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The rhizosphere constitutes a dynamic interface between plant hosts and their associated microbial communities. Despite the acknowledged potential for enhancing plant fitness by manipulating the rhizosphere, the engineering of the rhizosphere microbiome through inoculation has posed significant challenges. These challenges are thought to arise from the competitive microbial ecosystem where introduced microbes must survive, and the absence of adaptation to the specific metabolic and environmental demands of the rhizosphere. Here, we engineered a synthetic rhizosphere community (SRC1) with the anticipation that it would exhibit a selective advantage in colonizing the host Sorghum bicolor, thereby potentially fostering its growth. SRC1 was assembled from bacterial isolates identified either for their potential role in community cohesion through network analysis or for their ability to benefit from host-specific exudate compounds. The growth performance of SRC1 was assessed in vitro on solid media, in planta under gnotobiotic laboratory conditions, and in the field. Our findings reveal that SRC1 cohesion is most robust when cultivated in the presence of the plant host under laboratory conditions, with lineages being lost from the community when grown either in vitro or in a native field setting. We establish that SRC1 effectively promotes the growth of both above- and below-ground plant phenotypes in both laboratory and native field contexts. Furthermore, in laboratory conditions, these growth enhancements correlate with the transcriptional dampening of lignin biosynthesis in the host. Collectively, these results underscore the potential utility of synthetic microbial communities for modulating crop performance in controlled and native environments alike., (Published by Oxford University Press on behalf of the International Society for Microbial Ecology 2024.)

Plant response to drought stress involves fungi and bacteria that live on and in plants and in the rhizosphere, yet the stability of these myco- and micro-biomes remains poorly understood. We investigate the resistance and resilience of fungi and bacteria to drought in an agricultural system using both community composition and microbial associations. Here we show that tests of the fundamental hypotheses that fungi, as compared to bacteria, are (i) more resistant to drought stress but (ii) less resilient when rewetting relieves the stress, found robust support at the level of community composition. Results were more complex using all-correlations and co-occurrence networks. In general, drought disrupts microbial networks based on significant positive correlations among bacteria, among fungi, and between bacteria and fungi. Surprisingly, co-occurrence networks among functional guilds of rhizosphere fungi and leaf bacteria were strengthened by drought, and the same was seen for networks involving arbuscular mycorrhizal fungi in the rhizosphere. We also found support for the stress gradient hypothesis because drought increased the relative frequency of positive correlations., (© 2022. The Author(s).)

The microbiome plays an important role in shaping plant growth and immunity, but few plant genes and pathways impacting plant microbiome composition have been reported. In Arabidopsis thaliana, the phosphate starvation response (PSR) was recently found to modulate the root microbiome upon phosphate (Pi) starvation through the transcriptional regulator PHR1. Here, we report that A. thaliana PHR1 directly binds to the promoters of rapid alkalinization factor (RALF) genes, and activates their expression under phosphate-starvation conditions. RALFs in turn suppress complex formation of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) receptor through FERONIA, a previously-identified PTI modulator that increases resistance to certain detrimental microorganisms. Suppression of immunity via the PHR1-RALF-FERONIA axis allows colonization by specialized root microbiota that help to alleviate phosphate starvation by upregulating the expression of PSR genes. These findings provide a new paradigm for coordination of host-microbe homeostasis through modulating plant innate immunity after environmental perturbations., (© 2022 The Authors.)

