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1. Pangenome Evolution Reconciles Robustness and Instability of Rhizobial Symbiosis

   https://doi.org/10.1128/mbio.00074-22  

   Weisberg, Alexandra J.; Rahman, Arafat; Backus, Dakota; Tyavanagimatt, Parinita; Chang, Jeff H.; Sachs, Joel L. (June 2022, mBio)

   Cooper, Vaughn S. (Ed.)

   ABSTRACT Root nodulating rhizobia are nearly ubiquitous in soils and provide the critical service of nitrogen fixation to thousands of legume species, including staple crops. However, the magnitude of fixed nitrogen provided to hosts varies markedly among rhizobia strains, despite host legumes having mechanisms to selectively reward beneficial strains and to punish ones that do not fix sufficient nitrogen. Variation in the services of microbial mutualists is considered paradoxical given host mechanisms to select beneficial genotypes. Moreover, the recurrent evolution of non-fixing symbiont genotypes is predicted to destabilize symbiosis, but breakdown has rarely been observed. Here, we deconstructed hundreds of genome sequences from genotypically and phenotypically diverse Bradyrhizobium strains and revealed mechanisms that generate variation in symbiotic nitrogen fixation. We show that this trait is conferred by a modular system consisting of many extremely large integrative conjugative elements and few conjugative plasmids. Their transmissibility and propensity to reshuffle genes generate new combinations that lead to uncooperative genotypes and make individual partnerships unstable. We also demonstrate that these same properties extend beneficial associations to diverse host species and transfer symbiotic capacity among diverse strains. Hence, symbiotic nitrogen fixation is underpinned by modularity, which engenders flexibility, a feature that reconciles evolutionary robustness and instability. These results provide new insights into mechanisms driving the evolution of mobile genetic elements. Moreover, they yield a new predictive model on the evolution of rhizobial symbioses, one that informs on the health of organisms and ecosystems that are hosts to symbionts and that helps resolve the long-standing paradox. IMPORTANCE Genetic variation is fundamental to evolution yet is paradoxical in symbiosis. Symbionts exhibit extensive variation in the magnitude of services they provide despite hosts having mechanisms to select and increase the abundance of beneficial genotypes. Additionally, evolution of uncooperative symbiont genotypes is predicted to destabilize symbiosis, but breakdown has rarely been observed. We analyzed genome sequences of Bradyrhizobium, bacteria that in symbioses with legume hosts, fix nitrogen, a nutrient essential for ecosystems. We show that genes for symbiotic nitrogen fixation are within elements that can move between bacteria and reshuffle gene combinations that change host range and quality of symbiosis services. Consequently, nitrogen fixation is evolutionarily unstable for individual partnerships, but is evolutionarily stable for legume- Bradyrhizobium symbioses in general. We developed a holistic model of symbiosis evolution that reconciles robustness and instability of symbiosis and informs on applications of rhizobia in agricultural settings. 

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2. Host-Associated Rhizobial Fitness: Dependence on Nitrogen, Density, Community Complexity, and Legume Genotype

   https://doi.org/10.1128/aem.00526-22  

   Burghardt, Liana T.; Epstein, Brendan; Hoge, Michelle; Trujillo, Diana I.; Tiffin, Peter (August 2022, Applied and Environmental Microbiology)

   Glass, Jennifer B. (Ed.)

   ABSTRACT The environmental context of the nitrogen-fixing mutualism between leguminous plants and rhizobial bacteria varies over space and time. Variation in resource availability, population density, and composition likely affect the ecology and evolution of rhizobia and their symbiotic interactions with hosts. We examined how host genotype, nitrogen addition, rhizobial density, and community complexity affected selection on 68 rhizobial strains in the Sinorhizobium meliloti – Medicago truncatula mutualism. As expected, host genotype had a substantial effect on the size, number, and strain composition of root nodules (the symbiotic organ). The understudied environmental variable of rhizobial density had a stronger effect on nodule strain frequency than the addition of low nitrogen levels. Higher inoculum density resulted in a nodule community that was less diverse and more beneficial but only in the context of the more selective host genotype. Higher density resulted in more diverse and less beneficial nodule communities with the less selective host. Density effects on strain composition deserve additional scrutiny as they can create feedback between ecological and evolutionary processes. Finally, we found that relative strain rankings were stable across increasing community complexity (2, 3, 8, or 68 strains). This unexpected result suggests that higher-order interactions between strains are rare in the context of nodule formation and development. Our work highlights the importance of examining mechanisms of density-dependent strain fitness and developing theoretical predictions that incorporate density dependence. Furthermore, our results have translational relevance for overcoming establishment barriers in bioinoculants and motivating breeding programs that maintain beneficial plant-microbe interactions across diverse agroecological contexts. IMPORTANCE Legume crops establish beneficial associations with rhizobial bacteria that perform biological nitrogen fixation, providing nitrogen to plants without the economic and greenhouse gas emission costs of chemical nitrogen inputs. Here, we examine the influence of three environmental factors that vary in agricultural fields on strain relative fitness in nodules. In addition to manipulating nitrogen, we also use two biotic variables that have rarely been examined: the rhizobial community's density and complexity. Taken together, our results suggest that (i) breeding legume varieties that select beneficial strains despite environmental variation is possible, (ii) changes in rhizobial population densities that occur routinely in agricultural fields could drive evolutionary changes in rhizobial populations, and (iii) the lack of higher-order interactions between strains will allow the high-throughput assessments of rhizobia winners and losers during plant interactions. 

