Characterization of a complex nuclear restorer locus of Brassica napus

The plant trait of cytoplasmically-inherited male sterility (CMS) and its suppression by nuclear restorer-of-fertility (Rf) genes can be viewed as a genetic arms-race between the mitochondrial and nuclear genomes. The male sterilizing factors consist of unusual genes or open reading frames (ORFs) that usually contain a portion of functional mitochondrial genes derived sequences and sequences of unknown origin. These unusual ORFs are maternally inherited, transcribed and often effectively translated into novel proteins that are associated with the inability of the plant to produce functional pollen. Most nuclear Rf genes have been shown to encode P-type pentatricopeptide repeat proteins (PPRs). Phylogenetic analysis of P-class PPRs from sequenced plants genomes has shown that Rf-proteins cluster in a distinct clade of P-class PPRs, RFL-PPRs that display hallmarks of positive evolutionary selection. In the canola, (oilseed rape) species Brassica napus, previous work has indicated the nuclear restorer genes for the two native forms of CMS, Rfn (for nap CMS) and Rfp (pol CMS) represent alternate haplotypes, or alleles, of a single nuclear locus. The capacity of the Rfn haplotype to mediate the processing of multiple transcripts is of interest as it is unique among other known restorer alleles. I explored the different processing events that each Rfn transcript target undergoes by mapping their 3' and 5' termini in plants containing and lacking Rfn. CR-RT-PCR was used to distinguish 5' and 3' ends produced by post-transcriptional processing events, in particular Rfn specific processing events, in the key transcripts of interest orf222/nad5c/orf139, atp8 and nad4. This procedure consists of covalently ligating the 5' and 3' ends of total or mtRNAs using an RNA ligase. The reaction leads to the formation of circular RNA, which serves as template for RT-PCR. Sequence analysis between Arabidopsis thaliana, B. napus as well as between the different termini mapped, were used in order to explore probable conserved processing events and Rfn specific recognition sequences.Fine genetic mapping indicates that Rfn localizes to the same genomic region as Rfp as shown in previous studies. We find this region is enriched in RFL genes, four of which, based on their position and expression, represent potential candidates for Rfn; one of these genes, designated PPR4, is a preferred candidate in that it is not expressed in the nap CMS line. Comparison of the corresponding regions of the genomes of B. rapa, B. oleracea, Arabidopsis thaliana and A. lyrata provides insight into the expansion of this group of RFL genes in different lines of evolutionary descent. Unlike other nuclear restorer loci containing multiple RFL genes, the RFL genes in the Rf region of B. napus are not present in tandem arrays but rather are dispersed in genomic location. The genes do not share similar flanking non-coding regions and do not contain introns, indicating that they have duplicated primarily through a retrotransposition-mediated process. In contrast, segmental duplication has been responsible for the distribution of the 10 sequences I annotated as RFL genes in the corresponding region of the A. lyrata genome. My observations define the Brassica Rf locus and indicate that different mechanisms may be responsible for the proliferation of RFL genes even among closely related genomes. In the last chapter of this thesis, I describe the different efforts made towards the identification of the restorer of fertility of nap CMS and exploration of its biochemical proprieties in order to determine the mechanism of fertility restoration.La stérilité mâle cytoplasmique (SMC) chez les plantes et sa suppression par des restorateurs nucléaires (Rfs) peuvent être visualisés comme un bras de fer génétique entre les génomes mitochondrial et nucléaire. Chez les espèces sauvages, la SMC est observée par le phénomène de la gynodioécie, où les individus hermaphrodites et femelles coexistent dans une même population. Les facteurs de stérilisation consistent en des gènes inhabituels ou des phases de lecture ouvertes (ORFs), qui peuvent contenir une portion de gènes mitochondriaux ainsi que des séquences d'origines inconnues. Ces ORFs inhabituelles sont héritées maternellement, transcrites et souvent traduites efficacement en nouvelles protéines associées avec l'incapacitée de la plante à produire du pollen fonctionnel. Il a été démontré que la plupart des gènes Rfs codent pour des protéines à répétition de pentatricopeptides (PPRs). Les études phylogénétiques de la classe P des PPRs à partir de génomes de plantes séquencés ont montré que les protéines Rfs se retrouvent dans un groupe distinct de PPR de classe P, les RFL-PPRs, qui affiche des marques de sélection évolutive positive. Chez l'espèce Brassica napus (ou canola), des études antérieures ont indiqué que les gènes de restoration nucléaire pour les deux formes de SMC natives, Rfn (pour nap SMC) et Rfp (pol SMC), représentent deux haplotypes, ou allèles, d'un seul locus. La responsabilité du locus Rfn dans la formation de termini en 5' de multiples transcrits est unique parmi les capacités d'autres restaurateurs de fertilité connus. L'analyse des séquences des produits obtenus chez B. napus et la comparaison avec les séquences d'ARNs mitochondriaux d'Arabidopsis thaliana ont permis l'exploration d'évènements de modification post-transcriptionnelle conservés et possiblement dépendants de la présence du restorateur Rfn. Nous avons pu mettre en évidence la formation de multiples termini en 5', contrairement à la formation de terminus unique en 3' pour les ARN mitochondriaux étudiés sans détection d'une séquence commune. Cependant, la présence d'une homologie de séquence autour des extrémités 5' produites par Rfn, associées ou non à la restauration de la fertilité, m'a permis d'explorer la possibilité de l'existence d'une séquence cible sur les ARNs étudiés.La cartographie génétique fine présentée indique que Rfn est localisé dans la même région génomique que Rfp. Cette région est enrichie en gènes RFL, dont quatre, selon leurs positions et leurs expressions, représentent des candidats potentiels pour Rfn. Un de ces gènes, PPR4, est un candidat favori car il n'est pas exprimé dans la lignée nap SMC. La comparaison des régions correspondantes dans les génomes de B. rapa, B. oleracea, A. thaliana et A. lyrata a permis d'éclairer les mécanismes d'expansion du groupe de gènes RFL venant de lignées évolutives différentes. Contrairement aux autres loci nucléaires restaurateurs contenant de multiples gènes RFL, les gènes RFL dans la région Rf de B. napus ne sont pas présents en tandem mais ont plutôt des locations génomiques dispersées. Ces gènes ne partagent par leurs régions non-codantes encadrantes et ne contiennent pas d'introns, ce qui indiquent que la duplication a eu premièrement lieu par un processus de rétro-transposition, contrairement à la duplication segmentale responsable pour la distribution des 10 séquences annotées comme gènes RFL dans la région correspondante du génome de A. lyrata. Mes observations définissent le locus Rfn de Brassica et indiquent que différents mécanismes sont responsables pour la prolifération de gènes RFL parmi des génomes relativement proches

