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Deep sequencing of the small RNAs derived from two symptomatic variants of a chloroplastic viroid: implications for their genesis and for pathogenesis.

ABSTRACT: Northern-blot hybridization and low-scale sequencing have revealed that plants infected by viroids, non-protein-coding RNA replicons, accumulate 21-24 nt viroid-derived small RNAs (vd-sRNAs) similar to the small interfering RNAs, the hallmarks of RNA silencing. These results strongly support that viroids are elicitors and targets of the RNA silencing machinery of their hosts. Low-scale sequencing, however, retrieves partial datasets and may lead to biased interpretations. To overcome this restraint we have examined by deep sequencing (Solexa-Illumina) and computational approaches the vd-sRNAs accumulating in GF-305 peach seedlings infected by two molecular variants of Peach latent mosaic viroid (PLMVd) inciting peach calico (albinism) and peach mosaic. Our results show in both samples multiple PLMVd-sRNAs, with prevalent 21-nt (+) and (-) RNAs presenting a biased distribution of their 5' nucleotide, and adopting a hotspot profile along the genomic (+) and (-) RNAs. Dicer-like 4 and 2 (DCL4 and DCL2, respectively), which act hierarchically in antiviral defense, likely also mediate the genesis of the 21- and 22-nt PLMVd-sRNAs. More specifically, because PLMVd replicates in plastids wherein RNA silencing has not been reported, DCL4 and DCL2 should dice the PLMVd genomic RNAs during their cytoplasmic movement or the PLMVd-dsRNAs generated by a cytoplasmic RNA-dependent RNA polymerase (RDR), like RDR6, acting in concert with DCL4 processing. Furthermore, given that vd-sRNAs derived from the 12-14-nt insertion containing the pathogenicity determinant of peach calico are underrepresented, it is unlikely that symptoms may result from the accidental targeting of host mRNAs by vd-sRNAs from this determinant guiding the RNA silencing machinery.

SUBMITTER: Di Serio F

PROVIDER: S-EPMC2760764 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Deep sequencing of the small RNAs derived from two symptomatic variants of a chloroplastic viroid: implications for their genesis and for pathogenesis.

Di Serio Francesco F Gisel Andreas A Navarro Beatriz B Delgado Sonia S Martínez de Alba Angel-Emilio AE Donvito Giacinto G Flores Ricardo R

PloS one 20091021 10

Northern-blot hybridization and low-scale sequencing have revealed that plants infected by viroids, non-protein-coding RNA replicons, accumulate 21-24 nt viroid-derived small RNAs (vd-sRNAs) similar to the small interfering RNAs, the hallmarks of RNA silencing. These results strongly support that viroids are elicitors and targets of the RNA silencing machinery of their hosts. Low-scale sequencing, however, retrieves partial datasets and may lead to biased interpretations. To overcome this restra ...[more]

PMID: 19847296

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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.

Dataset Information

Deep sequencing of the small RNAs derived from two symptomatic variants of a chloroplastic viroid: implications for their genesis and for pathogenesis.

Publications

Deep sequencing of the small RNAs derived from two symptomatic variants of a chloroplastic viroid: implications for their genesis and for pathogenesis.

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