Cytoplasmic male sterility of rice with boro II cytoplasm is caused by a cytotoxic peptide and is restored by two related PPR motif genes via distinct modes of mRNA silencing

Abstract

Cytoplasmic male sterility (CMS) and nucleus-controlled fertility restoration are widespread plant reproductive features that provide useful tools to exploit heterosis in crops. However, the molecular mechanism underlying this kind of cytoplasmic-nuclear interaction remains unclear. Here, we show in rice (Oryza sativa) with Boro II cytoplasm that an abnormal mitochondrial open reading frame, orf79, is cotranscribed with a duplicated atp6 (B-atp6) gene and encodes a cytotoxic peptide. Expression of orf79 in CMS lines and transgenic rice plants caused gametophytic male sterility. Immunoblot analysis showed that the ORF79 protein accumulates specifically in microspores. Two fertility restorer genes, Rf1a and Rf1b, were identified at the classical locus Rf-1 as members of a multigene cluster that encode pentatricopeptide repeat proteins. RF1A and RF1B are both targeted to mitochondria and can restore male fertility by blocking ORF79 production via endonucleolytic cleavage (RF1A) or degradation (RF1B) of dicistronic B-atp6/orf79 mRNA. In the presence of both restorers, RF1A was epistatic over RF1B in the mRNA processing. We have also shown that RF1A plays an additional role in promoting the editing of atp6 mRNAs, independent of its cleavage function.

Figure 1.

Characterization of the CMS-Associated Gene in Rice with BT-Cytoplasm. (A) The structures of N-atp6 and B-atp6/orf79 transcripts, the sequence downstream of B-atp6, and the ORF79 peptide sequence. The primers P1, P2, and P3 were used for RT-PCR to examine the editing of N-atp6 and B-atp6 mRNAs. To determine the 5′ and 3′ ends of the primary (N-atp6) and processed RNA fragments containing B-atp6 or orf79 by a CR-RT-PCR method (Kuhn and Binder, 2002) (see Figure 7B), the primers P4 and P8 were used for the reverse transcription of the circularly ligated RNAs, and the reverse-directed primer pairs P5/P6, P7/P6, and P9/P10 for the PCR. The 5′ and 3′ termini of the processed B-atp6 and orf79 RNA fragments (∼1 and 0.45 kb) are indicated by vertical arrows and open triangles, respectively. The primary 5′ and 3′ termini of N-atp6 and B-atp6/orf79 mRNAs and their different downstream sequences (starting from the bent arrow) have been described (Iwabuchi et al., 1993). The nucleotides identical to cox1 and the encoding amino acids are underlined. The dotted underline indicates a segment identical to the 5′ UTR of a predicted mitochondrial gene orf91 (Itadani et al., 1994). Rectangles indicate the deleted nucleotides in fragment II and the amino acids. (B) and (C) Effect of orf79 expression with fragments I and II (Figure 1A), indicated by I and II, respectively, on the growth of E. coli cells on an agar plate (B) and in liquid cultures (C) with or without isopropylthio-β-d-galactoside (IPTG). In the liquid cultures (C), IPTG was added when the cell growth reached OD₅₅₀ = 0.6. (D) The expressed recombinant ORF79 protein (arrowed) with fragment II.

Figure 2.

Functional Test of orf79 in Male Sterility. (A) The binary construct for expression of recombinant orf79 in rice. P_35S, the CaMV35S promoter; Rf1b-5′, the 5′ segment of Rf1b encoding mitochondrion transit signal. (B) to (D) Pollen grains of a normal fertile rice line (B) and orf79 transgenic T0 rice plants with single (C) or two (D) T-DNA insertions. The darkly stained pollens were fertile and the lightly stained were sterile. Bars = 50 μm. (E) Cosegregation of the orf79 transgene and the semi-male-sterility (s) in a T1 family with single T-DNA insertion as assayed by RNA gel blot analysis of young panicle RNA using orf79 as a probe. f, full male fertility.

Figure 3.

