Title: A microbiome engineering framework to evaluate rhizobial symbionts of legumes
Abstract Background For well over a century, rhizobia have been recognized as effective biofertilizer options for legume crops. This has led to the widespread use of rhizobial inoculants in agricultural systems, but a recurring issue has emerged: applied rhizobia struggle to provide growth benefits to legume crops. This has largely been attributed to the presence of soil rhizobia and has been termed the ‘rhizobial competition problem.’ Scope Microbiome engineering has emerged as a methodology to circumvent the rhizobial competition problem by creating legume microbiomes that do not require exogenous rhizobia. However, we highlight an alternative implementation of microbiome engineering that focuses on untangling the complexities of the symbiosis that contribute to the rhizobial competition problem. We outline three approaches that use different starting inocula to test hypotheses to overcome the rhizobial competition problem. Conclusions The approaches we suggest are targeted at various stages of the legume-rhizobium symbiosis and will help us uncover underlying molecular mechanisms that contribute to the rhizobial competition problem. We conclude with an integrative perspective of these different approaches and suggest a path forward for future research on legumes and their complex microbiome. more »« less
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.
Liu, J; Yu, X; Qin, Q; Dinkins, Randy D; Zhu, H.
(, Frontiers in genetics)
Legumes are the second most important family of crop plants. One defining feature of legumes is their unique ability to establish a nitrogen-fixing root nodule symbiosis with soil bacteria known as rhizobia. Since domestication from their wild relatives, crop legumes have been under intensive breeding to improve yield and other agronomic traits but with little attention paid to the belowground symbiosis traits. Theoretical models predict that domestication and breeding processes, coupled with high‐input agricultural practices, might have reduced the capacity of crop legumes to achieve their full potential of nitrogen fixation symbiosis. Testing this prediction requires characterizing symbiosis traits in wild and breeding populations under both natural and cultivated environments using genetic, genomic, and ecological approaches. However, very few experimental studies have been dedicated to this area of research. Here, we review how legumes regulate their interactions with soil rhizobia and how domestication, breeding and agricultural practices might have affected nodulation capacity, nitrogen fixation efficiency, and the composition and function of rhizobial community. We also provide a perspective on how to improve legume-rhizobial symbiosis in sustainable agricultural systems.
Weisberg, Alexandra J.; Rahman, Arafat; Backus, Dakota; Tyavanagimatt, Parinita; Chang, Jeff H.; Sachs, Joel L.
(, 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.
Ghosh, Prithwi; Adolphsen, Katie N.; Yurgel, Svetlana N.; Kahn, Michael L.
(, Applied and Environmental Microbiology)
Stabb, Eric V.
(Ed.)
ABSTRACT Some soil bacteria, called rhizobia, can interact symbiotically with legumes, in which they form nodules on the plant roots, where they can reduce atmospheric dinitrogen to ammonia, a form of nitrogen that can be used by growing plants. Rhizobium-plant combinations can differ in how successful this symbiosis is: for example, Sinorhizobium meliloti Rm1021 forms a relatively ineffective symbiosis with Medicago truncatula Jemalong A17, but Sinorhizobium medicae WSM419 is able to support more vigorous plant growth. Using proteomic data from free-living and symbiotic S. medicae WSM419, we previously identified a subset of proteins that were not closely related to any S. meliloti Rm1021 proteins and speculated that adding one or more of these proteins to S. meliloti Rm1021 would increase its effectiveness on M. truncatula A17. Three genes, Smed_3503, Smed_5985, and Smed_6456, were cloned into S. meliloti Rm1021 downstream of the E. coli lacZ promoter. Strains with these genes increased nodulation and improved plant growth, individually and in combination with one another. Smed_3503, renamed iseA ( i ncreased s ymbiotic e ffectiveness), had the largest impact, increasing M. truncatula biomass by 61%. iseA homologs were present in all currently sequenced S. medicae strains but were infrequent in other Sinorhizobium isolates. Rhizobium leguminosarum bv. viciae 3841 containing iseA led to more nodules on pea and lentil. Split-root experiments with M. truncatula A17 indicated that S. meliloti Rm1021 carrying the S. medicae iseA is less sensitive to plant-induced resistance to rhizobial infection, suggesting an interaction with the plant’s regulation of nodule formation. IMPORTANCE Legume symbiosis with rhizobia is highly specific. Rhizobia that can nodulate and fix nitrogen on one legume species are often unable to associate with a different species. The interaction can be more subtle. Symbiotically enhanced growth of the host plant can differ substantially when nodules are formed by different rhizobial isolates of a species, much like disease severity can differ when conspecific isolates of pathogenic bacteria infect different cultivars. Much is known about bacterial genes essential for a productive symbiosis, but less is understood about genes that marginally improve performance. We used a proteomic strategy to identify Sinorhizobium genes that contribute to plant growth differences that are seen when two different strains nodulate M. truncatula A17. These genes could also alter the symbiosis between R. leguminosarum bv. viciae 3841 and pea or lentil, suggesting that this approach identifies new genes that may more generally contribute to symbiotic productivity.
