Evolutionary analysis of the exocyst in streptophytes links EXO70 diversification to dominance over SEC3 in membrane targeting

The exocyst is a conserved octameric vesicle-tethering complex essential for targeted secretion. It is organized into two modules (I: SEC3, SEC5, SEC6, SEC8; II: SEC10, SEC15, EXO70, EXO84). In plants, the module II subunits SEC15, EXO84, and especially EXO70 have diversified into multiple subfamilies, yet the evolutionary origins and functional consequences of this diversification remain unclear. Here we reconstruct exocyst evolution across streptophytes using phylogenomic, functional complementation, and structural modeling analyses. We show that the three major EXO70 subfamilies originated in anydrophytes--the common ancestor of Zygnematophyceae and land plants--indicating that EXO70 diversification had begun at the dawn of plant terrestrialization. Complementation of Arabidopsis exo70 mutants with EXO70 paralogs from the liverwort Marchantia polymorpha and the streptophyte alga Klebsormidium nitens demonstrates that the ancestral canonical function is retained in the EXO70.1 lineage, whereas other subfamilies have undergone substantial functional specialization. We further uncover an evolutionary shift in exocyst membrane targeting: while Klebsormidium SEC3 retains autonomous membrane-recruitment capacity, land-plant SEC3 subunits have lost this ability, rendering exocyst targeting increasingly dependent on EXO70. Together, these findings suggest that early EXO70 diversification, combined with the redistribution of membrane-targeting functions within the exocyst, enabled paralog-specific exocyst recruitment and facilitated the emergence of specialized secretion pathways during plant terrestrialization. Significance StatementThe exocyst is a conserved protein complex that targets secretory vesicles to the plasma membrane across eukaryotes. In land plants, the EXO70 subunit diversified extensively, but the origins and consequences of this diversification remained unclear. We show that three major EXO70 subfamilies arose in the common ancestor of land plants and their closest algal relatives, at the dawn of plant terrestrialization. Cross-species complementation reveals that the EXO70.1 lineage retains the ancestral canonical function, whereas other subfamilies specialized. We further uncover an evolutionary shift in the mechanism of exocyst membrane targeting, from SEC3 toward EXO70, that could have enabled distinct EXO70 paralogs to direct secretion to different cellular sites during the colonization of land.