Mapping and Cloning of the Rf Genes and Characterization of the PPR Subfamily. (A) Molecular mapping of an Rf gene (Rf1b) for the Rf-1 locus using an F2 population generated from a cross between a CMS line 731A and a restorer line C9083, in which Rf1b is functional. The numbers of recombinants between the markers and Rf1b are shown. (B) Physical maps based on BACs (Rice Chromosome 10 Sequencing Consortium, 2003) and TACs from a restorer line MH63 that contains two functional Rf genes (Rf1a and Rf1b). Rf1b was located to a 37-kb region by the molecular mapping. The pCAMBIA1300-based subclones M4047 and M1521 were able to restore the male fertility of a CMS-BT line by transformation. (C) The PPR gene cluster consisting of Rf1a and Rf1b and their homologs. The ORFs RRR791, PPR762, and PPR794 of a restorer line IR24 (Komori et al., 2004) are allelic to ORFs ^#3, ^#4, and ^#6 of MH63, respectively. In the ORF^#1 and ^#3-^#6 regions, the locations of the PPR ORFs in the genome of a japonica cultivar Nipponbare are the same as MH63. ORFs with predicted or experimentally confirmed mitochondrion transit signals are marked by an asterisk. (D) DNA gel blot analysis of HindIII-digested genomic DNA of rice (MH63) and other plant species using Rf1b as a probe. The fragments of 6.7, 4.8, and 1.3 kb correspond to fragments containing whole or parts of the ORFs ^#1, ^#6, and ^#3/^#4 (overlapped), respectively, according to the sequence of M-L19 and M-L10. (E) Phylogenetic analysis of the PPR protein subfamily. ORF^#10 is a gene located on chromosome 8. Numbers below the branches indicate the bootstrap proportions (%) for maximum parsimony (above) and maximum likelihood (below) analyses.

Figure 4.

Products of the Rf Genes Are Directed to Mitochondria. Green fluorescence spots in bombarded onion epidermal cells show the expression of GFP fused with the N-terminal sequences of ORF^#1 (A) or ORF^#3 (B) containing putative mitochondrion transit signals. The images observed by confocal scanning microscopy show parts of single cells. The GFP fusion proteins are targeted to the mitochondria, according to the colocalization of GFP images with those stained with mitochondrion-specific dye (MitoTracker Red) in the same cells. Bars = 18 μm.

Figure 5.

Functional Complementation Test of the Rf Gene Candidates. (A) Completely sterile pollen of a CMS-BT line KFA. (B) Fertility-restored pollen in dark staining (∼50%) of an ORF^#1 transgenic T0 plant of KFA. Bars in (A) and (B) = 50 μm. (C) and (D) Panicles of the transgenic T0 plants with ORF^#1 (Rf1b) and ORF^#3 (Rf1a) transgenes, respectively, showing restored spikelet fertility.

Figure 6.

The Rice Restorer Proteins and PPR Consensus Sequences of the Rice PPR Subfamily. (A) Alignment of RF1A and RF1B sequences from MH63. The PPR repeats are inside the rectangle, of which the second and third are PPR-like L motif. The functional alteration of Asn⁴¹²-to-Ser between proteins encoded by Rf1b and rf1b is shown. The underlined sequences correspond to mitochondrion transit signals predicted with probabilities of 0.95 and 0.97, respectively. (B) PPR consensus sequences for RF1A, RF1B, and all the members of the rice PPR subfamily.

Figure 7.

Processing of B-atp6/orf79 mRNA and Suppression of the ORF79 Production by the Rf Genes. (A) Transcript profile of B-atp6/orf79 in transgenic plants with Rf1a assayed by RNA gel blot analysis. The relatively low signal intensity of the 1.4- and 1.0-kb bands was due to the relatively smaller portion of the DNA fragment III probe (see Figure 1A) hybridizing to atp6. The 1.4-kb band (N-atp6) also served as a loading control. ML, maintainer line. (B) CR-RT-PCR analysis of the 5′ and 3′ termini of the primary N-atp6 transcript from a maintainer line (lanes 1 and 2), the processed B-atp6 (lanes 3 and 4), and orf79 (lanes 5 and 6) mRNA fragments from an Rf1a transgenic plant using the primer pairs P5/P6, P7/P6, and P9/P10, respectively (see Figure 1A). The RNA samples were treated with (lanes 1, 3, and 5) and without (lanes 2, 4, and 6) TAP before the RNA ligation reaction. M, molecular weight marker. (C) Transcript profile of B-atp6/orf79 in transgenic and fertility-restored hybrid plants with Rf1b. (D) Transcript profile of B-atp6/orf79 in fertility-restored hybrid plants with Rf1a alone or both of Rf1a and Rf1b. (E) Immunoblot analysis probed with an antibody to ORF79. Proteins were prepared from anthers containing microspores of a CMS-BT line (lanes 3 and 4), fertility-restored transgenic plants with Rf1a (lane 1) or Rf1b (lane 2), fertility-restored hybrid plants carrying Rf1a and Rf1b (lanes 5 and 6), and a maintainer line (lane 7). The specific band (lanes 3 and 4) for ORF79 is indicated with an arrow, and the top one marked with an asterisk is a cross-reacting unknown protein. (F) Immunoblot analysis of ORF79 using proteins prepared from microspores of fertility-restored transgenic plants with Rf1a (lane 1), mitochondria of seedling leaves of the CMS-BT line (lane 2), microspores of the CMS-BT line (lanes 3 and 4), and anther wall tissue not including the microspores (lanes 5 and 6). The cross-reacting unknown protein, as well as ORF79, was not detected in the mitochondria of the seedling leaves of the CMS-BT line (lane 2).