ACMV-derived siRNAs are phosphorylated at the 5′ end RNA gel blot analysis of 20 µg total RNA prepared from ACMV-infected wild-type and treated (+) or not (−) with alkaline phosphatase

<p><b>Copyright information:</b></p><p>Taken from "Molecular characterization of geminivirus-derived small RNAs in different plant species"</p><p>Nucleic Acids Research 2006;34(2):462-471.</p><p>Published online 18 Jan 2006</p><p>PMCID:PMC1342034.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> The blot was successively probed with DNA oligonucleotides corresponding to the ACMV DNA-A complementary (AC2 as) and virion (AC2 s) strand sequences in the AC2 coding region. Positions of the 21 and 24 nt RNAs are indicated

Maps of vsRNAs from CaLCuV-infected wild type (Col-0) and <i>rdr1/2/6</i> triple mutant plants at single-nucleotide resolution.

<p>The graphs plot the number of 20–25 nt vsRNA reads at each nucleotide position of the 2583 bp DNA-A (<b>A</b>) and the 2513 bp DNA-B (<b>B</b>); Bars above the axis represent sense reads starting at each respective position; those below represent antisense reads ending at the respective position (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002941#ppat.1002941.s009" target="_blank">Tables S2</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002941#ppat.1002941.s010" target="_blank">S3</a>). The genome organizations of DNA-A and DNA-B are shown schematically above the graphs, with leftward (AC1, AC4, AC2, AC3 and BC1) and rightward (AV1 and BV1) ORFs and common region (CR) indicated. The predicted rightward and rightward mRNAs are shown as respectively blue and red solid lines with arrowheads. Potential readthrough transcripts are shown as dotted thin lines.</p

Maps of primary and secondary siRNAs accumulating in L2 transgenic plants infected with CaLCuV::GFP viruses that target the <i>GFP</i> transcribed region.

<p>The graphs plot the number of 20–25 nt vsRNA reads at each nucleotide position of the L2 T-DNA-based 35S::GFP transgene; Bars above the axis represent sense reads starting at each respective position; those below represent antisense reads ending at the respective position (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002941#ppat.1002941.s012" target="_blank">Table S5</a>). The 35S-GFP transgene is shown schematically above the graphs. Positions of the duplicated 35S enhancer and core promoter, <i>GFP</i> mRNA elements and 35S terminator are indicated. Numbering is from the T-DNA left border (LB). The VIGS target sequences inserted in the CaLCuV::GFP viruses Lead, CodM, Trail and polyA are indicated with dotted boxes.</p

VIGS phenotypes and accumulation of primary and secondary siRNAs in L2 transgenic plants infected with CaLCuV::GFP viruses targeting the <i>GFP</i> transcribed region.

<p>(<b>A</b>) The L2 T-DNA region containing the 35S-GFP transgene is shown schematically. Positions of the duplicated CaMV 35S enhancer and core promoter elements, <i>GFP</i> mRNA elements including 5′UTR, translation start (AUG) and stop (UAA) codons and 3′UTR with poly(A) signal (AAUAAA), and 35S terminator sequences indicated. Numbering is from the T-DNA left border (LB). The VIGS target sequences, inserted in the CaLCuV::GFP viruses <i>Lead</i>, <i>CodB, CodM, CodE, Trail</i> and <i>polyA</i>, are indicated with dotted boxes; (<b>B</b>) Pictures under UV light of L2 transgenic plants infected with the above viruses; (<b>C</b>) Blot hybridization analysis of total RNA isolated from plants shown in Panel B. The blot was successively hybridized with short DNA probes specific for CaLCuV <i>AC4</i> gene (AC4_s) and 35S::GFP transgene sequences inserted in the CaLCuV::GFP viruses (<i>Lead, CodB, CodM, CodE, Trail</i> and <i>polyA</i>), the <i>GFP</i> mRNA 3′UTR non-target sequence (3′UTR) and <i>Arabidopsis</i> miR173 and Met-tRNA (the latter two serve as loading control).</p

Primary and secondary siRNAs in CaLCuV::Chl virus-infected wild type (Col-0) plants.