Plants form intimate associations with microorganisms, and these associations are directly impacted by the host genotype. However, identifying specific host genetic pathways that influence these microbial interactions has proved challenging. Genome-wide association-based approaches that use features of microbiome composition as a quantitative trait represent a novel and underutilized strategy to identify such pathways. Several recent studies have demonstrated the potential utility of plant microbiome-based genome-wide association studies (GWAS). In this chapter, we describe the process of implementing GWAS using the plant microbiome as the primary quantitative trait, considering experimental design, sample harvest, and processing, but with an emphasis on data filtering, data normalization, and statistical analyses., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Host genetics has recently been shown to be a driver of plant microbiome composition. However, identifying the underlying genetic loci controlling microbial selection remains challenging. Genome-wide association studies (GWAS) represent a potentially powerful, unbiased method to identify microbes sensitive to the host genotype and to connect them with the genetic loci that influence their colonization. Here, we conducted a population-level microbiome analysis of the rhizospheres of 200 sorghum genotypes. Using 16S rRNA amplicon sequencing, we identify rhizosphere-associated bacteria exhibiting heritable associations with plant genotype, and identify significant overlap between these lineages and heritable taxa recently identified in maize. Furthermore, we demonstrate that GWAS can identify host loci that correlate with the abundance of specific subsets of the rhizosphere microbiome. Finally, we demonstrate that these results can be used to predict rhizosphere microbiome structure for an independent panel of sorghum genotypes based solely on knowledge of host genotypic information., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Recent studies have demonstrated that drought leads to dramatic, highly conserved shifts in the root microbiome. At present, the molecular mechanisms underlying these responses remain largely uncharacterized. Here we employ genome-resolved metagenomics and comparative genomics to demonstrate that carbohydrate and secondary metabolite transport functionalities are overrepresented within drought-enriched taxa. These data also reveal that bacterial iron transport and metabolism functionality is highly correlated with drought enrichment. Using time-series root RNA-Seq data, we demonstrate that iron homeostasis within the root is impacted by drought stress, and that loss of a plant phytosiderophore iron transporter impacts microbial community composition, leading to significant increases in the drought-enriched lineage, Actinobacteria. Finally, we show that exogenous application of iron disrupts the drought-induced enrichment of Actinobacteria, as well as their improvement in host phenotype during drought stress. Collectively, our findings implicate iron metabolism in the root microbiome's response to drought and may inform efforts to improve plant drought tolerance to increase food security.

Host-microbiome interactions are recognized for their importance to host health. An improved understanding of the molecular underpinnings of host-microbiome relationships will advance our capacity to accurately predict host fitness and manipulate interaction outcomes. Within the plant microbiome research field, unlocking the functional relationships between plants and their microbial partners is the next step to effectively using the microbiome to improve plant fitness. We propose that strategies that pair host and microbial datasets-referred to here as holo-omics-provide a powerful approach for hypothesis development and advancement in this area. We discuss several experimental design considerations and present a case study to highlight the potential for holo-omics to generate a more holistic perspective of molecular networks within the plant microbiome system. In addition, we discuss the biggest challenges for conducting holo-omics studies; specifically, the lack of vetted analytical frameworks, publicly available tools, and required technical expertise to process and integrate heterogeneous data. Finally, we conclude with a perspective on appropriate use-cases for holo-omics studies, the need for downstream validation, and new experimental techniques that hold promise for the plant microbiome research field. We argue that utilizing a holo-omics approach to characterize host-microbiome interactions can provide important opportunities for broadening system-level understandings and significantly inform microbial approaches to improving host health and fitness. Video abstract.

While numerous studies implicate the microbiome in host fitness, contributions of host evolution to microbial recruitment remain largely uncharacterized. Past work has shown that plant polyploidy and domestication can influence plant biotic and abiotic interactions, yet impacts on broader microbiome assembly are still unknown for many crop species. In this study, we utilized three approaches-two field studies and one greenhouse-based experiment-to determine the degree to which patterns in bacterial community assembly in wheat (Triticum sp.) roots and rhizospheres are attributable to the host factors of ploidy level (2n, 4n, 6n) and domestication status (cultivated vs. wild). Profiling belowground bacterial communities with 16S rRNA gene amplicon sequencing, we analyzed patterns in diversity and composition. From our initial analyses of a subsetted dataset, we observed that host ploidy level was statistically significant in explaining variation in alpha and beta diversity for rhizosphere microbiomes, as well as correlated with distinct phylum-level shifts in composition, in the field. Using a reduced complexity field soil inoculum and controlled greenhouse conditions, we found some evidence suggesting that genomic lineage and ploidy level influence root alpha and beta diversity (p-value<0.05). However, in a follow-up field experiment using an expanded set of Triticum genomes that included both wild and domesticated varieties, we did not find a strong signal for either diploid genome lineages, domestication status, or ploidy level in shaping rhizosphere bacterial communities. Taken together, these results suggest that while host ploidy and domestication may have some minor influence on microbial assembly, these impacts are subtle and difficult to assess in belowground compartments for wheat varieties. By improving our understanding of the degree to which host ploidy and cultivation factors shape the plant microbiome, this research informs perspectives on what key driving forces may underlie microbiome structuring, as well as where future efforts may be best directed towards fortifying plant growth by microbial means. The greatest influence of the host on the wheat microbiome appeared to occur in the rhizosphere compartment, and we suggest that future work focuses on this environment to further characterize how host genomic and phenotypic changes influence plant-microbe communications., Competing Interests: The authors have declared that no competing interests exist.

