CMS restorer genes and proteins of Brassica napus (canola)

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Cytoplasmic male sterility (CMS) is a widespread trait among higher plants that results from the expression of novel open reading frames (ORFs) in the mitochondrial genome. In most cases, nuclear genes termed restorers-of-fertility (Rf ) have been identified that can suppress the CMS phenotype. Such nuclear restorer genes are specific for a particular form of CMS and function by down-regulating the expression of the novel, CMS-associated mitochondrial ORF. CMS – restorer gene systems offer a means of studying interactions between the nuclear and mitochondrial genomes and are of evolutionary interest because they represent a type of “genomic conflict” or evolutionary “arms race”: maternally inherited, CMS-conferring mtDNAs can spread in a population but as pollen becomes limiting, selective pressure builds for the evolution of a restorer gene that can suppress the CMS. We have discovered novel features of the two forms of CMS, pol and nap, native to the canola plant Brassica napus that suggest these systems evolved recently and hence can provide insight into the molecular mechanisms through which CMS – nuclear restorer systems evolve. In particular restorer genes for these systems, Rfp (for pol) and Rfn, map to the same chromosomal locus. Further understanding how nuclear restorer genes can evolve in response to the appearance of a CMS-associated mitochondrial gene will require a molecular characterization of the Rfn/Rfp locus and the identification of the individual genes that are responsible for restoration of the pol and nap CMS forms. The primary objective of this discovery grant is to complete our ongoing studies aimed at achieving this goal. My group was one of the first to isolate a nuclear restorer, in our case the Rfo gene which restores the ogu CMS of radish. We showed that Rfo encodes a P-type pentatricopeptide repeat (PPR) protein, a feature subsequently found for other restorers. Such proteins contain tandem copies of a canonical 35 amino acid repeat and specify sites of transcript modification such as nuclease processing. We have now found that the Rfp gene localizes to a region of a bacterial artificial chromosome (BAC) generated from the Rfn genotype. Higher resolution genetic mapping studies indicate that the Rfn gene maps to the same region. The Rfn genotype BAC contains 4 related Rf-like PPR encoding genes. The three most likely candidates are currently being introduced into B. napus plants in order to assess their capacity for conferring Rfn-specific transcript modifications and suppressing the nap form of CMS. Over the next two years, the expected duration of this Discovery Grant, we will complete the identification of the Rfn and Rfp genes through genetic transformation studies. We hope as well, through collaboration with scientists at INRA, Versailles, France to identify the RNAs that are specifically bound by Rfn and to identify its binding site on the target orf222/nad5c transcript.