Replication of temperature-sensitive mutants of alfalfa mosaic virus in protoplasts

Six mutants of alfalfa mosaic virus (AIMV), previously found to have conditionally lethal defects when inoculated to tobacco leaf discs, were assayed for a temperature-sensitive (ts) phenotype in cowpea protoplasts. At 30 degrees the virus production of four mutants was less than 10% of that obtained at 25 degrees , whereas wild-type AIMV multiplied with similar efficiencies at both temperatures. Supplementation experiments performed in protoplasts with two mutants indicated the presence of mutations on both RNA 1 and RNA 2. Viral plus- and minus-strand RNA synthesis induced by these mutants was monitored by the Northern blotting technique under various supplementation conditions. The ts defect was mainly in the production of viral minus-strand RNA whereas transcription of minus-strand into genomic and subgenomic RNAs was much less affected. In addition, results indicate that replication of RNA 1 requires a function not essential for the synthesis of the other RNAs. One of the mutants showed a host-dependent expression of ts mutations, probably reflecting that host-viral interactions play an important role in A1MV replication.