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• W2560851080 abstract "Article5 December 2016free access Transparent process Mitochondrial–nuclear co-evolution leads to hybrid incompatibility through pentatricopeptide repeat proteins Han-Ying Jhuang orcid.org/0000-0002-1918-5151 Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan Search for more papers by this author Hsin-Yi Lee Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan Molecular and Cell Biology, Taiwan International Graduate Program, Graduate Institute of Life Sciences, National Defense Medical Center and Academia Sinica, Taipei, Taiwan Search for more papers by this author Jun-Yi Leu Corresponding Author [email protected] orcid.org/0000-0001-5445-2799 Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan Search for more papers by this author Han-Ying Jhuang orcid.org/0000-0002-1918-5151 Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan Search for more papers by this author Hsin-Yi Lee Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan Molecular and Cell Biology, Taiwan International Graduate Program, Graduate Institute of Life Sciences, National Defense Medical Center and Academia Sinica, Taipei, Taiwan Search for more papers by this author Jun-Yi Leu Corresponding Author [email protected] orcid.org/0000-0001-5445-2799 Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan Search for more papers by this author Author Information Han-Ying Jhuang1,2, Hsin-Yi Lee1,3 and Jun-Yi Leu *,1,2 1Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan 2Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan 3Molecular and Cell Biology, Taiwan International Graduate Program, Graduate Institute of Life Sciences, National Defense Medical Center and Academia Sinica, Taipei, Taiwan *Corresponding author. Tel: +886 2 26519574; E-mail: [email protected] EMBO Rep (2017)18:87-101https://doi.org/10.15252/embr.201643311 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Mitochondrial–nuclear incompatibility has a major role in reproductive isolation between species. However, the underlying mechanism and driving force of mitochondrial–nuclear incompatibility remain elusive. Here, we report a pentatricopeptide repeat-containing (PPR) protein, Ccm1, and its interacting partner, 15S rRNA, to be involved in hybrid incompatibility between two yeast species, Saccharomyces cerevisiae and Saccharomyces bayanus. S. bayanus-Ccm1 has reduced binding affinity for S. cerevisiae-15S rRNA, leading to respiratory defects in hybrid cells. This incompatibility can be rescued by single mutations on several individual PPR motifs, demonstrating the highly evolvable nature of PPR proteins. When we examined other PPR proteins in the closely related Saccharomyces sensu stricto yeasts, about two-thirds of them showed detectable incompatibility. Our results suggest that fast co-evolution between flexible PPR proteins and their mitochondrial RNA substrates may be a common driving force in the development of mitochondrial–nuclear hybrid incompatibility. Synopsis Mitochondrial–nuclear incompatibility can cause reproductive isolation between species. This study shows that fast co-evolution between flexible PPR proteins and their mitochondrial RNA targets represents a common driving force in the development of hybrid incompatibility. Fast evolution of a PPR protein Ccm1 leads to symmetric incompatibility between two closely related yeast species. The CCM1 incompatibility causes reduced levels of mitochondrial 15S rRNA and protein translation. The CCM1 incompatibility can be rescued by mutations in the PPR motifs or mitochondrial DNA. Two-thirds of yeast PPR proteins have evolved mitochondrial–nuclear incompatibility among the Saccharomyces sensu stricto species. Introduction A critical step of speciation is the emergence of reproductive isolating barriers between diverging populations. These barriers are classified into two major forms: (i) prezygotic isolation caused by mating discrimination or unsuccessful gamete recognition, and (ii) postzygotic isolation caused by hybrid inviability or sterility. Identifying the molecular mechanisms underlying reproductive isolation would allow us to deduce the driving