Fig. 3. Complementation of the tom2-1 mutation by T-DNA clones 472 and 511 at the protoplast level. Time courses of the accumulation of tobamovirus-related RNAs in wild type (wt), B1-113, B1-234 and B1-113-derived transformants with the T-DNA clones 472 (A and C) or 511 (B and D) are shown. Protoplasts were isolated from T3 transgenic plants that carried the respective transgene at a single locus homozygously. Protoplasts were then inoculated with TMV-Cg (A and B) or TMV-L (C and D) virion RNA by electroporation. Total RNA was extracted at 2, 8, 16 or 24 h post-inoculation (h.p.i.) and analyzed by northern blot hybridization. A ³²P-labeled RNA probe synthesized by SP6 RNA polymerase from EcoRI-digested pCgP2 (Ishikawa et al., 1993) was used to detect positive-strand RNAs of TMV-Cg in (A) and (B). A ³²P -labeled RNA probe synthesized by T3 RNA polymerase from HindIII-digested pT732 (Ishikawa et al., 1991) was used to detect positive-strand RNAs of TMV-L in (C) and (D). Positions of the viral genomic RNA (G) and subgenomic mRNAs for MP and CP are indicated to the right of each panel. A band representing a cross-hybridization of the TMV-Cg probe to A.thaliana RNA is indicated by an asterisk. The quality of protoplasts in each preparation was confirmed by similar levels of accumulation of TCV-related RNAs (data not shown). A representative result of three independent repeats is shown. M, mock-inoculation; a, 100-fold dilution of RNA extracted at 24 h.p.i. from wild-type protoplasts inoculated with the respective virion RNA; b, 10-fold dilution of RNA extracted at 24 h.p.i. from wild-type protoplasts inoculated with the respective virion RNA. Note that the degree of viral RNA reduction in B1-113 protoplasts is weaker than that for CP shown in Figure 2, which was measured in systemically infected leaves. This is consistent with our previous observation (Ohshima et al., 1998) and may be due to differences in host defense functions between protoplasts and intact plants.