Détermination de la structure de tous les viroïdes

Les viroïdes sont des agents pathogènes subviraux qui infectent des plantes importantes en agriculture. Jusqu’à aujourd’hui, une trentaine d’espèces ont été découvertes. Celles-ci sont composées d’un brin d’ARN circulaire de longueur variant selon l'espèce de 245 à 400 nucléotides qui ne codent pour aucune protéine. Chaque viroïde dépend de sa structure pour interagir avec son hôte et effectuer toutes les étapes de son cycle biologique. Il est donc de la plus haute importance de bien la connaître. À ce jour, la plupart des études présentent la structure des viroïdes par une prédiction bio-informatique. Au début de mes études, les structures de seulement deux viroïdes étaient connues en solution. Leurs structures avaient été étudiées par cartographie enzymatique ou chimique, cependant ces techniques sont longues et fastidieuses. Malgré cela, des motifs importants et non prédits par les prédictions bio-informatiques ont été découverts. Ces résultats ont renforcé la nécessité de découvrir la structure des viroïdes en solution. C’est pour cette raison que la structure de chaque espèce de viroïdes a été étudiée dans cette thèse. Pour relever ce défi, la technique de SHAPE a été adaptée pour la cartographie rapide et précise des viroïdes. L’efficacité de la technique a été confirmée en comparant les résultats obtenus par SHAPE avec ceux des viroïdes étudiés précédemment. Par la suite, les deux polarités de toutes les espèces de la famille des Avsunviroidae ont été caractérisées. De plus, les structures des membres de la seconde famille de viroïdes nommée Pospiviroidae ont aussi été étudiées. En dernier lieu, lors d'infections virales chez la plante, des particules à ARN circulaire et simple brin peuvent co-infecter les plantes avec certains virus ; ce sont des ARN satellites. Étant très semblables aux viroïdes, deux représentants ont été étudiés afin de comparer leurs structures à celles des viroïdes. Chaque ARN cartographié en solution a précisé de façon non négligeable le modèle de la structure secondaire par rapport à ceux proposés par la prédiction bio-informatique seule. De plus, des motifs tertiaires ont aussi été trouvés pour quelques-uns de ces ARN. L’ensemble du travail a aussi permis de proposer des améliorations à la classification des viroïdes et de classer de nouvelles espèces. Pour terminer, ce compendium de structures des viroïdes servira de point de départ pour étudier les motifs structuraux importants pour leur biologie

Elucidation of the structures of all members of the Avsunviroidae family

Abstract: Viroids are small single-stranded RNA pathogens which cause significant damage to plants. As their nucleic acids do not encode for any proteins, they are dependant solely on their structure for their propagation. The elucidation of the secondary structures of viroids has been limited because of the exhaustive and timeconsuming nature of classic approaches. Here, the method of high-throughput selective 2'-hydroxyl acylation analysed by primer extension (hSHAPE) has been adapted to probe the viroid structure. The data obtained using this method were then used as input for computer-assisted structure prediction using RNAstructure software in order to determine the secondary structures of the RNA strands of both (+) and (–) polarities of all Avsunviroidae members, one of the two families of viroids. The resolution of the structures of all of the members of the family provides a global view of the complexity of these RNAs. The structural differences between the two polarities, and any plausible tertiary interactions, were also analysed. Interestingly, the structures of the (+) and (–) strands were found to be different for each viroid species. The structures of the recently isolated grapevine hammerhead viroid-like RNA strands were also solved. This species shares several structural features with the Avsunviroidae family, although its infectious potential remains to be determined. To our knowledge, this article represents the first report of the structural elucidation of a complete family of viroids

Small RNA derived from the virulence modulating region of the Potato spindle tuber viroid silences callose synthase genes of tomato plants

Abstract: The tomato (Solanum lycopersicum) callose synthase genes CalS11-like and CalS12-like encode proteins that are essential for the formation of callose, a major component of pollen mother cell walls; these enzymes also function in callose formation during pathogen infection. This article describes the targeting of these callose synthase mRNAs by a small RNA derived from the virulence modulating region of two Potato spindle tuber viroid variants. More specifically, viroid infection of tomato plants resulted in the suppression of the target mRNAs up to 1.5-fold, depending on the viroid variant used and the gene targeted. The targeting of these mRNAs by RNA silencing was validated by artificial microRNA experiments in a transient expression system and by RNA ligase-mediated rapid amplification of cDNA ends. Viroid mutants incapable of targeting callose synthase mRNAs failed to induce typical infection phenotypes, whereas a chimeric viroid obtained by swapping the virulence modulating regions of a mild and a severe variant of Potato spindle tuber viroid greatly affected the accumulation of viroids and the severity of disease symptoms. These data provide evidence of the silencing of multiple genes by a single small RNA derived from a viroid

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 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

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

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

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