force of speciation. Yeasts have proven to be an excellent model organism for studying postzygotic isolation, helping to demonstrate the involvement of anti-recombination induced by DNA sequence divergence, chromosomal rearrangements, and genetic incompatibility 12345. Apart from DNA sequence divergence, which can occur simply by mutation accumulation, development of postzygotic isolation is often deemed a by-product of adaptive selection 6. In the classical genetic incompatibility model, environmental adaptation is hypothesized to be an important force driving fixation of diverging alleles in different populations 7. However, many known instances of genetic incompatibility seem to be caused by susceptibility to mutation pressure and invasion of pathogens or selfish genetic elements 8. Fixation of divergent alleles involved in incompatibility can be achieved by repeated mutation compensation processes or during the resolution of genetic conflicts 910. Drawing a general picture of how genetic incompatibility evolved entails identification of more incompatibility genes. Mitochondrial–nuclear incompatibility is a specific form of Dobzhansky–Muller incompatibility 111213. Mitochondria play essential roles in the survival, growth, and sexual reproduction of organisms 14. During evolution, selective pressures for better maintenance or enhanced fixation of beneficial mutations in mitochondrial genes have led to transfer of most mitochondrial genes to the nuclear genome, leaving only a handful of protein-coding genes in modern mitochondrial genomes (mtDNA) 15161718. In the budding yeast S. cerevisiae, there are around 1,000 nucleus-encoded mitochondrial proteins, whereas the mtDNA encodes only eight proteins 19. Compared to the nuclear genome, the mutation rate of mtDNA is generally an order of magnitude higher 20. To maintain proper interactions, nucleus-encoded mitochondrial proteins may need to rapidly co-evolve with mtDNA 212223. Consequently, mismatched mitochondrial and nuclear genomes have been observed to cause inter- or intraspecific hybrid incompatibilities in a broad range of species, even though the molecular basis of incompatibility was not identified in most cases 12242526272829303132. Among the closely related Saccharomyces sensu stricto yeast species, several genes involved in mitochondrial–nuclear hybrid incompatibility have been characterized at the molecular level. Nucleus-encoded Mrs1 regulates intron removal of the mitochondrion-encoded COX1 mRNA 333435. Mrs1 splices two of the S. paradoxus COX1 introns, but one of these introns was lost in S. cerevisiae during evolution. S. cerevisiae Mrs1 fails to splice both introns of the S. paradoxus COX1 gene in hybrid cells, resulting in hybrid incompatibility between S. cerevisiae and S. paradoxus 36. Nucleus-encoded Aim22 is an enzyme required for lipoylation of mitochondrial targets 373839. S. cerevisiae Aim22 cannot function properly in an S. bayanus mitochondrial genomic background, though the incompatible interacting partners remain elusive 36. Similarly, Aep2 is a nucleus-encoded pentatricopeptide repeat (PPR) protein required for translation of OLI1 mRNA, which encodes for F0-ATP synthase subunit c 4041. S. bayanus Aep2 is incompatible with the S. cerevisiae OLI1 gene, so synthesis of the Oli1 protein is inhibited 29. Despite that the incompatibility of both Mrs1 and Aep2 involve protein–RNA interactions, it is necessary to identify more genes in order to elucidate whether this is a common mode of mitochondrial–nuclear incompatibility. Pentatricopeptide repeat proteins are often observed to regulate mitochondrial RNA and these proteins constitute one of the largest protein families in eukaryotes, mainly contributed by expanded plant PPR genes 42. The pentatricopeptide repeat is a degenerate 35-amino acid structural motif, and multiple tandem PPR motifs in the PPR protein act in a coordinated modular manner, which might allow PPR proteins to evolve rapidly 434445. In land plants, expansion of the PPR family has been speculated to have had important impacts on the evolution of organellar genome complexity 46. The S. cerevisiae genome contains 15 predicted PPR genes 4147. Deletion of PPR