Distinct subcellular localizations of DUF1218 proteins in Marchantia polymorpha and Nicotiana benthamiana reveal two different plasmodesmata-targeting mechanisms

Plasmodesmata are membrane-lined channels connecting plant cells to facilitate intercellular transport of molecules. Although many plasmodesmata-localized proteins have evolved throughout plant evolution, whether they use a conserved targeting system remains unclear. In the bryophyte Marchantia polymorpha , we identified two DUF1218-domain proteins homologous to the Arabidopsis plasmodesmata-localized AtTVA. When ectopically expressed, MpDUF1218-1 localized to plasmodesmata in both Nicotiana benthamiana and M. polymorpha , whereas MpDUF1218-2 formed cytoplasmic puncta in both species. Unexpectedly, AtTVA formed cytoplasmic puncta rather than localizing to plasmodesmata in M. polymorpha . Domain-swap analyses revealed that the first helix of MpDUF1218-1 is crucial for plasmodesmata localization in N. benthamiana , while the first two helices are required in M. polymorpha . In contrast, the second and third helices of AtTVA contribute to its plasmodesmata localization in N. benthamiana . Further domain dissection indicated that other regions of MpDUF1218-1 also contribute to accurate targeting by regulating its distribution among the ER, cytoplasmic puncta, and plasma membrane. Together, our findings suggest that MpDUF1218-1 is targeted by a mechanism shared between the two species, whereas AtTVA relies on a distinct mechanism present in N. benthamiana but absent in M. polymorpha , suggesting the emergence of alternative plasmodesmata-targeting pathways during land plant evolution.