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.