Usak, D.; Cifrova, P.; Danek, M.; Podmanicka, T. K.; Kocourkova, D.; Eeckhout, D.; Van Leene, J.; Zeiner, A.; Neubergerova, M.; Kraus, J. M.; Hudecek, R.; Garcia-Gonzalez, J.; Zhivaeva, A.; Antonova, A.; Kalachova, T.; De Jaeger, G.; Wrzaczek, M.; Van Damme, D.; Pleskot, R.

Callose synthase is responsible for the targeted deposition of the {beta}-1,3-glucan polymer, callose which underlines essential plant developmental processes, including cell division, pathogen defense or cell-cell communication. The architecture of the callose synthase complex (CALSC) as well as the molecular mechanisms of callose synthesis remain unknown. Here we report an integrative characterisation of the Arabidopsis CALS complex, with the most enriched subunits, CALS1, CALS2 and CALS3, forming its core. Structurally, CALSC assembles into a trimer, requiring the plant-specific Bag domain to mediate inter-subunit associations. The biological importance of CALSC assembly is highlighted by the simultaneous loss of CALS1 and CALS3, which abolishes plasmodesmal callose deposition and affects symplastic transport. Site-directed mutagenesis and molecular dynamics simulations depict the topology of the CALS1 active site in detail, including the components of the enzymatic reaction. We pinpoint the translocating tunnel through which the nascent glucan is delivered and mechanistically confirm the role of transmembrane helix 8 in regulating glucan export. Our work provides unprecedented insight into the molecular architecture of the CALSC and the distinct changes from maturation to activity at the plasma membrane, while showcasing the mechanisms involved in callose synthesis at the molecular level.