Isolation and transient transformation of protoplasts from Cuscuta campestris, an obligate parasitic plant

Parasitic plants are important agricultural pests and fascinating examples of plant evolution. Cuscuta campestris is able to locate other plants, coil around them, and produce haustoria that form vascular connections with a host plant. This obligate parasite feeds on the host and exchanges molecules that function in signaling and coordinated development. Research on parasitic plants is challenging because few techniques have been established for their study and genetic manipulation. In this work we aimed to develop a methodology for C. campestris protoplast isolation and transformation. The major challenge for isolation of C. campestris protoplasts was to determine the best starting tissue. Options are limited for a parasite that lacks expanded leaves or roots. We tested shoots from plants grown on a host, seedlings, and callus, but found the best success with shoots that developed from callus culture. Optimization of other parameters ultimately resulted in the ability to routinely isolate approximately one million protoplasts from 3 g starting material, with over 85% protoplast viability. Protoplasts were transiently transformed with multiple constructs, demonstrating robustness of the technique that routinely yields an approximate 10% transformation rate. The research value of C. campestris protoplasts was demonstrated by the expression of subcellular targeted fluorescent proteins and flow-cytometric quantification of the activation of an exogenous auxin-responsive reporter gene. This study describes a technique to produce protoplasts from an obligate parasitic plant and genetically transform the protoplasts. Protoplasts are efficient systems for analyses of cell-level events including gene expression, gene regulatory networks, gene editing, protein localization, and protein function. This method for generating C. campestris protoplasts has potential to accelerate research into the fundamental processes of parasitic plant cells and how they may contrast with autotrophic plants.