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

Article Comparative Transcriptome Analyses Reveal Core Parasitism Genes and Suggest Gene Duplication and Repurposing as Sources of Structural Novelty

Abstract The origin of novel traits is recognized as an important process underlying many major evolutionary radiations. We studied the genetic basis for the evolution of haustoria, the novel feeding organs of parasitic flowering plants, using comparative transcriptome sequencing in three species of Orobanchaceae. Around 180 genes are upregulated during haustorial development following host attachment in at least two species, and these are enriched in proteases, cell wall modifying enzymes, and extracellular secretion proteins. Additionally, about 100 shared genes are upregulated in response to haustorium inducing factors prior to host attachment. Collectively, we refer to these newly identified genes as putative "parasitism genes." Most of these parasitism genes are derived from gene duplications in a common ancestor of Orobanchaceae and Mimulus guttatus, a related nonparasitic plant. Additionally, the signature of relaxed purifying selection and/or adaptive evolution at specific sites was detected in many haustorial genes, and may play an important role in parasite evolution. Comparative analysis of gene expression patterns in parasitic and nonparasitic angiosperms suggests that parasitism genes are derived primarily from root and floral tissues, but with some genes co-opted from other tissues. Gene duplication, often taking place in a nonparasitic ancestor of Orobanchaceae, followed by regulatory neofunctionalization, was an important process in the origin of parasitic haustoria

Overall average hardness after a 7d simulated supply chain (in air at 20°C).

Overall average hardness after a 7d simulated supply chain (in air at 20°C).</p

Fig 3 -

PCA 1 and 2 of TPM transcriptomic data counts colored by A) treatment and B) days post harvest. PC1 primarily separates fruit based on temperature and days post-harvest of the warmer fruit whereas PC2 separates based on days post harvest of fruit treated with 1-MCP and CA.</p

Elastic net top 15 gene expression.

The expression level (y-axis) of genes using TPM normalized counts over time (x-axis) for the top 15 genes identified using the elastic net model. (PNG)</p

Fig 6 -

Model performance of a single random forest reduced model (RF-rm) and elastic net reduced model (EN-rm) training A and testing B. training C and testing D. Data for replicated 100 runs of these model is presented in Table 1. Reported r2 and m_rmse values in this figure represent a single run of a representative model, whereas data reported in Table 1 represents the average of 100 replicates.</p

Fig 5 -

Model stability of A) Random Forest (RF) Full Model (FM) and B) Elastic Net (EN) FM. The top 15 genes of each model are shown along the y-axis. Each point represents a single bootstrap re-run of the model and its position along the x-axis is the gene’s rank in importance from the re-run model. A rank of 1 is given to a gene if it is the most important in the respective model. A point is present for a re-run model only if the gene occurred in the top 500 important genes. Numbers on the right side y-axis of the graph indicate how many times the gene was selected by the model.</p

Harvest boxplots.

A) Differences in fruit diameter at harvest between 2018 and 2019. B) Differences in creep at harvest during the same period. (PNG)</p

RNA-Seq TPM vs qPCR.

Scatterplot of gene expression (y-axis) using TPM normalized counts vs qPCR levels (x-axis). (PNG)</p