Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

Université Laval

Frequency-dependent selection is a major force determining the evolutionary dynamics of alleles at the self-incompatibility locus (S-locus) in flowering plants. We introduce a general method using numerical simulations to test several alternative models of frequency-dependent selection on S-locus data from sporophytic systems, taking into account both genetic drift and observed patterns of dominance interactions among S-locus haplotypes (S-haplotypes). Using a molecular typing method, we estimated S-haplotype frequencies in a sample of 322 adult plants and of 245 offspring obtained from seeds sampled on 22 maternal plants, collected in a single population of Arabidopsis halleri (Brassicaceae). We found eight different S-haplotypes and characterized their dominance interactions by controlled pollinations. We then compared the likelihood of different models of frequency-dependent selection: we found that the observed haplotype frequencies and observed frequency changes in one generation best fitted a model with (1) the observed dominance interactions and (2) no pollen limitation. Overall, our population genetic models of frequency-dependent selection, including patterns of dominance interactions among S-haplotypes and genetic drift, can reliably predict polymorphism at the S-locus. We discuss how these approaches allow detecting additional processes influencing the evolutionary dynamics of the S-locus, such as purifying selection on linked loci.

Does frequency-dependent selection with complex dominance interactions accurately predict allelic frequencies at the self-incompatibility locus in Arabidopsis halleri?

Mycosphaerella graminicola populations were examined in France with microsatellite markers and PCR-SSCP analysis of partial actin and β-tubulin encoding sequences. A total of 363 isolates was sampled in 2005 from 17 provinces, and genotypes from corresponding strains were characterized. Unique haplotypes comprised 84% of the population, and gene diversity was high nationwide (0.70) and locally. A moderate genetic differentiation (G(ST) = 0.18) was found and indicated that in France the M. graminicola population was more structured than in other previously studied European countries. Bayesian structure analysis identified three genetic clusters distributed among the 17 provinces. Our results highlighted the potential for the adaptation of the fungus to local conditions, leading to genetic clusters among the French population of the fungus as well as genotype flow between regional clusters.

Genetic diversity and population structure in French populations of Mycosphaerella graminicola.

Despite the tremendous efforts devoted to the identification of genetic incompatibilities underlying hybrid sterility and inviability, little is known about the effect of inter-species hybridization at the protein interactome level. Here, we develop a screening platform for the comparison of protein-protein interactions (PPIs) among closely related species and their hybrids. We examine in vivo the architecture of protein complexes in two yeast species (Saccharomyces cerevisiae and Saccharomyces kudriavzevii) that diverged 5-20 million years ago and in their F1 hybrids. We focus on 24 proteins of two large complexes: the RNA polymerase II and the nuclear pore complex (NPC), which show contrasting patterns of molecular evolution. We found that, with the exception of one PPI in the NPC sub-complex, PPIs were highly conserved between species, regardless of protein divergence. Unexpectedly, we found that the architecture of the complexes in F1 hybrids could not be distinguished from that of the parental species. Our results suggest that the conservation of PPIs in hybrids likely results from the slow evolution taking place on the very few protein residues involved in the interaction or that protein complexes are inherently robust and may accommodate protein divergence up to the level that is observed among closely related species.

Evidence for the robustness of protein complexes to inter-species hybridization.

Over the past decade, the study of protein interaction networks (PINs) has shed light on the organizing principles of living cells. However, PINs have been mostly mapped in one single condition. We outline three of the most promising avenues of investigation in this field, namely the study of first, how PINs are rewired by mutations and environmental perturbations; secondly, how inter-species interactions affect PIN achitectures; thirdly, what mechanisms and forces drive PIN evolution. These investigations will unravel the dynamics and condition dependence of PINs and will thus lead to a better functional annotation of network architecture. One major challenge to reach these goals is the integration of PINs with other cellular regulatory networks in the context of complex cellular phenotypes.

Integrative avenues for exploring the dynamics and evolution of protein interaction networks.

We examined the northern limit of Saccharomyces cerevisiae and Saccharomyces paradoxus in northeast America. We collected 876 natural samples at 29 sites and applied enrichment methods for the isolation of mesophilic yeasts. We uncovered a large diversity of yeasts, in some cases, associated with specific substrates. Sequencing of the ITS1, 5.8S and ITS2 loci allowed to assign 226 yeast strains at the species level, including 41 S. paradoxus strains. Our intensive sampling suggests that if present, S. cerevisiae is rare at these northern latitudes. Our sampling efforts spread across several months of the year revealed that successful sampling increases throughout the summer and diminishes significantly at the beginning of the fall. The data obtained on the ecological context of yeasts corroborate what was previously reported on Pichiaceae, Saccharomycodaceae, Debaryomycetaceae and Phaffomycetaceae yeast families. We identified 24 yeast isolates that could not be assigned to any known species and that may be of taxonomic, medical, or biotechnological importance. Our study reports new data on the taxonomic diversity of yeasts and new resources for studying the evolution and ecology of S. paradoxus.

Exploring the northern limit of the distribution of Saccharomyces cerevisiae and Saccharomyces paradoxus in North America.

