T. versicolor and hosts were germinated and grown axenically in separate culture plates. To begin co-culture, hosts were transferred to fresh plates and T. versicolor were added and placed in close proximity (~1 mm) to host roots. The attachment rate of T. versicolor to host roots was ~90% for M. truncatula and ~50% for Z. mays. This difference was likely due to the more rapid growth rate of Z. mays (compared to M. truncatula) coupled with the confined dimensions of the co-culture petri dish rather than differential parasite-host compatibility. Where host roots remained more or less stationary on the agar growth medium during early phases of co-culture, the attachment rates of T. versicolor were high (>90%) and equivalent between Z. mays and M. truncatula.

The first step in sample preparation for LPCM was isolation and cryosectioning of haustoria formed on each host. The optimum section thickness was determined empirically by micro-dissecting samples from sections cut at 1μm section thickness intervals from 18-30 μm. For T. versicolor haustoria we determined that 25 μm cryosections were optimal to allow efficient tissue release from the adhesive coated StarFrost™ LPCM slides coupled with maximum tissue harvest volume. Parasite and host cells that were in contact with each other at the interface were difficult to separate, so to ensure capture of the entire parasite interface cell population we intentionally included a minimal amount of host tissue, knowing that host transcripts could be identified and removed informatically. Figure 1 shows a typical haustorium cross section before (A) and after (B) LPCM.

To generate representative interface-cell samples we pooled ~110 interface regions of interest (ROIs) from biological replicates (>8 haustoria). The average ROI for T. versicolor interface transcriptome samples grown on M. truncatula was 54,910 μm² with a total area of 6.1 million μm² that yielded 144 ng total RNA. This pooled sample had an RNA integrity number (RIN) of 7.6, an A₂₆₀/A₂₈₀ of 1.58 and an A₂₆₀/A₂₃₀ of 0.76. The average ROI for T. versicolor interface transcriptome samples grown on Z. mays was 56,079 μm² with a total area of 6.4 million μm² that yielded 160 ng total RNA with a RIN of 6.9, a A₂₆₀/A₂₈₀ of 1.68 and a A₂₆₀/A₂₃₀ of 0.11.

The first step of T7 based amplification is cDNA synthesis with an oligo-dT/T7 RNA primer/promoter. It is critical that this step is highly efficient to minimize the bias toward shorter fragment lengths in amplified samples 33 . We frequently observed a yellow-brown material at the host parasite interface that may have contributed to the low initial purity of the interface RNA samples, indicated by the low A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ ratios. Thus, we cleaned the interface total RNA with the Zymo™ RNA Clean and Concentrator kit. Subsequently, we observed consistent amplification performance between technical and biological replicates of the cleaned interface total RNA as well as performance consistent with positive control samples of A. thaliana young leaf RNA of high quality and purity (28s/18s ratio: 1.9; RIN: 8; A₂₆₀/A₂₈₀: 2.0, A₂₆₀/A₂₃₀: 2.1).

Amplification of ~100 ng of total RNA routinely yielded 50-100 ug of amplified RNA (aRNA) after two rounds of amplification, which was consistent with the positive control (Arabidopsis young leaf total RNA), the expected performance of the Message Amp™ II aRNA kit, and a previous report 34 . The aRNA yield after a single round of amplification was up to 100 ng, which is sufficient for construction of an Illumina sequencing library, yet we chose to amplify the samples for two rounds since it was desirable to have additional aRNA for further analyses including qRT-PCR validation of gene expression profiles. The fragment length profiles, as determined via Bioanalyzer™, were reduced from the first to the second round of amplification, which is consistent with a previous report 33 .

