Plant myo-inositol transport influences bacterial colonization phenotypes.

Plant microbiomes are assembled and modified through a complex milieu of biotic and abiotic factors. Despite dynamic and fluctuating contributing variables, specific host metabolites are consistently identified as important mediators of microbial interactions. We combine information from a large-scale metatranscriptomic dataset from natural poplar trees and experimental genetic manipulation assays in seedlings of the model plant Arabidopsis thaliana to converge on a conserved role for transport of the plant metabolite myo-inositol in mediating host-microbe interactions. While microbial catabolism of this compound has been linked to increased host colonization, we identify bacterial phenotypes that occur in both catabolism-dependent and -independent manners, suggesting that myo-inositol may additionally serve as a eukaryotic-derived signaling molecule to modulate microbial activities. Our data suggest host control of this compound and resulting microbial behavior are important mechanisms at play surrounding the host metabolite myo-inositol.

Optimization of Molecular Methods for Detecting Duckweed-Associated Bacteria.

The bacterial colonization dynamics of plants can differ between phylogenetically similar bacterial strains and in the context of complex bacterial communities. Quantitative methods that can resolve closely related bacteria within complex communities can lead to a better understanding of plant-microbe interactions. However, current methods often lack the specificity to differentiate phylogenetically similar bacterial strains. In this study, we describe molecular strategies to study duckweed-associated bacteria. We first systematically optimized a bead-beating protocol to co-isolate nucleic acids simultaneously from duckweed and bacteria. We then developed a generic fingerprinting assay to detect bacteria present in duckweed samples. To detect specific duckweed-bacterium associations, we developed a genomics-based computational pipeline to generate bacterial strain-specific primers. These strain-specific primers differentiated bacterial strains from the same genus and enabled the detection of specific duckweed-bacterium associations present in a community context. Moreover, we used these strain-specific primers to quantify the bacterial colonization of duckweed by normalization to a plant reference gene and revealed differences in colonization levels between strains from the same genus. Lastly, confocal microscopy of inoculated duckweed further supported our PCR results and showed bacterial colonization of the duckweed root-frond interface and root interior. The molecular methods introduced in this work should enable the tracking and quantification of specific plant-microbe associations within plant-microbial communities.

Additive genetic effects in interacting species jointly determine the outcome of caterpillar herbivory.

Plant-insect interactions are common and important in basic and applied biology. Trait and genetic variation can affect the outcome and evolution of these interactions, but the relative contributions of plant and insect genetic variation and how these interact remain unclear and are rarely subject to assessment in the same experimental context. Here, we address this knowledge gap using a recent host-range expansion onto alfalfa by the Melissa blue butterfly. Common garden rearing experiments and genomic data show that caterpillar performance depends on plant and insect genetic variation, with insect genetics contributing to performance earlier in development and plant genetics later. Our models of performance based on caterpillar genetics retained predictive power when applied to a second common garden. Much of the plant genetic effect could be explained by heritable variation in plant phytochemicals, especially saponins, peptides, and phosphatidyl cholines, providing a possible mechanistic understanding of variation in the species interaction. We find evidence of polygenic, mostly additive effects within and between species, with consistent effects of plant genotype on growth and development across multiple butterfly species. Our results inform theories of plant-insect coevolution and the evolution of diet breadth in herbivorous insects and other host-specific parasites.

A continental-scale survey of Wolbachia infections in blue butterflies reveals evidence of interspecific transfer and invasion dynamics.

Infections by maternally inherited bacterial endosymbionts, especially Wolbachia, are common in insects and other invertebrates but infection dynamics across species ranges are largely under studied. Specifically, we lack a broad understanding of the origin of Wolbachia infections in novel hosts, and the historical and geographical dynamics of infections that are critical for identifying the factors governing their spread. We used Genotype-by-Sequencing data from previous population genomics studies for range-wide surveys of Wolbachia presence and genetic diversity in North American butterflies of the genus Lycaeides. As few as one sequence read identified by assembly to a Wolbachia reference genome provided high accuracy in detecting infections in host butterflies as determined by confirmatory PCR tests, and maximum accuracy was achieved with a threshold of only 5 sequence reads per host individual. Using this threshold, we detected Wolbachia in all but 2 of the 107 sampling localities spanning the continent, with infection frequencies within populations ranging from 0% to 100% of individuals, but with most localities having high infection frequencies (mean = 91% infection rate). Three major lineages of Wolbachia were identified as separate strains that appear to represent 3 separate invasions of Lycaeides butterflies by Wolbachia. Overall, we found extensive evidence for acquisition of Wolbachia through interspecific transfer between host lineages. Strain wLycC was confined to a single butterfly taxon, hybrid lineages derived from it, and closely adjacent populations in other taxa. While the other 2 strains were detected throughout the rest of the continent, strain wLycB almost always co-occurred with wLycA. Our demographic modeling suggests wLycB is a recent invasion. Within strain wLycA, the 2 most frequent haplotypes are confined almost exclusively to separate butterfly taxa with haplotype A1 observed largely in Lycaeides melissa and haplotype A2 observed most often in Lycaeides idas localities, consistent with either cladogenic mode of infection acquisition from a common ancestor or by hybridization and accompanying mutation. More than 1 major Wolbachia strain was observed in 15 localities. These results demonstrate the utility of using resequencing data from hosts to quantify Wolbachia genetic variation and infection frequency and provide evidence of multiple colonizations of novel hosts through hybridization between butterfly lineages and complex dynamics between Wolbachia strains.

