Identification of the fertility restoration locus, Rfo, in radish, as a member of the pentatricopeptide-repeat protein family

Abstract

Ogura cytoplasmic male sterility (CMS) in radish (Raphanus sativus) is caused by an aberrant mitochondrial gene, Orf138, that prevents the production of functional pollen without affecting female fertility. Rfo, a nuclear gene that restores male fertility, alters the expression of Orf138 at the post-transcriptional level. The Ogura CMS/Rfo two-component system is a useful model for investigating nuclear-cytoplasmic interactions, as well as the physiological basis of fertility restoration. Using a combination of positional cloning and microsynteny analysis of Arabidopsis thaliana and radish, we genetically and physically delimited the Rfo locus to a 15-kb DNA segment. Analysis of this segment shows that Rfo is a member of the pentatricopeptide repeat (PPR) family. In Arabidopsis, this family contains more than 450 members of unknown function, although most of them are predicted to be targeted to mitochondria and chloroplasts and are thought to have roles in organellar gene expression.

Figure 1

Genetic map of the Rfo locus in radish. The data are based on the screening of 800 PstI–MseI primer combinations for amplified fragment length polymorphism (AFLP) markers linked to the Rfo locus, and 900 segregant plants for recombination events in the pVR1–pVR8 region.

Figure 2

Analysis of microsynteny around the Rfo locus between radish and Arabidopsis. The genetic map of Rfo is based on the analysis of 6,907 segregant plants. The Arabidopsis physical map was deduced from the AGI database (http://arabidopsis.org). On the right, the physical localization of the markers in the Arabidopsis BAC (bacterial artificial chromosome) contig are shown, and the arrows localize them on the radish genetic map, which is shown on the left.

Figure 3

High-resolution genetic and physical maps of the Rfo locus. (A) Arabidopsis thaliana BAC (bacterial artificial chromosome) contigs that are syntenic to the Rfo locus in radish. The positions of the markers that are shown in (B) are indicated by black triangles. (B) High-resolution genetic map of the Rfo locus in radish. The black arrow indicates the Rfo locus. (C) Radish BAC contigs that span the Rfo locus. Broken lines indicate the positions of the markers from (B) in the radish BAC clones. The Rfo locus is physically delimited to BAC64.

Figure 4

Representation of the annotated BAC64 sequence. The prediction of genes and the identification of their functions were carried out using GENSCAN software and BLAST analysis against predicted Arabidopsis proteins, respectively. Horizontal arrows indicate the positions and orientations of the predicted genes. Vertical arrows indicate the positions of the markers that are tightly linked to Rfo. BAC, bacterial artificial chromosome.

Figure 5

Alignment of predicted protein sequences of PPR-A, PPR-B and PPR-C. The sequences of the 17 PPR repeats (amino acids 78–684 of PPR-B) are shown. The four amino-acid deletions in the third PPR repeats of both PPR-A and PPR-C are indicated by dashes. The gene encoding PPR-C contains a 17-bp deletion in repeat 6 that leads to a frameshift and a premature stop codon. The PPR-C sequence shown here, with a 30-amino-acid deletion in PPR repeats six and seven, is the hypothetical sequence obtained if this frameshift is spliced out in an intron predicted by GENSCAN. Asterisks indicate amino-acid identity; colons indicate a high level of amino-acid similarity; dots indicate low levels of amino-acid similarity. PPR, pentatricopeptide repeat.

Figure 6

Relationships between PPR-A, PPR-B, PPR-C, petunia RF and several PPR homologues from Arabidopsis, radish and maize. CRP1 (chloroplast processing 1) is a maize protein that is involved in plastid messenger RNA processing (Fisk et al., 1999). p67 is a radish chloroplast protein of unknown function (Lahmy et al., 2000). At5g42310 and At4g16390 are the closest Arabidopsis homologues of these two proteins. The distance tree was produced using ClustalW to align the sequences and using a neighbour-joining algorithm to group them. The Arabidopsis homologues were identified by BLASTP searches against predicted Arabidopsis proteins. The lengths of the lines connecting the proteins indicate the mean number of estimated substitutions per site (corrected for multiple substitutions). Scale bar, 0.1 substitutions per site. PPR, pentatricopeptide repeat.