FIGURE 4.

Compensatory mutational analysis of SL5. (A) Depiction of SL5 in mutants. Nucleotide substitutions are in boldface. (B,C) Northern blot analysis of protoplast infections. Cucumber protoplasts were inoculated with H₂O (mock), T100 (3 μg), or T100 and wild-type or mutant DI RNAs (1 μg) as indicated above the lanes. Total nucleic acids were isolated 24 h postinoculation, separated in 8M urea-4.5% polyacrylamide gels, transferred to nylon membranes, and probed with a ³²P-end-labeled minus-sensed probe. Bands corresponding to TBSV genomic RNA (g), subgenomic mRNA1 (sg1), subgenomic mRNA2 (sg2), and DI RNA (DI) are indicated. The accumulation level of wild-type DI-72SXP was set at 100%, and mutant levels were normalized to this value. Relative DI RNA accumulation levels (with standard errors) are presented as histograms. The asterisk indicates the position of DI RNA head-to-tail dimers (Ray and White 1999, 2003).