In situ localization of the non-structural protein P25 encoded by beet necrotic yellow vein virus RNA 3

Abstract

The in situ localization of the non-structural protein P25 encoded by beet necrotic yellow vein virus (BNYVV) RNA 3 and of the BNYVV coat protein (CP) was studied by immunoelectron microscopy in infected leaf and root cells of Chenopodium murale and C. quinoa. The CP was detected in the cytoplasm of all cell types except xylem, sieve elements, and companion cells. P25 was detected in the cytoplasm and nuclei of the same cell types. The intensity of CP labelling varied depending upon the stage of infection of the cell, whereas the P25 labelling intensity was similar in newly infected cells and in cells at later stages of infection. These results suggest that P25 may be synthesized at an earlier stage of infection than CP. Its presence in the nuclei of newly infected cells may be related to the reported effect of P25 on leaf symptom development.

Beet necrotic yellow vein virus accumulates inside resting spores and zoosporangia of its vector Polymyxa betae BNYVV infects P. betae

“…In this study P25 and P31 are the only proteins that localize to the zoospore nucleus. Prior studies in plant cells also show P25 traffics to the nucleus [13]. If P25 and P31 are actively transported into the nucleus, this would suggest that they are actively interacting with cellular components and that they may be functional within the P. betae zoospore.…”

“…S. Bouzoubaa and D. Gilmer (IBMP, Strasbourg, France) provided antisera to each of the nine BNYVV proteins: replicase, coat, readthrough domain of the coat (RTD), P42, P13, P15, P14, P25, and P31 proteins [12]. These antisera have been well characterized and used for immunodetection of BNYVV proteins in infected plants [13-17]. Between 10 and 70 sporosori were treated with each antisera and a majority of resting spores tested positive for each BNYVV protein (Table 1).…”

“…If P25 and P31 are actively transported into the nucleus, this would suggest that they are actively interacting with cellular components and that they may be functional within the P. betae zoospore. It has been suggested that nuclear accumulation of P25 may play a role in symptom expression in plants [13]. It would be intriguing to learn if P25 has similar abilities to cause disease symptoms in P. betae .…”

Expression of the Beet necrotic yellow vein virus p25 protein induces hormonal changes and a root branching phenotype in Arabidopsis thaliana

“…The soluble proteins were heated to 95°C for 5 min, 10 ll were loaded on a 12% polyacrylamide gel, resolved by SDS-PAGE and transferred by electro blotting onto a polyvinylidene difluoride membrane (Immobilon-P, Millipore, Billerica, Massachusetts, USA). Immunodetection of the p25 protein was performed by incubating membranes with rabbit polyclonal anti-p25 antibody (NiesbachKlosgen et al 1990;Haeberle and Stussi-Garaud, 1995) diluted 1/40,000 in phosphate buffer saline (PBS) containing 1% Tween/5% skimmed milk overnight at 4°C. Membranes were rinsed 3 times with PBS/1% Tween and then incubated for 2 h at room temperature with the secondary goat anti-rabbit antiserum coupled to peroxidase (Molecular Probes, Eugene, Oregon, USA) diluted 1/10,000 in PBS/1% Tween/5% skimmed milk.…”

“…Ultrathin sections (90 lm) were cut using an Ultracut E microtome (Reichert, Depew, New-York, USA) and collected on grids coated with formvar (EMS, Washington). Immunolocalization of the p25 protein was performed by incubating the sections with a rabbit antip25 antibody (Niesbach-Klosgen et al 1990;Haeberle and Stussi-Garaud 1995) diluted 1/10,000 in 1% bovine serum albumin (BSA) in phosphate buffered saline (PBS) for 2 h at room temperature. The secondary goat anti-rabbit (GAR) coupled to 15 nm gold particles (Aurion EM reagents, EMS, Wageningen, The Netherlands) was diluted 1/50 in 0.1% BSA.…”