Double infection of tobacco protoplasts with brome mosaic virus and cowpea chlorotic mottle virus.

Mixed infections of tobacco protoplasts with Cowpea chlorotic mottle virus (CCMV) and brome mosaic virus (BMV) have been studied. Fluorescent antibody staining showed that capsids of both viruses were made in doubly infected protoplasts but only infectious BMV could be recovered, i.e., BMV dominated in mixed infections and prevented synthesis of infectious CCMV. Healthy protoplasts could be infected after several hours in culture but the susceptibility to infection decreased with time in culture; the decrease was much faster with BMV than with CCMV, probably because the difference in electrical charge of the two viruses affects the method of entry into the protoplasts. If inoculation with the second virus was delayed there was an increasing resistance to superinfection (interference or cross-protection) so that when the delay between inoculations was more than 8 hr BMV was no longer able to infect protoplasts previously infected with CCMV and vice versa. No recombinants between BMV and CCMV were observed. Attempts to rescue a temperature-sensitive strain of CCMV with BMV under restrictive conditions (35 degrees ) were unsuccessful; dominance by BMV prevented synthesis of infectious CCMV RNA.