Analysis of Tospovirus NSs Proteins in Suppression of Systemic Silencing

PLoS One. 2015 Aug 14;10(8):e0134517. doi: 10.1371/journal.pone.0134517. eCollection 2015.

Abstract

RNA silencing is a sequence-specific gene regulation mechanism that in plants also acts antiviral. In order to counteract antiviral RNA silencing, viruses have evolved RNA silencing suppressors (RSS). In the case of tospoviruses, the non-structural NSs protein has been identified as the RSS. Although the tomato spotted wilt virus (TSWV) tospovirus NSs protein has been shown to exhibit affinity to long and small dsRNA molecules, its ability to suppress the non-cell autonomous part of RNA silencing has only been studied to a limited extent. Here, the NSs proteins of TSWV, groundnut ringspot virus (GRSV) and tomato yellow ring virus (TYRV), representatives for three distinct tospovirus species, have been studied on their ability and strength to suppress local and systemic silencing. A system has been developed to quantify suppression of GFP silencing in Nicotiana benthamiana 16C lines, to allow a comparison of relative RNA silencing suppressor strength. It is shown that NSs of all three tospoviruses are suppressors of local and systemic silencing. Unexpectedly, suppression of systemic RNA silencing by NSsTYRV was just as strong as those by NSsTSWV and NSsGRSV, even though NSsTYRV was expressed in lower amounts. Using the system established, a set of selected NSsTSWV gene constructs mutated in predicted RNA binding domains, as well as NSs from TSWV isolates 160 and 171 (resistance breakers of the Tsw resistance gene), were analyzed for their ability to suppress systemic GFP silencing. The results indicate another mode of RNA silencing suppression by NSs that acts further downstream the biogenesis of siRNAs and their sequestration. The findings are discussed in light of the affinity of NSs for small and long dsRNA, and recent mutant screen of NSsTSWV to map domains required for RSS activity and triggering of Tsw-governed resistance.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Green Fluorescent Proteins / genetics*
  • Green Fluorescent Proteins / metabolism
  • Mutation
  • Nicotiana / genetics*
  • Nicotiana / virology
  • Plants, Genetically Modified / virology
  • RNA Interference
  • RNA, Double-Stranded / metabolism*
  • RNA, Plant / metabolism
  • RNA, Viral / genetics
  • RNA, Viral / metabolism
  • Tospovirus / genetics
  • Tospovirus / metabolism*
  • Viral Nonstructural Proteins / chemistry
  • Viral Nonstructural Proteins / genetics
  • Viral Nonstructural Proteins / metabolism*

Substances

  • RNA, Double-Stranded
  • RNA, Plant
  • RNA, Viral
  • Viral Nonstructural Proteins
  • Green Fluorescent Proteins

Grants and funding

The present research was financially supported by the Brazilian National Council for Scientific and Technological Development (CNPq; MH) and the Dutch Technology Foundation STW (DdR). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. www.stw.nl, www.cnpq.br.