Molecular studies of potato leafroll luteovirus multiplication

Abstract

Potato leafroll luteovirus is an aphid-transmissible virus which has isometric particles and is confined to the phloem tissue of infected plants. Its multiplication was investigated by using plant protoplasts as a model system. In protoplasts, net accumulation of PLRV ceased at approximately 48 his post-inoculation. Virus-specific products were detectable 15 hrs or more post-inoculation and remained detectable at approximately 100 hrs postinoculation. The amount of PLRV accumulated depended on the conditions in which protoplasts were incubated. Incubation at 25°C rather than 20°C and incubation in the dark for a period rather than continuous light resulted in more PLRV accumulation. RNA extracted from PLRV-infected protoplasts was identical on northern blots to that extracted from leaf tissue of PLRV-infected Maris Piper potato plants. Northern blots of RNA from other plants, some resistant, some susceptible to PLRV multiplication, were very similar. Resistant plants appeared to contain smaller quantities of subgenomic RNA. The genes at the 3'-end of the genome are expressed by translation of a subgenomic RNA. This was mapped to position 3376 on the PLRV genome and is therefore 2505 nucleotides long. The untranslated leader sequence of 212 nucleotides contains some putative promoter sequences although not in the same order as described for other viruses. A sequence of 8 nucleotides at the 5'-end of the genomic RNA was found to be repeated at the 5'-end of subgenomic RNA. The complement of this sequence may form part of an internal initiation site for the viral replicase complex in the minus strand RNA. The possibility of the untranslated leader sequence containing several promoters for both subgenomic RNA synthesis and ORF expression is discussed. Protoplast lysates contained a component that sedimented nearer the top of a sucrose gradient than virus particles. This contained subgenomic RNA and was detectable by ELISA but not by electron microscopy. It was not present in extracts of PLRV-infected plant tissue or in preparations of purified virus particles and may therefore be an unstable structure possibly - playing a role in particle assembly.