Targeted Gene Silencing in Plants Using Rna Interference

1 1 Distribution of this document in any form violates the copyrights of the publisher. Cosuppression, post-transcriptional gene silencing (PTGS), and virus-induced gene silencing (VIGS) all describe homology-dependent gene silencing phenomena in plants that involve what is more broadly known as RNA interference (RNAi). The study of these phenomena in plants has provided numerous key discoveries in the field of RNAi research. These include initial evidence of homology-dependent gene silencing by transgenes [1-3] , the finding that mobile silencing signal(s) can spread systemically cell-to-cell [4,5] , the demonstration (coincident with studies in C. elegans) that double-stranded RNA triggers RNAi [6] , the discovery of small interfering RNAs (siRNAs) corresponding to eliminated mRNAs [7] , and the discovery of RNA-mediated DNA methylation [8,9]. Furthermore, the insight that developmental processes probably make use of RNAi-like mechanisms occurred when subunits of the RNAi machinery in Drosophila were found to correspond to the plant developmental genes ARG-In this chapter, we have focused on the design and use of vectors for targeted gene silencing in plants using RNAi, providing only a brief historical perspective of RNAi research in plants. Readers interested in a more extensive discussion of the plant literature can consult a number of recent reviews [14-18]. The phenomenon of homology-dependent gene silencing leapt onto the scientific stage through serendipity. Two seminal studies were those of Napoli and Jorgensen [2] and Van der Krol et al.[3], each attempting to make purple petu-nias an even darker purple by introducing a transgene designed to overproduce the chalcone synthase enzyme. Much to their surprise, over-expression of chal-cone synthase transgenes did not increase coloration. Instead, some flowers were completely colorless (white), and others showed interesting patterns such as loss of pigmentation along the veins but full pigmentation elsewhere. It was the complete loss of color in some or all flower cells that indicated a new phenomenon had been stumbled upon, for not only was the transgene silent in these cells but the endogenous copy of the chalcone synthase gene was silenced, as well. Napoli and Jorgensen thus introduced the term " cosuppres-sion " to describe the coordinate silencing of a transgene and its endogenous homolog(s). Other early studies yielded related findings, such as the unexpected gene silencing that occurred upon introduction of a transgene that shared sequence homology with a pre-existing transgene [1]. Transgenes are commonly introduced into the genomes of most model plant species using Agrobacterium tumefaciens, …