Classification of the <i>Pospiviroidae</i> based on their structural hallmarks

<div><p>The simplest known plant pathogens are the viroids. Because of their non-coding single-stranded circular RNA genome, they depend on both their sequence and their structure for both a successful infection and their replication. In the recent years, important progress in the elucidation of their structures was achieved using an adaptation of the selective 2’-hydroxyl acylation analyzed by primer extension (SHAPE) protocol in order to probe viroid structures in solution. Previously, SHAPE has been adapted to elucidate the structures of all of the members of the family <i>Avsunviroidae</i>, as well as those of a few members of the family <i>Pospiviroidae</i>. In this study, with the goal of providing an entire compendium of the secondary structures of the various viroid species, a total of thirteen new <i>Pospiviroidae</i> members were probed in solution using the SHAPE protocol. More specifically, the secondary structures of eleven species for which the genus was previously known were initially elucidated. At this point, considering all of the SHAPE elucidated secondary structures, a classification system for viroids in their respective genera was proposed. On the basis of the structural classification reported here, the probings of both the <i>Grapevine latent viroid</i> and the <i>Dahlia latent viroid</i> provide sound arguments for the determination of their respective genera, which appear to be <i>Apscaviroid</i> and <i>Hostuviroid</i>, respectively. More importantly, this study provides the complete repertoire of the secondary structures, mapped in solution, of all of the accepted viroid species reported thus far. In addition, a classification scheme based on structural hallmarks, an important tool for many biological studies, is proposed.</p></div

The elucidated secondary structures of 4 viroids from the genus <i>Pospiviroid</i>.

<p>The color of the nucleotide represents the level of accessibility as determined by SHAPE: namely the black nucleotides are of low reactivity (0–0.40), the orange nucleotides are of intermediate reactivity (0.40–0.85) and those in red are of high reactivity (>0.85). The different regions are marked by either full lines or dashed lines depending on whether they were previously published or were determined in this report, respectively. The boxed sections are the motifs referred to in the text.</p

The determined secondary structures of two novel viroids.

<p>The most stable structures of GLVd and DLVd as elucidated by SHAPE probing. The color of the nucleotides represents the level of accessibility as determined by SHAPE: namely the black nucleotides are of low reactivity (0–0.40), the orange nucleotides are of intermediate reactivity (0.40–0.85) and those in red are of high reactivity (>0.85). The different regions are marked by either full lines or dashed lines depending on whether they were previously published or were determined in this report, respectively. The boxed sections are the motifs referred to in the text.</p

Classification of the <i>Pospiviroidae</i> members based on their structural hallmarks.

<p>The boxed structures are representative examples for each genus. The color of the nucleotide represents the level of accessibility as determined by SHAPE: namely the black nucleotides are of low reactivity (0–0.40), the orange nucleotides are of intermediate reactivity (0.40–0.85) and those in red are of high reactivity (>0.85). The underlined nucleotides are very reactive (>2.0). Structure of the CCR of the <i>Coleviroid</i> is presented as in previous report [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182536#pone.0182536.ref014" target="_blank">14</a>].</p

The determined secondary structures of viroids from the genus <i>Cocadviroid</i>.

<p>The color of the nucleotides represents the level of accessibility as determined by SHAPE: namely the black nucleotides are of low reactivity (0–0.40), the orange nucleotides are of intermediate reactivity (0.40–0.85) and those in red are of high reactivity (>0.85). The different regions are marked by either full lines or dashed lines depending on whether they were previously published or were determined in this report, respectively. The boxed sections are the motifs referred to in the text.</p

Schematic representation of a SHAPE probing experiment.

<p>The arrows show the primers used for the PCR amplification of the monomeric DNA templates 1 (full arrowheads) and 2 (white arrowheads). The RNA substrates were then produced by transcription from the T3 RNA polymerase promoter (represented by the raised extremity of the primers). The resulting RNA substrates 1 and 2 were then used in independent SHAPE reactions, and the reactivities of a sample of nucleotides for each RNA substrate are illustrated by the graphs. The black bars in the graphs represent nucleotides with low reactivities (0–0.40), the orange bars represent nucleotides with intermediate reactivities (0.40–0.85) and the red bars represent nucleotides with high reactivities (>0.85). Typical results for RNA species 1 and 2 were aligned on the original viroid sequence, and were then averaged to produce the final reactivity of each nucleotide and used in computer directed secondary structure prediction.</p

The determined secondary structures of viroids from the genus <i>Apscaviroid</i>.

<p>The color of the nucleotides represents the level of accessibility as determined by SHAPE: namely the black nucleotides are of low reactivity (0–0.40), the orange nucleotides are of intermediate reactivity (0.40–0.85) and those in red are of high reactivity (>0.85). The different regions are marked either by full lines or dashed lines depending on whether they were previously published or determined in this report, respectively. The boxed sections are the motifs referred to in the text. The circled nucleotides in the TL region of CVd-V mark the insertions as compared to ASSVd, and the boxed nucleotide represents a nucleotide variation. The arrowheads represent the position of the deleted block of nucleotides when compared to ASSVd. The possible interactions in ADFVd are represented by the dashed lines.</p

Alterations of the viroid regions that interact with the host defense genes attenuate viroid infection in host plant

<p>Understanding in intimate details how the viroid interaction with host's defense genes is a cornerstone for developing viroid resistant plants. In this present study, small RNAs (sRNA) derived from <i>Potato spindle tuber viroid</i> (PSTVd) were studied <i>in silico</i> in order to detect any interactions with the serine threonine kinase receptor, a transmembrane protein that plays a role in disease resistance in plants. Using molecular biology techniques, it was determined that PSTVd infection negatively affects at least three serine threonine kinase receptors as well as with three other genes that are known to be involved in the overall development of the tomato plants. The transient expression of these putative PSTVd-sRNAs, using the microRNA sequence as a backbone, in tomato plants induced phenotypes similar to viroid infection. Mutants created by altering the sequence of PSTVd in these regions failed to infect the tomato plant. The data presented here illustrates the importance of these regions in viroid survival, and suggests a possible avenue of exploration for the development of viroid resistant plants.</p

Global magnesium localization along the HDV Rz’s folding pathway studied by magnesium-induced cleavage.

<p>Different 5′-end-labeled <i>trans</i>-acting mutant ribozymes that halt the folding pathway at each known HDV Rz folding intermediate were folded either in the absence or the presence (+) of SdA-1 substrate or 3′-end product. The probings were then allowed to proceed for 48 h at room temperature in the presence of 50 mM Tris-HCL (pH 8.3) and 20 mM MgCl₂. A control reaction without MgCl₂ (−) was also performed. The resulting probings were analysed on 8% denaturing PAGE gels. The positions of bromophenol blue dye (BPB) and of the different regions of the Rz are indicated on the right of the gel. The lanes designated “Ladder” and “T1” represent an alkaline hydrolysis and a ribonuclease T1 (RNase T1) mapping of the wild-type version of the ribozyme, respectively. Representative guanosine residues are indicated on the left of the gel.</p

MC-sym structure depicting the formation of the <i>trans</i> Watson-Crick GU base pair after the cleavage step.

<p>(<b>A</b>) Representative structure of the post-cleavage HDV ribozyme. (<b>B</b>) Closer view of stereodiagrams of loop III both before (containing a <i>t</i>WH GU base pair, upper panel) and after (containing a <i>t</i>WW GU base pair, lower panel) the cleavage step. The colors are harmonized as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040309#pone-0040309-g001" target="_blank">Figure 1</a>.</p