Cosuppresion-associated RNA silencing pathway that is different from RNA interference

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A tobacco endoplasmic reticulum ω-3 fatty acid desaturase (NtFAD3) catalyzes the formation of α-linolenic acid (18:3). Introduction of the NtFAD3 gene under the control of strong promoter into tobacco plants caused increase of 18:3 content. However, one of the transgenic lines, termed S44 line, showed a cosuppressed phenotype, namely drastic decrease of 18:3 content. In the S44 plants, the expression of endogenous NtFAD3 gene was inhibited by interference of splicing of pre-mRNA. This mis-splicing was observed in the sturctual gene sequence corresponding to the introns 6, 7 and 8. In fact, the splicing efficiencies of these introns are shown to decrease in the S44 plants by in vivo splicing assay. The NtFAD3 siRNAs were detected in the nuclear fraction prepared from the S44 leaves, implying the involvement of these siRNAs in the inhibition of splicing.When another copy of the NtFAD3 transgene was introduced into the S44 plants, two phenotypes were observed in the resultant offsprings; one is cosuppressed phenotype and the other is increase of 18:3 content in leaves. The 18:3 content of latter plants was higher than that of the wild type plants, indicating that the NtFAD3 transgene was successfully overexpressed (revertant phenotype). Since methylation-sensitive restriction enzymes failed to cut the corresponding recognition sites in the promoter region of the transgenes, transcriptional activity of the transgene should be declined in the S44 revertant plants. Then, methylation of cytosine residues in the promoter region of the transgene was induced by introduction of a hairpin construct that harbored the promoter sequences. When this hairpin construct was introduced into the S44 plants, the revertant phenotype was observed in the offsprings. These results indicated that cosuppression was induced by highly active transcription of the transgene.