Normalized abundance of the three major classes of endogenous siRNAs.

<p><b>A</b>) ta-siRNAs. <b>B</b>) endoIR-siRNas. <b>C</b>) PolIV/PolV-siRNAs. Small RNAs from wild-type (Col), <i>35S</i>:<i>RTL1</i> (<i>RTL1</i>) transgenic plants and <i>dcl2dcl3dcl4 (dcl234)</i> mutants were classified as ta-siRNAs, endoIR-siRNas, or PolIV/PolV-siRNAs based on published annotation. Small RNA abundance was normalized to the total amount of conserved miRNAs. Each size of small RNA is indicated by a different color: 21 nt (blue), 22 nt (green), 23 nt (pink), and 24 nt (red).</p

Impact of RTL1 on virus infection.

<p><b>A</b>) Pictures of water (mock)-, TCV-, TVCV-, CMV-, and TYMV-inoculated wild-type (Col) and <i>35S</i>:<i>RTL1-Flag</i> (<i>RTL1-Flag</i>) plant #2. Ten-day-old plants were inoculated. Pictures were taken three weeks following inoculation. <b>B</b>) RNA gel blot detection of TCV, TVCV, CMV, and TYMV RNAs and siRNAs in total aerial parts of mock- and virus-inoculated wild-type (Col) and <i>35S</i>:<i>RTL1-Flag</i> (<i>RTL1-Flag</i>) plant #2. <i>U6</i> was used as a loading control. The <i>U6</i> panels for CMV and TVCV are similar to those in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002326#pbio.1002326.g007" target="_blank">Fig 7A</a> because the blots used in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002326#pbio.1002326.g007" target="_blank">Fig 7A</a> were stripped and rehybridized with CMV and TVCV probes to produce Fig 8B.</p

RTL1 cleaves dsRNA.

<p><b>A</b>) RNA gel blot detection of <i>IR71</i> precursor RNA in wild-type (Col), Col transformed with the <i>35S</i>:<i>RTL1</i> construct (<i>Col/RTL1</i>), <i>dcl234</i>, and <i>dcl234</i> transformed with the <i>35S</i>:<i>RTL1</i> construct (<i>dcl234/RTL1</i>). Transformants exhibiting a strong RTL1 developmental phenotype were analyzed. High molecular weight (HMW) RNAs extracted from flowers were hybridized with a probe complementary to the <i>IR71</i> RNA and with <i>25S</i> as loading control. <b>B</b>) RNAs extracted from wild-type seedlings were incubated or not with wild-type His-RTL1 and subjected to RT-PCR to detect IR71, IR2039, and At3g18145 (3’UTR) precursor RNAs. <b>C</b>) RNAs extracted from wild-type seedlings were incubated with wild-type or mutant His-RTL1 and subjected to RT-PCR reactions to detect At3g18145 (3’UTR) precursor RNAs. Comassie blue-stained gel shows approximately 200 ng of wild-type and mutant proteins. A schematic representation of RTL1 (residues 1–289) is shown at the top. Black and grey boxes correspond to RNaseIII and dsRBD (double stranded RNA Binding Domain) motifs, respectively. The conserved amino acids in the RNase III signature motif are highlighted in black and the residues E86, E89, E92, and D96 mutated in recombinant proteins indicated by an asterisk. <b>D</b>) RNAs extracted from wild-type seedlings were denaturated or not before incubation with His-RTL1 and subjected to RT-PCR reactions to detect IR71 precursor RNAs. RT-PCR amplification of U3 snoRNA sequences shows similar amount of RNA in each reaction.</p

The RTL1 RNaseIII domain is required for inhibition of transgene PTGS.