<p>(<b>A</b>) The 2300 bp region of the <i>Arabidopsis</i> genome, which contains <i>Chlorata I/CH42</i> gene (<i>ChlI</i>), is shown schematically with positions of <i>ChlI</i> promoter, pre-mRNA with two introns, and terminator sequences indicated; numbering starts 500 nucleotides upstream of the transcription start site. The VIGS target sequence (inserted in CaLCuV::Chl virus) is highlighted with grey. The graph plots the number of 20–25 nt siRNA reads at each nucleotide position of the <i>ChlI</i> gene; Bars above the axis represent sense reads starting at each respective position; those below represent antisense reads ending at the respective position (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002941#ppat.1002941.s011" target="_blank">Table S4</a>). (<b>B</b>) The left bar graph shows the total numbers of 20–25 nt primary (CaLCuV::Chl-derived) and secondary siRNAs derived from <i>ChlI</i> sequences outside of the VIGS target region, while the right bar graph shows the number of primary siRNAs for each size class and polarity.</p

Illumina deep-sequencing of sRNAs from mock-inoculated and CaLCuV-infected <i>Arabidopsis</i> wild-type (Col-0) and <i>rdr1/2/6</i> triple mutant plants.

<p>The graphs show the percentages of <i>Arabidopsis</i> and vsRNAs in the pool of 20–25 nt reads mapped to the <i>Arabidopsis</i> and CaLCuV DNA-A and DNA-B genomes with zero mismatches (<b>A</b>), of each size-class of 20–25 nt host sRNA reads mapped to the <i>Arabidopsis</i> genome with zero mismatches (<b>B</b>), and of each size-class of 20–25 nt vsRNA reads mapped to the CaLCuV DNA-A and DNA-B with zero mismatches (<b>C</b>).</p

VIGS phenotypes and primary siRNA accumulation in L2 transgenic plants infected with CaLCuV::GFP viruses that target the <i>GFP</i> promoter and terminator elements.

<p>(<b>A</b>) The L2 T-DNA region containing the 35S-GFP transgene is shown schematically. Positions of the duplicated CaMV 35S enhancer (<i>Enh</i>) and core promoter (<i>Core</i>) elements (CAAT and TATA boxes and transcription start <i>Plus1</i>), the <i>GFP</i> mRNA elements (5′UTR, AUG and UAA codons and 3′UTR, and 35S terminator are indicated. Numbering is from the T-DNA left border (LB). The VIGS target sequences, inserted in the CaLCuV::GFP viruses <i>ProFL, Enh, CAAT, TATA, Plus1, CodFL, Trail</i> and <i>Post</i> are indicated with dotted boxes; (<b>B</b>) and (<b>C</b>) Blot hybridization analysis of total RNA isolated from L2 plants infected with the above viruses. The two blots were successively hybridized with short DNA probes specific for CaLCuV <i>AC4</i> gene (AC4_s) and the 35S::GFP transgene sequences inserted in CaLCuV::GFP viruses and <i>Arabidopsis</i> miR173 and Met-tRNA (the latter two serve as loading control). (<b>D</b>) Pictures under UV light of L2 transgenic plans infected with the CaLCuV::GFP viruses (names indicated).</p

Maps of primary siRNAs accumulating in L2 transgenic plants infected with CaLCuV::GFP viruses that target the <i>GFP</i> promoter elements.

<p>The graphs plot the number of 20–25 nt vsRNA reads at each nucleotide position of the L2 T-DNA-based 35S::GFP transgene; Bars above the axis represent sense reads starting at each respective position; those below represent antisense reads ending at the respective position (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002941#ppat.1002941.s012" target="_blank">Table S5</a>). The 35S-<i>GFP</i> transgene is shown schematically above the graphs. Positions of the duplicated 35S enhancer and core promoter, <i>GFP</i> mRNA elements and 35S terminator are indicated. Numbering is from the T-DNA left border (LB). The VIGS target sequences inserted in the CaLCuV::GFP viruses <i>CodFL, Enh</i> and <i>Core</i> are indicated with dotted boxes. Note that the duplicated 35S promoter sequences Enhancer* and Enhancer (each 273 nt long) share 94% nucleotide identity, since they originate from two different strains of CaMV. Therefore, primary siRNA reads are unequally distributed between the two VIGS target regions.</p

Genetic requirements for primary and secondary siRNA accumulation in L2 transgenic plants.

<p>Blot hybridization analysis of total RNA isolated from L2, L2 x <i>rdr6</i> and L2 x <i>dcl4</i> plants infected with CaLCuV::GFP viruses <i>Lead, CodM</i> and <i>Trail</i>. The blot was successively hybridized with short DNA probes specific for CaLCuV genes AC4 (AC4_s and AC4_as) and AV1 (A1063_s and A1063_as), 35S::GFP transgene sequences inserted in the CaLCuV::GFP viruses (<i>Lead, CodM, Trail</i>), <i>GFP</i> mRNA 3′UTR non-target sequence (3′UTR_s) and <i>Arabidopsis</i> miR173 and Met-tRNA (the latter two serve as loading control).</p