Amplified interface RNA samples were sequenced on one lane each of Illumina’s Genome Analyzer IIx with an 83 × 83 bp paired-end cycle protocol. Sequencing data are available at http://ppgp.huck.psu.edu 23 . The T. versicolor interface transcriptome datasets (Table 1) contained 17.9 million read pairs on Z. mays and 19.1 million read pairs on M. truncatula. Host reads from each interface transcriptome dataset were mapped to their respective host genomes, leading to the removal of 1.5 million M. truncatula reads and 0.4 million Z. mays reads from each respective transcriptome data set. Reads were quality trimmed and filtered (see methods), leaving >26 million reads (orphans and mate pairs) for each sample that were then assembled separately using Inchworm (Trinity, 30 ) and post-processed to remove exact duplicate or non-translatable sequences. The interface transcriptome assembly of T. versicolor grown on Z. mays yielded 12.77 Mbp of assembled sequence represented by 28,126 unigenes with an N50 of 525 bp (Table 1). The interface transcriptome assembly of T. versicolor grown on M. truncatula yielded 12.25 Mbp of assembled sequence represented by 26,709 unigenes with an N50 of 536 bp (Table 1). Sequencing and assembly statistics were similar in all categories (Table 1) indicating that both data sets were of equivalent quality.

Low quality reads were filtered before assembly, and host sequences were filtered both before and after assembly. Unigenes remaining after removal of host plant and non-plant sequences were aligned with BLASTx to sequences detected in any other PPGP transcriptome library of Triphysaria versicolor (http://ppgp.huck.psu.edu/). Unigenes with less than 95% pairwise identity to either host or to other Triphysaria libraries were sorted further if a BLASTx search of the NR (http://www.ncbi.nlm.nih.gov) database yielded alignments of 1e-10 or stronger. The remaining unclassified unigenes were submitted to OrthoMCL DB and InterProScan. Unigenes that remained unclassified after the final screen are called “no hit” unigenes.

Unigenes were annotated using an objective classification of known plant genes from the PlantTribes 2.0 database 35 36 as described in Wickett et al. 37 . We assigned genes into a hierarchy of gene clusters, which includes approximate gene families (Tribes), and potentially narrower lineages (Orthogroups) which seek to represent descendants of a single ancestral gene in the collection of reference plant species 35 36 37 . We also classified unigenes from our experiment using BLAST to query sequence databases (Table 1). To identify host derived unigenes in the mixed-species transcriptomes, we established a pairwise nucleotide identity threshold of 95% by querying a collection of Z. mays ESTs for T. versicolor grown on M. truncatula and vice versa (Additional file 1: Figure S1). The plot of T. versicolor unigene identity to each host database is clearly divergent at 95% compared to the reciprocal host database query. To verify that the incident high identity was not due to cross contamination between samples, we also queried the host EST databases with a de novo transcriptome assembly of Lindenbergia philippensis, a non-parasitic member of the Orobanchaceae 23 . The BLAST identity plot of the Lindenbergia transcriptome shows a similar trend to the plot of Triphysaria interface transcriptomes queried against the respective non-host databases (Additional file 1: Figure S1).

In order to remove host genes that may have escaped detection during pre-assembly read mapping, we further screened the assembly based on sequence similarity to cDNA (Phytozome, 38 ) and EST databases (PlantGDB, 39 ). This screen removed 4,967 unigenes from the transcriptome of T. versicolor grown on Z. mays and 7,785 unigenes from the transcriptome of T. versicolor grown on M. truncatula (Table 1). The same reference transcripts used to screen the raw read data were also used to screen assemblies. The large number of putative host derived unigenes indicates that read screening with Mosaik at default values alone was insufficient to remove all host contamination. After the host screen the remaining unigenes were filtered for T. versicolor genes based on sequence similarity to genes detected in other PPGP libraries of Triphysaria versicolor 23 . We identified 17,887 unigenes from the transcriptome of T. versicolor grown on Z. mays and 14,352 unigenes from the transcriptome of T. versicolor grown on M. truncatula that had >95% identity at the nucleotide level to T. versicolor genes from the other PPGP libraries. After removing unigenes with high similarity to T. versicolor unigenes from other assemblies in the PPGP database, the remaining 5272 unigenes in the interface transcriptome of T. versicolor grown on Z. mays and 4572 unigenes in the interface transcriptome of T. versicolor grown on M. truncatula were used to query the non-redundant protein sequence database (NR) at NCBI 40 using BLASTx at a threshold e-value of 1e-10. Roughly half of the remaining unigenes in each interface transcriptome had best hits to plants including the model species Arabidopsis, Populus, and Vitis, other Orobanchaceae, or >30 other plant species (“Other Plant Hits” Table 1).