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3. Interspecific conflict and the evolution of ineffective rhizobia

   https://doi.org/10.1111/ele.13247  

   Gano‐Cohen, Kelsey A.; Wendlandt, Camille E.; Stokes, Peter J.; Blanton, Mia A.; Quides, Kenjiro W.; Zomorrodian, Avissa; Adinata, Eunice S.; Sachs, Joel L.; Johnson, ed., Nancy (March 2019, Ecology Letters)

   Abstract Microbial symbionts exhibit broad genotypic variation in their fitness effects on hosts, leaving hosts vulnerable to costly partnerships. Interspecific conflict and partner‐maladaptation are frameworks to explain this variation, with different implications for mutualism stability. We investigated the mutualist service of nitrogen fixation in a metapopulation of root‐nodule formingBradyrhizobiumsymbionts inAcmisponhosts. We uncoveredBradyrhizobiumgenotypes that provide negligible mutualist services to hosts and had superiorin plantafitness during clonal infections, consistent with cheater strains that destabilise mutualisms. Interspecific conflict was also confirmed at the metapopulation level – by a significant negative association between the fitness benefits provided byBradyrhizobiumgenotypes and their local genotype frequencies – indicating that selection favours cheating rhizobia. Legumes have mechanisms to defend against rhizobia that fail to fix sufficient nitrogen, but these data support predictions that rhizobia can subvert plant defenses and evolve to exploit hosts. 

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4. Comparative genomics reveals high rates of horizontal transfer and strong purifying selection on rhizobial symbiosis genes

   https://doi.org/doi.org/10.1098/rspb.2020.1804  

   Epstein, B; Tiffin, P (January 2021, Proceedings)

   null (Ed.)

   Horizontal transfer (HT) alters the repertoire of symbiosis genes in rhizobial genomes and may play an important role in the on-going evolution of the rhizobia–legume symbiosis. To gain insight into the extent of HT of symbiosis genes with different functional roles (nodulation, N-fixation, host benefit and rhizobial fitness), we conducted comparative genomic and selection analyses of the full-genome sequences from 27 rhizobial genomes. We find that symbiosis genes experience high rates of HT among rhizobial lineages but also bear signatures of purifying selection (low Ka : Ks). HT and purifying selection appear to be particularly strong in genes involved in initiating the symbiosis (e.g. nodulation) and in genome-wide association candidates for mediating benefits provided to the host. These patterns are consistent with rhizobia adapting to the host environment through the loss and gain of symbiosis genes, but not with host-imposed positive selection driving divergence of symbiosis genes through recurring bouts of positive selection. 

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5. Lotus japonicus regulates root nodulation and nitrogen fixation dependent on the molecular form of nitrogen fertilizer

   https://doi.org/10.1007/s11104-022-05762-1  

   Ortiz-Barbosa, G. S.; Torres-Martínez, L.; Rothschild, J.; Sachs, J. L. (November 2022, Plant and Soil)

   Purpose Legumes form root nodules to gain fixed nitrogen from rhizobia and can also access nitrogen in soil. Data suggest that plants might discriminate among these sources to optimize growth, but recogni- tion of symbiotically fixed nitrogen and its regulation remain poorly understood. Methods A greenhouse inoculation study manipu- lated the molecular form and concentration of nitro- gen available using two Lotus japonicus genotypes and the nitrogen-fixing symbiont, Mesorhizobium loti. Plants were supplied with sole organic and inorganic nitrogen sources to simulate forms that plants might receive from symbiotic nitrogen fixation or from the soil. Host benefit from and regulation of symbiosis was investigated by quantifying symbiotic trait varia- tion and isotopic analysis of nitrogen fixation. Results Host growth varied in response to fertili- zation with alanine, aspartic acid, ammonium, and nitrate, suggesting differences in catabolism effi- ciency. Net benefits of nodulation were reduced or eliminated under all forms of extrinsic fertilization. However, even when symbiosis imposed significant costs, hosts did not reduce investment into nodulation or nitrogen fixation when exposed to aspartic acid, unlike with other nitrogen sources. Conclusions L. japonicus can adaptively down- regulate investment into symbiosis in the presence of some but not all nitrogen sources. Failure to down- regulate any aspect of symbiosis in the presence of aspartic acid suggests that it might be jamming the main signal used by L. japonicus to detect nitrogen fixation. 

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