Replication analysis of TGMV revertants. (A) Replication of TGMV A replicons encoding AL1 revertants was analyzed in tobacco protoplasts by agarose gel blot hybridization. Lanes 1 to 6 are transfections with TGMV A replicons with either wild-type (wt) (lanes 1 and 3) or mutant AL1 genes corresponding to L148M (lane 2), L148I (lane 4), C128W L148V (lane 5), and R125G L148V I155L (lane 6). The position of the double-stranded (ds) TGMV A DNA is marked on the left, and levels of replication relative to wild-type TGMV (100) at each exposure are indicated at the bottom of each lane. (B) N. benthamiana plants were cobombarded with wild-type or mutant TGMV A and wild-type TGMV B DNA. At 7 dpi, total DNA was isolated from three individual plants infected with TGMV B and either wild-type TGMV A (lanes 1 to 3) or mutant replicons carrying the L148M (lanes 4 to 6), L148I (lanes 7 to 9), C128W L148V (lanes 10 to 12), or R125G L148V I155L (lanes 13 to 15) mutations. DNA accumulation was monitored by agarose gel blot hybridization. The positions of single-stranded (ss) and double-stranded forms of TGMV A are marked on the left.