Each T. versicolor interface transcriptome had ~2300 unigenes with no significant alignments to sequences in any of the above described external databases (Table 1). We took several additional steps to try to identify these unknown sequences. Though these unigenes are not classified by source, we identified potential plant gene orthologs for ~20-25% of the remaining unigenes via the query of the PlantTribes 2.0 database. We then queried the extensive InterProScan (IPS) 41 and OrthoMCL DB 42 databases with the translated sequences of the remaining, unclassified unigenes. The majority of these unigenes (>75% in each transcriptome) lacked significant similarity to genes in the OrthoMCL database, nor did they contain IPS peptide motifs (Additional file 2: Figure S2); they are thus referred to as “no hit” unigenes (Table 1). The scan of OrthoMCL DB resulted in identification of an additional 100 plant and 7 non-plant unigenes from the Zea grown T. versicolor and 85 plant and 16 non-plant unigenes from the Medicago grown T. versicolor (Table 1). Additionally, 370 Zea grown T. versicolor and 310 Medicago grown T. versicolor unigenes contained IPS motifs. Roughly half of the OrthoMCL DB hits lack descriptions or have minimal (e.g. one word) descriptions (Additional file 3). A similar pattern exists in the IPS search results, where about half of the unigenes with IPS motifs contain only putative secretion signals and/or transmembrane domains (Additional file 3). Overall our efforts to classify the unigenes in each assembly resulted in identification of potentially orthologous sequences for 82% of the Zea grown T. versicolor interface unigenes and 88% of the Medicago grown T. versicolor unigenes. We were able to assign a putative origin to >90% of unigenes in both transcriptomes and only 5% in each transcriptome remain unclassified. Of these, 493 unigenes from the T. versicolor grown on Medicago assembly and 536 unigenes from the T. versicolor grown on Zea assembly are longer than 300 bp and have read support.

The interface transcriptome of T. versicolor grown on Z. mays and M. truncatula contained a total of 127 and 329 unigenes, respectively, with best hits to non-plant species (Table 1). The non-plant component of each interface transcriptome included best hits to 16 taxa shared by both interface libraries. These included Escherichia, Aspergillus, Clavispora, Burkholderia, and others. Among this set, Burkholderia was the most highly represented taxon (>20 fold increase over any other species) in the non-plant component of both interface transcriptomes. These interface Burkholderia sequences were not detected in the reference transcriptome (TrVeBC1, sequence identity cutoff >90%).

The remaining “no hit” sequences, especially those >300 bp with read support, could represent unannotated host or parasite genes, uncharacterized associated symbionts, or incidental contamination. All remaining unigenes not assigned to a host plant or non-plant source in the NR database (23,032 for T. versicolor on Z. mays and 18,595 for T. versicolor on M. truncatula) are considered collectively as “putative T. versicolor derived unigenes.”

To examine the profiles of the T. versicolor interface transcriptomes, we used an annotated transcriptome from above ground tissues of autotrophically grown T. versicolor as a reference ( 23 Triphysaria assembly TrVeBC1). We sorted unigenes by (PlantTribes 2.0) Orthogroups to identify host-specific and shared components of the interface transcriptomes of T. versicolor grown on Z. mays and M. truncatula (Figure 2). As expected, the largest number of Orthogroups (5947, or 53.6% of the total detected in T. versicolor) were shared between all three transcriptomes, and likely represent expression of genes involved in processes common to a wide variety of cell types. A large number of Orthogroups (1124) were shared between the interface transcriptomes of T. versicolor interacting with both hosts. These genes likely include a putative core set of parasite genes that are active irrespective of the host plant species. Many additional Orthogroups were either exclusive to the interface and host-specific (677 for Z. mays and 361 for M. truncatula), or shared with above ground phases of growth (1066 for Z. mays and 314 for M. truncatula).

Our annotation strategy includes assignment of a GO Slim category term derived from the best BLAST hit in PlantTribes 2.0. GO Slim categories are the broadest designations of GO and are useful for transcriptome-wide comparisons. The host-specific component of the T. versicolor interface transcriptome is likely to contain genes that interact with unique aspects of host biology while those that are shared likely contain genes essential for parasitism. To determine if the annotation profiles were similar between the overlapping and unique transcriptome components we plotted the proportion of GO Slim categories of unigenes (Figure 3) represented by unique or overlapping Orthogroups in Figure 2. GO Slim category profiles between equivalent components of each interface transcriptome were generally similar to each other, yet often distinct from profiles of non-equivalent components of each interface transcriptome (Figure 3). For instance, interface-unique Orthogroup profiles were similar between both interface transcriptomes, yet distinct from the above ground Orthogroup profiles of both interface transcriptomes.

