RNA decay is an antiviral defense in plants that is counteracted by viral RNA silencing suppressors

<div><p>Exonuclease-mediated RNA decay in plants is known to be involved primarily in endogenous RNA degradation, and several RNA decay components have been suggested to attenuate RNA silencing possibly through competing for RNA substrates. In this paper, we report that overexpression of key cytoplasmic 5’–3’ RNA decay pathway gene-encoded proteins (5’RDGs) such as decapping protein 2 (DCP2) and exoribonuclease 4 (XRN4) in <i>Nicotiana benthamiana</i> fails to suppress sense transgene-induced post-transcriptional gene silencing (S-PTGS). On the contrary, knock-down of these 5’RDGs attenuates S-PTGS and supresses the generation of small interfering RNAs (siRNAs). We show that 5’RDGs degrade transgene transcripts via the RNA decay pathway when the S-PTGS pathway is disabled. Thus, RNA silencing and RNA decay degrade exogenous gene transcripts in a hierarchical and coordinated manner. Moreover, we present evidence that infection by turnip mosaic virus (TuMV) activates RNA decay and 5’RDGs also negatively regulate TuMV RNA accumulation. We reveal that RNA silencing and RNA decay can mediate degradation of TuMV RNA in the same way that they target transgene transcripts. Furthermore, we demonstrate that VPg and HC-Pro, the two known viral suppressors of RNA silencing (VSRs) of potyviruses, bind to DCP2 and XRN4, respectively, and the interactions compromise their antiviral function. Taken together, our data highlight the overlapping function of the RNA silencing and RNA decay pathways in plants, as evidenced by their hierarchical and concerted actions against exogenous and viral RNA, and VSRs not only counteract RNA silencing but also subvert RNA decay to promote viral infection.</p></div

Model for counter-defense by VSRs against cytoplasmic RNA decay- and RNA silencing-mediated plant defenses.

<p>After transcription, mRNAs are deadenylated, decapped and degraded by XRN4-mediated 5’–3’ decay or exosome-mediated 3’–5’ degradation. Deadenylated, decapped, or XRN4-partially degraded RNAs facilitate RDR6 to transform ssRNA into dsRNA to degrade the left RNAs by PTGS pathway. Potyviruses encode at least two VSRs: HC-Pro and VPg. HC-Pro interacts with XRN4 to inhibit its slicing activity and VPg disrupts the interaction between NbDCP1 and NbDCP2 by targeting NbDCP2 to the nucleus. As a result, RNA decay-mediated plant defense is compromised. In addition, VPg interacts with SGS3 and mediates its degradation via ubiquitination and autophagy pathways to block RDR6-mediated anti-viral response [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007228#ppat.1007228.ref036" target="_blank">36</a>].</p

Expression of NbDCP1, NbDCP2, NbXRN4, or NbPARN suppresses <i>GFP</i> expression in RDR6-deficient <i>N</i>. <i>benthamiana</i> plants.

<p>(<b>A</b>) Visualization of green fluorescence of representative agroinfiltrated leaves. Pictures of representative agroinfiltrated leaves were taken at 5 dpi under UV light. Wild type (Wt) <i>N</i>. <i>benthamiana</i> or dsRDR6 transgenic <i>N</i>. <i>benthamiana</i> (dsRDR6) plants were agroinfiltrated with three expression vectors including 35S-GFP, 35S-GF and one of the following vectors: an empty vector (Vec) as a control, Myc-tagged-NbDCP1, NbDCP2, NbXRN4, and NbPARN. Similar results were obtained from three independent experiments. (<b>B</b>) Accumulation of GFP protein, <i>GFP</i> mRNA and siRNAs in the infiltrated leaves shown in (A) at 5 dpi. CBB staining of the large subunit of Rubisco, ethidium bromide staining of rRNA, and U6 serve as a loading control for immunoblot, mRNA blot and siRNA blot, respectively. (<b>C</b>) Relative accumulation of <i>GFP</i> mRNAs analyzed by specific qRT-PCR in the infiltrated leaves shown in (A) at 5 dpi. <i>NbActin</i> serves as an internal standard. Each mean value was calculated based on three independent experiments (n = 3 samples). Values represent the mean ± SD. Double asterisks indicate a highly significant difference compared to 35S-GFP+35S-GF+Vec/dsRDR6 (<i>P</i> < 0.01, Student’s <i>t</i> test). The values of GFP siRNAs/U6 were quantified by ImageJ software and then were normalized against the mean value corresponding to the Vec treatment in Wt <i>N</i>. <i>benthamiana</i> plants, which was set to 1.00.</p

