This study extends biochemical analysis of RNA silencing to plants, demonstrating that wheat germ extracts contain Dicer-like enzymes that convert double-stranded RNA (dsRNA) into two classes of small interfering RNAs (siRNAs) and an RNA-dependent RNA polymerase (RdRP) activity that can synthesize dsRNA from single-stranded RNA. The endogenous microRNA (miRNA) miR165/166, which lacks perfect complementarity to its targets, acts as a siRNA in plant extracts, guiding an endonuclease to efficiently cleave wild-type *Arabidopsis PHAVOLUTA* mRNA but not a dominant mutant. This finding supports the view that plant miRNAs direct RNAi and that miRNA-mediated mRNA destruction is crucial for proper plant development. The study also reveals that different Dicer-like enzymes produce each class of siRNA, and that the RdRP activity in wheat germ extracts can synthesize dsRNA without a primer, preferentially converting it into the longer siRNA class. These findings provide insights into the biochemical mechanisms of RNA silencing in plants.This study extends biochemical analysis of RNA silencing to plants, demonstrating that wheat germ extracts contain Dicer-like enzymes that convert double-stranded RNA (dsRNA) into two classes of small interfering RNAs (siRNAs) and an RNA-dependent RNA polymerase (RdRP) activity that can synthesize dsRNA from single-stranded RNA. The endogenous microRNA (miRNA) miR165/166, which lacks perfect complementarity to its targets, acts as a siRNA in plant extracts, guiding an endonuclease to efficiently cleave wild-type *Arabidopsis PHAVOLUTA* mRNA but not a dominant mutant. This finding supports the view that plant miRNAs direct RNAi and that miRNA-mediated mRNA destruction is crucial for proper plant development. The study also reveals that different Dicer-like enzymes produce each class of siRNA, and that the RdRP activity in wheat germ extracts can synthesize dsRNA without a primer, preferentially converting it into the longer siRNA class. These findings provide insights into the biochemical mechanisms of RNA silencing in plants.