<p><b>A</b>) <i>N</i>. <i>benthamiana</i> leaves were infiltrated with a <i>35S</i>:<i>GU-UG</i> construct (<i>GU-UG</i>) together with either a wild-type <i>35S</i>:<i>RTL1</i> construct <i>(RTL1)</i>, a construct mutated in the RNaseIII domain (<i>RTL1mR3</i>) or a <i>35S</i>:<i>GFP</i> control <i>(GFP)</i>. Low molecular weight (LMW) RNAs were hybridized with a <i>GUS</i> probe and with <i>U6</i> as a loading control. <b>B</b>) <i>N</i>. <i>benthamiana</i> leaves were infiltrated with a <i>35S</i>:<i>GU-UG</i> construct (<i>GU-UG</i>) and a <i>35S</i>:<i>GUS</i> construct (<i>GUS)</i> together with either a wild-type <i>35S</i>:<i>RTL1</i> construct <i>(RTL1)</i>, a construct mutated in the RNaseIII domain (<i>RTL1mR3</i>), or a <i>35S</i>:<i>GFP</i> control <i>(GFP)</i>. LMW RNAs were hybridized with a <i>GUS</i> probe and with U6 as loading control. High molecular weight RNAs were hybridized with a <i>GUS</i> probe and with 25S as loading control. <b>C</b>) The <i>Arabidopsis</i> line <i>L1</i> carrying a <i>35S</i>:<i>GUS</i> transgene silenced by PTGS was transformed with a wild-type <i>35S</i>:<i>RTL1</i> construct <i>(RTL1)</i>. LMW <i>GUS</i> RNAs from two independent transformants were analyzed. Note that the images presented in this panel are internal to the images presented in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002326#pbio.1002326.s010" target="_blank">S9C Fig</a>. <b>D</b>) <i>N</i>. <i>benthamiana</i> leaves were infiltrated with a <i>35S</i>:<i>GU-UG</i> construct (<i>GU-UG</i>) together with either a wild-type <i>35S</i>:<i>RTL1</i> construct <i>(RTL1)</i>, tagged constructs (<i>RTL1-Myc</i>, <i>Myc-RTL1</i>, <i>RTL1-Flag</i>, <i>Flag-RTL1</i>), or a <i>35S</i>:<i>GFP</i> control <i>(GFP)</i>. LMW RNAs were hybridized with a <i>GUS</i> probe and with <i>U6</i> as a loading control. <b>E</b>) <i>N</i>. <i>benthamiana</i> leaves were infiltrated with a <i>35S</i>:<i>GU-UG</i> construct (<i>GU-UG</i>) together with either a wild-type tagged <i>35S</i>:<i>RTL1-Myc</i> construct <i>(RTL1-Myc)</i>, a construct mutated in the RNaseIII domain (<i>RTL1mR3-Myc</i>) or a <i>35S</i>:<i>GFP</i> control <i>(GFP)</i>. LMW RNAs were hybridized with a <i>GUS</i> probe and with <i>U6</i> as a loading control. Proteins were extracted and hybridized with an anti-Myc antibody. Ponceau staining serves as a loading control. <b>F</b>) The <i>Arabidopsis</i> line <i>L1</i> was transformed with either a wild-type-tagged <i>35S</i>:<i>RTL1</i> construct <i>(RTL1-Myc)</i> or a tagged construct mutated in the RNaseIII domain (<i>RTL1mR3-Myc</i>). Proteins were extracted from three independent <i>RTL1-Myc</i> transformants and eight independent <i>RTL1mR3-Myc</i> transformants and hybridized with an anti-Myc antibody. Ponceau staining serves as a loading control. <b>G</b>) LMW RNAs from <i>RTL1-Myc</i> and <i>RTL1mR3-Myc</i> transformants expressing comparable amount of proteins were hybridized with <i>GUS</i> and <i>TAS2</i> probes and with <i>U6</i> as a loading control.</p

RTL1 expression and localization.

<p><b>A)</b> RNA extracted from the total aerial part of wild-type plants (Col) three weeks after inoculation with water (mock), TCV, TVCV, CMV, or TYMV were subjected to oligo-dT reverse transcription followed by qPCR with primers specific to <i>RTL1</i>, <i>RTL2</i>, or <i>RTL3</i>. Analysis was done in triplicate. Results were normalized to <i>GAPDH</i>. <b>B)</b> RNA extracted from leaves and roots of 3 wk-old wild-type plants (Col) were subjected to oligo-dT reverse transcription followed by qPCR with RTL1 oligos. Analysis was done in triplicate. Results were normalized to <i>GAPDH</i>. <b>C)</b> RNA extracted from leaves of 3 wk-old wild-type plants (Col) and <i>35S</i>:<i>RTL1-Flag#2</i> (<i>RTL1-Flag</i> #2) plants were subjected to oligo-dT reverse transcription followed by qPCR with RTL1 oligos. Analysis was done in triplicate. Results were normalized to <i>GAPDH</i>. <b>D</b>) Onion epidermial cells transformed with a <i>35S</i>:<i>RTL1-GFP</i> construct were imaged using a Zeiss Axioskop 2 microscope and recorded using a Leica DC 300 FX digital camera (Leica). <b>E</b>) Proteins were extracted from 18-d-old seedlings of wild-type plants (Col) and <i>35S</i>:<i>RTL1-Flag</i> (<i>RTL1-Flag</i>) plants and hybridized with an anti-RTL1 antibody. Hybridization with an anti-RPL13 antibody serves as a loading control. <b>F)</b> Immunostaining of root cells from 8-d-old seedlings of wild-type plants (Col) and <i>35S</i>:<i>RTL1-Flag</i> (<i>RTL1-Flag</i>) plants was performed using an anti-RTL1 antibody and revealed with Alexa 488.</p

RTL1 activity is suppressed by VSRs.

<p><b>A</b>) RNAs extracted from water (mock)- or virus inoculated wild-type (Col) and <i>35S</i>:<i>RTL1-Flag</i> (<i>RTL1-Flag</i>) plant #2 were hybridized with a <i>TAS2</i> probe and <i>U6</i> as a loading control. <b>B</b>) <i>N</i>. <i>benthamiana</i> leaves were infiltrated with a <i>35S</i>:<i>GU-UG</i> construct (<i>GU-UG</i>) together with either a <i>35S</i>:<i>GFP</i> control <i>(GFP)</i>, a wild-type <i>35S</i>:<i>RTL1</i> construct <i>(RTL1)</i>, a <i>35S</i>:<i>VSR</i> construct (<i>P38</i>, <i>P38GA2</i>, <i>P1/HC-Pro</i>, <i>2b</i>, <i>or P69</i>), or a mix of the <i>35S</i>:<i>RTL1</i> and <i>35S</i>:<i>VSR</i> constructs. LMW RNAs were hybridized with a <i>GUS</i> probe and with <i>U6</i> as a loading control.</p