Replication of subterranean clover stunt virus in pea and subterranean clover protoplasts

Abstract

Subterranean clover stunt virus (SCSV) was previously found to be representative of a new type of single-stranded DNA virus. Purified SCSV particles did not infect subterranean clover when various attempts were made to transmit them to this host using aphids ( Aphis craccivora) which had previously been fed on preparations of the virus. To demonstrate that purified preparations of SCSV are capable of replication, pea and subterranean clover protoplasts were inoculated with the virus using PEG treatment or electroporation, and maintained for up to 13 days. Up to 40% of the protoplasts survived after PEG treatment but less than 10% survived after electroporation. Both enzyme-linked immunosorbent assay (ELISA) and nucleic acid hybridisation detected de novo synthesis of SCSV in the inoculated protoplasts. The amount of SCSV coat protein detected by ELISA decreased from day 0 to day 3 post-inoculation but increased thereafter over several days to a maximum at about 10 days. Nucleic acid hybridisation studies using strand-specific probes showed that the kinetics of synthesis of the virion and its complementary strand nucleic acid corresponded with the kinetics of accumulation of SCSV antigens. These results suggest that purified SCSV particles are capable of replication in transient protoplast systems. The protoplast assay could be used to characterise new or altered segments or functional domains of the SCSV genome and for the development of SCSV genome as a vector for foreign gene expression in plants.