Open Access Research article

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

Loren A Honaas1, Eric K Wafula2, Zhenzhen Yang1, Joshua P Der12, Norman J Wickett128, Naomi S Altman3, Christopher G Taylor4, John I Yoder5, Michael P Timko6, James H Westwood7 and Claude W dePamphilis12*

Author Affiliations

1 Intercollege Graduate Program in Plant Biology, Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, Pennsylvania 16802, USA

2 Department of Biology and Institute of Molecular Evolutionary Genetics, The Pennsylvania State University, University Park, Pennsylvania 16802, USA

3 Department of Statistics and Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, Pennsylvania, 16802, USA

4 Department of Plant Pathology, The Ohio State University, Ohio Agricultural Research and Development Center, Wooster, OH, 44691, USA

5 Department of Plant Sciences, University of California, Davis, Davis, California, 95616, USA

6 Department of Biology, University of Virginia, Charlottesville, VA, 22904, USA

7 Department of Plant Pathology, Physiology and Weed Science, Virginia Polytechnic Institute and State University, Blacksburg, VA, 24061, USA

8 Present address: Chicago Botanic Garden, Glencoe, IL, 60022, USA

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BMC Plant Biology 2013, 13:9  doi:10.1186/1471-2229-13-9

Published: 9 January 2013

Additional files

Additional file 1: Figure S1:

Unigene Pairwise Nucleotide Identity Plot. Sequence identity between unigenes considered in this study and reference EST sets (PlantGDB public ESTs, http://www.plantgdb.org/ webcite) for the hosts Z. mays and M. truncatula. Triphysaria unigenes were aligned to the host reference to identify host contaminants and aligned to the reciprocal non-host reference sets to identify the incidental nucleotide pairwise identity. A whole plant normalized transcriptome assembly of Lindenbergia philippensis (a non-parasitic member of the Orobanchaceae) was used to determine the distribution of pairwise identity for a non-parasite to each host and to control for high unigene identity to host ESTs from potential cross contamination. A threshold of 95% was chosen to balance exclusion of host transcripts with retention of Triphysaria unigenes that had incident high identity to host ESTs.

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Additional file 2: Figure S2:

VENN diagram summary of OrthoMCL DB and InterProScan (IPS) results. ESTScan ORF predictions from unigenes in each interface transcriptome that remained unclassified after extensive BLAST-based database searching were translated and submitted to OrthoMCL DB and InterProScan. The pattern is similar between unigenes from each transcriptome indicating equivalent unigene classification for T. versicolor grown on both hosts. The number of unigenes for which an ortholog or peptide motif was identified was relatively small, indicating our unigene classification using PlantTribes 2.0 and external database queries was robust. Approximately 25% of the known orthologs identified in the OrthoMCL database from each transcriptome are shared.A majority of the unigenes remain unknown, and these include many (~500 in each transcriptome) that are >300 nucleotide bp and have read support.

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Additional file 3:

OrthoMCL DB and InterProScan annotation summary spreadsheet of unigenes that remained after the screen of PPGP databases, host cDNA and EST sequences, and NCBI’s non-redundant protein sequences database.

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Additional file 4: Figure S3:

GO Slim category analysis. Chi-Square test (P<<0.0001) of GO Slim terms represented in the indicated regions of the Venn. The numbers of unigenes in each GO category for indicated regions are listed in the table. Cells with strongly positive residual values (>4) are indicated as bold+ and strongly negative residual values (<-4) are indicated as bold-. GO Slim Function (A), Component (B) and Process (C) category analysis for the interface transcriptome of T. versicolor grown on Z. mays. GO Slim Function (D), Component (E) and Process (F) category analysis for the interface transcriptome of T. versicolor grown on M. truncatula.

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Additional file 5: Figure S4:

Correlation of normalized read counts (RPKM) for unigenes in orthogroups shared between the interface transcriptomes and reference assembly TrVeBC1 (ppgp.huck.psu.edu). Reads from each interface transcriptome were mapped to a reference assembly (TrVeBC2, ppgp.huck.psu.edu) that included whole haustorium data from T. versicolor grown on M. truncatula. A subset of unigenes is more highly expressed in the interface transcriptome of T. versicolor grown on M. truncatula; a similar pattern is not observed for T. versicolor grown on Z. mays. This is due to a bias for Medicago grown Triphysaria unigenes in the reference dataset TrVeBC2, which was constructed with reads from Medicago grown Triphysaria. For unigenes in shared orthogroups, the RPKM values are highly correlated (Pearson’s R = 0.81) between interface transcriptomes indicating that technical and biological variation is low.

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Additional file 6: Figure S5:

Highly Expressed Interface Unigenes. The 20 most highly expressed (RPKM) unigenes (ID) in each indicated portion of the transcriptome Venn diagram for the interaction of T. versicolor with each host species. NR BLASTx – description, species and %id.: the description, species of origin, and percent pairwise identify, respectively, of the best unigene alignment (<1e-10) resulting from the NR database query, 17 genomes BLAST and %id.: best hit species in a BLAST database of 17 annotated plant genomes with the percent pairwise identity in the nucleotide BLAST (N) or translated nucleotide BLAST (P). TXXX = IPS transmembrane prediction, SXXX = IPS secretion signal prediction.

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Additional file 7:

Text file (BLAST default output format) of BLAST results from the query of NCBI’s non-redundant protein sequences database with Z. mays grown T. versicolor unigenes listed in Additional file 6.

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Additional file 8:

Text file (BLAST default output format) of BLAST results from the query of NCBI’s non-redundant protein sequences database with M. truncatula grown T. versicolor unigenes listed in Additional file 6.

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Additional file 9:

Results from the InterProScan analysis of Z. mays grown T. versicolor unigenes listed in Additional file 6.

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Additional file 10:

Results from the InterProScan analysis of M. truncatula grown T. versicolor unigenes listed in Additional file 6.

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Additional file 11: Figure S6:

RaxML analysis of A: Triphysaria beta expansin gene TvEXPB1 (TrVeIntZeamaGB1_772, green text), and B: alpha expansin gene TvEXPA4 (TrVeIntMedtrGB1_11, green text). Bootstrap proportions are given above each node. Taxon abbreviations for A: Arabidopsis thaliana (AT), Oryza sativa (Os), Mimulus guttatus (Mg), Triphysaria versicolor (TrVe), Striga hermonthica (StHe), Phelipanche (=Orobanche) aegyptiaca (OrAe), Selaginella moellendorffii (Smoellendorffii). Taxon Abbreviations for B:Oryza sativa (Os), Sorghum bicolor (Sb), Striga hermonthica (StHe), Phelipanche (=Orobanche) aegyptiaca (OrAe), Triphysaria versicolor (TrVe), Carica papaya (Carpa), Populus trichocarpa (Poptr), Medicago truncatula (Medtr), Vitis vinifera (Vitvi), Arabidopsis thaliana (AT), Selaginella moellendorffii (Selmo), Physcomitrella patens (Phypa).

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