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The capacity of beneficial microbes to compete for host infection-and the ability of hosts to discriminate among them-introduces evolutionary conflict that is predicted to destabilize mutualism. We investigated fitness outcomes in associations between legumes and their symbiotic rhizobia to characterize fitness impacts of microbial competition. Diverse Bradyrhizobium strains varying in their capacity to fix nitrogen symbiotically with a common host plant, Acmispon strigosus, were tested in full-factorial coinoculation experiments involving 28 pairwise strain combinations. We analyzed the effects of interstrain competition and host discrimination on symbiotic-interaction outcomes by relativizing fitness proxies to clonally infected and uninfected controls. More than one thousand root nodules of coinoculated plants were genotyped to quantify strain occupancy, and the Bradyrhizobium strain genome sequences were analyzed to uncover the genetic bases of interstrain competition outcomes. Strikingly, interstrain competition favored a fast-growing, minimally beneficial rhizobia strain. Host benefits were significantly diminished in coinoculation treatments relative to expectations from clonally inoculated controls, consistent with competitive interference among rhizobia that reduced both nodulation and plant growth. Competition traits appear polygenic, linked with inter-strain allelopathic interactions in the rhizosphere. This study confirms that competition among strains can destabilize mutualism by favoring microbes that are superior in colonizing host tissues but provide minimal benefits to host plants. Moreover, our findings help resolve the paradox that despite efficient host control post infection, legumes nonetheless encounter rhizobia that vary in their nitrogen fixation.

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.

Acquisition of mobile genetic elements can confer novel traits to bacteria. Some integrative and conjugative elements confer upon members of Bradyrhizobium the capacity to fix nitrogen in symbiosis with legumes. These so-called symbiosis integrative conjugative elements (symICEs) can be extremely large and vary as monopartite and polypartite configurations within chromosomes of related strains. These features are predicted to impose fitness costs and have defied explanation. Here, we show that chromosome architecture is largely conserved despite diversity in genome composition, variations in locations of attachment sites recognized by integrases of symICEs, and differences in large-scale chromosomal changes that occur upon integration. Conversely, many simulated nonnative chromosome-symICE combinations are predicted to result in lethal deletions or disruptions to architecture. Findings suggest that there is compatibility between chromosomes and symICEs. We hypothesize that the size and structural flexibility of symICEs are important for generating combinations that maintain chromosome architecture across a genus of nitrogen-fixing bacteria with diverse and dynamic genomes.

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.

Specialization in mutualisms is thought to be a major driver of diversification, but few studies have explored how novel specialization evolves, or its relation to the evolution of other niche axes. A fundamental question is whether generalist interactions evolve to become more specialized (i.e., oscillation hypothesis) or if partner switches evolve without any change in niche breadth (i.e., musical chairs hypothesis). We examined alternative models for the evolution of specialization by estimating the mutualistic, climatic, and edaphic niche breadths of sister plant species, combining phylogenetic, environmental, and experimental data on Acmispon strigosus and Acmispon wrangelianus genotypes across their overlapping ranges in California. We found that specialization along all three niche axes was asymmetric across species, such that the species with broader climatic and edaphic niches, Acmispon strigosus, was also able to gain benefit from and invest in associating with a broader set of microbial mutualists. Our data are consistent with the oscillation model of specialization, and a parallel narrowing of the edaphic, climatic, and mutualistic dimensions of the host species niche. Our findings provide novel evidence that the evolution of specialization in mutualism is accompanied by specialization in other niche dimensions.

Bacterial mutualists generate major fitness benefits for eukaryotes, reshaping the host phenotype and its interactions with the environment. Yet, microbial mutualist populations are predicted to generate mutants that defect from providing costly services to hosts while maintaining the capacity to exploit host resources. Here, we examined the mutualist service of symbiotic nitrogen fixation in a metapopulation of root-nodulating Bradyrhizobium spp. that associate with the native legume Acmispon strigosus. We quantified mutualism traits of 85 Bradyrhizobium isolates gathered from a 700 km transect in California spanning 10 sampled A. strigosus populations. We clonally inoculated each Bradyrhizobium isolate onto A. strigosus hosts and quantified nodulation capacity and net effects of infection, including host growth and isotopic nitrogen concentration. Six Bradyrhizobium isolates from five populations were categorized as ineffective because they formed nodules but did not enhance host growth via nitrogen fixation. Six additional isolates from three populations failed to form root nodules. Phylogenetic reconstruction inferred two types of mutualism breakdown, including three to four independent losses of effectiveness and five losses of nodulation capacity on A. strigosus. The evolutionary and genomic drivers of these mutualism breakdown events remain poorly understood.

