Replication of Cauliflower Mosaic Virus ORF I Mutants in Turnip Protoplasts

Abstract

We succeeded in infecting turnip protoplasts with a cloned cauliflower mosaic virus (CaMV) DNA, pCa122, which contains 1.2 copy of CaMV genomic DNA and a plasmid expressing open reading frame (ORF) VI products (pEXP6) using polyethylene glycol. Fluorescent antibody staining showed that up to 50% of protoplasts were infected. It was difficult to detect the progeny DNA and the viral protein in protoplasts inoculated with pCa122 alone. Co-transfection with the plasmid pEXP6 produced larger fluorescing specks in each infected protoplasts and increased the accumulation of the progeny DNA and some other viral proteins to detectable levels. Using this protoplast system, three CaMV ORF I insertional mutants which were not infectious on turnip plants were tested for their infectivity on turnip protoplasts. Viral DNA and products accumulated in infected protoplasts to the same extent of the wild type DNA-infected protoplasts. These results indicate that ORF I product is not required for multiplication of CaMV in protoplasts, but is indispensable for infection on whole plants, strongly supporting that ORF I product is involved in cell-to-cell movement of CaMV.