Article Figures & Data
Figures
- Figure 1. RNAi pathways in whitefly (B. tabaci).
(A) Relatedness of argonaute and Piwi (Ago/Piwi) proteins from whitefly (Bta) to orthologs in Drosophila (Dme), Tribolium (Tca), and select family members from C. elegans (Cel). Ago and Piwi clades highlighted by colored boxes and whitefly genes in red text. Phylogenetic tree was constructed using the maximum likelihood method. Branch support values shown at nodes. (B) Expression determined by RPKM of whitefly Ago/Piwi proteins in whole body, gut, and salivary gland. (C) Numbers of miRNAs annotated in this study. Loci are categorized into those conserved with Drosophila, novel highly confident, and lower confidence candidates. (D) Distribution of small RNA read sizes mapping to the whitefly genome (MEAM v1.2) and piRNA biogenesis modes. Left inset shows read overlap Z-scores to demonstrate the ping-pong piRNA signature of 10-nt overlaps, and right panel distance to trailing 1U reads showing the phasing signature. Bars in the size distribution are colored based on the portion of reads with the sequence identity indicated in the inset legend.
- Figure 2. Whitefly small RNA expressing loci.
(A) Comparison of 3,878 small RNA loci is annotated in whitefly by locus size, number of mapped reads, and the ratio of short (19–23 nt) to long (25–30 nt) mapping reads. (B) Visualization of small RNA sizes and piRNA biogenesis signatures for all 3,878 loci. Each row of the heat map represents a locus, which is arranged by read size bias with short read bias at the top and long bias at the bottom. Left panel shows size distribution. Nucleotide sizes are indicated below. Arrows at top show sizes expected to represent siRNAs (si) and piRNAs (pi). Middle panel shows read overlaps quantified by Z-score, arrow shows the 10-nt overlap size. Right panel shows distance of trailing 1U reads; arrow shows the 2-nt proximal read distance. Dashed line box highlights the ∼100 loci that do not have piRNA signatures in terms of read size, overlaps, or phasing. This group of loci have more reads at the 22 nt (siRNA) size. (B, C) Matrix of Dicer-2 nt overhang signature calculated for loci in the dashed box in panel (B). Read pairs where the query read overlapped by 2 minus its total length were quantified and plotted in the heat map. Line of boxes below the matrix show the read size distribution for reads mapping to the analyzed loci (dashed box in part B). (D, E) Number of mRNA and transposable element targets for the 50 most high expressing (Fig S1) (D) loci biased to long reads or (E) loci biased toward short reads.
- Figure S1. Characterization of the 50 most highly expressed small read biases and long read biased loci.
(A) 50 most high expressing long read (25–30 nt) biased loci. Heat map shows distribution of reads between 16 and 40 nt. (B) 50 most high expressing short read (19–23 nt). si, siRNA sized; pi, piRNA sized biased-loci. Heat map shows distribution of reads between 16 and 40 nt Yellow shows density of all reads. (A, B, C) 3D scatterplot for the loci in (A, B) assessed by locus length, expression, and 1U bias. (D) 50 top long (25–31 nt) read loci where orange shows multi-mapping long reads and violet uniquely mapping long reads. (E) 50 top short (19–23 nt) read loci where orange shows multi-mapping short reads and violet uniquely mapping short reads.
- Figure S2. Visualization of RNA structure and small RNA expression at curated hairpin RNA loci.
Top part of each panel depicts RNA structure with lines connecting one or more bases indicating pairing. Bottom panel is a density plot showing relative read depth across the locus. Red color indicates accumulation of 20–23-nt reads that map to more than one position in the genome. Blue indicates 20–23-nt reads that map uniquely to the locus. Yellow shows density of all read sizes.
- Figure 3. Characterization of whitefly loci with Dicer cleavage signature.
(A) Intersection of Dicer processing loci showing 2-nt overhangs for reads sized 20–23 nt with long and short read loci. The sequence biases of Dicer read loci are shown below in the seqlogo graphic. (B) Appearance of Dicer produced small RNAs (siRNAs) at sites of convergent transcription. Top panel shows expression of siRNAs in a piRNA cluster. Bottom panel is a cis-natural antisense transcript (cis-NAT). Blue trace shows all reads mapping to locus. Read trace shows reads with Dicer-2-nt overhang cleavage pattern. (C) Read size distribution and biogenesis pattern of small RNAs produced at 76 Dicer signature loci. Length of reads in heat maps is indicated below. Curated identities are shown on the left. The leftmost heat map shows the distribution of reads sizes, middle shows z-scores for 2-nt overhangs (siRNAs), and right heat maps show z-scores for 10-nt overlaps (piRNAs).
