Ultrastructural Impact of Tobacco Rattle Virus on Tobacco and Pepper Ovary and Anther Tissues

Abstract

The one-third of plant viruses are seed transmitted, and this has significant economic consequences. Tobacco rattle virus (TRV), belonging to the genus Tobravirus and family Virgaviridae, has one of the widest host range of any known plant viruses. TRV infects vegetative organ and effects seed and pollen development that results in a decrease in crop yield. The mechanisms by which Tobravirus is transmissible to seeds are still poorly understood. The presence of the virus in pollen grains and inside ovaries is linked with seed transmission and can have effects on virus particles' transport during the pollination and fertilization process. This paper focuses on the significant impact of TRV on pepper and tobacco anthers and ultrastructure changes in ovaries. The presence of two types of TRV particles in ovary wall parenchyma and vascular tissues as well as in placenta cells was demonstrated via ultrastructural analysis. For the first time, the regular inclusion of virus particles was reported in both ovule integuments and nucellus parenchyma cells. Immunolocalization of TRV capsid proteins indicated the deposition of TRV CP epitope in ovary vascular bundles and in placenta cells. Moreover, the presence of virus particles was demonstrated inside pepper seeds in endothelium and integument parenchyma layers as well as on the embryo cell wall. Virus particles were found not only on the surface of pollen grains but also inside pepper pollen protoplasts in mature anthers. Also, this is the first time where TRV particles are reported in both differentiated endothecium cells and the remaining tapetum cells. Moreover, the detection of TRV capsid protein epitope in tobacco and pepper vascular anther tissues as well as in tapetum and endothecium cells was correlated with TRV distribution in infected anthers. Demonstrated analyses indicated that pollen grains and ovaries with ovules as well as could be a natural source of TRV transmission.

Subcelullar localization of proteins associated with Prune dwarf virus replication

“…Tobacco plants were inoculated mechanically with Carborundum using leaf tissue extracts of plum infected with PDV isolate 0599. Tissue extraction buffer was 0.01 M potassiumphosphate buffer (pH 7.2-7.4) containing Na-DIECA (according to Waterworth and Fulton 1964 Samples of tobacco leaf blades were collected 15 days after inoculation from plants infected with PDV-0599 and from healthy plants, washed in 0.1 M MSB buffer (pH 6.9) then fixed in 4% (w/v) paraformaldehyde on 0.1 M MSB (pH 6.9), dehydrated in ethanol with DTT and supersaturated with BMM (butyl-methyl-acrylate) resin (Sigma-Aldrich, Germany) (according with procedure presented by Barthels et al 1997;Otulak et al 2016). The material embedded in resin was polymerized for 20 h at −20°C using UV lamps (Rafińska et al 2014).…”

“…A series of macro-sections of leaf blades fragments of a thickness of 2-3 μm were made using glass knives on RM 2065 microtome (Reichert-Jung). The sections were placed on a silylated slide (Thermo) in drops of water as described by Otulak et al (2016) with one modification. Sections were glued on thermoblocks at 45°C by 24 h. Next steps in procedure (acetone and distilled water washing, slide preparation, PBS preincubation and PBS-BSA blocking) were performed according to method already presented in Otulak et al (2016).…”

“…The sections were placed on a silylated slide (Thermo) in drops of water as described by Otulak et al (2016) with one modification. Sections were glued on thermoblocks at 45°C by 24 h. Next steps in procedure (acetone and distilled water washing, slide preparation, PBS preincubation and PBS-BSA blocking) were performed according to method already presented in Otulak et al (2016). The slides were then washed in PBS, incubated for one hour with primary purified rabbit polyclonal antibody anti-CP-PDV (Bioreba, Switzerland) or P1-PDV (detecting TRQVGPKAQYERGYTVN fragment of P1 protein of the reference isolate PDV-Salmo BC cherry, NCBI Protein: YP_611154.1, GeneCust, Luxemburg) in dilution of 1: 100 and 1:50, respectively, in PBS.…”

“…After incubation the sections were washed by PBS-Tween and PBS and stained by DAPI (4′, 6-diamidino-2-phenylindole). The observations were carried out by use of light-fluorescent microscope (Otulak and Garbaczewska 2010;Otulak et al 2016). Parallel to the immunolabeling, the tests using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) were performed to verify the specificity of P1 antibodies for virus detection.…”

“…Material was fixed, dehydrated and embedded in accordance with the procedure presented by Otulak et al (2016). Ultrathin sections (90-110 nm) were cut on an ultramicrotome (Reichert-Jung Ultracut E), transferred to nickel grids with formvar, and then subjected to labeling (Hayat 1986).…”