AbstractMitochondrial genes encode key components of the cellular energy machinery, but their genetic analysis is difficult or impossible in most organisms (including plants) because of the lack of viable transformation approaches. We report here a method to block the expression of the mitochondrial nad6 gene encoding a subunit of respiratory complex I in Arabidopsis thaliana, via the modification of the specificity of the RNA-binding protein RNA PROCESSING FACTOR 2 (RPF2). We show that the modified RPF2 binds and specifically induces cleavage of nad6 RNA, almost eliminating expression of the Nad6 protein and consequently complex I accumulation and activity. To our knowledge, this is the first example of a targeted block in expression of a specific mitochondrial transcript by a custom-designed RNA-binding protein. This opens the path to reverse genetics studies on mitochondrial gene functions and leads to potential applications in agriculture.