Knock-down of <i>NbDCP1</i>, <i>NbDCP2</i>, <i>NbXRN4</i> or <i>NbPARN</i> facilitates TuMV infection.

<p><b>(A)</b> GFP fluorescence in <i>N</i>. <i>benthamiana</i> leaves co-infiltrated with TuMV-GFP and one of the following expression vectors: an empty vector (Vec) as a control, NbDCP1, dsNbDCP1, dsNbDCP2, dsNbXRN4 and dsNbPARN at 4 dpi. <b>(B)</b> Relative TuMV RNA levels determined by qRT-PCR. RNA was extracted from the infiltrated patches shown in (A) at 4 dpi. Each value was normalized against <i>NbActin</i> transcripts in the same sample. Error bars represent SD (n = 3 independent biological repeats). *, <i>P</i> < 0.05; **, <i>P</i> < 0.01 (Student’s <i>t</i> test). (<b>C</b>) GFP fluorescence in systemic leaves of plants pre-treated with TRV-GUS, TRV-NbDCP1, TRV-NbDCP2, TRV-NbXRN4, or TRV-NbPARN and then infected by TuMV-GFP was photographed under UV light at 6 dpi. (<b>D</b>) Relative TuMV RNA levels determined by qRT-PCR. RNA was extracted from plants in (C) at 14 dpi. *, <i>P</i> < 0.05; **, <i>P</i> < 0.01 (Student’s <i>t</i> test). (<b>E</b>) Accumulation of GFP protein and TuMV siRNAs in the systemic leaves of plants in (C) at 14 dpi. CBB staining of the large subunit of Rubisco and U6 serve as a loading control for immunoblot, mRNA blot and siRNA blot, respectively. The values of GFP siRNAs/U6 were quantified by ImageJ software and then were normalized against the mean value corresponding to the TRV-GUS treatment, which was set to 1.00.</p

Expression of NbDCP1, NbDCP2, NbXRN4, or NbPARN reduces TuMV RNA accumulation.

<p><b>(A, B)</b> GFP fluorescence in Wt or RDR6-deficient (dsRDR6) <i>N</i>. <i>benthamiana</i> leaves co-infiltrated with TuMV-GFP and one of the following vectors: Vec, NbDCP1, NbDCP2, NbXRN4 or NbPARN at 3 dpi. <b>(C)</b> qRT-PCR analyses of TuMV RNA levels. RNA was extracted from the infiltrated patches shown in (A) at 3 dpi. Each value was normalized against <i>NbActin</i> transcripts in the same sample. Error bars represent SD (n = 3 independent biological repeats). Double asterisks indicate a highly significant difference compared to the treatment of Vec (<i>P</i> < 0.01, Student’s <i>t</i> test). <b>(D, E)</b> GFP fluorescence in Wt or RDR6-deficient (dsRDR6) <i>N</i>. <i>benthamiana</i> leaves co-infiltrated with TuMV-GFP-ΔGDD (a replication-defective mutant) and one of the following vectors: Vec, NbDCP1, NbDCP2, NbXRN4 or NbPARN at 3 dpi. (<b>F</b>) Accumulation of TuMV siRNAs in the infiltrated patches shown in (A, B, D, E) at 3 dpi. Northern blotting was performed using DIG-labeled DNA probes complementary to the TuMV genome. U6 serves as a loading control for siRNA blot, respectively. The values of GFP siRNAs/U6 were quantified by ImageJ software and then were normalized against the mean value corresponding to the Vec treatment, which was set to 1.00.</p

Interactions and subcellular co-localization of NbDCP1, NbDCP2 and NbXRN4.

