Dataset Information

An element of the tertiary structure of Peach latent mosaic viroid RNA revealed by UV irradiation.

ABSTRACT: Following UV irradiation, denaturing polyacrylamide gel electrophoresis and Northern blot hybridization revealed a cross-link in Peach latent mosaic viroid (PLMVd) plus-strand RNA. Primer extension and partial alkaline hydrolysis of the UV-irradiated PLMVd plus-strand RNA resulting from the hammerhead-mediated self-cleavage mapped the cross-link at U81 and at the 3'-terminal C289 (or at a very proximal nucleotide). Supporting this notion, in vitro-synthesized PLMVd plus-strand RNAs with short insertions/deletions at their 3' termini failed to cross-link. Because U81 and C289 are conserved in PLMVd variants and because the initiation site of PLMVd minus-strand RNA maps at a short double-stranded motif containing C289, the UV-photo-cross-linkable element of tertiary structure may be functionally significant. A second cross-linked species similar in size and sequence to the monomeric circular PLMVd form, observed in some PLMVd variants, probably derives from UV-induced ligation of the two termini resulting from self-cleavage.

SUBMITTER: Hernandez C

PROVIDER: S-EPMC1563920 | biostudies-literature | 2006 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

An element of the tertiary structure of Peach latent mosaic viroid RNA revealed by UV irradiation.

Hernández Carmen C Di Serio Francesco F Ambrós Silvia S Daròs José-Antonio JA Flores Ricardo R

Journal of virology 20060901 18

Following UV irradiation, denaturing polyacrylamide gel electrophoresis and Northern blot hybridization revealed a cross-link in Peach latent mosaic viroid (PLMVd) plus-strand RNA. Primer extension and partial alkaline hydrolysis of the UV-irradiated PLMVd plus-strand RNA resulting from the hammerhead-mediated self-cleavage mapped the cross-link at U81 and at the 3'-terminal C289 (or at a very proximal nucleotide). Supporting this notion, in vitro-synthesized PLMVd plus-strand RNAs with short in ...[more]

PMID: 16940546

Similar Datasets

Project description:BackgroundIt has been observed that following viroid infection, there is an accumulation of viroid-derived siRNAs in infected plants. Some experimental results suggest that these small RNAs may be produced by the plant defense system to protect it from infection, indicating that viroids can elicit the RNA-silencing pathways. The objective of this study is to identify in the peach latent mosaic viroid (PLMVd), a model RNA genome, the regions that are most susceptible to RNA interference machinery.ResultsThe RNA isolated from an infected tree have been used to sequence in parallel viroid species and small non-coding RNA species. Specifically, PLMVd RNAs were amplified, cloned and sequenced according to a conventional approach, while small non-coding RNAs were determined by high-throughput sequencing. The first led to the typing of 18 novel PLMVd variants. The second provided a library of small RNAs including 880 000 sequences corresponding to PLMVd-derived siRNAs, which makes up 11.2% of the sequences of the infected library. These siRNAs contain mainly 21-22 nucleotide RNAs and are equivalently distributed between the plus and the minus polarities of the viroid. They cover the complete viroid genome, although the amount varies depending on the regions. These regions do not necessarily correlate with the double-stranded requirement to be a substrate for Dicer-like enzymes. We noted that some sequences encompass the hammerhead self-cleavage site, indicating that the circular conformers could be processed by the RNA-silencing machinery. Finally, a bias in the relative abundance of the nature of the 5' nucleotides was observed (A, U >> G, C).ConclusionsThe approach used provided us a quantitative representation of the PLMVd-derived siRNAs retrieved from infected peach trees. These siRNAs account for a relatively large proportion of the small non-coding RNAs. Surprisingly, the siRNAs from some regions of the PLMVd genome appear over-represented, although these regions are not necessarily forming sufficiently long double-stranded structures to satisfy Dicer-like criteria for substrate specificity. Importantly, this large library of siRNAs gave several hints as to the components of the involved silencing machinery.

Project description:Viroids are small circular single-stranded infectious RNAs characterized by a relatively high mutation level. Knowledge of their sequence heterogeneity remains largely elusive and previous studies, using Sanger sequencing, were based on a limited number of sequences. In an attempt to address sequence heterogeneity from a population dynamics perspective, a GF305-indicator peach tree was infected with a single variant of the Avsunviroidae family member Peach latent mosaic viroid (PLMVd). Six months post-inoculation, full-length circular conformers of PLMVd were isolated and deep-sequenced. We devised an original approach to the bioinformatics refinement of our sequence libraries involving important phenotypic data, based on the systematic analysis of hammerhead self-cleavage activity. Two distinct libraries yielded a total of 3,939 different PLMVd variants. Sequence variants exhibiting up to ∼17% of mutations relative to the inoculated viroid were retrieved, clearly illustrating the high level of divergence dynamics within a unique population. While we initially assumed that most positions of the viroid sequence would mutate, we were surprised to discover that ∼50% of positions remained perfectly conserved, including several small stretches as well as a small motif reminiscent of a GNRA tetraloop which are the result of various selective pressures. Using a hierarchical clustering algorithm, the different variants harvested were subdivided into 7 clusters. We found that most sequences contained an average of 4.6 to 6.4 mutations compared to the variant used to initially inoculate the plant. Interestingly, it was possible to reconstitute and compare the sequence evolution of each of these clusters. In doing so, we identified several key mutations. This study provides a reliable pipeline for the treatment of viroid deep-sequencing. It also sheds new light on the extent of sequence variation that a viroid population can sustain, and which may give rise to a quasispecies.

Project description:BackgroundMicroarray profiling is a powerful technique to investigate expression changes of large amounts of genes in response to specific environmental conditions. The majority of the studies investigating gene expression changes in virus-infected plants are limited to interactions between a virus and a model host plant, which usually is Arabidopsis thaliana or Nicotiana benthamiana. In the present work, we performed microarray profiling to explore changes in the expression profile of field-grown Prunus persica (peach) originating from Chile upon single and double infection with Prunus necrotic ringspot virus (PNRSV) and Peach latent mosaic viroid (PLMVd), worldwide natural pathogens of peach trees.ResultsUpon single PLMVd or PNRSV infection, the number of statistically significant gene expression changes was relatively low. By contrast, doubly-infected fruits presented a high number of differentially regulated genes. Among these, down-regulated genes were prevalent. Functional categorization of the gene expression changes upon double PLMVd and PNRSV infection revealed protein modification and degradation as the functional category with the highest percentage of repressed genes whereas induced genes encoded mainly proteins related to phosphate, C-compound and carbohydrate metabolism and also protein modification. Overrepresentation analysis upon double infection with PLMVd and PNRSV revealed specific functional categories over- and underrepresented among the repressed genes indicating active counter-defense mechanisms of the pathogens during infection.ConclusionsOur results identify a novel synergistic effect of PLMVd and PNRSV on the transcriptome of peach fruits. We demonstrate that mixed infections, which occur frequently in field conditions, result in a more complex transcriptional response than that observed in single infections. Thus, our data demonstrate for the first time that the simultaneous infection of a viroid and a plant virus synergistically affect the host transcriptome in infected peach fruits. These field studies can help to fully understand plant-pathogen interactions and to develop appropriate crop protection strategies.

Dataset Information

An element of the tertiary structure of Peach latent mosaic viroid RNA revealed by UV irradiation.

Publications

An element of the tertiary structure of Peach latent mosaic viroid RNA revealed by UV irradiation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets