thaliana and Oryza sativa. Proteomic analysis revealed that the highly enriched proteins were associated with vesicular trafficking, including multiple coatomer subunits, while no cellulose synthase components were detected. Live-cell imaging using FM4-64 dye demonstrated that indaziflam does not cause a complete inhibition of clathrin-mediated endocytosis. AlphaFold3-based protein–ligand modeling further identified relatively stable predicted indaziflam poses with select trafficking-associated proteins, including coatomer subunit β. Collectively, our results support a model in which indaziflam indirectly disrupts cellulose biosynthesis by interfering with intracellular trafficking processes required for proper cellulose synthase complex homeostasis.