P1/HC-Pro and P38 Enhance ΔGUS-SUG Loop mRNA and Primary siRNA Accumulation, but Eliminate Secondary siRNAs.

<p>(A) Gel blot analysis of HMW and LMW RNA is shown for progeny of crosses between the ΔGUS-SUG hairpin line 306-1 and lines expressing the indicated viral suppressors of silencing. All procedures and designations are as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001755#pone-0001755-g002" target="_blank">Figure 2</a>. Grouped lanes are all from the same gel, blot, and exposure. (B) RNA gel blot analysis is shown for lines that express the indicated viral suppressors and are transgenic for both the ΔGUS-SUG hairpin and 6b4 GUS expressing locus. Controls that carry 6b4 GUS alone (lane 10) or ΔGUS-SUG alone (lanes 1, 2, 9) are also included. All procedures and designations are as described in the legends to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001755#pone-0001755-g002" target="_blank">Figures 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001755#pone-0001755-g003" target="_blank">3</a>. A longer exposure of the loop band in lanes 7–9 is also shown. Accumulation of viral suppressor mRNA in lanes 1–6 is shown for a duplicate gel that was blotted to one membrane, cut into sections corresponding to the indicated viral suppressors, and hybridized with an RNA probe specific for that suppressor; otherwise, grouped lanes are all from the same gel, blot, and exposure. (C) RNA gel blot analysis is shown for progeny of crosses between the silenced L1 GUS line and lines expressing the indicated viral suppressor. All procedures and designations are as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001755#pone-0001755-g001" target="_blank">Figure 1</a> except that only RNA probes were used. Grouped lanes are all from the same gel, blot, and exposure.</p

Sense Transgene-induced Silencing is Impaired in <i>dcl2</i> Mutant Plants, but Enhanced in <i>dcl4</i> Mutants.

<p>(A) The diagram shows the coding region of the silenced GUS sense transgene in line L1, plus the coordinates and positions of the probes used throughout this work for detecting GUS mRNA and siRNA. Antisense polarity probes will be indicated by an asterisk. The position and length in base pairs (bp) of the stem, deletion, and loop regions of the ΔGUS-SUG hairpin construct (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001755#pone-0001755-g002" target="_blank">Figures 2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001755#pone-0001755-g003" target="_blank">3</a>) are also shown. (B) Accumulation of GUS mRNA, GUS siRNAs, and siR255 in wild type (wt) and mutant plants carrying the L1 GUS locus was determined using RNA gel blot analysis. ³²P-labelled RNA probes were used for hybridization except that a DNA oligonucleotide probe was used for the siR255 blots and a full length GUS cDNA probe was used for the high molecular weight (HMW) RNA blot of lanes 18–21. Otherwise, probe 3*, which has antisense polarity, was used to detect GUS mRNA. Probes for GUS siRNA all had sense polarity and, therefore, detected the antisense strand. The positions of 21- and 22-nt RNA size markers (Ambion Decade™ Marker system) are indicated on the right of the low molecular weight (LMW) RNA blots. Grouped lanes are all from the same gel, blot, and exposure. A longer exposure of the GUS mRNA band in lanes 3–6 and 9–12 is shown directly below the rRNA band. LMW RNA blots were successively stripped and hybridized with the indicated probes. Genotypes and the zygosity of the L1 locus are indicated at the top of the lanes. The designation “mix” indicates that a segregating F2 population that was a mix (theoretically about 2:1) of L1 GUS hemizygotes and homozygotes was used for RNA isolation; +/− and +/+ indicate hemizygous and homozygous for L1 GUS, respectively. Ethidium bromide (EtBr) stained rRNA and the major RNA species in LMW RNA are shown as loading controls. (C) Relative levels of transcription of the GUS transgene in plants wild type for <i>DCL2</i> (lane 1) and in <i>dcl2-1</i> mutant plants homozygous (lane 2) or hemizygous (lane 3) for L1 GUS were determined in isolated nuclei. Nuclear transcripts from these plants were labeled with ³²P by run-off transcription and then hybridized to slot blots loaded with plasmid DNA containing GUS, actin, and pUC19 empty vector sequence.</p

<i>DCL2</i> and <i>DCL4</i> have Distinct Roles in Self-silencing of the ΔGUS-SUG Hairpin Transgene.

<p>(A) The diagram shows the coding region of the ΔGUS-SUG hairpin (hp) transgene drawn to illustrate the double-stranded configuration of the transcript. The locations of the hybridization probes and the 231-bp deletion are indicated. Probes 1 and 3 are as specified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001755#pone-0001755-g001" target="_blank">Figure 1A</a>. Accumulation of GUS mRNA in the ΔGUS-SUG line 306-1 was determined using RNA gel blot analysis and RNA probes. Duplicate samples of two different RNA preparations were run on one gel and blotted onto one membrane, after which the membrane was cut in half. One half was hybridized with probe 1 and the other with probe 3*; probe 1* gave the same result as probe 1 (data not shown). Ethidium bromide stained rRNA is shown as a loading control. (B) Accumulation of GUS mRNA and siRNAs in wild type (wt) and mutant lines carrying the ΔGUS-SUG transgene of line 306-1 was determined using RNA gel blot analysis with RNA probes. Probe 3* was used to detect GUS mRNA, while probes for GUS siRNAs all had sense polarity. Grouped lanes are all from the same gel, blot, and exposure. LMW RNA blots were successively stripped and hybridized with the indicated probes. The positions of 21- to 24-nt RNA size markers are indicated. Ethidium bromide stained rRNA and the major RNA species in LMW RNA are shown as loading controls.</p

Secondary siRNAs in Hairpin-induced Silencing are Eliminated by <i>dcl2</i> but greatly Increased by <i>dcl4</i> Mutations.

<p>(A) Diagrams of the coding regions of the 6b4 GUS expressing locus and the ΔGUS-SUG hairpin show the regions and probes first described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001755#pone-0001755-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001755#pone-0001755-g002" target="_blank">2</a>. The solid bar in the 6b4 GUS diagram corresponds to the 231-bp deletion in the ΔGUS-SUG hairpin construct. (B) Accumulation of GUS mRNA and siRNAs in wt and mutant lines transgenic for both 6b4 GUS and the ΔGUS-SUG hairpin was determined using RNA gel blot analysis with RNA probes. Line 6b4, which carries only the 6b4 GUS transgene, was used as a control. Probe 3* was used to detect GUS mRNA, while probes for GUS siRNAs all had sense polarity. Grouped lanes are all from the same gel, blot, and exposure. LMW RNA blots were successively stripped and hybridized with the indicated probes. The positions of 21- to 24-nt RNA size markers are indicated. Ethidium bromide stained rRNA and the major RNA species in LMW RNA are shown as loading controls.</p