genes often leads to decreased respiratory growth in yeast 4748. Although these proteins have been shown to evolve more rapidly than the entire genome background in general, their evolutionary trajectories in closely related species have not been characterized 41. In the present study, we employed a chromosomal replacement strategy to identify another PPR gene, CCM1, involved in hybrid incompatibility between S. cerevisiae and S. bayanus. This incompatibility was bidirectional and could be rescued by a variety of mutations in the PPR motifs. Subsequent systematic replacements of all yeast PPR genes with orthologs from closely related species revealed that PPR genes prevalently contribute to hybrid incompatibility among the Saccharomyces sensu stricto yeasts. Our results demonstrate that evolution of mitochondrial–nuclear incompatibility is prevalent in yeast species and PPR proteins play a large role in fast co-evolution of the two genomes. Results Chromosome 7 of Saccharomyces bayanus is incompatible with the Saccharomyces cerevisiae genome To identify genetic incompatibility between the two closely related yeast species, S. bayanus and S. cerevisiae, we previously constructed chromosome replacement lines in which one or two chromosomes were derived from S. bayanus, and the remaining chromosomes and mtDNA were from S. cerevisiae 29. When all the chromosome replacement lines were examined, we noticed that the replacement line carrying S. bayanus chromosome 7 (Sc + Sb-chr7) exhibited obvious defects in both vegetative growth and sporulation, suggestive of defects in mitochondrial respiration. To address that we grew cells in medium containing only the nonfermentable carbon source (i.e., glycerol in the following experiments). The Sc + Sb-chr7 strain showed much reduced growth on glycerol-containing plates, indicating that the mitochondrial function of Sc + Sb-chr7 was compromised (Fig 1A). However, if we crossed Sc + Sb-chr7 with a S. cerevisiae haploid strain without mtDNA (Sc-ρ⁰), the growth defect was fully rescued, suggesting that the mtDNA of Sc + Sb-chr7 is intact and the observed respiratory defect is recessive (Fig 1A). Figure 1. CCM1 is involved in the incompatibility between S. bayanus chromosome 7 and the S. cerevisiae mitochondrial genome Chromosome 7 of S. bayanus is incompatible with the S. cerevisiae genome. The chromosome 7 replacement stain (Sc + Sb-chr7) exhibited serious growth defects when grown on the nonfermentable carbon source (glycerol). The respiration defects could be partially rescued when a whole set of S. bayanus chromosomes were provided (Sc + Sb-chr7 × Sb-ρ⁰), or fully rescued with the addition of S. bayanus mtDNA (Sc + Sb-chr7-ρ⁰ × Sb). Cell cultures were serially diluted and spotted onto YPD (glucose) or YPG (glycerol) plates. The plates were then incubated at 28°C until colonies were easily observed. Ectopically expressing Sc-CCM1 rescues the respiration defect of Sc + Sb-chr7. Spot assays for the wild-type S. cerevisiae (Sc) and the chromosome 7 replacement stain (Sc + Sb-chr7) carrying an empty plasmid (+ vector) or the plasmid with the S. cerevisiae CCM1 gene (+ Sc-CCM1). Sb-CCM1 incompatibility could only be rescued in the presence of S. bayanus mtDNA. The native CCM1 ORF in S. cerevisiae was replaced by either Sb-CCM1 (Sc + Sb-CCM1) or Sc-CCM1 (Sc + Sc-CCM1, as a control) coding regions. Crossing the Sc + Sb-CCM1 strain with the mtDNA-less Sb-ρ⁰ strain (Sc + Sb-CCM1 × Sb-ρ⁰) could not rescue the growth defect. CCM1 incompatibility is symmetric between S. cerevisiae and S. bayanus. The CCM1 ORF in S. bayanus was replaced by either Sc-CCM1 (Sb + Sc-CCM1) or Sb-CCM1 (Sb + Sb-CCM1, as a control) coding regions. Only the Sb + Sc-CCM1 strain exhibited respiration defects and the defect could not be rescued even when a whole set of S. cerevisiae chromosomes were provided (Sb + Sc-CCM1 × Sc-ρ⁰). CCM1 incompatibility probably occurred during the divergence between S. bayanus and the common ancestor of S. cerevisiae, S. paradoxus, S. mikatae, and S. kudriavzevii. The endogenous CCM1 in S. cerevisiae was replaced with its orthologous alleles from other Saccharomyces sensu stricto yeasts. Only the strain carrying