GO Slim category profiles in overlapping and unique sets of Orthogroups within each interface transcriptome were tested for proportionality by a Chi-Square test (Additional file 4: Figure S3A-F). The results of all 6 tests showed disproportionality and were strongly significant (P<<0.0001). The number of unigenes in each GO Slim category, with strong residual values (strongly positive or strongly negative, thus disproportionate), is indicated in Additional file 4: Figures S3A-F. The results of this analysis are concordant with the plot of GO Slim category profiles (Figure 3). The most striking result is the consistently strong over-representation of unigenes lacking GO Slim categories in interface-unique Orthogroups (Additional file 4: Figure S3A-C: columns A and B, Figure S3D-F: columns E and F) compared to the consistent weak representation of unigenes lacking GO Slim categories in the shared Orthogroups (Additional file 4: S3A-C: column D, Figure S3D-F: column H). Interestingly the Orthogroups shared in the interaction of T. versicolor with both hosts are overrepresented by “transcription factor activity” GO Slim Function terms (Additional file 4: Figure S3A: column B and 3D: column F) and underrepresented by “transport” GO Slim Process terms (Additional file 4: Figure 3: column B and 3F: column F). This indicates that there are transcription factor genes active at the parasite-host interface that are not expressed in the above ground reference transcriptome. In contrast, the transporter gene families expressed at the interface are expressed in the above ground reference transcriptome as well.

An advantage of using a de novo assembled transcriptome for RNA-Seq is an intrinsic threshold for transcript detection. If the transcript is represented by sufficient reads for de novo assembly, the presence of a target for de novo RNA-Seq is evidence for the presence of a transcript. The reference assembly TrVeBC2 23 includes data from the haustorium of T. versicolor grown on M. truncatula and was used as a reference to map reads from each interface transcriptome. We correlated normalized reads (reads/kilobase/million mappable reads (RPKM)) from unigenes belonging to Orthogroups shared between the interface transcriptomes and the above ground reference transcriptome, TrVeBC1 (Additional file 5: Figure S4). For unigenes detected in both interface transcriptomes the correlation was high (Pearson’s R= 0.81), which indicates low technical and biological variability between the interface transcriptomes.

To determine the expression level of each unigene we also mapped reads to each respective interface de novo assembly. The 20 most highly expressed unigenes in each set of shared and unique Orthogroups from the two transcriptomes are presented in Additional file 6: Figures S5A-C. We queried this set of 120 unigenes against the NR database using BLASTx (e-value threshold: 1e-10) and 17 annotated plant genomes using BLASTn and BLASTx. The results of the database queries using BLAST are presented in Additional file 6: Figures S5A-C. The best-hit descriptions from searches in NR were concordant with the annotations assigned using PlantTribes 2.0 (See BLAST results in Additional files 7 and 8). We also used InterProScan to predict signal peptides and transmembrane domains for the unigenes listed in Additional file 6: Figure 5A-C (Additional files 9 and 10). The motif prediction tools frequently identified putative transmembrane domains in unigenes annotated as transporters and secretion signals in unigenes annotated as secretory proteins. Of the 120 unigenes listed (Additional file 6: Figures S5A-C), nine had no hit when queried against NR. The remaining unigenes had best hits to plant species. About 30% of these 120 unigenes have either no BLAST hits in NR or align to predicted, hypothetical, or otherwise uncharacterized sequences. This result is consistent our finding that the interface is enriched with unigenes that lack GO Slim category assignments (thus functional annotations).

Among the most highly expressed genes in the shared orthogroups of interface samples of T. versicolor grown on in both Z. mays and M. truncatula (Additional file 6: Figure S5A) are a β-expansin gene (see below), genes for several other cell wall modifying enzymes, and a gene encoding a putative ap2-erf domain transcription factor. A striking pattern in the shared interface Orthogroups was 10 unigenes (including six of the most strongly expressed unigenes from T. versicolor grown on Medicago) with sequence identity to annotated pathogenesis-related proteins in other eudicot species. A single M. truncatula unigene passed through the host plant removal process in the common interface component (ID 5537); this also shared high sequence identity with a pathogenesis-related protein.