Auxin-Producing Bacteria from Duckweeds Have Different Colonization Patterns and Effects on Plant Morphology.

The role of auxin in plant-microbe interaction has primarily been studied using indole-3-acetic acid (IAA)-producing pathogenic or plant-growth-promoting bacteria. However, the IAA biosynthesis pathway in bacteria involves indole-related compounds (IRCs) and intermediates with less known functions. Here, we seek to understand changes in plant response to multiple plant-associated bacteria taxa and strains that differ in their ability to produce IRCs. We had previously studied 47 bacterial strains isolated from several duckweed species and determined that 79% of these strains produced IRCs in culture, such as IAA, indole lactic acid (ILA), and indole. Using Arabidopsis thaliana as our model plant with excellent genetic tools, we performed binary association assays on a subset of these strains to evaluate morphological responses in the plant host and the mode of bacterial colonization. Of the 21 tested strains, only four high-quantity IAA-producing Microbacterium strains caused an auxin root phenotype. Compared to the commonly used colorimetric Salkowski assay, auxin concentration determined by LC-MS was a superior indicator of a bacteria's ability to cause an auxin root phenotype. Studies with the auxin response mutant axr1-3 provided further genetic support for the role of auxin signaling in mediating the root morphology response to IAA-producing bacteria strains. Interestingly, our microscopy results also revealed new evidence for the role of the conserved AXR1 gene in endophytic colonization of IAA-producing Azospirillum baldaniorum Sp245 via the guard cells.

Bacterial communities of the Salvia lyrata rhizosphere explained by spatial structure and sampling grain.

Advancements in molecular technology have reduced the constraints that the grain of observation, or the spatial resolution and volume of the sampling unit, has on the characterization of plant-associated microbiomes. With discrete ecological sampling and massive parallel sequencing, we can more precisely portray microbiome community assembly and microbial recruitment to host tissue over space and time. Here, we differentiate rarefied community richness and relative abundance in bacterial microbiomes of Salvia lyrata dependent on three spatial depths, which are discrete physical distances from the soil surface within the rhizosphere microhabitat as a proxy for the root system zones. To assess the impact of sampling grain on rarefied community richness and relative abundance, we evaluated the variation of these metrics between samples pooled prior to DNA extraction and samples pooled after sequencing. A distance-based redundancy analysis with the quantitative Jaccard distance revealed that rhizosphere microbiomes vary in richness between rhizosphere soil depths. At all orders of diversity, rarefied microbial richness was consistently lowest at the deepest samples taken (approximately 4 cm from soil surface) in comparison with other rhizosphere soil depths. We additionally show that finer grain sampling (i.e., three samples of equal volume pooled after sequencing) recovers greater microbial richness when using 16S rRNA gene sequencing to describe microbial communities found within the rhizosphere system. In summary, to further elucidate the extent host-specific microbiomes assemble within the rhizosphere, the grain at which bacterial communities are sampled should reflect and encompass fine-scale heterogeneity of the system.

Duckweed hosts a taxonomically similar bacterial assemblage as the terrestrial leaf microbiome.

Culture-independent characterization of microbial communities associated with popular plant model systems have increased our understanding of the plant microbiome. However, the integration of other model systems, such as duckweed, could facilitate our understanding of plant microbiota assembly and evolution. Duckweeds are floating aquatic plants with many characteristics, including small size and reduced plant architecture, that suggest their use as a facile model system for plant microbiome studies. Here, we investigated the structure and assembly of the duckweed bacterial microbiome. First, a culture-independent survey of the duckweed bacterial microbiome from different locations in New Jersey revealed similar phylogenetic profiles. These studies showed that Proteobacteria is a dominant phylum in the duckweed bacterial microbiome. To observe the assembly dynamics of the duckweed bacterial community, we inoculated quasi-gnotobiotic duckweed with wastewater effluent from a municipal wastewater treatment plant. Our results revealed that duckweed strongly shapes its bacterial microbiome and forms distinct associations with bacterial community members from the initial inoculum. Additionally, these inoculation studies showed the bacterial communities of different duckweed species were similar in taxa composition and abundance. Analysis across the different duckweed bacterial communities collected in this study identified a set of "core" bacterial taxa consistently present on duckweed irrespective of the locale and context. Furthermore, comparison of the duckweed bacterial community to that of rice and Arabidopsis revealed a conserved taxonomic structure between the duckweed microbiome and the terrestrial leaf microbiome. Our results suggest that duckweeds utilize similar bacterial community assembly principles as those found in terrestrial plants and indicate a highly conserved structuring effect of leaf tissue on the plant microbiome.