S. bayanus CCM1 displayed growth defects on glycerol-containing plates. Data information: Sc, S. cerevisiae. Sb, S. bayanus. ρ⁰, mtDNA-less strains. Download figure Download PowerPoint The respiratory defect of Sc + Sb-chr7 might result from incompatibility between S. bayanus chromosome 7 and other S. cerevisiae chromosomes or mtDNA. To address this issue, we generated two hybrid diploids: In the first one (Sc + Sb-chr7 × Sb-ρ⁰), a whole set of S. bayanus chromosomes were provided but mtDNA was from S. cerevisiae, and in the second hybrid (Sc + Sb-chr7-ρ⁰ × Sb), it contained both S. bayanus chromosomes and mtDNA. The chr 7 incompatibility was fully rescued in the second hybrid, but only partially rescued in the first one (Fig 1A). These results indicate that the incompatibility is mainly between S. bayanus chromosome 7 and S. cerevisiae mtDNA (Sc-mtDNA), and the interactions between different chromosomes only contribute minor effects. CCM1 is involved in the incompatibility between Saccharomyces bayanus chromosome 7 and the Saccharomyces cerevisiae mitochondrial genome We performed a genomic DNA library screen to search for S. cerevisiae gene(s) that could rescue the growth defect of Sc + Sb-chr7 on glycerol plates. Two different clones were obtained from the screen and both of them contained the full-length S. cerevisiae CCM1 gene (Sc-CCM1), which is also located on chromosome 7. To verify the involvement of CCM1 in hybrid incompatibility, we PCR-amplified the CCM1 gene from S. cerevisiae genomic DNA, cloned it into a single-copy plasmid, and tested its ability to rescue the growth defect of Sc + Sb-chr7. Expression of Sc-CCM1 in Sc + Sb-chr7 exhibited a considerable rescue effect (Fig 1B). We also constructed allele replacement strains in which the native CCM1 ORF in S. cerevisiae was replaced by the S. bayanus CCM1 orthologous allele and a nutrient marker (HIS3) or simply placed the nutrient marker in the downstream of CCM1 as a control (Sc + Sb-CCM1 and Sc + Sc-CCM1, respectively). Only the Sc + Sb-CCM1 strain showed substantial growth defects on glycerol plates, suggesting that the Sb-Ccm1 protein is incompatible with the S. cerevisiae genetic background (Fig 1C). In S. cerevisiae, CCM1 encodes a mitochondrial protein essential for pre-mRNA intron removal of two mtDNA-encoded genes, COX1 and COB 49. In addition, the Ccm1 protein directly interacts and stabilizes mitochondrial 15S rRNA 50. Therefore, CCM1 incompatibility is likely related to its functions in the mitochondria. We tested this idea by crossing the Sc + Sb-CCM1 strain to S. bayanus with or without mtDNA. Sb-CCM1 incompatibility could only be rescued in the presence of the S. bayanus mtDNA (Sc + Sb-CCM1-ρ⁰ × Sb), indicating that Sb-CCM1 is incompatible with Sc-mtDNA (Fig 1C). CCM1 incompatibility is symmetric between Saccharomyces cerevisiae and Saccharomyces bayanus Hybrid incompatibility caused by two interacting genetic loci may be unidirectional (asymmetric) if one of the loci remains the ancestral form in parental populations. Alternatively, if both loci have changed, as might be the case of fast-evolving genes, the incompatibility could be bidirectional (symmetric). The mitochondrial–nuclear incompatibilities identified in previous yeast studies are all asymmetric 2936. To characterize the symmetry of CCM1 incompatibility, we replaced the native CCM1 ORF in S. bayanus with the coding region of Sc-CCM1 and a nutrient marker (HIS3), or simply placed the nutrient marker downstream of Sb-CCM1 as a control (Sb + Sc-CCM1 or Sb + Sb-CCM1, respectively), and examined their fitness. The Sb + Sc-CCM1 strain exhibited growth defects when cultured on glycerol-containing plates (Fig 1D). Furthermore, the incompatibility could be rescued by crossing Sb + Sc-CCM1 with the S. bayanus ρ⁰ strain to supply a whole set of S. bayanus chromosomes, but was not rescued when crossed with the S. cerevisiae ρ⁰ strain (Fig 1D). The results indicate that CCM1 is involved in a symmetric mito-nuclear incompatibility between S. cerevisiae and S. bayanus. Both S. cerevisiae and S. bayanus belong to the Saccharomyces sensu stricto group. To fine-map in which branch CCM1 incompatibility evolved, we replaced native