Evolutionary analysis of the exocyst complex in streptophytes links early EXO70 diversification with dominance over SEC3 in membrane targeting

The exocyst is an evolutionarily conserved vesicle-tethering complex composed of two modules (I: SEC3, SEC5, SEC6, SEC8; II: SEC10, SEC15, EXO70, EXO84). In plants, the module II subunits SEC15, EXO84, and particularly EXO70 have diversified into distinct subfamilies, yet the evolutionary origins and functional consequences of this diversification remain unclear. Here, we reconstruct the evolutionary history of the exocyst across streptophytes and combine phylogenetic analyses with cross-species functional complementation and structural modeling to investigate how exocyst membrane targeting evolved. We show that the three major EXO70 subfamilies emerged already in anydrophytes--the common ancestor of Zygnematophyceae and Embryophyta--and subsequently expanded independently in multiple lineages. Complementation experiments using the liverwort Marchantia polymorpha and the streptophyte alga Klebsormidium nitens demonstrate that canonical EXO70 function is deeply conserved and retained in the EXO70.1 lineage, whereas other subfamilies exhibit pronounced functional specialization. Importantly, we uncover an evolutionary shift in exocyst membrane targeting: whereas Klebsormidium SEC3 retains autonomous exocyst membrane-recruitment capacity, land-plant SEC3 subunits have lost this ability, rendering exocyst membrane targeting increasingly dependent on EXO70. We propose that this shift, together with early EXO70 diversification, enabled paralog-specific exocyst targeting and facilitated the evolution of specialized secretion pathways during plant terrestrialization.