Assembly of double-shelled, virus-like particles in transgenic rice plants expressing two major structural proteins of rice dwarf virus

Abstract

Rice dwarf virus (RDV) is a double-shelled particle that contains a major capsid protein (P8), a major core protein (P3), several minor core proteins, and viral genomic double-stranded RNA. Coexpression of P8 and P3 in transgenic rice plants resulted in formation of double-shelled, virus-like particles (VLPs) similar to the authentic RDV particles. The VLPs were not detected in transgenic rice plant cells expressing P8 alone. This in vivo result suggests that P8 interacted with P3 and that these two proteins provide the structural integrity required for the formation of VLPs in rice cells independently of other structural proteins, nonstructural proteins, or viral genomic double-stranded RNAs.

FIG. 1

Genomic DNA Southern blot analysis. Genomic DNAs isolated from the independent transgenic rice plant lines 1 and 2 and from nontransformed control plants (NC) were digested with restriction enzyme XhoI (A) or SalI (B) and hybridized with a radiolabeled 1.4-kb fragment of the RDV S8 gene (A) or 3.2-kb fragment of the RDV S3 coding region (B). The molecular size markers (in kilobases) are on the right.

FIG. 2

Western blot analysis showing the coexpression of RDV P8 and P3 in transgenic rice plants. Total proteins extracted from independent transgenic rice plant lines 1 and 2, from nontransformed control plants (NC), and from RDV-infected rice plants (P) were detected with an antiserum against RDV P8 (A) or P3 (B).

FIG. 3

Electron micrographs of uranyl acetate-stained RDV particles. Shown are double-shelled VLPs assembled in cells of transgenic rice lines 1 (a) and 2 (b) coexpressing the P3 and P8 structural proteins. Also shown is immunogold probe detection of P8 in VLPs from transgenic rice plant lines 1 (c) and 2 (d). (e) RDV particle purified from RDV-infected rice plants. Bars = 10 nm.