Approximately 1% of all extant angiosperm species are parasitic, deriving all or part of the water and nutrients from host plant species using specialized feeding structures known as haustoria. Among families containing parasites, only the Orobanchaceae contain species representing the full spectrum of parasitism from potentially free-living facultative forms to non-photosynthetic, obligate parasites . Lindenbergia is a non-parasitic lineage of Orobanchaceae sister to all parasitic species ; therefore the family represents an ideal comparative framework to study the evolution of parasitism. Parasitic Orobanchaceae growing in Africa and the Mediterranean include the devastating agricultural pests witchweed (Striga) and broomrape (Orobanche and Phelipanche), respectively. The Striga infestation covers 123.5 million acres resulting in annual yield losses greater than US$7 billion [3, 4]. Broomrapes threaten nearly 40 million acres, though yield losses are difficult to assess due to the frequent abandonment of infested fields and unreliable data on yield loss . Striga is one of the primary biotic constraints to agriculture in Sub-Saharan Africa and the affected areas are increasing in size . The weedy, parasitic Orobanchaceae also threaten parts of Asia, Europe, and North America .
Motivated by the agronomic threat presented by some parasitic Orobanchaceae, Triphysaria versicolor has been developed as a model parasitic plant for the family. As a transformable  and tractable facultative generalist parasite, T. versicolor represents an excellent species to investigate the evolution of parasitism, haustorium development, plant-plant communication, host-parasite interactions, and many other facets of parasite biology [9, 10]. To discover processes important in parasitic plant biology, we focused our analysis on the unifying anatomical feature of parasitic plants, the haustorium. This modified root structure is adapted to enable feeding on the host and is unique to parasitic plants, thus it is a focal point for interactions between the parasite and host .
Heide-Jorgensen and Kuijt [12, 13] showed that the haustorium of T. versicolor contains many specialized cells including haustorial hairs, a xylem bridge between the host and parasite, and transfer-like cells adjacent to vessel elements at the host-parasite interface. Although histological evidence for xylem connectivity between the haustorium of T. versicolor and its host is well documented [12, 13], there is no evidence for phloem connectivity. However, there is evidence that phloem-mobile virus particles move between host and parasite in the holoparasite Phelipanche (syn. Orobanche) ramosa and phloem continuity has been observed in Orobanche crenata. The mechanisms of transport between host phloem and parasite phloem likely vary in different parasites from direct phloem connections  to transport via apoplastic pathways. Bi-directional movement of small RNAs between host and parasite has been documented in T. versicolor attacking transgenic lettuce . The anatomy of the haustorial interface cells and empirical evidence for bi-directional transport point to the host-parasite interface as an epicenter of host-parasite dialogue.
Intimate symbioses tend towards specialization (e.g. parasitism) . A true generalist strategy, where a parasite routinely feeds on many distantly related host species, is relatively uncommon in parasitic organisms . At face value, this is surprising, because a broad host range provides more feeding opportunities. Seedlings of most parasitic plants, for example, must contact and parasitize a suitable host plant soon after germination , and access to a wider range of potential host plants should increase the likelihood of survival, regardless of the specific plants growing nearby . Although less common than host plant specialists, many parasitic plant families do contain generalists, including some or all parasitic members of Orobanchaceae, Lauraceae, Convolvulaceae, Krameriaceae, and most of the 18 families of Santalales (sandalwoods, mistletoes and their relatives ).
If mutations that increase specialized feeding strategies increase in frequency when specific host resources are predictable , then traits associated with maintenance of generalist abilities are likely to decrease in frequency. If a generalist strategy involves the evolution of a general-purpose suite of genes that are necessary and sufficient to successfully parasitize a wide range of hosts, then such a trend could lead to a long-term stable generalist strategy. Alternatively, if generalists maintain distinct sets of genes specific to different hosts, then the long-term maintenance of gene sets for attacking different hosts may be unlikely unless there is frequent reinforcement by a diverse range of hosts.
Triphysaria (Orobanchaceae) is a generalist parasite that feeds on a highly diverse collection of angiosperms in nature, including at least 30 species in 17 families of monocot and eudicot host plants . We reasoned that sequencing transcriptomes from the haustorium of T. versicolor grown on distantly related hosts would maximize the potential to identify both shared and host-specific patterns of gene expression. The transcriptome datasets of T. versicolor provide a unique opportunity to leverage newly established genomic resources of the Parasitic Plant Genome Project (PPGP, ) with well developed functional protocols including parasite-host co-culture [9, 24], haustorium induction assays , and parasite transformation [8, 16, 26]. By characterizing the molecular signature of host-parasite interactions, we stand to gain insight into the processes underway in a generalist parasite that facilitate a broad host range and learn about the molecular mechanisms that can facilitate the generalist parasite strategy.
Two substantial hurdles emerge when characterizing the transcriptomes of T. versicolor haustoria. The first is that gene expression profiles of specialized cells in the haustorium become diluted when harvesting even the tiny haustorium (1–2 mm diameter) of T. versicolor. The excellent histology and electron microscopy work by Heide-Jorgensen and Kuijt [12, 13] revealed cells residing at the host parasite interface that had transfer cell-like morphology. The anatomy of these specialized cells includes dense cytoplasm, numerous small vacuoles, a highly invaginated cell membrane, and a labyrinthine cell wall (for a review see ). We hypothesized that the small collection of interface cells, including those with transfer-cell like morphology, facilitate the elusive molecular interaction between host and parasite, making them excellent candidates for transcriptome analysis. The second hurdle is that discovery of genes and subsequent gene expression analysis on a genome-wide scale is difficult without a sequenced and well-annotated genome, which is currently lacking for T. versicolor. Next Generation Sequencing (NGS) technologies have emerged as powerful tools for exploring new genomes because the cost per base is substantially lower than traditional dye-terminator or even pyro-sequencing (454) methods . In the wake of the NGS revolution several tools for data analysis (for a review see ), including high performance de novo transcriptome assemblers like Trinity , have emerged to facilitate transcriptome analysis in uncharacterized model systems.
To overcome the limitations of reference independent transcriptome analysis of small numbers of difficult to harvest cells, we developed methods to sample parasite-host interface cells from T. versicolor grown on the distantly related and sequenced model hosts Zea mays (B73) (monocot) and Medicago truncatula (A17) (eudicot) via Laser Pressure Catapult Microdissection (LPCM). We extracted and then amplified exceedingly small RNA samples via T7-based linear amplification and then deeply sequenced each of the amplified parasite-host interface transcriptomes. We assembled millions of paired-end Illumina reads de novo, annotated each assembly and then estimated levels of gene expression via read mapping to the de novo assembled transcriptome. Using this approach, we identified genes that were part of a host-specific response as well as those that are part of a shared response of T. versicolor to the different hosts. We also verified the host-specific differential expression pattern of two Triphysaria expansin genes. Expansins are among the few genes known to be differentially regulated in haustoria [31, 32]. Analysis of expansin genes allowed us to verify the differential gene expression pattern present in the interface sequence data, and demonstrate the first evidence that a β-expansin is highly upregulated in T. versicolor when grown on the Z. mays host. Our results suggest that the maintenance of a generalist feeding strategy in Triphysaria involves both generalized and specialized gene responses that help us understand Triphysaria’s generalist feeding abilities.