Compensatory sequence variation between trans-species small RNAs and their target sites
- The Pennsylvania State University, United States
Figures
Figure 1 with 2 supplements
Haustorium-induced small RNAs (HI-sRNAs) are present in multiple Cuscuta species.
(A) Phylogeny of select Cuscuta species. Size distribution of HI-sRNAs for each sequenced isolate and acronyms are shown. (B) Sampling and sequencing schematic to discern HI-sRNAs. (C) HI-sRNA …
Figure 1—figure supplement 1
Host preference in Cuscuta species in the United States.
(A) Pipeline for processing herbaria data from the mid-atlantic herbaria consortium (MAHC; http://midatlanticherbaria.org) on interactions with each Cuscuta species of interest. (B) Ranked list of …
Figure 1—figure supplement 2
Genome-free HI-sRNA discovery pipeline.
(A) Discovery of HI-sRNAs in Cuscuta isolates. Three major steps include condensing reads to representative sRNAs in a genome-free manner, filtering reads which could have originated from A. thaliana…
Figure 2 with 2 supplements
Host targets of Cuscuta HI-sRNAs.
(A) Modeled sRNA-target interaction for A. thaliana CRCK2. (B) Secondary siRNA accumulation from CRCK2. (C) Phasing analysis of secondary siRNAs from CRCK2. Expected phase for cut-site shown in red. …
Figure 2—figure supplement 1
Summary of Cuscuta HI-sRNA and host gene target relationships.
(A) Complete list of target interactions between sRNAs and host genes. Confirmation status diagram indicates in what species the interaction is confirmed. Target gene information includes the number …
Figure 2—figure supplement 2
Most common GO terms for confirmed target genes.
(A) GO terms for molecular function with a nodescore ≥5.0, demonstrating the species for which the interaction is confirmed with colored bars. Locations where bars overlap indicate genes where both …
Figure 3
Analysis of mRNA accumulation in host-parasite interfaces.
Cumulative density plots of interface/control stem ratios for host mRNAs expressed in Cuscuta-host interfaces, assessed by RNA-seq. All mRNAs shown with black line. Colored lines and dots indicate …
Figure 4 with 1 supplement
Predicted trans-species and self-targeting in C. campestris homologs of target A. thaliana mRNAs.
Target prediction scores for confirmed A. thaliana mRNA targets (black) and best-blast-hit homologs in C. campestris (red). All sRNAs with predicted targeting are shown.
Figure 4—figure supplement 1
Experimental flowchart for confirming self-targeting of C.campestris mRNAs by HI-sRNAs.
(A) Pipeline for confirmation by the presence of secondary siRNAs. (B) Pipeline for confirmation by the 5’ transcript sequencing (NanoPARE). (C) List of all mRNAs with strong evidence for …
Figure 5 with 2 supplements
Cuscuta HI-sRNAs form superfamilies that co-vary with target sites across eudicots.
(A) sRNA superfamily count and membership for each Cuscuta isolate. Colors indicate general groupings of superfamilies. (B) An example HI-sRNA superfamily aligned to target sites from homologs in 36 …
Figure 5—figure supplement 1
Clustering method for forming HI-sRNA superfamilies.
(A) Example demonstrating implementation of the ‘modified hamming distance’ (mHD) when comparing strings. Levenshtein edit distance is tolerant of insertions and deletions, yet the mHD does not …
Figure 5—figure supplement 2
Testing distance cutoff parameters for superfamily formation.
(A) Experimental pipeline for testing cutoff. sRNA libraries are shuffled using UShuffle maintaining dinucleotide composition. (B) Number of superfamilies formed for real HI-sRNAs and shuffled …
Figure 6
Superfamilies compensate for variation in N.benthamiana target homologs.
(A) Accumulation of N. benthamiana target mRNAs. Interface (IN, red) and control stem (CS, black) are shown relative to average CS expression. Points represent biological replicates (N = 5 to 6). P …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (A. thaliana) | xrn4 | Rymarquis et al., 2011 | xrn4-5; CS68822; SAIL_681_E01 | T-DNA insertion mutation in Col-0 background |
| Commercial assay or kit | Nextera DNAflex kit | Illumina | Product: 20018704 | |
| Commercial assay or kit | NEB primers set 1 | New England Biolabs | Product: E7335S | |
| Commercial assay or kit | NEB primers set 2 | New England Biolabs | Product: E7500S | |
| Commercial assay or kit | NEB primers set 3 | New England Biolabs | Product: E7710S | |
| Commercial assay or kit | NEB primers set 4 | New England Biolabs | Product: E7730S | |
| Software, algorithm | ShortStack | (Johnson et al., 2016) | v3.8.5 | https://github.com/MikeAxtell/ShortStack |
| Software, algorithm | DESeq2 | (Love et al., 2014) | v1.24.0 | https://bioconductor.org/packages/release/bioc/html/DESeq2.html |
| Biological sample (C. campestris) | ccm | Shahid et al., 2018; Jim Westwood, Virginia Tech | ‘doddi’ | |
| Biological sample (C. pentagona) | cpe-2017 | Ebay, seller: eden_wilds | 2017 collection | |
| Biological sample (C. pentagona) | cpe-2015 | Ebay, seller: eden_wilds | 2015 collection | |
| Biological sample (C. gronovii) | cgr-dp | Claude dePamphilis, Penn State | Provenance unknown | |
| Biological sample (C. gronovii) | cgr-mass | Jim Westwood, Virginia Tech | massachusetts isolate | Origin: A Massachusetts cranberry bog |
| Biological sample (C. gronovii) | cgr-pm | Wild collection | purdue mountain isolate | Origin: Roadside near State College, PA (Coordinates: 40.866 N, 77.888 W) |
| Biological sample (C. indecora) | cin | www.ars-grin.gov | PI 675068 | Origin: Texas |
Additional files
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Supplementary file 1
Unabridged phylogeny of Cuscuta Phylogeny based on TrnL-F sequencing using vouchered samples and primers (Stefanovic et al., 2007; Costea et al., 2015).