<p><b>(A)</b> BiFC assays between NbDCP1, NbDCP2 and NbXRN4 in H2B-RFP transgenic <i>N</i>. <i>benthamiana</i> leaves at 32 hpi. Yellow fluorescence (green) resulted from the interaction of two tested proteins tagged by the C-terminal half of YFP (C-YFP) or the N-terminal half of YFP (N-YFP). Nuclei of tobacco leaf epidermal cells are indicated by expression of the H2B-RFP transgene (red). P3N-PIPO tagged by C-YFP or N-YFP serves as a negative control. Bars = 50 μm. <b>(B)</b> Co-localization of NbDCP1, NbDCP2 and NbXRN4 in the H2B-RFP transgenic <i>N</i>. <i>benthamiana</i> leaf cells at 32 hpi. The yellow signals result from the overlapping of NbDCP1-CFP (green) with YFP-NbDCP2 (red) or YFP-NbXRN4 (red), or NbDCP2-CFP (green) with YFP-NbXRN4 (red). Insets are the enlarged images of the areas in white boxes in the corresponding panels. H2B-RFP is shown in blue. Bars = 50 μm.</p

TuMV infection upregulates the RNA decay pathway in <i>N</i>. <i>benthamiana</i>.

<p><b>(A, B)</b> The expression levels of <i>NbDCP1</i>, <i>NbDCP2</i>, <i>NbXRN4</i> and <i>NbPARN</i> were analyzed by qRT-PCR in mock (infiltration buffer) or TuMV-infiltrated <i>N</i>. <i>benthamiana</i> leaves at 3 dpi (A) or upper new leaves at 10 dpi (B). <i>NbActin</i> was used as an internal standard. Each mean value was calculated based on three independent biological repeats (n = 3 samples). Values represent the mean ± SD. Double asterisks indicate a highly significant difference compared to mock at 3 dpi (A) or 10 dpi (B) (<i>P</i> < 0.01, Student’s <i>t</i> test). (<b>C</b>) Confocal microscopy analysis of cells co-expressing NbDCP1-CFP (green) and 6K2-YFP (panel I, red), or 6K2-NIa-VPg-YFP (panel II, red), or NIb-YFP (pane III, red) at 32 hpi. Bars, 25 μm. <b>(D)</b> Confocal microscopy of cells co-expressing NbDCP1-YFP (green) and TuMV-6K2-mCherry (panel I and panel II, red) or TuMV-CFP-NIb (panel III, red) at 72 hpi. The enlarged image of the area in the white box in panel I is shown in panel II. Bars = 50 μm.</p

Silencing of <i>NbDCP1</i>, <i>NbDCP2</i>, <i>NbXRN4</i>, or <i>NbPARN</i> suppresses S-PTGS, but not IR-PTGS in <i>N</i>. <i>benthamiana</i> plants.

<p>(<b>A, D</b>) Visualization of green fluorescence of representative agroinfiltrated leaves. Leaf patches were agroinfiltrated with three expression vectors including 35S-GFP, 35S-GF (or 35S-dsGF) and one of the following vectors: an empty vector (Vec), dsNbDCP1, dsNbDCP2, dsNbXRN4, dsNbPARN, P19, Myc-tagged-NbDCP1, NbDCP2, NbXRN4, and NbPARN. Pictures were taken at 5 dpi under UV light. Similar results were obtained from three independent experiments. (<b>B, E</b>) Relative accumulation of <i>GFP</i> mRNAs in agroinfiltrated patches of the infiltrated leaves indicated in (A) and (D), respectively. Total RNA was isolated at 5 dpi and <i>GFP</i> mRNA was analyzed by qRT-PCR. <i>NbActin</i> serves as an internal standard. Each mean value was derived from three independent experiments (n = 3 samples). Values represent the mean ± SD. Double asterisks indicate a highly significant difference compared to 35S-GFP+35S-GF+Vec (B) or 35S-GFP+35S-dsGF+Vec (E) (<i>P</i> < 0.01, Student’s <i>t</i> test). (<b>C, F</b>) Accumulation of GFP protein, <i>GFP</i> mRNA and siRNAs in the infiltrated leaves shown in (A) and (D), respectively. Samples were collected at 5 dpi. CBB staining of the large subunit of Rubisco, ethidium bromide staining of rRNA, and U6 serve as a loading control for immunoblot, mRNA blot and siRNA blot, respectively. The values of GFP siRNAs/U6 were quantified by ImageJ software and then were normalized against the mean value corresponding to the Vec treatment, which was set to 1.00.</p

