Distinct Effects of p19 RNA Silencing Suppressor on Small RNA Mediated Pathways in Plants

RNA silencing is one of the main defense mechanisms employed by plants to fight viruses. In change, viruses have evolved silencing suppressor proteins to neutralize antiviral silencing. Since the endogenous and antiviral functions of RNA silencing pathway rely on common components, it was suggested that viral suppressors interfere with endogenous silencing pathway contributing to viral symptom development. In this work, we aimed to understand the effects of the tombusviral p19 suppressor on endogenous and antiviral silencing during genuine virus infection. We showed that ectopically expressed p19 sequesters endogenous small RNAs (sRNAs) in the absence, but not in the presence of virus infection. Our presented data question the generalized model in which the sequestration of endogenous sRNAs by the viral suppressor contributes to the viral symptom development. We further showed that p19 preferentially binds the perfectly paired ds-viral small interfering RNAs (vsiRNAs) but does not select based on their sequence or the type of the 5’ nucleotide. Finally, co-immunoprecipitation of sRNAs with AGO1 or AGO2 from virus-infected plants revealed that p19 specifically impairs vsiRNA loading into AGO1 but not AGO2. Our findings, coupled with the fact that p19-expressing wild type Cymbidium ringspot virus (CymRSV) overcomes the Nicotiana benthamiana silencing based defense killing the host, suggest that AGO1 is the main effector of antiviral silencing in this host-virus combination

Tomato Yellow Leaf Curl Sardinia Virus Rep-Derived Resistance to Homologous and Heterologous Geminiviruses Occurs by Different Mechanisms and Is Overcome if Virus-Mediated Transgene Silencing Is Activated

The replication-associated protein (Rep) of geminiviruses is involved in several biological processes brought about by the presence of distinct functional domains. Recently, we have exploited the multifunctional character of the Tomato yellow leaf curl Sardinia virus (TYLCSV) Rep to develop a molecular interference strategy to impair TYLCSV infection. We showed that transgenic expression of its N-terminal 210 amino acids (Rep-210) confers resistance to the homologous virus by inhibiting viral transcription and replication. We have now used biochemical and transgenic approaches to carry out a fuller investigation of the molecular resistance mechanisms in transgenic plants expressing Rep-210. We show that Rep-210 confers resistance through two distinct molecular mechanisms, depending on the challenging virus. Resistance to the homologous virus is achieved by the ability of Rep-210 to tightly inhibit C1 gene transcription, while that to heterologous virus is due to the interacting property of the Rep-210 oligomerization domain. Furthermore, we present evidence that in Rep-210-expressing plants, the duration of resistance is related to the ability of the challenging virus to shut off transgene expression by a posttranscriptional homology-dependent gene silencing mechanism. A model of Rep-210-mediated geminivirus resistance that takes transgene- and virus-mediated mechanisms into account is proposed

p19 protein expressing transgenic <i>Nicotiana benthamiana</i> (p19syn) plants.

<p>(A) Specific developmental phenotype of p19syn plants compared to wild type plants. Three representative independent transgenic lines are shown alongside wild-type <i>N</i>. <i>benthamiana</i> plant. (B) Western blot of p19syn transgenic and wild-type plants from lines shown in (A). The presence of p19 transgene does not impact NbAGO1 or NbAGO2 protein levels. Protein loading is shown below (StainFree). (C) Stem length from cotyledons to the fourth and last leaf insertion in wt and T1 p19syn plants at 8–9 leaf stage (lines:1–29 and 1–57) were used to show the elongated internode trait of p19syn plants. Bars: standard deviation. (D) Wild type and p19syn plants agroinfiltrated with GFP and GFP+p19 constructs as indicated. Pictures were taken at 4 dpi. (E) p19-mediated inhibition of RNA silencing of tobacco magnesium protoporphyrin chelatase subunit I <i>(ChlI)</i> (a key chlorophyll biosynthetic gene) induced by CMV + Y-satRNA infection (14 dpi, left panel), silencing of <i>ChlI</i> causes yellowing on wt plants. Northern blot hybridization of CMV + Y-satRNA infected wt or p19syn plants; nucleolar small RNA U6 was used as an internal control (right panel).</p

p19 interaction with sRNAs <i>in vivo</i>.

