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

PLoS One. 2009 Oct 21;4(10):e7539. doi: 10.1371/journal.pone.0007539.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blotting, Northern
  • Chloroplasts / virology*
  • Cytoplasm / metabolism
  • Genetic Techniques
  • Genetic Variation
  • Models, Genetic
  • Nucleic Acid Conformation
  • Oligonucleotides / genetics
  • Prunus / genetics*
  • Prunus / virology*
  • RNA Interference*
  • RNA* / metabolism
  • RNA-Dependent RNA Polymerase / genetics
  • Sequence Analysis, RNA / methods*
  • Software
  • Viroids / genetics*

Substances

  • Oligonucleotides
  • RNA
  • RNA-Dependent RNA Polymerase