A selective bottleneck during host entry drives the evolution of new legume symbionts

During the emergence of new host-microbe symbioses, multiple selective pressures-acting at the different steps of the microbial life cycle–shape the phenotypic traits that jointly determine microbial fitness. However, the relative contribution of these different selective pressures to the adaptive trajectories of microbial symbionts are still poorly known. Here we characterized the dynamics of phenotypic adaptation and its underlying genetic bases during the experimental evolution of a plant pathogenic bacterium into a legume symbiont. We observed that fast adaptation was predominantly driven by selection acting on competitiveness for host entry, which outweighed selection acting on within-host proliferation. Whole-population sequencing of evolved bacteria revealed that phenotypic adaptation was supported by the continuous accumulation of new mutations and the sequential sweeps of cohorts of mutations with similar temporal trajectories. The identification of adaptive mutations within the fixed mutational cohorts showed that all of them improved competitiveness for host entry, while only a subset of those also improved within host proliferation. Computer simulations predict that this effect emerges from the presence of a strong selective bottleneck at host entry. Together, these results show how selective bottlenecks can alter the relative influence of selective pressures acting during bacterial adaptation to multistep infection processes

A selective bottleneck during host entry drives the evolution of new plant symbionts

International audienceThe acquisition of mobile genetic elements (MGEs) can trigger radical shifts in microbial lifestyles, for example by converting a mutualist into a pathogen or vice versa. These transitions often involve changes in host range, infection strategies, or ecological niches, which typically require specific adaptations to the different stages of the new life cycle. While comparative genomic studies have documented several MGE-associated lifestyle transitions, a detailed understanding of the tempo and mode of adaptation to new symbiotic lifestyles is still lacking.Here, we sought to evolve new nitrogen-fixing bacterial symbionts of the legume plant Mimosa pudica from an ancestral plant pathogenic bacterium, Ralstonia solanacearum, which was engineered with a plasmid carrying essential symbiosis genes. We experimentally evolved this strain for over 900 generations during serial passages through a simplified symbiotic life cycle (comprising rhizosphere inoculation, root entry, and within-plant proliferation) and characterized the dynamics of molecular and phenotypic adaptation in the evolved populations.Multiple independent lineages exhibited rapid and parallel fitness increase during their interaction with M. pudica. Whole-population sequencing revealed a continuous accumulation of mutations, facilitated by a plasmid-induced transient hypermutagenesis phase before host entry. We observed sequential sweeps of mutational cohorts, with multiple adaptive mutations co-occurring within these cohorts. Notably, all adaptive mutations enhanced host entry, while only a subset also improved within-host proliferation. Computational modelling suggests that this pattern is a consequence of the strong selective bottleneck occurring at host entry and immediately following the hypermutagenesis phase. Our findings highlight how selective bottlenecks shape adaptive trajectories of symbiotic bacteria experiencing complex infection processes

A selective bottleneck during host entry drives the evolution of new legume symbionts

AbstractDuring the emergence of new host-microbe symbioses, multiple selective pressures-acting at the different steps of the microbial life cycle–shape the phenotypic traits that jointly determine microbial fitness. However, the relative contribution of these different selective pressures to the adaptive trajectories of microbial symbionts are still poorly known. Here we characterized the dynamics of phenotypic adaptation and its underlying genetic bases during the experimental evolution of a plant pathogenic bacterium into a legume symbiont. We observed that fast adaptation was predominantly driven by selection acting on competitiveness for host entry, which outweighed selection acting on within-host proliferation. Whole-population sequencing of evolved bacteria revealed that phenotypic adaptation was supported by the continuous accumulation of new mutations and the sequential sweeps of cohorts of mutations with similar temporal trajectories. The identification of adaptive mutations within the fixed mutational cohorts showed that all of them improved competitiveness for host entry, while only a subset of those also improved within host proliferation. Computer simulations predict that this effect emerges from the presence of a strong selective bottleneck at host entry. Together, these results show how selective bottlenecks can alter the relative influence of selective pressures acting during bacterial adaptation to multistep infection processes.</jats:p

Spatial distribution of genotypes of <i>M. bovis</i> isolated from multiple hosts.

<p>Spatial distribution of genotypes of <i>M. bovis</i> isolated from multiple hosts.</p

Definition of “F4-family”.

