Non-functional allele of RESTORER OF FERTILITY 4 is functional for the reduction of orf288 RNA in japonica rice

Cytoplasmic male sterility (CMS) is driven by the incompatibility between a mitochondrial gene and the nuclear RESTORER OF FERTILITY (Rf) gene. Previously, we identified orf288 as the cryptic CMS-causing gene in the mitochondrial genome of japonica rice (Oryza sativa) Taichung 65 (T65) through the analysis of CMS lines of O. glaberrima carrying the cytoplasm of japonica rice; orf288 RNA was greatly reduced in the presence of a T65-derived nuclear Rf gene, which was linked to a region harboring rf4 encoding a pentatricopeptide repeat (PPR) protein and other Rf-like PPR genes. In this study, we knocked out Rf-like PPR genes in T65 to identify the Rf-like PPR gene regulating the reduction of orf288 RNA and pollen fertility. Among the CRISPR-Cas9-mediated mutations in PPR461 (Os10g0495100), PPR782/rf4 (Os10g0495200), and PPR794 (Os10g0497300), mutations in PPR782/rf4 (Os10g0495200) allowed the expression of orf288; however, the orf288 RNA level was lower than that in the O. glaberrima CMS line and did not affect pollen fertility. Our results indicate that PPR782/rf4, a non-functional allele of the RF4 gene responsible for fertility restoration in wild-abortive (WA)-type CMS, is functional and suppresses the accumulation of orf288 RNA in the japonica rice.