Tubular Structure Induced by a Plant Virus Facilitates Viral Spread in Its Vector Insect

Abstract

Rice dwarf virus (RDV) replicates in and is transmitted by a leafhopper vector in a persistent-propagative manner. Previous cytopathologic and genetic data revealed that tubular structures, constructed by the nonstructural viral protein Pns10, contain viral particles and are directly involved in the intercellular spread of RDV among cultured leafhopper cells. Here, we demonstrated that RDV exploited these virus-containing tubules to move along actin-based microvilli of the epithelial cells and muscle fibers of visceral muscle tissues in the alimentary canal, facilitating the spread of virus in the body of its insect vector leafhoppers. In cultured leafhopper cells, the knockdown of Pns10 expression due to RNA interference (RNAi) induced by synthesized dsRNA from Pns10 gene strongly inhibited tubule formation and prevented the spread of virus among insect vector cells. RNAi induced after ingestion of dsRNA from Pns10 gene strongly inhibited formation of tubules, preventing intercellular spread and transmission of the virus by the leafhopper. All these results, for the first time, show that a persistent-propagative virus exploits virus-containing tubules composed of a nonstructural viral protein to traffic along actin-based cellular protrusions, facilitating the intercellular spread of the virus in the vector insect. The RNAi strategy and the insect vector cell culture provide useful tools to investigate the molecular mechanisms enabling efficient transmission of persistent-propagative plant viruses by vector insects.

Development of Continuous Cell Culture of Brown Planthopper To Trace the Early Infection Process of Oryzaviruses in Insect Vector Cells

“…IgG isolated from polyclonal antisera was directly conjugated to fluorescein isothiocyanate (FITC), rhodamine, or Alexa Fluor 647 carboxylic acid according to the instructions of the user manual (Invitrogen). BPH cells infected with RRSV or Sf9 cells infected with recombinant bacmids on coverslips were fixed for 30 min in 4% paraformaldehyde, permeabilized for 5 min in 0.1% Triton X-100, and then processed for immunofluorescence microscopy, as described previously (2,23). Cells on coverslips were incubated with a 50-fold-diluted solution of the directly conjugated IgG.…”

“…Cells on coverslips were incubated with a 50-fold-diluted solution of the directly conjugated IgG. Samples were then imaged by a Leica TCS SP5 confocal microscope, as described previously (2,23).…”

“…BPH cells infected with RRSV or Sf9 cells on coverslips were fixed in 2% paraformaldehyde plus 2% glutaraldehyde, and then processed for immunoelectron microscopy, as described previously (2,4,23). Cell sections were treated with antibodies to P3, P8, Pns6, and Pns10 of RRSV and then with anti-rabbit IgG conjugated to 15-nm gold particles (Sigma).…”

“…Cell sections were treated with antibodies to P3, P8, Pns6, and Pns10 of RRSV and then with anti-rabbit IgG conjugated to 15-nm gold particles (Sigma). Samples were observed with a Hitachi H-7650 electron microscope as described previously (2,4,23).…”

“…A DNA fragment spanning a 1,000-bp segment of Pns6 gene or a 793-bp segment of Pns10 gene of RRSV was used for dsRNA synthesis according to the protocol of a T7 RiboMAX Express RNAi system kit (Promega), as described previously (20). BPH cells on coverslips were transfected with 0.5 g of dsRNAs/l from the Pns6 gene (dsPns6), from the Pns10 gene (dsPns10), or from the green fluorescence protein (GFP)-encoding gene (dsGFP) via Cellfectin reagent (Invitrogen), as reported by Chen et al (23). After 24 h, the cells were inoculated with RRSV and grown further in culture medium.…”