<p>(A) Geographical location: in the map, shades of grey indicate the percentage of isolates of the“F4 family” spoligotypes from the total <i>M</i>. <i>bovis</i> population, showing a principal localization in the South of France. (B) Spoligotype particularity: the chosen spoligotype, SB0818, the likely common ancestor of this family, is characterized by classic <i>M</i>. <i>bovis</i> missing spacers 3, 9, 16, and 39 to 43 plus the absence of the spacer 33, specific to the “F4-family”. (C) VNTR characteristics: “F4-family” possesses a truncated repetition (30 bp instead of 56 bp) in locus 4052, which is counted for the allele definition. In the example, other strains possess five repetitions but in the case of “F4 family”, the fifth repetition is truncated and is represented as a “5s”.</p

Minimum spanning tree (MST) of the 2,332 <i>Mycobacterium bovis</i> isolates between 1978 and 2013.

<p>706 genotypes were obtained by combination of spoligotyping and VNTR typing. The colored nodes show the principal families of <i>M</i>. <i>bovis</i>, SB0120 (Pink), SB0121 (Blue), SB0134 (Green) and “F4-family” (Grey). Distances between nodes represent distances between profiles.</p

Comparative phylotranscriptomics reveals a 110 million years-old symbiotic program

Summary Symbiotic interactions have structured past and present ecosystems and shaped the evolution of life. As any trait, the symbiotic state observed in extant species builds on ancestral and conserved features, and lineage-specific innovations. From these mixed origins, defining the ancestral state of symbiotic associations is challenging although it is instrumental for understanding how symbiotic abilities emerge from non-symbiotic states. Here we aimed at reconstructing the intermediate steps leading to the root-nodule nitrogen-fixing symbiosis (RNS) observed in some extant flowering plants. For this, we compared the transcriptomic responses of nine host plants in response to symbiotic bacteria. We included the mimosoid legume Mimosa pudica for which we assembled a chromosome-level genome and generated the transcriptomic response to experimentally evolved bacterial symbionts. With this dataset, we reconstructed the ancestral RNS transcriptome, composed of most already described symbiotic genes together with hundreds of novel candidates. We found that the response to the chemical signals produced by the symbiont, nodule organogenesis and nitrogen-fixation are predominantly linked to ancestral responses, although these traits have diversified in the different nitrogen-fixing lineages. We detected a clear signature of recent and convergent evolution for the ability to release intracellular symbiosomes in two legume lineages, exemplified by the expression of different classes of small proteins in each group, potentially leading to the convergent gain of symbiotic evolutionary stability. Our findings demonstrate that most of the novelties for RNS were mostly in place in the most recent common ancestor of the RNS-forming species that lived on Earth 110 million years ago. Graphical abstract A little graphical/nice phylogeny with nodes of interest Highlights We sequenced a high-quality genome of the Mimosoideae Mimosa pudica The nitrogen-fixing root-nodule symbiosis relies on an ancestral transcriptomic response All symbiotic traits involve genes of the ancestral symbiotic program Symbiont perception, nodule organogenesis and nitrogen-fixation are essentially ancestral processes Convergent evolution of intracellular accommodation of symbionts additionally involves lineage-specific gene

Detailed spoligotypes: number of strains, discrimination by MLVA MIRU-VNTR typing.

<p>Among the 154 MIRU-VNTR genotypes identified between 1978 and 2013, two which account for more than 52% of the totality stand out (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117103#pone.0117103.t002" target="_blank">Table 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117103#pone.0117103.s002" target="_blank">S1A Fig.</a>): profile-5 3 5 3 9 4 5 6, mainly present in Dordogne and neighboring regions (South-west), and profile-5 5 4 3 11 4 5 6, specifically located in Côte d’Or (Center-east) albeit being widely represented in the last period.</p><p>Detailed spoligotypes: number of strains, discrimination by MLVA MIRU-VNTR typing.</p

Geographical distribution of endemic outbreaks of bovine tuberculosis in France.

<p>Color intensity reflects the percentage of <i>M</i>. <i>bovis</i> strains isolated in each “department” compared to the totality of <i>M</i>. <i>bovis</i> isolated in the whole territory for the given period of time. The first map represents the 377 <i>M</i>. <i>bovis</i> strains isolated in France from 1978 to 1990. The second map represents the 1,176 <i>M</i>. <i>bovis</i> strains isolated in France from 1991 to 2000. The third map represents the 3,101 <i>M</i>. <i>bovis</i> isolated in France from 2001 to 2013.</p