Fig. 2. Identification of TOM2A and TOM2B by functional complementation. (A) T-DNA contig covering the deletion detected in the tom2-1 mutant. The arrows above chromosome 1 show the regions determined to be deleted or translocated. Lines under the chromosome show the position of the RDA fragment and the Y2 and Y5 probes appearing in Figure 1. T-DNA clones shown as gray boxes with plus (+) signs complemented the tom2-1 mutation, while those shown as open boxes with minus (–) signs did not. (B) Position, orientation and intron–exon structures of TOM2A and TOM2B. Coding and non-coding regions in exons are shown as filled and open boxes respectively, whereas introns are marked with diagonal lines. (C and D) Complementation of the tom2-1 mutation with the T-DNA clones 472 (C) and 511 (D). Each T-DNA clone was used to genetically transform B1-113 plants. T2 plants derived from each single T1 plant were inoculated with TMV-Cg or TMV-L. At 11 (for TMV-Cg) and 21 days (for TMV-L) post-inoculation, upper uninoculated leaves of inoculated plants were harvested and CP accumulation was examined by SDS–PAGE and Coomassie Blue staining (panels marked Cg CP or L CP). The positions of TMV-Cg and TMV-L CP are indicated. A representative result from one of the T1 lines for each construct is shown in each set of panels, whereas lanes labeled i–vi represent single individual T2 plants. Genomic DNA was also prepared from each T2 plant and a transgene-specific DNA fragment was amplified by PCR, separated by agarose gel electrophoresis and stained by ethidium bromide (panels marked T-DNA). As T1 plants should carry transgenes heterozygously, transgene segregation was observed in the T2 generation. Therefore most T2 plants carry transgenes but some do not. Note that complementation occurs only if the T2 plants carry the transgenes.