Isolates used in this study are in bold and indicated with arrows. Samples identified as members of species examined in this study are highlighted with color; red - C. campestris, purple - C. pentagona, green - C. gronovii, pink - C. indecora. Format: PDF
- https://cdn.elifesciences.org/articles/49750/elife-49750-supp1-v1.pdf
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Supplementary file 2
List of all libraries and tissues prepared or used in this study.
- https://cdn.elifesciences.org/articles/49750/elife-49750-supp2-v1.xlsx
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Supplementary file 3
Testing alternative p-value cutoffs for HI-sRNA detection.
Format: xlsx
- https://cdn.elifesciences.org/articles/49750/elife-49750-supp3-v1.xlsx
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Supplementary file 4
Comprehensive list of haustorium-induced small RNAs (HI-sRNAs) discovered in this study.
Format: xlsx
- https://cdn.elifesciences.org/articles/49750/elife-49750-supp4-v1.xlsx
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Supplementary file 5
Predicted secondary structures of miRNA hairpins producing HI-sRNAs in C. campestris.
Predicted RNA secondary structures and expression profiles of loci that produce HI-sRNAs and have an apparent miRNA hairpin. Format: PDF
- https://cdn.elifesciences.org/articles/49750/elife-49750-supp5-v1.pdf
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Supplementary file 6
Target confirmation data for every confirmed HI-sRNA-target interaction.
Details of confirmed HI-sRNA targets including HI-sRNA-target complementarity, site, score, superfamily and the status of C. campestris superfamily members as a confirmed miRNA. Targeting confirmation for target mRNA is shown in upper right, with confirmed interactions in species highlighted in red. sRNA distribution at target locus is shown for experimental interface and control, demonstrating secondary siRNA phasing and size distribution for up-regulated loci. Degradome sequencing is shown where confirmed hits were discovered in NanoPARE data. Format: PDF
- https://cdn.elifesciences.org/articles/49750/elife-49750-supp6-v1.pdf
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Supplementary file 7
Target interactions with significant correlation of variation in superfamily and target site.
Multiple sequence alignments of HI-sRNA superfamilies which have significant correlations between sRNA positional variation and target site variation. Alignment of eudicot homologs around target site also shown, with nucleotide and amino acid Shannon entropy shown as bits. Vertical red lines indicate the frame. Dots indicate the number of possible synonymous nucleotides at a position for the confirmed target’s sequence. Nucleotide positions are in reference to the position in the multiple sequence alignment. Format: PDF
- https://cdn.elifesciences.org/articles/49750/elife-49750-supp7-v1.pdf
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Supplementary file 8
Eudicot genomic resources used in this study.
All available in Phytozome version v12.1.6. Format: xlsx
- https://cdn.elifesciences.org/articles/49750/elife-49750-supp8-v1.xlsx
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Supplementary file 9
Target confirmation data for every confirmed HI-sRNA-target interaction in N. benthamiana.
Details of confirmed C. campestris HI-sRNA targets in N. benthamiana, including HI-sRNA-target complementarity, site, score, superfamily and the status of C. campestris superfamily members as a confirmed miRNA. sRNA distribution at target locus is shown for experimental interface and control, demonstrating secondary siRNA phasing and size distribution for up-regulated loci. Format: PDF
- https://cdn.elifesciences.org/articles/49750/elife-49750-supp9-v1.pdf
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Supplementary file 10
N. benthamiana targets of HI-sRNAs Based on N. benthamiana genome v1.0.1.
Format: xlsx
- https://cdn.elifesciences.org/articles/49750/elife-49750-supp10-v1.xlsx
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Supplementary file 11
Target interactions of A. thaliana homologs with conserved target motifs.
Multiple sequence alignments sRNA of superfamilies and conserved target motifs found in Arabidopsis transcriptome, with nucleotide and amino acid Shannon entropy shown as bits. Vertical red lines indicate the frame. Dots indicate the number of possible synonymous nucleotides at a position for the confirmed target’s sequence. Nucleotide positions are in reference to the position in the multiple sequence alignment. Color of gene names indicates if there is evidence for targeting in NanoPARE data (black - 0 replicates; orange - 1 or two replicates; red - three replicates, confirmed interaction). Format: PDF
- https://cdn.elifesciences.org/articles/49750/elife-49750-supp11-v1.pdf
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Supplementary file 12
List of primers used in this study.
Format: xlsx
- https://cdn.elifesciences.org/articles/49750/elife-49750-supp12-v1.xlsx
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Supplementary file 13
Alignment of TrnL-F sequences from Cuscuta.
These were the basis for the phylogenetic tree presented in Supplementary file 1. Format: FASTA (plain text).
- https://cdn.elifesciences.org/articles/49750/elife-49750-supp13-v1.fasta
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Transparent reporting form
- https://cdn.elifesciences.org/articles/49750/elife-49750-transrepform-v1.docx