CCM1 in S. cerevisiae with its orthologous alleles from other Saccharomyces sensu stricto yeasts, including S. paradoxus, S. mikatae, or S. kudriavzevii. We found that only the CCM1 orthologous allele from S. bayanus was incompatible with the S. cerevisiae genetic background (Fig 1E). These results suggest that CCM1 incompatibility probably occurred during the divergence between S. bayanus and the common ancestor of S. cerevisiae, S. paradoxus, S. mikatae, and S. kudriavzevii. Sb-CCM1 incompatibility leads to reduced levels of mitochondrial 15S rRNA One possible explanation for the Sb-CCM1 incompatibility is that the Sb-Ccm1 protein cannot be transported efficiently to Sc-mitochondria. We used subcellular fractionation to determine the localization of Ccm1. Mitochondria from wild-type S. cerevisiae strains carrying Sc-CCM1-13Myc or Sb-CCM1-13Myc were isolated and examined using Western blotting. Although a low level of Sb-Ccm1 was detected in the cytosolic fraction, the majority of Sb-Ccm1 localized to mitochondria, indicating that mislocalization was not the cause of hybrid incompatibility (Fig 2A). Figure 2. CCM1 incompatibility results in reduced levels of 15S rRNA and mtDNA-encoded proteins Sb-Ccm1 protein is transported efficiently to S. cerevisiae mitochondria. Total cell extracts (total), cytosolic fractions (cyto), or mitochondrial fractions (mito) were hybridized with different antibodies to detect the proteins. G6PDH (glucose-6-phosphate dehydrogenase) and Cox2 served as cytosolic and mitochondrial markers, respectively. Both Sc-Ccm1 and Sb-Ccm1 were fused with c-Myc at the C-terminus and expressed from a single-copy plasmid. The Myc-tagged Ccm1 proteins consistently appeared as double bands, which was probably due to some unknown protein modifications. The level of 15S rRNA is reduced in Sc + Sb-CCM1 cells. RNA isolated from Sc + Sb-CCM1 (labeled as Sb) or Sc + Sc-CCM1 (labeled as Sc) cells was hybridized with gene-specific probes using Northern blots. Sb-Ccm1 interacts weakly with S. cerevisiae 15S rRNA. The level of Ccm1-bound 15S rRNA was measured using quantitative PCR analysis following mitochondrial RNA immunoprecipitation of S. cerevisiae cells expressing Sc-CCM1-13Myc or Sb-CCM1-13Myc. Data were normalized to the control group without the Myc tag to obtain the relative fold enrichment. Graph was plotted using data from three independent repeats for each strain, and P-value was calculated by unpaired, two-sided Student's t-test. Error bars indicate SD. ***P-value < 0.001. Overexpression of Sb-CCM1 rescues the respiration defect of Sc + Sb-CCM1 cells. Cell cultures were serially diluted and spotted onto YPD (glucose) or YPG (glycerol) plates. The plates were then incubated at 28°C until colonies were easily observed. The steady-state level of mtDNA-encoded proteins is reduced in Sc + Sb-CCM1 cells. Immunoblotting for the mitochondrial proteins in Sc + Sb-CCM1 (labeled as Sb) or Sc + Sc-CCM1 (labeled as Sc) cells. Cox1, Cox2, and Cox3 are mtDNA-encoded complex IV subunits. Tom20 is a nucleus-encoded mitochondrial protein. Download figure Download PowerPoint Because Ccm1 affects both pre-mRNA splicing of COX1 and COB, and stability of 15S rRNA 495051, we used Northern blots to examine whether the steady-state levels or maturation of the transcripts from mtDNA-encoded genes was affected in the Sc + Sb-CCM1 strain. For all the examined mtDNA-encoded genes, only 15S rRNA showed a reduced level in Sc + Sb-CCM1 cells (Fig 2B). No differences were detected in the mRNA levels of COX1 and COB between the Sc + Sb-CCM1 and Sc + Sc-CCM1 strains. To elucidate the mechanism underlying reduced levels of 15S rRNA, we tested whether the interaction between 15S rRNA and the Sb-Ccm1 protein is impeded in Sc + Sb-CCM1 cells. We expressed either C-terminally Myc-tagged Sc-Ccm1 or Sb-Ccm1 in S. cerevisiae strains, isolated the mitochondria, and then immunoprecipitated Ccm1 via the anti-Myc antibody. The level of Ccm1-bound 15S rRNA was then measured using real-time quantitative PCR, and the mRNAs of a nucleus-encoded gene TDH3 and a mtDNA-encoded gene COB were used as the negative controls (see Materials and Methods). We found that levels of