Fungi play a central role in both ecosystems and human societies. This is in part because they have adopted a large diversity of life history traits to conquer a wide variety of ecological niches. Here, I review recent fungal genomics studies that explored the molecular origins and the adaptive significance of this diversity. First, macro-ecological genomics studies revealed that fungal genomes were highly remodelled during their evolution. This remodelling, in terms of genome organization and size, occurred through the proliferation of non-coding elements, gene compaction, gene loss and the expansion of large families of adaptive genes. These features vary greatly among fungal clades, and are correlated with different life history traits such as multicellularity, pathogenicity, symbiosis, and sexual reproduction. Second, micro-ecological genomics studies, based on population genomics, experimental evolution and quantitative trait loci approaches, have allowed a deeper exploration of early evolutionary steps of the above adaptations. Fungi, and especially budding yeasts, were used intensively to characterize early mutations and chromosomal rearrangements that underlie the acquisition of new adaptive traits allowing them to conquer new ecological niches and potentially leading to speciation. By uncovering the ecological factors and genomic modifications that underline adaptation, these studies showed that Fungi are powerful models for ecological genomics (eco-genomics), and that this approach, so far mainly developed in a few model species, should be expanded to the whole kingdom.

Ecological genomics of adaptation and speciation in fungi.

Exploring the ability of organisms to locally adapt is critical for determining the outcome of rapid climate changes, yet few studies have addressed this question in microorganisms. We investigated the role of a heterogeneous climate on adaptation of North American populations of the wild yeast Saccharomyces paradoxus. We found abundant among-strain variation for fitness components across a range of temperatures, but this variation was only partially explained by climatic variation in the distribution area. Most of fitness variation was explained by the divergence of genetically distinct groups, distributed along a north-south cline, suggesting that these groups have adapted to distinct climatic conditions. Within-group fitness components were correlated with climatic conditions, illustrating that even ubiquitous microorganisms locally adapt and harbour standing genetic variation for climate-related traits. Our results suggest that global climatic changes could lead to adaptation to new conditions within groups, or changes in their geographical distributions.

Local climatic adaptation in a widespread microorganism.

Self-incompatibility (SI) is a genetic system that prevents self-fertilization in many Angiosperms. Although plants from the Brassicaceae family present an apparently unique SI system that is ancestral to the family, investigations at the S-locus responsible for SI have been mostly limited to two distinct lineages (Brassica and Arabidopsis-Capsella, respectively). Here, we investigated SI in a third deep-branching lineage of Brassicaceae: the tribe Biscutelleae. By coupling sequencing of the SI gene responsible for pollen recognition (SRK) with phenotypic analyses based on controlled pollinations, we identified 20 SRK-like sequences functionally linked to 13 S-haplotypes in 21 individuals of Biscutella neustriaca and 220 seedlings. We found two genetic and phylogenetic features of SI in Biscutelleae that depart from patterns observed in the reference Arabidopsis clade: (1) SRK-like sequences cluster into two main phylogenetic lineages interspersed within the many SRK lineages of Arabidopsis; and (2) some SRK-like sequences are transmitted by linked pairs, suggesting local duplication within the S-locus. Strikingly, these features also were observed in the Brassica clade but probably evolved independently, as the two main SRK clusters in Biscutella are distinct from those in Brassica. In the light of our results and of what has been previously observed in other Brassicaceae, we discuss the ecological and evolutionary implications on SI plant populations of the high diversity and the complex dominance relationships we found at the S-locus in Biscutelleae.

Self-incompatibility in Brassicaceae: identification and characterization of SRK-like sequences linked to the S-locus in the tribe Biscutelleae.

Reproductive isolation is a critical step in the process of speciation. Among the most important factors driving reproductive isolation are genetic incompatibilities. Whether these incompatibilities are already present before extrinsic factors prevent gene flow between incipient species remains largely unresolved in natural systems. This question is particularly challenging because it requires that we catch speciating populations in the act before they reach the full-fledged species status. We measured the extent of intrinsic postzygotic isolation within and between phenotypically and genetically divergent lineages of the wild yeast Saccharomyces paradoxus that have partially overlapping geographical distributions. We find that hybrid viability between lineages progressively decreases with genetic divergence. A large proportion of postzygotic inviability within lineages is associated with chromosomal rearrangements, suggesting that chromosomal differences substantially contribute to the early steps of reproductive isolation within lineages before reaching fixation. Our observations show that polymorphic intrinsic factors may segregate within incipient species before they contribute to their full reproductive isolation and highlight the role of chromosomal rearrangements in speciation. We propose different hypotheses based on adaptation, biogeographical events and life history evolution that could explain these observations.

Jean-Baptiste Leducq

Département de Biologie,
Institut de biologie intégrative et des systémes & PROTEO,
Département de Biologie, Institut de biologie intégrative et des systémes & PROTEO

Université Laval

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Jean-Baptiste Leducq

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Département de Biologie,Institut de biologie intégrative et des systémes & PROTEO,Département de Biologie, Institut de biologie intégrative et des systémes & PROTEO

Université Laval

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Département de Biologie,
Institut de biologie intégrative et des systémes & PROTEO,
Département de Biologie, Institut de biologie intégrative et des systémes & PROTEO