Abstract
Legumes preferentially associate with and reward beneficial rhizobia in root nodules, but the processes by which rhizobia evolve to provide benefits to novel hosts remain poorly understood. Using cycles of in planta and in vitro evolution, we experimentally simulated lifestyles where rhizobia repeatedly interact with novel plant genotypes with which they initially provide negligible benefits. Using a full-factorial replicated design, we independently evolved two rhizobia strains in associations with each of two Lotus japonicus genotypes that vary in regulation of nodule formation. We evaluated phenotypic evolution of rhizobia by quantifying fitness, growth effects and histological features on hosts, and molecular evolution via genome resequencing. Rhizobia evolved enhanced host benefits and caused changes in nodule development in one of the four host–symbiont combinations, that appeared to be driven by reduced costs during symbiosis, rather than increased nitrogen fixation. Descendant populations included genetic changes that could alter rhizobial infection or proliferation in host tissues, but lack of evidence for fixation of these mutations weakens the results. Evolution of enhanced rhizobial benefits occurred only in a subset of experiments, suggesting a role for host–symbiont genotype interactions in mediating the evolution of enhanced benefits from symbionts.
Highlights
Plants interact with diverse soil microbes that can enhance their health and fitness [1,2]
We simulated an agronomic lifecycle in rhizobia where they recurrently interacted with the same L. japonicus genotypes, similar to settings where crops are replanted in fields over multiple seasons
Each rhizobia strain had a different deficiency in association with L. japonicus, and faced distinct hurdles to evolve enhanced benefits
Summary
Plants interact with diverse soil microbes that can enhance their health and fitness [1,2]. MAG is a relatively low nutrient medium, with 1 gm l−1 of yeast, or about 20% compared to rich media [31] This in vitro growth phase represents a free-living state experienced by rhizobia between host infections and allowed us to inoculate the round of hosts with a consistent number of cells every passage. To measure evolutionary changes in symbiont effectiveness on hosts, seedlings were planted in sterilized Conetainers (SC10; Steuwe and Sons) filled with autoclaved inert calcined clay (Pro League; Turface Athletics), and grown in a controlled facility with daily mist-watering until true leaves formed and thereafter were fertilized weekly with 5 ml of nitrogen-free Jensens solution. Each host genotype (MG-20, har1) received inoculation treatments, including the ancestral or derived rhizobial populations from each rhizobia strain (CE3, NGR; 5 × 108 cells in 5 ml, dripped directly onto soil) and experimental replicate (a, b), or water as a control. SnpEff v. 4.3t and the CFN 42 reference genome sequence were used to annotate variants for predicted functional effects [39]
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