The propensity of pentatricopeptide repeat genes to evolve into restorers of cytoplasmic male sterility

Cytoplasmic male sterility (CMS) is a widespread phenotype in plants, which present a defect in the production of functional pollen. The male sterilizing factors usually consist of unusual genes or open reading frames encoded by the mitochondrial genome. CMS can be suppressed by specific nuclear genes called restorers of fertility (Rfs). In the majority of cases, Rf genes produce proteins that act directly on the CMS conferring mitochondrial transcripts by binding them specifically and promoting processing events. In this review, we explore the wide array of mechanisms guiding fertility restoration. PPR proteins represent the most frequent protein class among identified Rfs and they exhibit ideal characteristics to evolve into restorer of fertility when the mechanism of restoration implies a post-transcriptional action. Here, we review the literature that highlights those characteristics and help explain why PPR proteins are ideal for the roles they play as restorers of fertility

Additional file 5: Figure S2. of Comparative genomic analysis of the compound Brassica napus Rf locus

Complementary DNA (cDNA) sequences of transcripts of B. napus genes PPR1-4. (PDF 36 kb

Additional file 4: Figure S1. of Comparative genomic analysis of the compound Brassica napus Rf locus

Phylogenetic analysis of proteins encoded in the Rf-region of B. napus/rapa and the orthologous segment of A. thaliana. Numbers in red type indicate bootstrap values. (PPTX 68 kb

Additional file 2: Table S2. of Comparative genomic analysis of the compound Brassica napus Rf locus

Annotation of B. napus BAC NO202E11. (XLS 20 kb

The propensity of Pentatricopeptide Repeat genes to evolve into restorers of cytoplasmic male sterility

Cytoplasmic male sterility (CMS) is a widespread phenotype in plants, which present a defect in the production of functional pollen. The male sterilizing factors usually consist of unusual genes or open reading frames (ORFs) encoded by the mitochondrial genome. CMS can be suppressed by specific nuclear genes called restorers of fertility (Rfs). In the majority of cases, Rf genes produce proteins that act directly on the CMS conferring mitochondrial transcripts by binding them specifically and promoting processing events. In this review, we explore the wide array of mechanisms guiding fertility restoration. PPR proteins represent the most frequent protein class among identified Rfs and they exhibit ideal characteristics to evolve into restorer of fertility when the mechanism of restoration implies a post-transcriptional action. Here, we review the literature that highlights those characteristics and help explain why PPR proteins are ideal for the roles they play as restorers of fertility