Two DYW subclass PPR proteins are Involved in RNA editing of ccmFc and atp9 transcripts in the moss physcomitrella patens: First complete set of PPR editing factors in plant mitochondria

The moss Physcomitrella patens has 11 RNA editing sites in mitochondrial transcripts. We previously identified six DYW subclass pentatricopeptide repeat (PPR) proteins as RNA editing factors for nine out of 11 sites. In this study, we identified two novel DYW subclass PPR proteins, PpPPR-65 and PpPPR-98, as RNA editing factors. Disruption of the PpPPR-65 gene resulted in a complete loss of RNA editing at two neighboring sites, ccmFc-C103 and ccmFc-C122, in the mitochondrial ccmFc transcript. To confirm this result, we further generated PpPPR-65 knockdown (KD) mutants by an inducible RNA interference (RNAi) system. The generated RNAi lines displayed reduced levels of RNA editing at both ccmFc-C103 and ccmFc-C122 sites. Next, we characterized the function of PpPPR-98 by constructing a KD mutant of PpPPR-98 expression. The KD mutant showed a 30% reduction in the level of atp9-C92 editing. When PpPPR-98 cDNA was introduced into the KD mutant, RNA editing levels were restored to the wild-type level. This indicates that PpPPR-98 is an editing factor for the atp9-C92 site. The recombinant PpPPR-98 protein bound to the upstream sequence of the editing site that was created by splicing of atp9 transcript. This suggests that atp9 RNA editing occurs after splicing of atp9 transcript. Our present and previous data provide the first evidence that all 11 known editing events require at least eight DYW subclass PPR proteins in the moss mitochondria.