New Construct Approaches for Efficient Gene Silencing in Plants

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Abstract
An important component of conventional sense, antisense, and double-strand RNA-based gene silencing constructs is the transcriptional terminator. Here, we show that this regulatory element becomes obsolete when gene fragments are positioned between two oppositely oriented and functionally active promoters. The resulting convergent transcription triggers gene silencing that is at least as effective as unidirectional promoter-to-terminator transcription. In addition to short, variably sized, and nonpolyadenylated RNAs, terminator-free cassette produced rare, longer transcripts that reach into the flanking promoter. These read-through products did not influence the efficacy and expression levels of the neighboring hygromycin phosphotransferase gene. Replacement of gene fragments by promoter-derived sequences further increased the extent of gene silencing. This finding indicates that genomic DNA may be a more efficient target for gene silencing than gene transcripts.