mSphere of Influence: Peering through a Keyhole into the Unseen World.

Sarah Lebeis studies the assembly and function of plant microbiomes. In this mSphere of Influence article, she reflects on how the paper "Functional Overlap of the Arabidopsis Leaf and Root Microbiota" (Y. Bai, D. B. Müller, G. Srinivas, R. Garrido-Oter, et al., Nature 528:364-369, 2015, https://doi.org/10.1038/nature16192) provided a roadmap for how large culture collections composed of well-characterized bacterial isolates provide essential resources to test hypotheses concerning microbial communities.

Bridging the Gap Between Single-Strain and Community-Level Plant-Microbe Chemical Interactions.

Although the influence of microbiomes on the health of plant hosts is evident, specific mechanisms shaping the structure and dynamics of microbial communities in the phyllosphere and rhizosphere are only beginning to become clear. Traditionally, plant-microbe interactions have been studied using cultured microbial isolates and plant hosts but the rising use of 'omics tools provides novel snapshots of the total complex community in situ. Here, we discuss the recent advances in tools and techniques used to monitor plant-microbe interactions and the chemical signals that influence these relationships in above- and belowground tissues. Particularly, we highlight advances in integrated microscopy that allow observation of the chemical exchange between individual plant and microbial cells, as well as high-throughput, culture-independent approaches to investigate the total genetic and metabolic contribution of the community. The chemicals discussed have been identified as relevant signals across experimental spectrums. However, mechanistic insight into the specific interactions mediated by many of these chemicals requires further testing. Experimental designs that attempt to bridge the gap in biotic complexity between single strains and whole communities will advance our understanding of the chemical signals governing plant-microbe associations in the rhizosphere and phyllosphere.

Distinguishing nutrient-dependent plant driven bacterial colonization patterns in alfalfa.

To understand factors that influence the assembly of microbial communities, we inoculated Medicago sativa with a series of nested bacterial synthetic communities and grew plants in distinct nitrogen concentrations. Two isolates in our eight-member synthetic community, Williamsia sp. R60 and Pantoea sp. R4, consistently dominate community structure across nitrogen regimes. While Pantoea sp. R4 consistently colonizes plants to a higher degree compared to the other six organisms across each community and each nutrient level, Williamsia sp. R60 exhibits nutrient specific colonization differences. Williamsia sp. R60 is more abundant in plants grown at higher nitrogen concentrations, but exhibits the opposite trend when no plant is present, indicating plant-driven influence over colonization. Our research discovered unique, repeatable colonization phenotypes for individual microbes during plant microbiome assembly, and identified alterations caused by the host plant, microbes, and available nutrients.

Microbial Ecology: How to Fight the Establishment.

Creating microbial consortia capable of consistently producing desired qualities requires a detailed understanding of community interactions. A new paper demonstrates the role of historical contingency in Arabidopsis thaliana leaf-microbiome formation using an adaptable experimental approach, which could be applied to other host organisms.

Genome-Resolved Proteomic Stable Isotope Probing of Soil Microbial Communities Using 13CO2 and 13C-Methanol.

Stable isotope probing (SIP) enables tracking the nutrient flows from isotopically labeled substrates to specific microorganisms in microbial communities. In proteomic SIP, labeled proteins synthesized by the microbial consumers of labeled substrates are identified with a shotgun proteomics approach. Here, proteomic SIP was combined with targeted metagenomic binning to reconstruct metagenome-assembled genomes (MAGs) of the microorganisms producing labeled proteins. This approach was used to track carbon flows from 13CO2 to the rhizosphere communities of Zea mays, Triticum aestivum, and Arabidopsis thaliana. Rhizosphere microorganisms that assimilated plant-derived 13C were capable of metabolic and signaling interactions with their plant hosts, as shown by their MAGs containing genes for phytohormone modulation, quorum sensing, and transport and metabolism of nutrients typical of those found in root exudates. XoxF-type methanol dehydrogenases were among the most abundant proteins identified in the rhizosphere metaproteomes. 13C-methanol proteomic SIP was used to test the hypothesis that XoxF was used to metabolize and assimilate methanol in the rhizosphere. We detected 7 13C-labeled XoxF proteins and identified methylotrophic pathways in the MAGs of 8 13C-labeled microorganisms, which supported the hypothesis. These two studies demonstrated the capability of proteomic SIP for functional characterization of active microorganisms in complex microbial communities.

