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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - HARNESS (Harnessing experimental evolution of rhizobia for an integrative view of endosymbiosis)

Teaser

Summary

Rhizobia are soil bacteria that can establish a mutualistic nitrogen-fixing symbiosis with leguminous plants. Their impact on agro-ecosystems is fundamental, since leguminous plants do not rely on synthetic nitrogen fertilizers for their growth. A better understanding of the functioning and evolution of these symbioses may thus help improve strategies favouring sustainable plant nitrogen nutrition.
In bacteria, the ability to perform symbiotic nitrogen fixation evolved several times independently via the acquisition of essential nodulation and nitrogen fixation genes by horizontal gene transfer (HGT), sometimes occurring between distantly related bacteria. The symbiotic capacity conferred by these genes may need to be optimized through genomic remodeling of the recipient â€˜proto-rhizobiaâ€™ strain. This optimisation occurs under plant-mediated selection and allows bacterial adaptation to their new ecological niche via the modification of metabolic or signalling pathways. To better understand the dynamics of genome remodeling in proto-rhizobia, an evolution experiment was launched in the host team to replay the evolution of a new rhizobium from a pathogenic ancestor. The symbiotic plasmid from the Î²-rhizobium Cupriavidus taiwanensis was transferred to the plant pathogen Ralstonia solanacearum. The resulting chimeric strain was evolved during successive inoculation-nodulation cycles on the plant Mimosa pudica, the natural host of C. taiwanensis. This protocol mimicked â€“on an accelerated basisâ€“ the alternation of ex planta and in planta periods that bacteria encounter in the wild, and provided an ecological framework to select bacteria with improved symbiotic phenotypes. Evolved bacteria thus constitute an ideal collection of strains to dissect the genetic bases of the stepwise acquisition of symbiotic properties.
Research carried on in this project aims at harnessing this evolution experiment in order to understand how bacteria adapt to life within plant cells, a defining but poorly understood aspect of nitrogen-fixing symbioses. Three distinct work-packages were designed to address the following questions. First, what are the genetic routes to the acquisition and amelioration of intracellularity (WP1)? Second, what are the bacterial functions involved in intracellular infection and persistence within plant cells (WP2)? Third, how do plant transcriptional responses adjust to accommodate bacterial strains that differ in their intracellular properties (WP3)?

Work performed

HARNESS was terminated prematurely 5 months after its beginning. Therefore, the full research plan could not be completed but the following results were obtained. I sequenced bacterial populations from two lineages which had been evolved for 35 cycles and identified genetic mutations that occurred during this experiment. I observed cohorts of mutations with similar evolutionary trajectories over successive cycles (see Figure). Some of these cohorts reached fixation at the end of the 35 cycles, suggesting the presence of adaptive mutation(s) within each of these cohorts. From these cohorts, I selected a list a candidate adaptive mutations that are currently being tested for their effect on symbiotic phenotypes. Moreover, I initiated the RNA-sequencing experiments that will allow the description of plant responses to intracellular bacterial infection. Mimosa pudica seedlings were infected with bacteria showing different infection phenotypes and nodules were collected at different times post-infection. Plant RNAs were extracted from these samples and have recently been sent for sequencing. The analysis of these data will provide a list of genes whose expression correlates with the different infection phenotypes. Finally, I also took advantage of this post-doctoral fellowship to strengthen my research network. I initiated a new collaboration with an evolutionary biologist from Toulouse, with expertise in the modelling of host-pathogen interaction. I attended and/or presented my work at several meetings, including the Labex Tulip meeting, a working-group dedicated to experimental evolution in south-western France, and the Symbifix meeting (national network of scientists interested in nitrogen-fixing symbioses)

Final results

Although fundamental for the functioning of nitrogen-fixing symbioses, the evolutionary steps that led to the acquisition of intracellular infection are poorly understood. Having replayed the evolution of intracellular infection in the laboratory, we are now in position to further analyze its molecular and selective bases. Ongoing RNA sequencing experiments should identify plant genes whose transcriptional activation allows or prevents intracellular bacterial infection.
Moreover, using whole-population sequencing, I identified all mutations present in two evolutionary lineages (beyong a threshold of 5% frequency) as well as their change in frequency along evolution cycles. Mutations that reach fixation often belong to cohorts containing multiple mutations (see Figure). The fitness effect of mutations comprised in these cohorts will be assessed experimentally in order to identify adaptive mutations. These data, which will be expanded by sequencing additional experimental populations in the future, will provide a better description of bacterial evolutionary dynamics in this system and may reveal new insights into the mechanisms and selective pressures controlling bacterial adaptation during symbiotic interactions with their host plants.
Together, this work will improve our understanding of the legume-rhizobium symbiosis, an important mutualistic interaction that provides a sustainable nitrogen source both in natural environments and in agricultural settings.