Of the unigenes listed in Additional file 6: Figures S5A-C, 42 from the interface transcriptome of T. versicolor grown on Z. mays and 34 from the interface transcriptome of T. versicolor grown on M. truncatula had strongest BLAST hits to Asterid genomes (including Mimulus guttatus). When we queried the 17 plant genomes database there were slightly more best hits to legumes in the Medicago grown Triphysaria data set, perhaps because there is less sequence divergence between the eudicots Triphysaria and Medicago than between the more distantly related Triphysaria and Zea. This results in a somewhat broader range of ambiguous sequence identity between host and parasite. Despite rigorous filtering, a single putative Z. mays transcript and three putative M. truncatula transcripts persevered (indicated in bold) in the highly expressed gene list in Additional file 6: Figure S5.

Among the highly expressed unigenes observed in the interface transcriptome of T. versicolor grown on Z. mays was a putative β-expansin (Additional file 6: Figure S5A). Manual curation of the read mapping data indicated that it was highly expressed when grown on Z. mays, and a nearly identical unigene from the M. truncatula-grown Triphysaria interface was lowly expressed. This apparently host-specific gene expression pattern was of interest because expansins are cell-wall loosening proteins (for a review, see 43 ) that have been implicated in the interaction between parasitic plants and their hosts 31 32 . While the β-expansin gene was expressed in both samples, read mapping evidence suggested that this gene (unigene 772) was highly differentially expressed. As a point of comparison, we investigated a putative α-expansin, unigene 11, which showed a reciprocal pattern of high expression in the interface transcriptome of T. versicolor grown on M. truncatula. We verified the nucleotide sequence of unigenes 772 and 11 via dye-terminator sequencing of PCR products amplified from interface aRNA.

Phylogenetic analysis of β-expansin unigene 772 shows that it is nested within a supported clade of dicot β-expansin sequences (Additional file 11: Figure S6A) indicating that unigene 772 is a dicot β-expansin and not a Z. mays derived sequence. Annotation via InterProScan supports an expansin identity for 772 (Additional file 9) and shows a putative 5’ signal peptide (Additional file 6: Figure S5A), consistent with a role in the apoplast that is typical for expansins. Additionally, the results of all of the BLAST searches suggest that unigene 772 is a T. versicolor derived sequence. Phylogenetic evidence for unigene 11 does not yield a well-resolved tree of α-expansins (Additional file 11: Figure S6B), but the BLAST results suggest that the pairwise nucleotide identity to known, or putative (e.g. ESTs) M. truncatula genes is <70%, while unigene 11 has high identity (>95% pairwise nucleotide identity) to Triphysaria unigenes in other PPGP assemblies.

We sought to verify the reciprocal expression patterns of these two expansins via qRT-PCR. Unigenes 772 and 11 were assigned formal names TvEXPB1 and TvEXPA4, respectively. We verified that primers were specific to their targets by melting curve analysis. To further verify that the TvEXPB1, TvEXPA4, and TvActin primers were specific to parasite transcripts, we harvested portions of host roots that were immediately adjacent to mature T. versicolor attachments and interrogated them via qRT-PCR. In these host root samples we were able to detect ZmActin in Z. mays root samples and MtActin in M. truncatula samples, while the parasite primers yielded signal consistent with background.

We interrogated biological replicates of the T. versicolor host-parasite interface cells grown on both Z. mays and M. truncatula via qRT-PCR for expression of the reference gene, TvActin, TvEXPA4, and TvEXPB1 (Figure 4). TvEXPB1 is up-regulated >120 fold (P=0.024) in T. versicolor haustorial interface cells grown on Z. mays relative to T. versicolor grown on M. truncatula. TvEXPA4 shows a weak reciprocal pattern (P=0.17). The expression patterns of TvEXPA4 and TvEXPB1 are concordant with our de novo RNA-Seq results. Additionally, when we examined TvEXPB1 expression in whole haustorium samples the signal was indistinguishable from background, suggesting that the massive upregulation of TvEXBP1 is specific to a small number of interface cells.

