A pentatricopeptide repeat-containing gene restores fertility to cytoplasmic male-sterile plants

Abstract

Known in over 150 species, cytoplasmic male sterility is encoded by aberrant mitochondrial genes that prevent pollen development. The RNA- or protein-level expression of most of the mitochondrial genes encoding cytoplasmic male sterility is altered in the presence of one or more nuclear genes called restorers of fertility that suppress the male-sterile phenotype. Cytoplasmic male sterility/restorer systems have been proven to be an invaluable tool in the production of hybrid seeds. Despite their importance for both the production of major crops such as rice and sunflower and the study of organelle/nuclear interactions in plants, none of the nuclear fertility-restorer genes that reduce the expression of aberrant mitochondrial proteins have previously been cloned. Here we report the isolation of a gene directly involved in the control of the expression of a cytoplasmic male sterility-encoding gene. The Petunia restorer of fertility gene product is a mitochondrially targeted protein that is almost entirely composed of 14 repeats of the 35-aa pentatricopeptide repeat motif. In a nonrestoring genotype we identified a homologous gene that exhibits a deletion in the promoter region and is expressed in roots but not in floral buds.

Fig 1.

The Rf locus contains two tandem mitochondrially targeted PPR motif genes. (A) Genomic organization of the region containing the Rf-PPR592 and Rf-PPR591 genes. Duplicated blocks are indicated by similar colors. Arrows indicate the direction of transcription. 1 and 2 show locations of the primers used to amplify the rf-PPR592 gene from a CMS plant. (B) A single onion epidermal cell expressing a known mitochondrially targeted GFP after DNA bombardment. (C) A single onion epidermal cell transiently expressing 44 N-terminal amino acids of Rf-PPR592 fused to GFP. (D) Comparison of PPR motifs found in Rf-PPR592 with the MEME-derived consensus from 1,303 PPR motifs. The 14 PPR repeats are sorted by decreasing statistical significance, with PPR 230–264 showing the highest match to the consensus motif that is generated by retaining only the amino acids that occur at least in 6 of the 14 repeats. The color code is taken from the MEME output.

Fig 2.

Genetic structure of the rf-PPR592 gene. (A) Comparison of Rf-PPR592 and rf-PPR592 reveals a size polymorphism. The first lane was loaded with the Rf-PPR592 PCR amplicon obtained from a restorer line (Rf/Rf), the adjacent lane was loaded with the rf-PPR592 PCR amplicon obtained with the same primer pair from a CMS line (rf/rf). (B) Comparison of Rf-PPR592, Rf-PPR591, and rf-PPR592 reveals five similarity blocks. For each block, (I to V), the two blocks that exhibit the greatest similarity are shown with the same shading. Overall all three sequences are greater than 90% identical at the nucleotide level except in block V, where Rf-PPR591 exhibits only 23% identity to the other two genes. The locations of 47- and 49-nt deletions in Rf-PPR591 and 47- and 530-nt deletions in rf-PPR592 with respect to the Rf-PPR592 sequence in blocks I and II are shown as lines.

Fig 3.

Expression pattern of rf-PPR592 and Rf-PPR592. (A) Examination of floral bud RNA for expression of rf-PPR592 and Rf-PPR592. RT-PCR of floral bud RNA of a CMS plant (S) with primers specific to rf-PPR592, and RT-PCR of floral bud RNA of an RfRf (nontransgenic) fertile plant with primers specific for Rf-PPR592 (R). DNA, positive control for the amplification where the substrate is leaf DNA from a CMS plant; M, mass markers; 0, no template added, negative control. (B) Examination of different tissues for expression of rf-PPR592. RT-PCR of RNA from different tissues of a CMS plant with primers specific to rf-PPR592. DNA, M, and 0 same as in A.

Fig 4.

Restoration of fertility to CMS Petunia lines by transformation with a 4.6-kb genomic sequence carrying Rf-PPR592. (A) Flower of P. parodii CMS line 3688. (B) Regenerant carrying Rf-PPR592. (C) P. hybrida CMS line 2423. (D) Regenerant carrying Rf-PPR592.

Fig 5.

Cosegregation of the Rf-PPR592 transgene, restoration of fertility, and reduction of PCF. (A) DNA blot hybridized with an npt II transgene-specific probe. Lane 1, P. parodii CMS line 3688; lanes 2-and 3, sterile T₁ progeny of transformed P. parodii; lanes 4–9, fertile T₁ progeny. (B) Immunoblot of floral bud proteins probed with anti-PCF antibody. Lanes as in A.