co-immunoprecipitated S. cerevisiae 15S rRNA were more enriched in the strains carrying Sc-Ccm1 than those with Sb-Ccm1 (330.0 ± 15.4 vs. 7.1 ± 0.4 fold enrichment relative to controls without tagging, Fig 2C), while the transcripts of the negative controls were not detected in the elutes. These results suggest that Sb-CCM1 may function as a hypomorphic mutant of Sc-CCM1, and the weakened interaction between Sb-Ccm1 and 15S rRNA might be responsible for the reduced level of 15S rRNA. To further test this weak allele idea, we overexpressed Sb-CCM1 in S. cerevisiae using a 2-micron multi-copy plasmid (pRS426). Indeed, the respiratory defect of Sc + Sb-CCM1 cells was largely rescued by overexpression of Sb-CCM1 (Fig 2D). 15S rRNA is an essential component of mitochondrial ribosomes. The decrease in 15S rRNA is likely to cause a reduction in translation of mitochondrion-encoded proteins. We used Western blots to examine Cox1, Cox2, and Cox3, that is, the subunits of mitochondrial complex IV (cytochrome c oxidase) that are encoded on the mtDNA. The steady-state protein levels of Cox1, Cox2, and Cox3 were all reduced in the Sc + Sb-CCM1 strain (Fig 2E). In contrast, the nucleus-encoded mitochondrial protein, Tom20, was maintained at similar levels in the Sc + Sb-CCM1 and Sc + Sc-CCM1 strains. Experimental evolution to isolate mutations that can rescue CCM1 incompatibility Sc-Ccm1 is a large protein (864 a.a.) and it only shares 72% protein identity with Sb-Ccm1. To dissect the molecular detail of CCM1 incompatibility, we used an experimental evolution approach to isolate the mutations that could rescue the incompatibility. Single colonies of Sc + Sb-CCM1 cells were used to initiate 98 independent cell cultures, and the cultures were continuously propagated in glycerol-containing medium (see Materials and Methods). Under this selective regime, cells containing spontaneous mutations that rescued the respiratory defects of Sc + Sb-CCM1 would grow more quickly and be enriched in the population. After selection, only one colony with improved fitness was selected from each cell culture for further analysis. Therefore, mutations identified from each colony were likely to represent an independent event. We collected 94 mutant clones in total because four of the cell cultures were contaminated during the experiment. Next, we crossed the mutant clones with an isogenic Sc + Sb-CCM1-ρ⁰ strain in which mtDNA had been deleted and a different nutrient marker (URA3) was inserted next to the Sb-CCM1 gene. The diploid cells were then induced to enter meiosis, and their tetrads were analyzed. If the suppressor mutation occurred in mtDNA, the spores would display a non-Mendelian 4:0 segregation pattern for improved respiration (Fig 3A). In contrast, a 2:2 segregation pattern would be observed if the rescue effect involved a single nuclear mutation. Since the two alleles of Sb-CCM1 were next to different nutrient markers (URA3 or HIS3), we could easily check whether the suppressor mutations co-segregated with the Sb-CCM1 gene. The evolved Sb-CCM1 gene was sequenced if it was linked with the suppressor mutation. Among 94 evolved clones, two clones carried mtDNA suppressors, 20 clones carried mutations in the Sb-CCM1 gene, and the remainder contained mutations in unknown nuclear genes. Figure 3. Incompatibility of Sb-CCM1 can be relieved by mutations in the mitochondrial genome Tetrad analysis reveals that the respiration defect of Sc + Sb-CCM1 cells can be rescued by mutations in mitochondrial or nuclear genomes. The suppressor clones were crossed with the Sc + Sb-CCM1-ρ⁰ strain and the tetrads from the cross were analyzed. A non-Mendelian 4:0 segregation pattern for improved respiration is expected if the suppressor mutation is in mtDNA (left panel). Otherwise, a 2:2 segregation pattern would be observed if the rescue effect involved a single nuclear mutation (right panel). Partial sequence alignment of 15S rRNA from S. bayanus, S. cerevisiae, and two mitochondrial suppressor clones (#1 and #2) of Sc + Sb-CCM1. A single nucleotide deletion at position 188 and four nucleotide substitutions at positions 1,524–1,527 found in both suppressor clones are labeled in red. Download