Molecular characterization of 5’RDGs.

<p><b>(A)</b> qRT-PCR analysis of <i>NbDCP1</i>, <i>NbDCP2</i>, <i>NbXRN4</i> and <i>NbPARN</i> expression levels in different tissues of <i>N</i>. <i>benthamiana</i>. Expression was normalized against <i>NbActin</i> transcripts, which serve as an internal standard. Each mean value was derived from three independent experiments (n = 3 samples). Values represent the mean ± standard deviation (SD). <b>(B)</b> Micrographs showing cells from leaves of H2B-RFP transgenic <i>N</i>. <i>benthamiana</i> expressing YFP-NbDCP1, YFP-NbDCP2, YFP-NbXRN4 or YFP-NbPARN. Bars = 50 μm. (<b>C</b>) Western Blot (WB) analysis of total protein extracts from infiltrated leaves as indicated in (B) at 32 hours post infiltration (hpi), antibody against GFP (WB:GFP) was applied. Red asterisks indicate the expected band sizes. Coomassie brilliant blue-stained Rubisco large subunit was used as a loading control.</p

Expression of NbDCP1, NbDCP2, NbXRN4, or NbPARN fails to suppress GFP-induced RNA silencing in <i>N</i>. <i>benthamiana</i> plants.

<p><b>(A)</b> Schematic representation of the RNA fragment derived from expression vectors GFP, GF and dsGF. (<b>B, C</b>) Pictures of representative agroinfiltrated leaves taken at 5 dpi under UV light. Leaf patches were agroinfiltrated with three vectors including 35S-GFP, 35S-GF (or 35S-dsGF) and one of the following vectors: an empty vector (Vec), Myc-tagged-NbDCP1, NbDCP2, NbXRN4, NbPARN, and TBSV p19 (B). Similar results were obtained from three independent experiments. (<b>D, E</b>) Analyses of relative accumulations of <i>GFP</i> mRNAs by specific qRT-PCR in the infiltrated leaves shown in (B, C) at 5 dpi. <i>NbActin</i> serves as an internal standard. Each mean value was calculated based on three independent experiments (n = 3 samples). Values represent the mean ± SD. Double asterisks indicate a highly significant difference compared to 35S-GFP+35S-GF+Vec (D) or 35S-GFP+35S-dsGF+Vec (E) (<i>P</i> < 0.01, Student’s <i>t</i> test). (<b>F, G</b>) Accumulations of GFP protein, Myc-tagged-NbDCP1, NbDCP2, NbXRN4, NbPARN protein, <i>GFP</i> mRNAs and siRNAs in the infiltrated leaves shown in (B, C) at 5 dpi. Protein levels were analyzed by immunoblot analysis using antibodies against GFP (WB:GFP) or Myc (WB:Myc). Coomassie brilliant blue (CBB) staining of the large subunit of Rubisco, ethidium bromide staining of rRNA, and U6 serve as a loading control for immunoblot, mRNA blot and siRNA blot, respectively. The values of GFP siRNAs/U6 were quantified by ImageJ software and then normalized against the mean value corresponding to the Vec treatment, which was set to 1.00.</p