Experimental drought has been shown to delay the development of the root microbiome and increase the relative abundance of Actinobacteria, however, the generalizability of these findings to natural systems or other diverse plant hosts remains unknown. Bacterial cell wall thickness and growth morphology (e.g., filamentous or unicellular) have been proposed as traits that may mediate bacterial responses to environmental drivers. Leveraging a natural gradient of water-availability across the coast redwood (Sequoia sempervirens) range, we tested three hypotheses: (a) that site-specific water-availability is an important predictor of bacterial community composition for redwood roots and rhizosphere soils; (b) that there is relative enrichment of Actinobacteria and other monoderm bacterial groups within the redwood microbiome in response to drier conditions; and (c) that bacterial growth morphology is an important predictor of bacteria response to water-availability, where filamentous taxa will become more dominant at drier sites compared to unicellular bacteria. We find that both α- and β-diversity of redwood bacterial communities is partially explained by water-availability and that Actinobacterial enrichment is a conserved response of land plants to water-deficit. Further, we highlight how the trend of Actinobacterial enrichment in the redwood system is largely driven by the Actinomycetales. We propose bacterial growth morphology (filamentous vs. unicellular) as an additional mechanism behind the increase in Actinomycetales with increasing aridity. A trait-based approach including cell-wall thickness and growth morphology may explain the distribution of bacterial taxa across environmental gradients and help to predict patterns of bacterial community composition for a wide range of host plants., (© 2020 John Wiley & Sons Ltd.)

Despite growing interest in utilizing microbial-based methods for improving crop growth, much work still remains in elucidating how beneficial plant-microbe associations are established, and what role soil amendments play in shaping these interactions. Here, we describe a set of experiments that test the effect of a commercially available soil amendment, VESTA, on the soil and strawberry (Fragaria x ananassa Monterey) root bacterial microbiome. The bacterial communities of the soil, rhizosphere, and root from amendment-treated and untreated fields were profiled at four time points across the strawberry growing season using 16S rRNA gene amplicon sequencing on the Illumina MiSeq platform. In all sample types, bacterial community composition and relative abundance were significantly altered with amendment application. Importantly, time point effects on composition are more pronounced in the root and rhizosphere, suggesting an interaction between plant development and treatment effect. Surprisingly, there was slight overlap between the taxa within the amendment and those enriched in plant and soil following treatment, suggesting that VESTA may act to rewire existing networks of organisms through an, as of yet, uncharacterized mechanism. These findings demonstrate that a commercial microbial soil amendment can impact the bacterial community structure of both roots and the surrounding environment.

