Deep sequencing of the small RNAs derived from two symptomatic variants of a chloroplastic viroid: implications for their genesis and for pathogenesis.
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.
Istituto di Virologia Vegetale del CNR, Bari, Italy.
Francesco
F
Di Serio
Andreas
A
Gisel
Beatriz
B
Navarro
Sonia
S
Delgado
Angel-Emilio
AE
Martínez de Alba
Giacinto
G
Donvito
Ricardo
R
Flores
D015152:Blotting, Northern; D002736:Chloroplasts / Q000821:virology*; D003593:Cytoplasm / Q000378:metabolism; D005821:Genetic Techniques; D014644:Genetic Variation; D008957:Models, Genetic; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000235:genetics; D027861:Prunus / Q000235:genetics* / Q000821:virology*; D012313:RNA / Q000378:metabolism; D034622:RNA Interference; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics; D017423:Sequence Analysis, RNA / Q000379:methods*; D012984:Software; D014772:Viroids / Q000235:genetics*
D009841:Oligonucleotides; D012313:RNA; D012324:RNA-Dependent RNA Polymerase