figure Download PowerPoint For those clones carrying mtDNA suppressors, the rescue effect was further confirmed by reintroducing their mtDNA into an ancestral Sc + Sb-CCM1-ρ⁰ strain, indicating that no nuclear mutations were involved. Because our experiments had shown that S. cerevisiae 15S rRNA interacted with Sb-Ccm1 more weakly than its interaction with Sc-Ccm1, we directly sequenced evolved mitochondrial 15S rRNA to see whether it had been modified. Interestingly, both mitochondrial suppressor clones shared similar mutations, a single nucleotide deletion at position 188 and four nucleotide substitutions at positions 1,524–1,527 (Fig 3B). Although we cannot rule out the possibility that the rescue effects arose from unidentified mutations elsewhere in the evolved mtDNA, these results clearly show that the compatibility status of mito-nuclear interactions can be changed by evolving either side of the interaction. Changes in the PPR domain of Sb-CCM1 rescue the incompatibility For the Sb-CCM1 mutant clones, we first PCR-amplified the Sb-CCM1 sequences from the suppressor genomes, reintroduced them into wild-type S. cerevisiae cells, and tested for their compatibility by growing the cells on glycerol plates. All the Sb-CCM1 mutants were confirmed to have regained compatibility with the S. cerevisiae genome (Fig 4A). Interestingly, most of the mutations were de novo amino acid changes and only two mutants had changed their residues from the S. bayanus sequence to the S. cerevisiae sequence (Sb-CCM1F294L and Sb-CCM1D400N). Because the D residue at position 400 is conserved between S. bayanus and the outgroup species Naumovia castellii, and it has become N in S. cerevisiae, S. paradoxus, S. mikatae, and S. kudriavzevii, it suggests that the D and N residues probably represent the ancestral and derived amino acid states, respectively (Fig 4B). The unequivocal correlation between the compatibility and the amino acid states of the 400th residue in S. cerevisiae prompted us to test the importance of this residue in S. bayanus. Indeed, the D400N substitution, but not E375Q (a de novo substitution that repeatedly appeared in three independent clones), in Sb-CCM1 caused a severe respiratory defect in S. bayanus (Fig 4C), suggesting that the 400th residue plays a determinant role in S. cerevisiae–S. bayanus incompatibility. Figure 4. Changes in the PPR domain of Sb-CCM1 rescue the incompatibility Reconstituted Sb-CCM1 mutants can rescue the incompatibility of Sb-CCM1. Among 94 suppressor clones, 20 of them carry mutations in the Sb-CCM1 gene. These mutants were reconstructed in Sc + Sb-CCM1 cells to test their function. Superscripts next to Sb-CCM1 indicate the respective amino acid substitutions, and numbers in parentheses indicate the number of independent clones in our suppressor screen. Sc, wild-type S. cerevisiae. Sequence alignment shows that most of the Sb-CCM1 suppressor mutants contain de novo amino acid changes and only two mutants have switched their residues from the S. bayanus sequence to the S. cerevisiae sequence (F294L and D400N). Letters beside the phylogenetic tree (in which the line lengths are not proportional to the evolutionary distances) indicate the amino acid state at the 400th residue and parentheses contain the codons encoding for the amino acids. The 400th residue plays a determinant role in the S. cerevisiae–S. bayanus incompatibility. Both Sb-CCM1E375Q and Sb-CCM1D400N mutants relieved the incompatibility in S. cerevisiae, but only Sb-CCM1D400N caused respiration defects in S. bayanus. The Sb-CCM1E375Q mutant was selected as a control in this experiment since it was a charged-to-polar substitution similar to Sb-CCM1D400N and also appeared three times in our screen. Sb, wild-type S. bayanus. Protein structural analysis shows that all intramolecul" @default.
• W2560851080 created "2016-12-16" @default.
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• W2560851080 date "2016-12-05" @default.
• W2560851080 modified "2023-10-11" @default.
• W2560851080 title "Mitochondrial–nuclear co‐evolution leads to hybrid incompatibility through pentatricopeptide repeat proteins" @default.
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