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RNA interference, or RNAi, is arguably one of the most significant discoveries in biology in the last several decades. First recognized in plants (where it was called post-transcriptional gene silencing, PTGS) RNAi is a gene down-regulation mechanism since demonstrated to exist in all eukaryotes. In RNAi, small RNAs (of about 21–24 nucleotides) function to guide specific effector proteins (members of the Argonaute protein family) to a target nucleotide sequence by complementary base pairing. The effector protein complex then down-regulates the expression of the targeted RNA or
DNA
. Small RNA-directed gene regulation systems were independently discovered (and named) in plants, fungi, worms, flies, and mammalian cells. Collectively, PTGS, RNA silencing, and co-suppression (in plants); quelling (in fungi and algae); and RNAi (in
Caenorhabditis elegans
,
Drosophila
, and mammalian cells) are all examples of small RNA-based gene regulation systems. From the very beginning, plant research has had a major impact on our understanding of RNAi. The purpose of this chapter is to provide an historical perspective and overview on the discovery, characterization, and applications of RNAi in plants.