ABSTRACT
PTGS was originally reported in transgenic plants as an undesired outcome occurring when researchers attempted to express to a high level the product of a sense transgene conferring a desirable trait. In several cases, plants expressing the introduced transgene could not be obtained, and, instead, only plants that lacked both the expression of the transgene and any gene sharing homology to the transgene were identifi ed (Napoli et al. 1990, Smith et al. 1990, van der Krol et al. 1990). This observation puzzled researchers, but subsequent investigations led to the identifi cation of double-stranded RNA (dsRNA) as the sequence-specifi c molecule inducing RNA silencing (Fire et al. 1998, Waterhouse et al. 1998). Following this discovery, several techniques leading to the direct and effi cient production of dsRNA after transgene transcription, such as the expression of an inverted-repeat (IR) transgene, were developed to trigger silencing of a desired gene. In contrast to IR transgenes, the way by which sense transgenes produce dsRNA is not well understood, but unlike IR transgenes, sense transgenes require the activity of an RNA dependent RNA polymerase to induce silencing (see below). Based largely on this fundamental mechanistic difference, PTGS triggered by sense transgenes was renamed S-PTGS to distinguish it from IR-PTGS (Beclin et al. 2002). Both sense and invertedrepeat transgene-induced PTGS are powerful systems for identifying PTGS components through genetic screens. Moreover, the identifi cation of small RNA as effectors of RNA silencing (Hamilton et al. 1999) and the discovery that several classes of endogenous small RNA exists have blurred the lines between the mechanistic differences among the various PTGS pathways and, thus, have necessitated precise methods to defi ne the components of each small RNA pathway. Both forward and reverse genetic screens have had major roles in elucidating the protein requirements of these diverse small RNA pathways.
