Methods of double-stranded RNA-mediated gene inactivation in Arabidopsis and their use to define an essential gene in methionine biosynthesis

Controlled down-regulation of endogenous plant gene expression is a useful tool, but antisense and sense silencing lack predictability. Recent studies show that expression of both antisense and sense RNA together is an effect ive means of inactivating reporter and viral genes in plants. We created tr ansgenic plants expressing antisense and sense RNA together in a single 'do uble-stranded RNA' (dsRNA) transcript. This approach shows great promise as a highly effective means for reducing gene function. With this approach, w e demonstrated that the Arabidopsis cystathionine beta -lyase gene, which e ncodes a methionine biosynthetic enzyme, is essential for viability. Inacti vation of this gene was rescued by the addition of methionine to the growth medium. Compared to antisense and sense constructs, the dsRNA construct sh owed a much more consistent and complete suppression of gene activity. Addi tionally, expression of a transcript with a spacer sequence containing an u nrelated gene between antisense and sense luciferase gene fragments led to stronger inactivation of a second luciferase transgene than did constructs with a minimal spacing between sense and antisense fragments. However, the gene in the spacer region was neither functionally expressed nor functional in silencing a second, unlinked homologous transgene.