A fertility restorer gene, Rf4, widely used for hybrid rice breeding encodes a pentatricopeptide repeat protein

Background 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. Findings 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. Conclusions 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. Electronic supplementary material The online version of this article (doi:10.1186/s12284-014-0028-z) contains supplementary material, which is available to authorized users.

Rf4.We also showed that the cloned Rf4 reduced the orf352-containing transcripts and restored pollen fertility.
To identify the Rf4 gene for WA-CMS, we performed map-based cloning of Rf4 using a cultivar IR24 (Additional file 1: "Methods"), because this cultivar is known to be a strong restorer line for WA-CMS (Jing et al. 2001).We delimited the Rf4 candidate region between two SSR markers, SSR1045 and AT801, that corresponded to a 213-kb region of Nipponbare genome (Additional file 2: Table S1).While we were conducting the fine mapping, Ngangkham et al. (2010) reported that Rf4 was located in a region between RM6737 and RM6100 with a distance of 104 kb in the Nipponbare genome, which further narrowed down the candidate region.We isolated a bacterial artificial chromosome (BAC) clones covering the corresponding region from IR24 genomic libraries (Figure 1) and determined their nucleotide sequences (Additional file 1: "Methods").Because the presence of Rf4 reduced the orf352-containing transcripts in the mitochondria, we identified the candidate gene according to the following two criteria, (i) it would encode a protein whose function is related to RNA metabolism, and (ii) it would encode a mitochondrial-targeting protein.The candidate genes that fulfilled the criteria were four new pentatricopeptide repeat (PPR)-encoding genes, PPR454, PPR782a, PPR782b, and PPR458 (Figure 1).These genes were named on the number of amino acids that they encoded.PPR proteins are, in general, known to be involved in RNA regulation in the mitochondria and plastids (Schmitz-Linneweber and Small 2008).We obtained genomic fragments containing PPR454, PPR782a, PPR782b, or PPR458 from the IR24 BAC clone (Additional file 1: "Methods"; Additional file 3: Figure S1).We introduced each genomic fragment into a WA-CMS line, WAA, which carries a Taichung 65 nuclear background, and obtained at least twelve transgenic lines (Additional file 4: Figure S2).
The anthers of WA-CMS are milky white, slender, and stunted (Additional file 4: Figure S2) and contain shrunken pollen grains lacking starch accumulation ability, because of which they are not stained with 1% potassium iodide (Figure 2a).In contrast, anthers of fertile Taichung 65 are yellow and engorged and contain darkly stained pollen grains.In four of the thirteen transgenic plants with PPR782a, restorations of anther and pollen development and starch accumulation in pollen grains were observed (Figure 2a; Additional file 4: Figure S2).The percentage of stainable pollen grains (pollen stainability) was 48% for plant Nos. 9, 10, and 12, and 79% for plant No. 13 (Figure 2b).Especially, one of the plants (No. 13) showed 47% seed setting rate (Figure 2c).Restorations of anther and pollen development were not observed in the remaining nine plants.Introduction of three other genomic fragments, which contained PPR782b, PPR454, or PPR458, did not recover the anther and pollen morphology (Additional file 4: Figure S2), and resulted in 0% of

PPR454
PPR782a PPR782b PPR458 RM6737 pollen stainability and no seed setting.These data indicate that Rf4 is the PPR782a gene.T 1 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. 2012).
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 2003;Akagi et al. 2004;Komori et al. 2004;Kazama et al. 2008).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. 2002) and radish (Brown et al. 2003;Desloire et al. 2003;Koizuka et al. 2003).
Expression of Rf4 was assessed using RT-PCR and gene-specific primers (Additional file 1: "Methods").Rf4 was expressed weakly in leaf blades.Expression was increased in the course of anther development, with the highest levels noted in the anthers at the tri-cellular pollen stage (Figure 3).Rf4 has been proposed to express highly in the tapetum, because the fertility restoration was sporophytically controlled.Our results of expression analysis indicated that Rf4 might play a gametophytic role in the maturation of pollen grains after tapetum degradation, as well as a sporophytic role.
In a recent report on the WA-CMS-associated mitochondrial gene, orf352 (WA352), RF4 was proposed to function post-transcriptionally, leading to the degradation of orf352-containing transcripts, half of which were co-transcribed with the upstream gene rpl5 encoding the ribosomal protein large subunit 5 (rpl5-orf352 transcripts; Figure 4a), whereas RF3 acted post-translationally and suppressed translation of orf352 (Luo et al. 2013).To determine whether the cloned Rf4 reduced rpl5-orf352 transcripts, we performed northern blot analysis by using RNA extracted from the leaf blades (Additional file 1: "Methods").In a WA-CMS plant, WAA, we detected two strong signals of 4.5-and 2.7-kb bands corresponding to the rpl5-orf352 and orf352 transcripts, respectively (Figure 4b), as reported (Luo et al. 2013).In a fertility restorer line, WAR, the signals of both the bands almost disappeared (Figure 4b).In four transgenic plants (Nos. 9,10,12,and 13) showing recovery of anther morphology and pollen stainability, the amount of the two transcripts decreased (lanes 9, 10, 12, and 13; Figure 4b).These four plants showed higher expression of the introduced PPR782a gene as detected by RT-PCR (Figure 4c).Northern blot analyses performed using cox1 and atp9 probes revealed that the introduction of PPR782a did not affect the accumulation of these transcripts (Additional file 7: Figure S5).These results suggested that Rf4 was involved in the reduction of orf352-containing transcripts.
RF4 is considered to recognize and bind orf352containing transcripts and promote the degradation of orf352-containing transcripts to avoid ORF352-mediated premature programmed cell death and consequent male sterility (Luo et al. 2013).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. (2014) 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 selfpollinated progeny.In contrast, our study used the genomic clones isolated from BAC libraries of IR24 and examined the segregation in the T 1 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. (2014).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. (2014), but further provides more information.

Accession codes
The nucleotide sequences of PPR454, PPR782a, PPR782b, and PPR458 have been deposited at the DDBJ under accession numbers [AB900791 to AB900794].

Figure 1 Figure 2
Figure 1 Candidate genes present in the IR24 BAC clone.Candidate genes and SSR markers in the IR24 BAC clone are indicated.

Figure 3 Figure 4
Figure3PPR782a is expressed in various tissues with the highest expression in mature anthers.RT-PCR analysis of PPR782a in anthers at the meiotic stage (Mei), uni-nucleate microspore stage (Uni), bi-cellular pollen stage (Bi), and tri-cellular pollen stage (Tri), and in the leaf blades.PCR results obtained using genomic DNA is also shown.