<p>(A) Western blots of p19 (upper panel), Northern blot of p19-bound vsiRNAs (middle panel) and miR159 (lower panel) during the CymRSV and Cym19stop virus infection of p19syn and wt <i>N</i>. <i>benthamiana</i> plants as indicated. The ratio of p19-bound miR159 to the input (IP/input) is indicated below the panels. (B) The percent of vsiRNA sequences derived from the positive and negative viral strands in the input and in p19-IP. (C) Global accumulation of different size classes of vsiRNAs in p19syn and wt <i>N</i>. <i>benthamiana</i> plants infected by CymRSV and Cym19stop as indicated. Size classes of vsiRNAs are shown by color codes presented on the right side of the panel. (D) Ratio of full length and truncated miRNA matching reads. The columns show the percent of full-length (fl), 5’-truncated (5’tr) and 3’-truncated (3’tr) miRNAs in the input and p19-IP of p19syn and wild type plants. Values above the columns indicate the normalized read count ratio of p19-IP/input for each miRNA in a log2 scale.</p

Deep sequencing analysis of AGO1- and AGO2-bound sRNAs.

<p><i>N</i>. <i>benthamiana</i> specific reads from mock-inoculated (A), CymRSV- (C) and Cym19stop-infected wild-type plants (E). The vsiRNA reads of the same samples are presented in (B), (D) and (F) respectively. 5’ nucleotides of vsiRNAs are indicated by color code on the right. Size classes (nucleotide) of sRNAs are indicated by numbers. Read counts were normalized to 10⁶ total reads. Note the different scale in panel A.</p

Northern blot analysis of NbAGO1- and NbAGO2-bound vsiRNAs and miRNAs.

<p>(<b>A</b>) Serum- NbAGO1- and NbAGO2-immunoprecipitations from mock-, CymRSV- or Cym19stop-infected wt <i>N</i>. <i>benthamiana</i> plants as shown: upper panels, Western blots for AGO1, AGO2 and p19 are shown (StainFree gel picture shown as loading control); lower panels, Northern blots of AGO1-specific miR159, AGO2-specific miR390 and vsiRNAs (ethidium-bromide stain shown as loading control). (B) Serum- NbAGO1- and NbAGO2-immunoprecipitations from mock- or Cym19stop-infected transgenic p19syn lines as shown: upper panels, Western blots for AGO1, AGO2 and p19 (StainFree gel picture shown as loading control); lower panels, Northern blots of AGO1-specific vsiRNAs and miR159 (ethidium-bromide stain shown as loading control).</p

Affinity of p19 to perfect or mismatch-containing duplex si/miRNAs <i>in vitro</i>.

<p>Band shift assay of perfect duplex siR171 (A), and mismatched miR171a (B), miR171b (C) and miR171c (D) duplex RNAs’ with p19 protein. The structure of dsRNAs is shown above the gel pictures. Direct measurement of the absolute apparent dissociation constant Kd values (F) were calculated as previously described [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005935#ppat.1005935.ref057" target="_blank">57</a>] based on the quantification of band intensities of p19:dsRNA bound fraction as a function of p19 protein concentration (E). Relative dissociation constant (Krel) was calculated by normalization of Kd values to Kd of siR171 (F).</p

Endogenous and viral sRNAs sequestration preferences of p19.

<p>(A) Total and p19-bound vsiRNAs and endogenous sRNAs derived from <i>N</i>. <i>benthamiana</i> and p19syn plants infected with wt (CymRSV) and suppressor deficient Cym19stop viruses and mock-inoculated plants. (B) input and p19-bound endogenous sRNA duplexes in mock-inoculated plants. (C-F) inputs and p19-bound vsiRNAs and endogenous sRNAs when p19 was expressed in <i>trans</i> (C and D) or <i>in cis</i> (E and F) during virus infections as indicated. The size classes of sRNAs between 18 and 24nt are indicated by numbers The 5’ nucleotides are indicated by color codes. The percentages of specific 5’-nucleotide sRNAs in input and p19 IP are shown in brackets at the right side of the panel (input%; IP%). Note that B, C, E scales differ from D and F ones. Read counts were normalized to 1 million reads.</p