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Abstract We present the first chromosome-level genome assembly and annotation for the genus Cuscuta, a twining and leafless parasitic plant of the morning glory family (Convolvulaceae). C. campestris, the study species, is a widely studied model parasite, due in part to its worldwide occurrence as a weed of agricultural and natural plant communities. The species has served as a model parasite for studies of parasite biology, haustorium development, growth responses to chemical and light stimuli, gene content and expression, horizontal gene transfer, and interspecies RNA movement and has a recently developed transformation system. The 505 Mb (1C) genome is assembled into 31 chromosomes and supports annotation of 47,199 protein-coding genes, 214 small RNA loci (including 146 haustoria-specific miRNAs), and 3,238 interspecies mobile mRNA loci. C. campestris is a recent tetraploid with a high retention of duplicated genes and chromosomes, with less than 8% nucleotide divergence between homoeologous chromosomes. We also show that transformation of C. campestris with the RUBY marker system allows visualization of transformed Cuscuta-derived fluorescent mobile molecules that have entered the host stem. This genome, with an associated genome browser and BLAST server, will be of value for scientists performing fundamental research in a wide range of molecular, developmental, population, and evolutionary biology, as well as serve as a research tool for studying interspecies mobile molecules, generating genetic markers for species and genotype identification, and developing highly specific herbicides. 

Summary 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 toC. campestrisresearch is that a method to generate stable transgenic plants has not yet been developed.Here, we describe the development of aCuscutatransformation protocol using various reporter genes (GFP, GUS, or RUBY) and morphogenic genes (CcWUS2andCcGRF/GIF), leading to a robust protocol forAgrobacterium‐mediatedC. campestristransformation.The stably transformed and regenerated RUBYC. campestrisplants produced haustoria, the signature organ of parasitic plants, and these were functional in forming host attachments. The locations of T‐DNA integration in the parasite genome were confirmed through TAIL‐PCR. TransformedC. campestrisalso produced flowers and viable transgenic seeds exhibiting betalain pigment, providing proof of germline transmission of the RUBY transgene. Furthermore, RUBY is not only a useful selectable marker for theAgrobacterium‐mediated transformation, but may also provide insight into the movement of molecules fromC. campestristo the host during parasitism.Thus, the protocol for transformation ofC. campestrisreported here overcomes a major obstacle toCuscutaresearch and opens new possibilities for studying parasitic plants and their interactions with hosts.