Hill, Caleb A; McMullen, John G; Lennon, Jay T
(, bioRxiv)
1 Abstract Mutualisms evolve over time when individuals belonging to different species derive fitness benefits through the exchange of resources and services. Although prevalent in natural and managed ecosystems, mutualisms can be destabilized by environmental fluctuations that alter the costs and benefits of maintaining the symbiosis. In the rhizobia-legume mutualism, bacteria provide reduced nitrogen to the host plant in exchange for photosynthates that support bacterial metabolism. However, this relationship can be disrupted by the addition of external nitrogen sources to the soil, such as fertilizers. While the molecular mechanisms underpinning the rhizobia-legume symbiosis are well-characterized, the genome-wide fitness effects of nitrogen enrichment on symbiotic rhizobia are less clear. Here, we inoculated a randomly barcoded transposon-site sequencing (RB-TnSeq) library of the bacteriumEnsifer(Sinorhizobium)melilotiinto soils containing a host plant, alfalfa (Medicago sativa), under conditions of low and high nitrogen availability. Although plant performance remained robust to fertilization, nitrogen enrichment altered gene fitness for specific traits and functions in the rhizobial partner. Genes involved in carbohydrate metabolism showed increased fitness irrespective of soil nutrient content, whereas fitness gains in quorum-sensing genes were only observed in high-nitrogen environments. We also documented reductions in the fitness of nucleotide metabolism and cell-growth genes, while genes from oxidative phosphorylation and various amino-acid biosynthesis pathways were detrimental to fitness under elevated soil nitrogen, underscoring the complex trade-offs in rhizobial responses to nutrient enrichment. Our experimental functional genomics approach identified gene functions and pathways across allE. melilotireplicons that may be associated with the disruption of an agronomically important mutualism. 2ImportanceUnderstanding the evolutionary dynamics of the rhizobia-legume mutualism is important for elucidating how plant-soil-microbe interactions operate in natural and managed ecosystems. Legumes constitute a significant portion of global food production and generate 25% of all terrestrially fixed nitrogen. The application of chemical fertilizers can disrupt the mutualism by altering the selective pressures experienced by symbiotic rhizobia, potentially affecting gene fitness throughout the microbial genome and leading to the evolution of less productive or cooperative mutualists. To investigate how exogenous nitrogen inputs influence gene fitness during the complex rhizobial lifecycle, we used a barcoded genome-wide mutagenesis screen to quantify gene-level fitness across the rhizobial genome during symbiosis and identify metabolic functions affected by nitrogen enrichment. Our findings provide genomic insight into potential eco-evolutionary mechanisms by which symbioses are maintained or degraded over time in response to changing environmental conditions.
Quides, Kenjiro W., and Atamian, Hagop S. A microbiome engineering framework to evaluate rhizobial symbionts of legumes. Retrieved from https://par.nsf.gov/biblio/10294575. Plant and Soil 463.1-2 Web. doi:10.1007/s11104-021-04892-2.
Quides, Kenjiro W., & Atamian, Hagop S. A microbiome engineering framework to evaluate rhizobial symbionts of legumes. Plant and Soil, 463 (1-2). Retrieved from https://par.nsf.gov/biblio/10294575. https://doi.org/10.1007/s11104-021-04892-2
@article{osti_10294575,
place = {Country unknown/Code not available},
title = {A microbiome engineering framework to evaluate rhizobial symbionts of legumes},
url = {https://par.nsf.gov/biblio/10294575},
DOI = {10.1007/s11104-021-04892-2},
abstractNote = {Abstract Background For well over a century, rhizobia have been recognized as effective biofertilizer options for legume crops. This has led to the widespread use of rhizobial inoculants in agricultural systems, but a recurring issue has emerged: applied rhizobia struggle to provide growth benefits to legume crops. This has largely been attributed to the presence of soil rhizobia and has been termed the ‘rhizobial competition problem.’ Scope Microbiome engineering has emerged as a methodology to circumvent the rhizobial competition problem by creating legume microbiomes that do not require exogenous rhizobia. However, we highlight an alternative implementation of microbiome engineering that focuses on untangling the complexities of the symbiosis that contribute to the rhizobial competition problem. We outline three approaches that use different starting inocula to test hypotheses to overcome the rhizobial competition problem. Conclusions The approaches we suggest are targeted at various stages of the legume-rhizobium symbiosis and will help us uncover underlying molecular mechanisms that contribute to the rhizobial competition problem. We conclude with an integrative perspective of these different approaches and suggest a path forward for future research on legumes and their complex microbiome.},
journal = {Plant and Soil},
volume = {463},
number = {1-2},
author = {Quides, Kenjiro W. and Atamian, Hagop S.},
editor = {null}
}
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