Invasive microbes causing diseases such as sudden oak death negatively affect ecosystems and economies around the world. The deployment of resistant genotypes for combating introduced diseases typically relies on breeding programs that can take decades to complete. To demonstrate how this process can be accelerated, we employed a genome-wide association mapping of ca 1,000 resequenced Populus trichocarpa trees individually challenged with Sphaerulina musiva, an invasive fungal pathogen. Among significant associations, three loci associated with resistance were identified and predicted to encode one putative membrane-bound L-type receptor-like kinase and two receptor-like proteins. A susceptibility-associated locus was predicted to encode a putative G-type D-mannose-binding receptor-like kinase. Multiple lines of evidence, including allele analysis, transcriptomics, binding assays, and overexpression, support the hypothesized function of these candidate genes in the P. trichocarpa response to S. musiva.

Plants and animals detect biomolecules termed microbe-associated molecular patterns (MAMPs) and induce immunity. Agricultural production is severely impacted by pathogens which can be controlled by transferring immune receptors. However, most studies use a single MAMP epitope and the impact of diverse multicopy MAMPs on immune induction is unknown. Here, we characterized the epitope landscape from five proteinaceous MAMPs across 4,228 plant-associated bacterial genomes. Despite the diversity sampled, natural variation was constrained and experimentally testable. Immune perception in both Arabidopsis and tomato depended on both epitope sequence and copy number variation. For example, Elongation Factor Tu is predominantly single copy, and 92% of its epitopes are immunogenic. Conversely, 99.9% of bacterial genomes contain multiple cold shock proteins, and 46% carry a nonimmunogenic form. We uncovered a mechanism for immune evasion, intrabacterial antagonism, where a nonimmunogenic cold shock protein blocks perception of immunogenic forms encoded in the same genome. These data will lay the foundation for immune receptor deployment and engineering based on natural variation. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is a foodborne pathogen of concern in dairy processing facilities, with the potential to cause human illness and trigger regulatory actions if found in the product. Monitoring for Listeria spp. through environmental sampling is recommended to prevent establishment of these microorganisms in dairy processing environments, thereby reducing the risk of product contamination. To inform on L. monocytogenes diversity and transmission, we analyzed genome sequences of L. monocytogenes strains (n = 88) obtained through the British Columbia Dairy Inspection Program. Strains were recovered from five different dairy processing facilities over a 10 year period (2007-2017). Analysis of whole genome sequences (WGS) grouped the isolates into nine sequence types and 11 cgMLST types (CT). The majority of isolates (93%) belonged to lineage II. Within each CT, single nucleotide polymorphism (SNP) differences ranged from 0 to 237 between isolates. A highly similar (0-16 SNPs) cluster of over 60 isolates, collected over 9 years within one facility (#71), was identified suggesting a possible persistent population. Analyses of genome content revealed a low frequency of genes associated with stress tolerance, with the exception of widely disseminated cadmium resistance genes cadA1 and cadA2. The distribution of virulence genes and mutations within internalin genes varied across the isolates and facilities. Further studies are needed to elucidate their phenotypic effect on pathogenicity and stress response. These findings demonstrate the diversity of L. monocytogenes isolates across dairy facilities in the same region. Findings also showed the utility of using WGS to discern potential persistence events within a single facility over time. [ABSTRACT FROM AUTHOR]

Members of the agrobacteria-rhizobia complex (ARC) have multiple and diverse plasmids. The extent to which these plasmids are shared and the consequences of their interactions are not well understood. We extracted over 4000 plasmid sequences from 1251 genome sequences and constructed a network to reveal interactions that have shaped the evolutionary histories of oncogenic virulence plasmids. One newly discovered type of oncogenic plasmid is a mosaic with three incomplete, but complementary and partially redundant virulence loci. Some types of oncogenic plasmids recombined with accessory plasmids or acquired large regions not known to be associated with pathogenicity. We also identified two classes of partial virulence plasmids. One class is potentially capable of transforming plants, but not inciting disease symptoms. Another class is inferred to be incomplete and non-functional but can be found as coresidents of the same strain and together are predicted to confer pathogenicity. The modularity and capacity for some plasmids to be transmitted broadly allow them to diversify, convergently evolve adaptive plasmids and shape the evolution of genomes across much of the ARC. [ABSTRACT FROM AUTHOR]