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In plants, post-transcriptional gene silencing (PTGS) is mediated by small RNAs that guide the RNA-induced silencing complex (RISC) to target transcripts for cleavage or translational repression. PTGS can be classified into an RNA-dependent RNA polymerase-dependent and an RNA-dependent RNA polymerase-independent pathway. To date, only a small number of endogenous genes have been identified that are silenced in both pathways. We have conducted a genome-wide survey of RNA silencing in Arabidopsis by examining gene-specific and transgene-derived small RNAs and the small RNA-associated Argonaute proteins. We found that hundreds of endogenous genes are silenced by either RNA-dependent RNA polymerase-dependent or RNA-dependent RNA polymerase-independent pathways, with no single gene silenced by both pathways. No small RNA was detected that guides the silencing of either of these pathways, which may explain why genes silenced by RNA-dependent RNA polymerase-independent pathways do not have a requirement for a functional RNA-dependent RNA polymerase (RDR). In contrast, almost half of the transgenic lines examined produced small RNAs that were able to guide the silencing of at least one endogenous gene. Transgenic plants with mutations in the putative RDR gene, RDR2, or the putative RDR6-like gene, RDR6L, show an altered silencing phenotype, indicating that the PTGS of the testis-expressed gene is not RDR2- or RDR6L-dependent. Furthermore, certain AGOs were associated with different classes of small RNAs, suggesting that these Argonautes might have a role in the different silencing pathways.Düsseldorf-Heerdt Düsseldorf-Heerdt is a municipality in the district of Düsseldorf in the state of North Rhine-Westphalia, Germany. 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