In vivo functional analysis of a nuclear restorer PPR protein

BACKGROUND: Nuclear restorers of cytoplasmic male fertility (CMS) act to suppress the male sterile phenotype by down-regulating the expression of novel CMS-specifying mitochondrial genes. One such restorer gene is Rfo, which restores fertility to the radish Ogura or ogu CMS. Rfo, like most characterized restorers, encodes a pentatricopeptide repeat (PPR) protein, a family of eukaryotic proteins characterized by tandem repeats of a 35 amino acid motif. While over 400 PPR genes are found in characterized plant genomes and the importance of this gene family in organelle gene expression is widely recognized, few detailed in vivo assessments of primary structure-function relationships in this protein family have been conducted. RESULTS: In contrast to earlier studies, which identified 16 or 17 PPR domains in the Rfo protein, we now find, using a more recently developed predictive tool, that Rfo has 18 repeat domains with the additional domain N-terminal to the others. Comparison of transcript sequences from pooled rfo/rfo plants with pooled Rfo/Rfo plants of a mapping population led to the identification of a non-restoring rfo allele with a 12 bp deletion in the fourth domain. Introduction into ogu CMS plants of a genetic construct in which this deletion had been introduced into Rfo led to a partial loss in the capacity to produce viable pollen, as assessed by vital staining, pollen germination and the capacity for seed production following pollination of CMS plants. The degree of viable pollen production among different transgenic plants roughly correlated with the copy number of the introduced gene and with the reduction of the levels of the ORF138 CMS-associated protein. All other constructs tested, including one in which only the C-terminal PPR repeat was deleted and another in which this repeat was replaced by the corresponding domain of the related, non-restoring gene, PPR-A, failed to result in any measure of fertility restoration. CONCLUSIONS: The identification of the additional PPR domain in Rfo indicates that the protein, apart from its N-terminal mitochondrial targeting presequence, consists almost entirely of PPR repeats. The newly identified rfo allele carries the same 4 amino acid deletion as that found in the neighboring, related, non-restoring PPR gene, PPR-A. Introduction of this four amino acid deletion into a central domain the Rfo protein, however, only partially reduces its restoration capacity, even though this alteration might be expected to alter the spacing between the adjoining repeats. All other tested alterations, generated by deleting specific PPR repeats or exchanging repeats with corresponding domains of PPR-A, led to a complete loss of restorer function. Overall we demonstrate that introduction of targeted alterations of Rfo into ogu CMS plants provides a sensitive in vivo readout for analysis of the relationship between primary structure and biological function in this important family of plant proteins.

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