A Selective Bottleneck During Host Entry Drives the Evolution of New Legume Symbionts.

During the emergence of new host-microbe symbioses, microbial fitness results from the ability to complete the different steps of symbiotic life cycles, where each step imposes specific selective pressures. However, the relative contribution of these different selective pressures to the adaptive trajectories of microbial symbionts is still poorly known. Here, we characterized the dynamics of phenotypic adaptation to a simplified symbiotic life cycle during the experimental evolution of a plant pathogenic bacterium into a legume symbiont. We observed that fast adaptation was predominantly explained by improved competitiveness for host entry, which outweighed adaptation to within-host proliferation. Whole-population sequencing of bacteria at regular time intervals along this evolution experiment revealed the continuous accumulation of new mutations (fuelled by a transient hypermutagenesis phase occurring at each cycle before host entry, a phenomenon described in previous work) and sequential sweeps of cohorts of mutations with similar temporal trajectories. The identification of adaptive mutations within the fixed mutational cohorts showed that several adaptive mutations can co-occur in the same cohort. Moreover, all adaptive mutations 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 occurring before within-host proliferation and just after the hypermutagenesis phase in the rhizosphere. Together, these results show how selective bottlenecks can alter the relative influence of selective pressures acting during bacterial adaptation to multistep infection processes.

Experimental Evolution of Legume Symbionts: What Have We Learnt?

Rhizobia, the nitrogen-fixing symbionts of legumes, are polyphyletic bacteria distributed in many alpha- and beta-proteobacterial genera. They likely emerged and diversified through independent horizontal transfers of key symbiotic genes. To replay the evolution of a new rhizobium genus under laboratory conditions, the symbiotic plasmid of Cupriavidus taiwanensis was introduced in the plant pathogen Ralstonia solanacearum, and the generated proto-rhizobium was submitted to repeated inoculations to the C. taiwanensis host, Mimosa pudica L.. This experiment validated a two-step evolutionary scenario of key symbiotic gene acquisition followed by genome remodeling under plant selection. Nodulation and nodule cell infection were obtained and optimized mainly via the rewiring of regulatory circuits of the recipient bacterium. Symbiotic adaptation was shown to be accelerated by the activity of a mutagenesis cassette conserved in most rhizobia. Investigating mutated genes led us to identify new components of R. solanacearum virulence and C. taiwanensis symbiosis. Nitrogen fixation was not acquired in our short experiment. However, we showed that post-infection sanctions allowed the increase in frequency of nitrogen-fixing variants among a non-fixing population in the M. pudica-C. taiwanensis system and likely allowed the spread of this trait in natura. Experimental evolution thus provided new insights into rhizobium biology and evolution.

Spatio-temporal control of mutualism in legumes helps spread symbiotic nitrogen fixation.

Mutualism is of fundamental importance in ecosystems. Which factors help to keep the relationship mutually beneficial and evolutionarily successful is a central question. We addressed this issue for one of the most significant mutualistic interactions on Earth, which associates plants of the leguminosae family and hundreds of nitrogen (N2)-fixing bacterial species. Here we analyze the spatio-temporal dynamics of fixers and non-fixers along the symbiotic process in the Cupriavidus taiwanensis-Mimosa pudica system. N2-fixing symbionts progressively outcompete isogenic non-fixers within root nodules, where N2-fixation occurs, even when they share the same nodule. Numerical simulations, supported by experimental validation, predict that rare fixers will invade a population dominated by non-fixing bacteria during serial nodulation cycles with a probability that is function of initial inoculum, plant population size and nodulation cycle length. Our findings provide insights into the selective forces and ecological factors that may have driven the spread of the N2-fixation mutualistic trait.