Abstract text: Callose, a β-1,3-glucan polymer, is an indispensable yet often overlooked component of plant cell walls that plays essential roles in cytokinesis, regulation of plasmodesmata, defence, and plant reproduction. Although plant cells must rapidly and precisely direct callose deposition, the mechanisms governing its spatial targeting and activation remain poorly understood. Callose is produced by 12 distinct CALLOSE SYNTHASES (CALSs), which belong among the largest integral membrane proteins in Arabidopsis. CALS has a size of around 200 kDa and is believed to form larger complex assemblies. In this work, we identified the CALS1 interactome, providing unprecedented insight into the regulatory network governing CALS folding, trafficking, and activity. Furthermore, we localised CALS1 in BY-2 cells and Arabidopsis plants, revealing the plasmodesmata and the cell plate to be the most prominent sites of its targeting. Finally, the pharmacological and genetic evidence revealed that anionic phospholipids act as platforms for the recruitment and/or activation of CALS complexes, ensuring precise and dynamic callose deposition at specific cellular sites.