Translation downstream of the RTSV and RTBV leaders is regulated by the first sORF.

<p>Relative values of CAT expression downstream of the wild type and mutated versions (‘KO start’ and ‘KO stop”) of the RTSV (top panel) and RTBV (bottom panel) leaders in the two translation systems are given. Expression from the wild type RTSV construct in <i>O. sativa</i> (rice) protoplasts and in the wheat germ (WG) <i>in vitro</i> system is set to 100%. The sORF1 region of the leaders in each construct is shown schematically; point mutations are indicated with crosses and sORFs with boxes.</p

Conserved shunt configurations in the RTBV and RTSV leader sequences.

<p>The primary and secondary structures of RTBV (<b>A</b>) and RTSV (<b>B</b>) leaders preceding the first large viral ORF (ORF I) are shown schematically. Short ORFs within the leaders are indicated by boxes, with internal AUGs indicated by vertical lines. Arrows under the leader line define the ascending and descending arms that form the base section of the large stem-loop structure. The stem-loop structures are predicted by the MFold program (Wisconsin GCG package) at 25°C and schematically drawn below the leader primary structures. The 5′- and 3′-sequences flanking the main structure are shown in open conformation. The stable structural element at the stem base (stem basal helix) and adjacent regions, are enlarged and their sequences shown. The nucleotide numbering is from the RNA 5′-end. The 5′-proximal short ORF (sORF1) is boxed. The sORF1 AUG and the non-AUG start codons in the shunt landing site are underlined. The identical nucleotide stretches/motifs in the shunt take-off and landing sites are highlighted in bold. Nucleotide substitutions that occur in five isolates of RTSV are indicated with arrows.</p

Integrity of the stem base secondary structure is essential for RTSV shunting.

<p>Twelve point mutations in either ascending (Disrupt L) or descending (Disrupt R) arm of the RTSV stem base secondary structure are shown on the left side. A combination of these mutations (Restore L+R) restores stable secondary structure. On the right side, relative values of CAT expression downstream of the wild type (“Wild type”) and the stem base-mutated versions (“Stem Disrupt L”, “Stem Disrupt R” and “Stem Restore L+R”) of the RTSV leader [or its variant with the Kozak stem (KS) sequence in the middle part] in <i>O. sativa</i> (rice) protoplasts are given. CAT expression from the wild-type leader construct is set to 100%.</p

Translation downstream of the RTSV leader is initiated by shunting but not internal initiation.

<p>Relative values of CAT expression downstream of the wild type (“Wild type”) and sORF1-mutated versions (“KO start” and “KO stop”) of the RTSV leader carrying the KS at the 5′ end or in the middle region in <i>O. sativa</i> (rice) protoplasts are given. CAT expression from the wild-type leader construct in the absence of KS is set to 100%. For each construct, the RTSV leader preceding the polyprotein ORF (ORF I) fused to the CAT reporter ORF is depicted as thick line: the sORFs are indicated by boxes, point mutations shown with crosses, KS insertions indicated with thick vertical lines.</p

Maps of viral siRNAs and their contigs.

<p>The graphs plot the number of 20–25 nt viral siRNA reads (redundant and non-redundant) at each nucleotide position of the genomes of CaMV, ORMV and CaLCuV (DNA-A and DNA-B); Bars above the axis represent sense reads starting at respective positions; those below the antisense reads ending at respective positions. Circular DNA genomes of CaMV and CaLCuV and linear RNA genome of ORMV are shown below the graphs, with the siRNA contigs covering the genomes depicted as green lines with arrowheads. Mismatches between the ORMV contig and the reference genome are indicated.</p

Bioinformatics algorithms for <i>de novo</i> reconstruction of viral/viroid genomes from siRNAs.

<p>The <i>de novo</i> assembler programs are boxed. Green arrows indicate the algorithm which in many cases generated the longest contigs.</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

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

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