The power to develop a comprehensive picture of any biological system lies with understanding the myriad processes underway in complex organs and tissues. A primary hurdle to revealing this picture is the ability to identify, separate, capture and analyze tissues and cells of interest. Several authors have emphasized the importance of high resolution, high through-put investigations of gene expression in a tissue- and cell-specific manner as well as the need to survey gene expression in a global manner, and why LM (including LPCM) is emerging as a powerful tool for genomics 44 45 46 47 .

LM generated samples from some model systems have been examined using microarrays 48 49 50 51 52 53 allowing investigators to analyze global gene expression patterns in specific tissues and cell types. More recently, LM has been increasingly coupled with NGS to sequence the transcriptomes of various tissues in Z. mays 54 55 56 and S. lycopersicum 34 . The advent of de novo transcriptome assembly now makes global surveys of gene expression in specific tissues and cells of non-sequenced organisms a logical next step. To examine the parasite-host interface transcriptome of T. versicolor, we combined LPCM with robust T7 based linear RNA amplification 33 57 58 59 60 in concert with Illumina mRNA-Seq, high performance de novo transcriptome assembly 30 , and various assembly post-processing tools.

For the haustorium of T. versicolor our sampling strategy was based largely on detailed histology and transmission electron microscopy work done with field-collected specimens 12 13 . This critical background information allowed us to identify cells of interest within the haustorium and then subsequently identify regions of the haustorium in cryosections that contained these cells. Plant tissues must be embedded prior to LPCM and preparatory steps can have an impact on the quantity and quality of RNA preparation 61 . However, the ability to identify tissues of interest must be balanced with downstream usability. The histological quality of the section is an important consideration that may determine the sample preparation method. Paraffin embedded sections generally provide high histological quality at the expense of RNA quality and yield 62 63 . Histological quality increases with thinner sections for sampling at a finer spatial resolution and the efficiency of pressure catapulting increases with thinner sections, yet Kerk et al. 64 report increased RNA yield from thicker sections. We found that the optimum section thickness for capturing interface cells of the T. versicolor haustorium was 20-25 μm. This was determined based on a balance of our ability for histological identification of tissues and cells of interest with efficient tissue release from the slide during the pressure catapult phase of LPCM. The integrity of plant tissues that are susceptible to damage by flash freezing can be preserved by infusion with a cryoprotectant 65 . Cryosectioning with the CryoJane™ (a cryosection transfer system) allowed us to easily capture serial cryosections of T. versicolor haustorium that routinely yielded high quality RNA from carefully chosen samples.

By design, our sampling strategy minimized the likelihood that differences in gene expression arose from temporal or spatial sampling artifacts. The co-culture of T. versicolor is not highly synchronous, so our sample of haustoria represents a broad temporal window of connections that are ~8-10 days old. We collected ~110 interface ROIs which diminishes the likelihood of spatial sampling artifacts. Furthermore, highly similar statistics throughout sample processing and data analysis, including a high correlation of read counts for unigenes in shared Orthogroups (Additional file 5: Figure S4) and verification of the expression pattern of TvEXPB1 in additional experiments with biological replicates (Figure 4), indicates low variation from either biological or technical sources.

Orobanchaceae include the pernicious weeds Striga, Orobanche and Phelipanche, as well as the model parasite Triphysaria. We reasoned that by discovering a subset of genes that likely include those central to the response of T. versicolor to distantly related hosts, we would move closer to identifying a core set of parasitism genes operating in the weedy Orobanchaceae. The molecular dialogue of the parasitic plant with its host is largely uncharacterized 66 . Here we report the identification of the subset of genes expressed at the host-parasite interface by T. versicolor in response to both of the distantly related hosts Z. mays and M. truncatula. Additionally, we identified host-specific patterns of gene expression indicating that the generalist parasitic plant T. versicolor maintains suites of genes for use with different hosts.