- Figure 4. Metabolism of exogenous dsRNAs in whitefly.
(A) Accumulation of reads mapping to dsRNA sites (green boxes) in the context of the originating transcript from potato psyllid (B. cockerelli). Blue trace shows all reads mapping to the locus. Read trace shows reads with Dicer cleavage pattern. (A, B) Size distribution of read derived from the three off-target dsRNAs (shown in (A)). Red arrow shows the expected size of siRNAs (22 nt). (C) Balloon plot showing characterization of sequence biases in exogenous siRNAs. Read sizes are indicated below. Color and diameter of circle scale with Z-scores quantifying different size reads. On left, the sequence identities of small RNA subsets are indicated for the first base of the read and the 20th base of the read. N = any residue, H = U/A/C, D = A/U/G, and W = A/U. The left group of balloons show the abundance of reads, and the right group of circles abundance of reads with 2-nt overhangs. (D, E) Differential expression of (D) small RNA loci and (E) mRNAs between whiteflies treated with water or the three off-target dsRNAs. Data points colored by identity. Circles represent nonsignificant change in expression, triangles significant. Dashed circle shows location of Dicer and Ago proteins in the scatterplot.
- Figure S3. Proposed mechanism for piRNA/siRNA trigger.
(A) Configuration of piRNA trigger where piRNA locus regions are modified to house the sequence of the target gene. (B) Single-stranded and dsRNA piRNA triggers developed in the work.
- Figure 5. Exogenous piRNA-mediated gene silencing in whitefly.
(A) Relative expression of AQP1 and AGLU1 genes determined by qRT-PCR after feeding with synthetic RNAs generated from piRNA triggers. Blue bar graphs are results when target gene sequences are fused to sequence from piRNA-biased locus 6 (piRB-6) sequences. Green graphs are when they are fused to No Bias-14 sequences. At least three independent biological replicates were used for each type of feeding. Error bars show standard error, and letters indicate significance groups determined by Tukey’s HSD test. *P ≤ 0.05. (B, C, D, E, F) Analysis of small RNA-sequencing data from animals fed piRB-6–based piRNA triggers that map to the synthetic RNAs. (B) Portion of small RNA-sequencing reads with 1U residues shows biased to long (piRNA) sized reads. Black bars are from double-stranded (DS) triggers and gray from single-stranded (SS) versions. (C). Enrichment of ping-pong piRNA pairs in longer sized RNAs (28–30 nt) in the target gene region of the piRNA triggers. Sequence identities are indicated in the legend. DS, double-stranded triggers; SS, single-stranded triggers. 1U-10A reads, which are characteristic of bona fide ping-pong piRNAs show the greatest abundance. (D, E) Phasing signature plots separated by off-target and on-target strands for (D) single-stranded piRNA triggers and (E) double-stranded triggers. (F) Balloon plot showing reads with Dicer-2 nt overhangs for the DS and SS triggers. Color and size of circles scale with the abundance of 2-nt overhang pairs. Left shows the sequence identities of small RNAs analyzed (N = any residue, H = U/A/C, D = A/T/G, and W = A/T).
- Figure 6. Processing of piRNA triggers.
(A, B, C, D) Characterization of the piRB-6 locus. (A) Phasing analysis of trailing 1U reads shows greater phasing signature on the plus strand of the locus compared to the antisense strand. (B) Overlap analysis for the piRB-6 locus showing a peak at 10-nt overlaps. (C) Enrichment of 22-nt reads that overlap by 2 nt at piRB-6. (D) Read accumulation at piRB-6. Alignments are colored by identity. Blue represents phasing piRNAs characterized by long 28–20-nt 1U reads that do not overlap by 10 with antisense reads and therefore unlikely to be involved in ping-pong. Red are ping-pong piRNAs being 28–30-nt reads that have 1U/10A sequences that also overlap by 10. Orange is siRNAs being 22-nt reads that have 2-nt overhangs with a 1U/A and 10A/U. The region cloned for the piRNA triggers indicated by dashed line box. The site of target sequence insertion is shown by the gray line. Y-axis shows read density. (E) Read accumulation using the color coding in part D at the sequence target region of piRNA triggers. Similarly, y-axis represents read density. Positive strand depicted at top of graphs is complementary to target. (E, F) Quantification of read identities by strand for plots shown in part (E). (D, E) Color scheme same as used in (D, E). AG = AGLU1 and AQ = AQP1. (G) Diagram showing the consequences of using different piRNA trigger configuration. Blue represents phasing strand of piRB-6 and red the complementary. (D) Same color scheme in (D) used to represent reads.
Supplementary Materials
Supplemental Data 1.