Plant-associated microbial communities contribute to host fitness, and perturbations in the plant microbiome can have a major impact on plant health. Drought has recently been shown to lead to enrichment of monoderm bacteria within the roots of many plant species across many environments. However, the underlying causes of this shift, and the consequences for plant fitness, remain largely unexplored. We present the hypotheses that drought-induced shifts in plant metabolism may be responsible for the observed monoderm enrichment, and that increased monoderm abundance may promote increased drought tolerance in the host. Finally, we discuss how these recent discoveries may inform ongoing efforts to use microbially mediated strategies to improve crop productivity., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Drought stress is a major obstacle to crop productivity, and the severity and frequency of drought are expected to increase in the coming century. Certain root-associated bacteria have been shown to mitigate the negative effects of drought stress on plant growth, and manipulation of the crop microbiome is an emerging strategy for overcoming drought stress in agricultural systems, yet the effect of drought on the development of the root microbiome is poorly understood. Through 16S rRNA amplicon and metatranscriptome sequencing, as well as root metabolomics, we demonstrate that drought delays the development of the early sorghum root microbiome and causes increased abundance and activity of monoderm bacteria, which lack an outer cell membrane and contain thick cell walls. Our data suggest that altered plant metabolism and increased activity of bacterial ATP-binding cassette (ABC) transporter genes are correlated with these shifts in community composition. Finally, inoculation experiments with monoderm isolates indicate that increased colonization of the root during drought can positively impact plant growth. Collectively, these results demonstrate the role that drought plays in restructuring the root microbiome and highlight the importance of temporal sampling when studying plant-associated microbiomes., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Root endophytes have been shown to have important roles in determining host fitness under periods of drought stress, and yet the effect of drought on the broader root endosphere bacterial community remains largely uncharacterized. In this study, we present phylogenetic profiles of bacterial communities associated with drought-treated root and rhizosphere tissues of 18 species of plants with varying degrees of drought tolerance belonging to the Poaceae family, including important crop plants. Through 16S rRNA gene profiling across two distinct watering regimes and two developmental time points, we demonstrate that there is a strong correlation between host phylogenetic distance and the microbiome dissimilarity within root tissues, and that drought weakens this correlation by inducing conserved shifts in bacterial community composition. We identify a significant enrichment in a wide variety of Actinobacteria during drought within the roots of all hosts, and demonstrate that this enrichment is higher within the root than it is in the surrounding environments. Furthermore, we show that this observed enrichment is the result of an absolute increase in Actinobacterial abundance and that previously hypothesized mechanisms for observed enrichments in Actinobacteria in drought-treated soils are unlikely to fully account for the phenomena observed here within the plant root.

Motivation: A key component of the analysis of microbiome datasets is the identification of OTUs shared between multiple experimental conditions, commonly referred to as the core microbiome., Results: We present a web platform named MetaCoMET that enables the discovery and visualization of the core microbiome and provides a comparison of the relative abundance and diversity patterns between subsets of samples within a microbiome dataset. MetaCoMET provides an efficient and interactive graphical interface for analyzing each subset defined by the union or disjunction of groups within the Venn diagram, and includes a graphical taxonomy summary, alpha diversity metrics, Principal Coordinate analysis, abundance-based heatmaps, and a chart indicating the geographic distribution of each sample., Availability and Implementation: MetaCoMET is a user-friendly and efficient web platform freely accessible at http://probes.pw.usda.gov/MetaCoMET or http://aegilops.wheat.ucdavis.edu/MetaCoMET CONTACT: devin.coleman-derr@ars.usda.govSupplementary information: Supplementary data are available at Bioinformatics online., (Published by Oxford University Press 2016. This work is written by US Government employees and is in the public domain in the US.)

Desert plants are hypothesized to survive the environmental stress inherent to these regions in part thanks to symbioses with microorganisms, and yet these microbial species, the communities they form, and the forces that influence them are poorly understood. Here we report the first comprehensive investigation of the microbial communities associated with species of Agave, which are native to semiarid and arid regions of Central and North America and are emerging as biofuel feedstocks. We examined prokaryotic and fungal communities in the rhizosphere, phyllosphere, leaf and root endosphere, as well as proximal and distal soil samples from cultivated and native agaves, through Illumina amplicon sequencing. Phylogenetic profiling revealed that the composition of prokaryotic communities was primarily determined by the plant compartment, whereas the composition of fungal communities was mainly influenced by the biogeography of the host species. Cultivated A. tequilana exhibited lower levels of prokaryotic diversity compared with native agaves, although no differences in microbial diversity were found in the endosphere. Agaves shared core prokaryotic and fungal taxa known to promote plant growth and confer tolerance to abiotic stress, which suggests common principles underpinning Agave-microbe interactions., (No claim to US Government works. New Phytologist © 2015 New Phytologist Trust.)