Elucidation of the structures of all members of the Avsunviroidae family
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...
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| Published in: | Molecular plant pathology Vol. 15; no. 8; pp. 767 - 779 |
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| Main Authors: | , , , |
| Format: | Journal Article |
| Language: | English |
| Published: |
England
Blackwell Science in collaboration with the British Society of Plant Pathology
01-10-2014
Blackwell Publishing Ltd John Wiley & Sons, Inc John Wiley and Sons Inc |
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| Online Access: | Get full text |
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| Summary: | 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 time‐consuming 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. |
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| Bibliography: | http://dx.doi.org/10.1111/mpp.12130 Natural Sciences and Engineering Research Council of Canada - No. 155219-07 ArticleID:MPP12130 Fig. S1 The normalized selective 2′-hydroxyl acylation analysed by primer extension (SHAPE) reactivities for the nucleotides of Peach latent mosaic viroid (PLMVd) (-). The reactions were performed in either the presence (black) or absence (red) of MgCl2. The results are presented as a function of nucleotide position.Fig. S2 Comparison of the two most stable structures of (+) polarity Chrysanthemum chlorotic mottle viroid (CChMVd). Plausible structural models of the most stable structure (A) and the second most stable structure (B) of CChMVd (+) obtained by selective 2′-hydroxyl acylation analysed by primer extension (SHAPE) and folded by RNAstructure. The nucleotides in black possess low SHAPE reactivities (0-0.40), those in orange intermediate reactivities (0.40-0.85) and those in red are highly reactive (>0.85). The nucleotides forming the hammerhead are underlined, the cleavage site is indicated by an arrow and the mutated nucleotides are identified by stars.Fig. S3 The normalized selective 2′-hydroxyl acylation analysed by primer extension (SHAPE) reactivities for the nucleotides of Chrysanthemum chlorotic mottle viroid (CChMVd) (+) (A) and CChMVd (-) (B). The reactions were performed in either the presence (black) or absence (red) of MgCl2. The results are presented as a function of nucleotide position.Fig. S4 The normalized selective 2′-hydroxyl acylation analysed by primer extension (SHAPE) reactivities for the nucleotides of Avocado sunblotch viroid (ASBVd) (+) (A) and ASBVd (-) (B). The reactions were performed in either the presence (black) or absence (red) of MgCl2. The results are presented as a function of nucleotide position.Fig. S5 The normalized selective 2′-hydroxyl acylation analysed by primer extension (SHAPE) reactivities for the nucleotides of Eggplant latent viroid (ELVd) (+) (A) and ELVd (-) (B). The reactions were performed in either the presence (black) or absence (red) of MgCl2. The results are presented as a function of nucleotide position.Fig. S6 The normalized selective 2′-hydroxyl acylation analysed by primer extension (SHAPE) reactivities for the nucleotides of grapevine hammerhead viroid-like RNA (GHVd) (+) (A) and GHVd (-) (B). The reactions were performed in either the presence (black) or absence (red) of MgCl2. The results are presented as a function of nucleotide position.Fig. S7 Principal component analysis (PCA) of the suboptimal structures proposed for Peach latent mosaic viroid (PLMVd) (+) by RNAstructure. The graph shows the differences between the structures obtained after folding of the PLMVd (+) structure. Each point represents a distinct structure, and they are numbered in decreasing order of stability. The distance between any two points correlates with the differences between the two structures in question. The outline for each structure is shown, with the region that is different from the most stable structure being depicted in red.Fig. S8 Primers used for the amplification of the monomeric DNA and subsequent transcription. The underlined sequences are either the T7 or T3 promoter. Unless specified, oligonucleotides of the same sequences as used in the amplification, and that do not contain the T7 or T3 promoter, were used for the primer extension reactions performed during the selective 2′-hydroxyl acylation analysed by primer extension (SHAPE) experiments. Université de Sherbrooke istex:3AFC4923457E469CEFA0620C32FBE0C7F11048A2 ark:/67375/WNG-0P3WZ88N-D |
| ISSN: | 1464-6722 1364-3703 |
| DOI: | 10.1111/mpp.12130 |