A fertility restorer gene, Rf4, widely used for hybrid rice breeding encodes a pentatricopeptide repeat protein
© Kazama and Toriyama; licensee Springer. 2014
Received: 16 June 2014
Accepted: 26 September 2014
Published: 1 November 2014
Uncontrolled expression of a certain mitochondrial gene often causes cytoplasmic male sterility (CMS) in plants. This phenotype is prevented by the presence of a fertility restorer (Rf) gene in the nuclear genome. Such CMS/Rf systems have been successfully used for breedings of F1 hybrid cultivars. In rice, approximately 99% of F1 hybrid cultivars have been developed using a wild abortive type of CMS (WA-CMS) and its Rf genes. Recently, a newly identified mitochondrial gene, orf352, was reported as a WA-CMS-causing gene.
We cloned and functionally characterized Rf4, a major Rf gene for WA-CMS. We revealed that Rf4 encoded a pentatricopeptide repeat-containing protein and reduced the orf352-containing transcripts, thereby restoring pollen fertility.
Through a map-based cloning, we have independently identified an allele of a recently reported Rf4 gene and demonstrated that the fertility restoration is controlled sporophytically.
KeywordsCytoplasmic male sterility Fertility restorer Rice
A wild abortive (WA)-type CMS has been almost exclusively used for breeding three-line hybrid rice and contributes to 10% of the total rice cultivated area worldwide (Li et al. ; Barclay ). Because of the great impact of WA-type CMS on agriculture, many studies have attempted to elucidate the CMS-causing gene in WA mitochondria and to determine the fertility restorer genes for WA-CMS. In 2013, a mitochondrial orf352 (WA352) gene that confers WA-CMS was discovered; this gene encodes 352-amino-acids protein (Luo et al. ). We also found a sequence variant of orf352 in an RT102 CMS line derived from O. rufipogon (Okazaki et al. 2013). However, the Rf genes in nuclear genome have not yet been cloned, although two major Rf genes, Rf3 and Rf4, have been mapped on the chromosomes 1 and 10, respectively (Lu et al. ; Yao et al. ; Tan et al. ; Jing et al. ; Ahmadikhah and Karlov ; Ngangkham et al. ; Suresh et al. ). In this study, we report the cloning of Rf4. We also showed that the cloned Rf4 reduced the orf352-containing transcripts and restored pollen fertility.
T1 seeds were set by self-pollination. The progeny segregated into 8 fertile plants, all of which carried the introduced gene, and 7 male-sterile plants, all of which did not carry the introduced gene (Additional file 5: Figure S3). The appearance of null segregants indicated that PPR782a controlled the fertility restoration sporophytically. The pollen fertility was not completely restored by the introduced Rf4 gene. Other fertility restorer genes, such as the Rf3 gene on chromosome 1, might be necessary for fully restoration of WA-CMS (Suresh et al. ).
The predicted amino acid sequence of PPR782A carries 18 repeats of PPR motif (Additional file 6: Figure S4) and is highly similar to RF1A for BT-CMS showing 86% identity (Kazama and Toriyama ; Akagi et al. ; Komori et al. ; Kazama et al. ). Amino acid identity between PPR782A and PPR782B was 94%, with a completely identical region in the N- and C-terminal ends. A non-restorer line, Nipponbare, contained a putative allele of PPR782a, which is encoded by the Rice Annotation Project (RAP) locus ID Os10g0495200. The amino acid sequence of PPR782A_Nipponbare shows 95% identity to that of PPR782A_IR24 (Additional file 6: Figure S4). Some amino acid substitutions might be crucial for the function of the PPR protein, as was reported for other PPR-type Rf genes in petunia (Bentolila et al. ) and radish (Brown et al. ; Desloire et al. ; Koizuka et al. ).
RF4 is considered to recognize and bind orf352-containing transcripts and promote the degradation of orf352-containing transcripts to avoid ORF352-mediated premature programmed cell death and consequent male sterility (Luo et al. ). Finding a nuclear Rf factor provide novel insights into reconciliation between mitochondria and nuclei in agronomically important crops, and has practical implications for production of hybrid rice.
During preparation of this manuscript, Tang et al. () have reported a short letter entitled as "The rice restorer Rf4 for wild-abortive cytoplasmic male sterility encodes a mitochondrial-localized PPR protein that functions in reduction of WA352 transcripts". Their study turned to be essentially the same to ours. They used a PCR-amplified genomic clone of cv. Minghui 63 for a complementation test, but did not investigate the self-pollinated progeny. In contrast, our study used the genomic clones isolated from BAC libraries of IR24 and examined the segregation in the T1 plants, showing that the fertility restoration was controlled sporophytically (Additional file 5: Figure S3). The nucleotide sequence of the RF4 allele of IR24 in our study is completely identical to that presented in Supplementary Figure of Tang et al. (). An adjacent gene, PPR782b, identified in our study has not been reported in their study. Thus our study is not just a confirmation of Tang et al. (), but further provides more information.
The nucleotide sequences of PPR454, PPR782a, PPR782b, and PPR458 have been deposited at the DDBJ under accession numbers [AB900791 to AB900794].
TK performed the experiments and drafted the manuscript. KT designed and supervised the study and revised the manuscript. Both authors read and approved the final draft of the manuscript.
Bacterial artificial chromosome
Cytoplasmic male sterility
Reverse-transcriptase-polymerase chain reaction
Simple sequence repeat
This work was supported by MEXT/JSPS KAKENHI Grant Numbers 2338002, 24117502 and 26292002, and by Science and technology research promotion program for agriculture, forestry, fisheries and food industry (No. 26010A) and a grant from the Ministry of Agriculture, Forestry and Fisheries of Japan (Genomics-based Technology for Agricultural Improvement, QTL-4008). We thank Dr. Masahiro Yano (National Institute of Agrobiological Sciences, Tsukuba, Japan) for providing BAC clones. The RIL accessions used in this study were obtained from the National Institute of Genetics supported by the National BioResource Project, MEXT, Japan.
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