Translation and replication of Rhopalosiphum padi virus RNA in a plant cellular environment.

摘要

Dicistroviruses are small, monopartite, positive-strand RNA viruses that infect arthropods. Unlike other members of the order Picornavirales , viruses in the Dicistroviridae, family possess two open reading frames (ORFs), each preceded by a distinct internal ribosome entry site (IRES). Availability of an infectious clone of Rhopalosiphum padi virus (RhPV) provides a useful molecular tool for the investigation of dicistrovirus translation and replication. Cross-kingdom analysis of translation and replication of this insect virus in a plant cellular environment could elucidate key factors involved in these processes and prove vital in efforts to apply dicistroviruses in transgenically expressed biopesticide management strategies. Intergenic region (IGR) IRES-mediated translation was tested in wheat germ extract (WGE) and oat protoplasts. IGR IRES translation was observed in WGE, but translation in oat protoplasts was insufficient to show IGR IRES function. Replication of RhPV infectious transcripts was assayed in oat protoplasts using strand-specific RT-PCR and northern blot hybridization. Negative-strand replication products were detected 48 hours post-electroporation of protoplasts with strand-specific RT-PCR, but were undetected via northern blot analysis. Though translation was not shown in this study, other preliminary experiments suggest that, with slight protocol modifications, IGR IRES-mediated translation may be detectable in oat protoplasts. Low levels of RhPV replication in planta may be beneficial in a transgenically-expressed biopesticide strategy by supporting virion packaging fidelity while avoiding RNAi silencing.