Agrobacterium -mediated Cuscuta campestris transformation as a tool for understanding plant-plant interactions

Cuscuta campestris , a stem parasitic plant, has served as a valuable model plant for the exploration of plant-plant interactions and molecular trafficking. However, a major barrier to C. campestris research is that a method to generate stable transgenic plants has not yet been developed. Here, we describe the development of a Cuscuta transformation protocol using various reporter genes (GFP, GUS, or RUBY) and morphogenic genes ( CcWUS2 and CcGRF/GIF ), ultimately leading to a robust protocol for Agrobacterium -mediated C. campestris transformation. The stably transformed and regenerated RUBY C. campestris plants produced haustoria, the signature organ of parasitic plants, and these were functional in forming host attachments. Transformed C. campestris also produced flowers and transgenic seeds exhibiting betalain pigment, providing proof of germline transmission of the RUBY transgene. The integration locations of T-DNAs were confirmed through TAIL-PCR. Furthermore, the RUBY reporter is not only a useful selectable marker for the Agrobacterium -mediated transformation but also provides insight into the movement of molecules from C. campestris to the host during parasitism. Thus, the protocol to generate transgenic C. campestris reported here overcomes a major obstacle to Cuscuta research and opens up new possibilities for studying parasitic plant interactions.