The genes of T. versicolor expressed at the host-parasite interface in response to both Z. mays and M. truncatula included a substantial set of genes annotated in other plant species as pathogenesis- (or pathogen-response) related proteins. This included six of the most highly expressed genes in the Triphysaria interface grown on M. truncatula, and three when grown on Z. mays, but a number of additional unigenes were also putative homologs of genes that are upregulated during pathogen invasion (dirigent-like, acidic endochitinase, disease resistance proteins, etc.). While upregulation of genes of these classes would be expected as a defense response to pathogens, this observation suggests that pathways commonly involved in plant protection are also turned on by the parasite during the process of host invasion. It has been already suggested that parasitic plants may have recruited pathways from allelochemical detoxification for use during haustorium signal transduction 67 . Whether Triphysaria is defending itself during the invasion process with pathogen resistance (PR) genes, or has recruited PR pathways as offensive ‘weapons’ is not yet clear, but it does appear that a portion of the core gene set for Triphysaria parasitizing either host has been derived from pathways that plants use for defense against pathogens.

There is no consensus for why true generalist parasites occur or persist, even infrequently, despite evidence for an evolutionary trend toward specialization ( 18 and references there-in). We hypothesized that a successful generalist feeding strategy might rely heavily on general-purpose genes that were deployed for feeding on any host. Instead, we see large components of the interface transcriptome that are detected only when feeding on one of the tested host plants. Of the 2162 Orthogroups detected only in interface transcriptomes, about half (52%) were expressed on both host species, but a substantial fraction of interface Orthogroups were detected only when grown on maize (31%), or on Medicago (17%). Long-term maintenance of extensive genetic machinery directed toward parasitism of subsets of possible hosts will require maintenance of selective pressures on the genes that are deployed in a host-specific manner. Unless the parasite routinely encounters and parasitizes a wide range of host plants, selective pressure on some host-specific genes will be relaxed and the gene functions eventually lost, limiting the plant’s potential to parasitize some hosts, despite the potential advantages of a large host range. The evidence for host specific gene expression suggests that T. versicolor regularly parasitizes across a broad host range, maintaining selective pressure on genes used in a host-specific manner. Indeed the advantage of a broad host range has been explored where host populations are moderately variable in space and time 19 . This study serves as a starting point for a broader survey of putative and potential hosts for Triphysaria as well as a first look at gene expression profiles of highly specialized tissues in parasitic plant haustoria.

It is intriguing that the host-specific and shared-interface gene families (Orthogroups) were over-represented by unigenes with no GO Slim category term assignment compared to the above ground transcriptome. Because Orthogroups were defined based on a classification of sequenced and annotated plant genomes 35 36 37 to which the parasite genes were assigned, this observation highlights the fact that genes expressed in the haustorium include many that have been recruited from the subset of genes whose function is not yet known in any plant. In addition, approximately 1300 unigenes from each interface transcriptome lack strong homology to any known sequence, though 25% of these unigenes are high identity reciprocal best hits in the interface data sets from each host. Further, because such patterns are reproducible in the interface transcriptomes of T. versicolor when grown on different host plants, these data suggest that genes of unknown function are expressed in the haustorium in a host-specific manner. Our data also suggest that underground phases of growth in T. versicolor are enriched for genes of unknown function.

Of those unigenes with GO Slim category assignments, the shared interface-specific Orthogroups are overrepresented for the GO Slim Function category “transcription factor” and underrepresented for the Go Slim Process category “transport.” This indicates that there are transcription factor Orthogroups unique to the interface (relative to the above ground reference transcriptome), yet Orthogroups involved in transport processes are active in all three transcriptomes examined. The latter observation regarding transport does not rule out differential expression of particular genes that are expressed in all three transcriptomes in this study.

The expansin gene family includes 4 main groups: α, β, expansin-like A and expansin-like B 43 . Expansins are thought to loosen plant cell walls by allowing slippage of cell wall polymers 43 . Their activity is non-enzymatic, but they are distantly related to glycoside hydrolase family proteins 43 . Expansins are involved with cell growth 68 and have been implicated in the interaction of bacterial plant pathogens 69 70 , plant-parasitic nematodes 71 72 and parasitic plants 31 32 with their plant hosts. In each each case, expansins are suggested to play a role in host invasion.

