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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

AuthorsDi Serio, Francesco; Gisel, Andreas; Navarro, Beatriz ; Delgado, Sonia ; Martínez de Alba, Ángel-Emilio ; Donvito, Giacinto; Flores, Ricardo
KeywordsInfected tomato plants
Dicer-like proteins
Silencing pathways
Issue Date21-Oct-2009
PublisherPublic Library of Science
CitationPlos One 4/10:e7539 (2009)
AbstractNorthern-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 vdsRNAs 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 (2) RNAs presenting a biased distribution of their 59 nucleotide, and adopting a hotspot profile along the genomic (+) and (2) 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.
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