The benyvirus RNA silencing suppressor is essential for long-distance movement, requires both zinc-finger and NoLS basic residues but not a nucleolar localization for its silencing-suppression activity

Mol Plant Microbe Interact. 2013 Feb;26(2):168-81. doi: 10.1094/MPMI-06-12-0142-R.

Abstract

The RNA silencing-suppression properties of Beet necrotic yellow vein virus (BNYVV) and Beet soil-borne mosaic virus (BSBMV) cysteine-rich p14 proteins have been investigated. Suppression of RNA silencing activities were made evident using viral infection of silenced Nicotiana benthamiana 16C, N. benthamiana agroinfiltrated with green fluorescent protein (GFP), and GF-FG hairpin triggers supplemented with viral suppressor of RNA silencing (VSR) constructs or using complementation of a silencing-suppressor-defective BNYVV virus in Chenopodium quinoa. Northern blot analyses of small-interfering RNAs (siRNAs) in agroinfiltration tests revealed reduced amounts of siRNA, especially secondary siRNA, suggesting that benyvirus VSR act downstream of the siRNA production. Using confocal laser-scanning microscopy imaging of infected protoplasts expressing functional p14 protein fused to an enhanced GFP reporter, we showed that benyvirus p14 accumulated in the nucleolus and the cytoplasm independently of other viral factors. Site-directed mutagenesis showed the importance of the nucleolar localization signal embedded in a C4 zinc-finger domain in the VSR function and intrinsic stability of the p14 protein. Conversely, RNA silencing suppression appeared independent of the nucleolar localization of the protein, and a correlation between BNYVV VSR expression and long-distance movement was established.

MeSH terms

  • Amino Acid Sequence
  • Beta vulgaris / virology
  • Cell Nucleolus / metabolism
  • Chenopodium quinoa / virology
  • Cytoplasm / metabolism
  • Gene Expression Regulation, Plant
  • Gene Expression Regulation, Viral
  • Green Fluorescent Proteins
  • Mutagenesis, Site-Directed
  • Nicotiana / ultrastructure
  • Nicotiana / virology*
  • Plant Diseases / virology*
  • Plant Leaves / ultrastructure
  • Plant Leaves / virology
  • Plant Viruses / genetics
  • Plant Viruses / physiology*
  • Protein Stability
  • Protein Transport
  • RNA Interference
  • RNA Viruses / genetics
  • RNA Viruses / physiology
  • RNA, Small Interfering
  • RNA, Viral / genetics
  • Recombinant Fusion Proteins
  • Viral Proteins / chemistry
  • Viral Proteins / genetics*
  • Viral Proteins / metabolism
  • Zinc Fingers

Substances

  • RNA, Small Interfering
  • RNA, Viral
  • Recombinant Fusion Proteins
  • Viral Proteins
  • small cysteine-rich protein P14, plant virus
  • Green Fluorescent Proteins