Expansin activity has been assayed in the cell walls of both monocots 73 74 75 and dicots 76 77 . β-expansins have activity that is specific to the cell walls of grasses, but not dicot or most other monocot cell walls 78 . The reverse pattern of action is found for α-expansins, which suggests substrate specificity for both α- and β-expansin proteins 43 79 . Typically, expansins are found at low concentrations 43 with the exception of grass pollen that secretes massive amounts of β-expansins 76 that likely serve to loosen stylar tissue during pollen tube growth 80 81 . Although the exact mechanism of action remains unknown, grass cell walls have relatively small amounts of xyloglucan and pectin; these are replaced with β-(1→3),(1→4)-D-glucan and glucuronoarabinoxylan. Both of these grass cell wall components are potential targets of β-expansins in their wall-loosening activity 82 . Throughout more than a decade of research, the accumulation of β-expansins to high levels was thought to be specific only to grass pollen 82 .

In this study, we have shown that the transcript level for the β-expansin TvEXPB1 is among the most highly expressed genes in the interface transcriptome of T. versicolor grown on Z. mays. Relative to the interface transcriptome of T. versicolor grown on M. truncatula, the expression of TvEXPB1 is greatly up-regulated (>120-fold) in the parasite-host interface tissues of T. versicolor grown on Z. mays. The massive and host-specific expression of TvEXPB1 is suggestive of a role in a dicot parasite’s interaction with a grass host. Additionally, the signal for TvEXPB1 in whole haustorium samples of T. versicolor grown on Z. mays was undetectable relative to the interface samples suggesting that TvEXPB1 is highly specific to the host-parasite interface.

Taken together, the evidence for grass cell wall-specific activity of β-expansins and the massive upregulation of a parasite β-expansin at the parasite-host interface suggests that T. versicolor expresses TvEXPB1 when interacting with the monocot host Z. mays in an effort to manipulate host cell walls. Heide-Jørgensen and Kuijt 12 13 observed that host cortical and epidermal cells seemed crushed and displaced at the host parasite interface in the haustorium of T. versicolor. The mechanism of host tissue displacement may include cell wall modifying proteins like expansins that have host cell wall-specific activity. Such a mechanism would allow the parasite to soften or separate host cell walls without affecting the integrity of its own cell walls in the penetrating haustorium. The specific role that expansins play in the host parasite interaction will only be uncovered through detailed functional analysis. This includes focused gene expression analysis, targeted silencing of T. versicolor expansin genes and biochemical characterization to determine the substrate specificity of the expansin proteins encoded by these genes.

The best represented genus in the non-plant transcriptome component (~60% of non-plant hits in T. versicolor grown on Z. mays and ~45% non-plant hits in T. versicolor grown on M. truncatula) was Burkholderia, a common genus of soil bacteria. The high frequency of hits to this bacterium is surprising for three reasons: (1) the relative frequency of other non-plant genera was much lower, (2) T. versicolor seeds were aggressively surface sterilized prior to axenic co-culture, and (3) Burkholderia-derived unigenes were not detected in the above ground reference assembly. The low frequency of hits to other genera (including plant pathogenic fungi, human and other bacteria) could be explained by incidental contamination from the lab environment, however the preponderance of Burkholderia hits only in the interface samples suggest the presence of an organism belonging to this genus in the co-culture system. The unigenes were generally <1 kbp (indicating transcript-sized unigenes) and RNA samples were DNase treated, diminishing the likelihood that these unigenes originated from genomic DNA contamination.

The genus Burkholderia has received increasing attention in the last two decades due in part to a diverse catalog of host interactions ranging from human pathogen to plant-growth promoting rhizosphere fauna 83 . Of particular interest here is their potential role as beneficial plant endosymbionts 83 . Attsat 84 noted the presence of filamentous bacteria-like structures in haustorial cells and that application of terramycin significantly reduced haustorium formation in Orthocarpus purpurascens (syn. Castilleja exserta), a hemi-parasite that is a close relative of T. versicolor 85 . The presence of Burkholderia in an axenic co-culture system points to an intriguing possibility: a species of Burkholderia was carried through surface sterilization with the seeds of T. versicolor. Evidence that may suggest a role for a prokaryotic endosymbiont in parasitic plant biology 84 considered with evidence for the presence of a genus of beneficial soil bacteria hints at a symbiosis between Burkholderia and Triphysaria. The presence and possible roles of Burkholderia at the host-parasite interface in T. versicolor are currently under investigation.