RNA interference (RNAi) is a gene regulatory mechanism that limits transcript levels by either suppressing transcription (transcriptional gene silencing [TGS]) or activating sequence-specific RNA degradation (posttranscriptional gene silencing [PTGS]/RNAi). PTGS is a rapidly expanding field, while TGS is still emerging. RNAi has been observed in various eukaryotic organisms, including plants, fungi, animals, and insects. Three distinct forms of RNAi—cosuppression in plants, quelling in fungi, and RNAi in animals—have been identified. Recent studies have revealed additional facets of naturally occurring RNAi, such as micro-RNA formation and heterochromatinization. During RNAi/PTGS, double-stranded RNA (dsRNA) molecules cleave inducer molecules into smaller pieces, eventually destroying cellular or viral mRNA. Target mRNAs cannot accumulate in the cytosol, though they remain detectable by nuclear run-on assays. In some cases, DNA expressing the target mRNA undergoes methylation as a by-product of the degradation process. The natural functions of RNAi and related processes seem to be protection of the genome against invasion by mobile genetic elements such as viruses and transposons, as well as the orchestrated functioning of developmental programs in eukaryotic organisms. RNAi has been observed in plants, fungi, and animals, with the process involving dsRNA as an inducer. In plants, the loss of cytosolic mRNA was not associated with reduced transcription, as demonstrated by run-on transcription tests. This phenomenon, called cosuppression, was originally observed in plants but has also been demonstrated in metazoans and mammals. In plants, the expression of transgenes led to the formation of dsRNA, which initiated PTGS. For example, in cosuppressed petunia plants, *chsA* mRNA formed a partial duplex due to self-complementarity between the 3' coding region and 3' untranslated region. This was revealed by DNA sequence analysis and experimental detection of in vitro-transcribed, RNase-resistant duplex *chsA* RNA. In an independent study, a p35S-ACC sense transgene with an inverted repeat in the 5' untranslated region was introduced into tomato to test the role of dsRNA as an inducer of PTGS. Cosuppression of the endogenous *acc* gene occurred at a higher frequency in these plants than in those harboring only the p35S-ACC sense transgene. The loss in steady-state accumulation of the target mRNA is almost total if the transgene produces the nuclear transcript in the duplex conformation. Recent studies have shown that the expression of self-cRNA of plum pox virus under the control of *rolC* promoter caused degradation of transgenic viral RNA and systemic disease resistance to challenge inoculum of plum pox virus in transgenic *Nicotiana benthamiana*. This evidence points out thatRNA interference (RNAi) is a gene regulatory mechanism that limits transcript levels by either suppressing transcription (transcriptional gene silencing [TGS]) or activating sequence-specific RNA degradation (posttranscriptional gene silencing [PTGS]/RNAi). PTGS is a rapidly expanding field, while TGS is still emerging. RNAi has been observed in various eukaryotic organisms, including plants, fungi, animals, and insects. Three distinct forms of RNAi—cosuppression in plants, quelling in fungi, and RNAi in animals—have been identified. Recent studies have revealed additional facets of naturally occurring RNAi, such as micro-RNA formation and heterochromatinization. During RNAi/PTGS, double-stranded RNA (dsRNA) molecules cleave inducer molecules into smaller pieces, eventually destroying cellular or viral mRNA. Target mRNAs cannot accumulate in the cytosol, though they remain detectable by nuclear run-on assays. In some cases, DNA expressing the target mRNA undergoes methylation as a by-product of the degradation process. The natural functions of RNAi and related processes seem to be protection of the genome against invasion by mobile genetic elements such as viruses and transposons, as well as the orchestrated functioning of developmental programs in eukaryotic organisms. RNAi has been observed in plants, fungi, and animals, with the process involving dsRNA as an inducer. In plants, the loss of cytosolic mRNA was not associated with reduced transcription, as demonstrated by run-on transcription tests. This phenomenon, called cosuppression, was originally observed in plants but has also been demonstrated in metazoans and mammals. In plants, the expression of transgenes led to the formation of dsRNA, which initiated PTGS. For example, in cosuppressed petunia plants, *chsA* mRNA formed a partial duplex due to self-complementarity between the 3' coding region and 3' untranslated region. This was revealed by DNA sequence analysis and experimental detection of in vitro-transcribed, RNase-resistant duplex *chsA* RNA. In an independent study, a p35S-ACC sense transgene with an inverted repeat in the 5' untranslated region was introduced into tomato to test the role of dsRNA as an inducer of PTGS. Cosuppression of the endogenous *acc* gene occurred at a higher frequency in these plants than in those harboring only the p35S-ACC sense transgene. The loss in steady-state accumulation of the target mRNA is almost total if the transgene produces the nuclear transcript in the duplex conformation. Recent studies have shown that the expression of self-cRNA of plum pox virus under the control of *rolC* promoter caused degradation of transgenic viral RNA and systemic disease resistance to challenge inoculum of plum pox virus in transgenic *Nicotiana benthamiana*. This evidence points out that