Functional genomics of a generalist parasitic plant: laser microdissection of host-parasite interface reveals host-specific patterns of parasite gene expression

Abstract

Background: Orobanchaceae is the only plant family with members representing the full range of parasitic lifestyles plus a free-living lineage sister to all parasitic lineages, Lindenbergia. A generalist member of this family, and an important parasitic plant model, Triphysaria versicolor regularly feeds upon a wide range of host plants. Here, we compare de novo assembled transcriptomes generated from laser micro-dissected tissues at the host-parasite interface to uncover details of the largely uncharacterized interaction between parasitic plants and their hosts.

Results: The interaction of Triphysaria with the distantly related hosts Zea mays and Medicago truncatula reveals dramatic host-specific gene expression patterns. Relative to above ground tissues, gene families are disproportionally represented at the interface including enrichment for transcription factors and genes of unknown function. Quantitative Real-Time PCR of a T. versicolor β-expansin shows strong differential (120x) upregulation in response to the monocot host Z. mays; a result that is concordant with our read count estimates. Pathogenesis-related proteins, other cell wall modifying enzymes, and orthologs of genes with unknown function (annotated as such in sequenced plant genomes) are among the parasite genes highly expressed by T. versicolor at the parasite-host interface.

Conclusions: Laser capture microdissection makes it possible to sample the small region of cells at the epicenter of parasite host interactions. The results of our analysis suggest that T. versicolor's generalist strategy involves a reliance on overlapping but distinct gene sets, depending upon the host plant it is parasitizing. The massive upregulation of a T. versicolor β-expansin is suggestive of a mechanism for parasite success on grass hosts. In this preliminary study of the interface transcriptomes, we have shown that T. versicolor, and the Orobanchaceae in general, provide excellent opportunities for the characterization of plant genes with unknown functions.

Figure 1

Laser Microdissected Haustorium. LPCM allows highly tissue- and cell-specific harvest after histological identification of tissues or cells of interest. A) Representative 25 μm cross-section of T. versicolor haustorium on the host M. truncatula approximately 9 days post infestation, and prior to LPCM. The mature haustorium contains the xylem bridge that connects the parasite and host vasculature and is visible in the penetration peg. B) The same section after LCPM shows the cleared interface tissue from the user-defined region of interest (ROI). The flakes of tissue are catapulted by a photonic cloud resulting from pulses of laser light focused between the tissue and glass slide. Multiple pulses of laser light raster across the ROI causing tissue in the selected region to be catapulted and then captured in the adhesive coated cap of a 0.5 mL tube held by a robotic arm in very close proximity (< 0.5 mm) to the upper surface of the section affixed to the slide.

Figure 2

Transcriptome Orthogroup Venn. Venn diagram showing the number of Orthogroups in the interface transcriptomes of T. versicolor with hosts Z. mays and M. truncatula and an above ground, autotrophically grown T. versicolor transcriptome (TrVeBC1) constructed from leaves, stems and inflorescences. Also shown are the numbers of host-derived Orthogroups. The lack of overlap between host and parasite transcriptomes does not imply lack of shared Orthogroups, but indicates the total number of host Orthogroups for a point of comparison.

Figure 3

GO Slim category summary. GO Slim category terms of unigenes in interface transcriptomes of T. versicolor and the above ground reference assembly of T. versicolor. Each series displays the average number of unigenes in equivalent transcriptome components with a given GO Slim term. For instance, “Interface Unique” indicates the average number of unigenes from interface unique components in both Medicago and Zea grown T. versicolor transcriptomes. Error bars are standard error of the mean. “Interface Unique” = unigenes from Orthogroups that are host and interface specific, “Interface Shared” = unigenes from Orthogroups that are interface specific and shared between interface transcriptomes, “Shared All” = unigenes from Orthogroups shared between both interface transcriptomes and the above ground transcriptome, “Interface/Above Ground Shared” = unigenes from Orthogroups that are shared between the above ground, autotrophic transcriptome and the host-specific interface transcriptome.

Figure 4

Differential expansin expression. qRT-PCR analysis of TvEXPA4 and TvEXPB1 expression relative to TvActin in parasite-host interface cells harvested by LPCM from the haustoria of T. versicolor *P<0.05.