The specific binding to 21-nt double-stranded RNAs is crucial for the anti-silencing activity of Cucumber vein yellowing virus P1b and perturbs endogenous small RNA populations

Adrian Valli*, Juan Carlos Oliveros, Attila Molnar, David Baulcombe, Juan Antonio Garcia

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract / Description of output

RNA silencing mediated by siRNAs plays an important role as an anti-viral defense mechanism in plants and other eukaryotic organisms, which is usually counteracted by viral RNA silencing suppressors (RSSs). The ipomovirus Cucumber vein yellowing virus (CVYV) lacks the typical RSS of members of the family Potyviridae, HCPro, which is replaced by an unrelated RSS, P1b. CVYV P1b resembles potyviral HCPro in forming complexes with synthetic siRNAs in vitro. Electrophoretic mobility shift assays showed that P1b, like potyviral HCPro, interacts with double-stranded siRNAs, but is not able to bind single-stranded small RNAs or small DNAs. These assays also showed a preference of CVYV P1b for binding to 21-nt siRNAs, a feature also reported for HCPro. However, these two potyvirid RSSs differ in their requirements of 2-nucleotide (nt) 3' overhangs and 5' terminal phosphoryl groups for siRNA binding. Copurification assays confirmed in vivo P1b-siRNA interactions. We have demonstrated by deep sequencing of small RNA populations interacting in vivo with CVYV P1b that the size preference of P1b for small RNAs of 21 nt also takes place in the plant, and that expression of this RSS causes drastic changes in the endogenous small RNA populations. In addition, a site-directed mutagenesis analysis strongly supported the assumption that P1b-siRNA binding is decisive for the anti-silencing activity of P1b and localized a basic domain involved in the siRNA-binding activity of this protein.

Original languageEnglish
Pages (from-to)1148-1158
Number of pages11
JournalRNA
Volume17
Issue number6
Early online date29 Apr 2011
DOIs
Publication statusPublished - 30 Jun 2011

Keywords / Materials (for Non-textual outputs)

  • RNA silencing suppressors
  • short interfering RNAs
  • RNA binding
  • siRNA sequestering
  • SMALL INTERFERING RNA
  • PLANT IMMUNE-SYSTEM
  • DICER-LIKE PROTEINS
  • VIRAL SUPPRESSOR
  • FAMILY POTYVIRIDAE
  • HC-PRO
  • NICOTIANA-BENTHAMIANA
  • MOSAIC-VIRUS
  • ANTIVIRAL DEFENSE
  • ARABIDOPSIS

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