Synthesis of potyviral RNA and proteins in tobacco mesophyll protoplasts inoculated by electroporation

Abstract

Tobacco (Nicotiana tabacum L.) mesophyll protoplasts were inoculated with the RNAs of the potyviruses tobacco vein mottling virus (TVMV), tobacco etch virus or potato virus Y (PVY) by electroporation. Analysis by enzyme-linked immunosorbent assay indicated that the TVMV coat and helper component proteins accumulated to similar extents over a 72-h period after inoculation. No evidence of infection was observed in protoplasts electroporated in the absence of TVMV RNA or in protoplasts mixed with TVMV RNA without electroporation. Immunostaining of electroporated protoplasts with antibodies to the non-structural TVMV cylindrical inclusion protein indicated that the majority of protoplasts became infected. Replication of viral RNA, measured by molecular hybridization to 32p-TVMV cDNA in a quantitative dot blot assay, was most rapid between 3 and 15 h after electroporation. Electroporation resulted in greater accumulation of progeny viral proteins and RNA and less damage to the protoplasts than did fusion with liposomes containing TVMV RNA or treatment with a complex of TVMV particles and poly-l-ornithine (PLO).