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. 2017 May 10;8:766.
doi: 10.3389/fpls.2017.00766. eCollection 2017.

Mal De Río Cuarto Virus Infection Triggers the Production of Distinctive Viral-Derived siRNA Profiles in Wheat and Its Planthopper Vector

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Free PMC article

Mal De Río Cuarto Virus Infection Triggers the Production of Distinctive Viral-Derived siRNA Profiles in Wheat and Its Planthopper Vector

Luis A de Haro et al. Front Plant Sci. .
Free PMC article

Abstract

Plant reoviruses are able to multiply in gramineae plants and delphacid vectors encountering different defense strategies with unique features. This study aims to comparatively assess alterations of small RNA (sRNA) populations in both hosts upon virus infection. For this purpose, we characterized the sRNA profiles of wheat and planthopper vectors infected by Mal de Río Cuarto virus (MRCV, Fijivirus, Reoviridae) and quantified virus genome segments by quantitative reverse transcription PCR We provide evidence that plant and insect silencing machineries differentially recognize the viral genome, thus giving rise to distinct profiles of virus-derived small interfering RNAs (vsiRNAs). In plants, most of the virus genome segments were targeted preferentially within their upstream sequences and vsiRNAs mapped with higher density to the smaller genome segments than to the medium or larger ones. This tendency, however, was not observed in insects. In both hosts, vsiRNAs were equally derived from sense and antisense RNA strands and the differences in vsiRNAs accumulation did not correlate with mRNAs accumulation. We also established that the piwi-interacting RNA (piRNA) pathway was active in the delphacid vector but, contrary to what is observed in virus-infected mosquitoes, virus-specific piRNAs were not detected. This work contributes to the understanding of the silencing response in insect and plant hosts.

Keywords: Fijivirus; MRCV; RNA silencing; piRNAs; planthopper; sRNAs; vsiRNAs; wheat.

Figures

FIGURE 1
FIGURE 1
Schematic representation of the experimental design for sRNA analysis of planthoppers and plants infected with MRCV. Step 1: 500 D. kuscheli nymphs were allowed to feed on a single MRCV-infected wheat plant for 48 h. Step 2: the insects were moved to chambers containing non-infected wheat plants for 17 days (latency period). During this period, upon sap ingestion, MRCV enters and multiplies in the planthopper midgut epithelial cells until reaching a certain threshold, disseminates into midgut muscles cells, hemolymph and eventually reaches the salivary glands and the insect becomes infective. Step 3: 1:1 infection of 165 wheat seedlings in individual cages. Steps 4 and 5: individual insect and plant (young systemic leaves) samplings. Step 6: infected plants were identified by the observation of viral symptoms and enzyme-linked immunosorbent assay (ELISA) tests followed by absolute RT-qPCR analysis to measure virus RNA titters. Individual transmitting planthoppers were also identified based on infected plants. Step 7: pooling of samples. Step 8: insect and plant sRNAs extraction and sequencing. Steps 1–3 were performed in growing chambers. Step 6 was performed in a greenhouse with controlled light and temperature conditions. The experiment was repeated twice.
FIGURE 2
FIGURE 2
Size distribution of total sRNAs and vsiRNAs in planthopper and wheat hosts. Total D. kuscheli (A) and wheat (B) sRNAs after MRCV infection. D. kuscheli (C) and wheat (D) vsiRNAs after MRCV infection. Control insects were fed in non-infected plants. Control plants were treated with non-viruliferous planthoppers. Insect sRNA samples were analyzed at 19 days post-acquisition (dpa). Wheat sRNA samples were analyzed at 12 and 21 days post-infection (dpi). Reads are redundant and normalized (reads per million). Error bars: SD.
FIGURE 3
FIGURE 3
Distribution of vsiRNAs from infected D. kuscheli (A) and wheat (B) along the 10 dsRNAs segments of MRCV genome. Average per-base coverage of vsiRNAs is represented in the y-axis and the nucleotide position of MRCV genomic segments are represented across the x-axis. vsiRNAs identical (dark gray) or complementary (light gray) to the positive strands are displayed above and below of each segment, respectively. A schematic representation of the predicted ORFs is shown across the x-axis. Next to each panel, proportion of vsiRNAs reads mapping to the positive (upper) or negative (lower) strands of each segment. A red line at 50% is shown. Error bars: percent SD.
FIGURE 4
FIGURE 4
Average vsiRNAs reads per kilobase per million reads (RPKM) values of the individual MRCV segments (S01–S10) in D. kuscheli (A) and wheat (B). One-way ANOVA grouping is shown with letters. Absolute quantification of the 10 MRCV genome segments (S01–S10) by RT-qPCR in D. kuscheli (C) and wheat (D). Error bars: SD.
FIGURE 5
FIGURE 5
Identification of piRNAs in D. kuscheli. Size distribution of D. kuscheli sRNAs mapping to Drosophila gypsy 2 TE (A); sequence logo showing ping-pong amplification loop signature of piRNAs of 10 nt overlapped reads mapping to Drosophila gypsy 2 sense (B, upper panel) or antisense (B, lower panel) TE; coverage graph of sRNAs mapping to sense (up) and antisense (low) strands of Drosophila gypsy 2 TE (C).

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