Soil indigenous microbiome and plant genotypes cooperatively modify soybean rhizosphere microbiome assembly.

BACKGROUND: Plants have evolved intimate interactions with soil microbes for a range of beneficial functions including nutrient acquisition, pathogen resistance and stress tolerance. Further understanding of this system is a promising way to advance sustainable agriculture by exploiting the versatile benefits offered by the plant microbiome. The rhizosphere is the interface between plant and soil, and functions as the first step of plant defense and root microbiome recruitment. It features a specialized microbial community, intensive microbe-plant and microbe-microbe interactions, and complex signal communication. To decipher the rhizosphere microbiome assembly of soybean (Glycine max), we comprehensively characterized the soybean rhizosphere microbial community using 16S rRNA gene sequencing and evaluated the structuring influence from both host genotype and soil source. RESULTS: Comparison of the soybean rhizosphere to bulk soil revealed significantly different microbiome composition, microbe-microbe interactions and metabolic capacity. Soil type and soybean genotype cooperatively modulated microbiome assembly with soil type predominantly shaping rhizosphere microbiome assembly while host genotype slightly tuned this recruitment process. The undomesticated progenitor species, Glycine soja, had higher rhizosphere diversity in both soil types tested in comparison to the domesticated soybean genotypes. Rhizobium, Novosphingobium, Phenylobacterium, Streptomyces, Nocardioides, etc. were robustly enriched in soybean rhizosphere irrespective of the soil tested. Co-occurrence network analysis revealed dominant soil type effects and genotype specific preferences for key microbe-microbe interactions. Functional prediction results demonstrated converged metabolic capacity in the soybean rhizosphere between soil types and among genotypes, with pathways related to xenobiotic degradation, plant-microbe interactions and nutrient transport being greatly enriched in the rhizosphere. CONCLUSION: This comprehensive comparison of the soybean microbiome between soil types and genotypes expands our understanding of rhizosphere microbe assembly in general and provides foundational information for soybean as a legume crop for this assembly process. The cooperative modulating role of the soil type and host genotype emphasizes the importance of integrated consideration of soil condition and plant genetic variability for future development and application of synthetic microbiomes. Additionally, the detection of the tuning role by soybean genotype in rhizosphere microbiome assembly provides a promising way for future breeding programs to integrate host traits participating in beneficial microbiota assembly.

Bacterial Production of Indole Related Compounds Reveals Their Role in Association Between Duckweeds and Endophytes.

Duckweed farming can be a sustainable practice for biofuel production, animal feed supplement, and wastewater treatment, although large scale production remains a challenge. Plant growth promoting bacteria (PGPB) have been shown to improve plant health by producing phytohormones such as auxin. While some of the mechanisms for plant growth promotion have been characterized in soil epiphytes, more work is necessary to understand how plants may select for bacterial endophytes that have the ability to provide an exogenous source of phytohormones such as auxin. We have isolated and characterized forty-seven potentially endophytic bacteria from surface-sterilized duckweed tissues and screened these bacterial strains for production of indole related compounds using the Salkowski colorimetric assay. Indole-3-acetic acid (IAA), indole-3-lactic acid (ILA), and indole produced by various bacterial isolates were verified by mass spectrometry. Using the Salkowski reagent, we found that 79% of the isolated bacterial strains from our collection may be capable of producing indole related compounds to various extents during in vitro growth. Of these bacteria that are producing indole related compounds, 19% are additionally producing indole. There is an apparent correlation between the type of indole related compound produced by a particular bacteria and the duckweed genus from which the bacterial strain is derived. These results suggest the possible association between different duckweed genera and endophytes that are producing distinct types of secondary metabolites. Understanding the role of indole related compounds during interaction between endophytes and the plant host may be useful to help design synthetic bacterial communities that could target specific or multiple species of duckweed in the future to sustainably enhance plant growth.

Genomic features of bacterial adaptation to plants.

Plants intimately associate with diverse bacteria. Plant-associated bacteria have ostensibly evolved genes that enable them to adapt to plant environments. However, the identities of such genes are mostly unknown, and their functions are poorly characterized. We sequenced 484 genomes of bacterial isolates from roots of Brassicaceae, poplar, and maize. We then compared 3,837 bacterial genomes to identify thousands of plant-associated gene clusters. Genomes of plant-associated bacteria encode more carbohydrate metabolism functions and fewer mobile elements than related non-plant-associated genomes do. We experimentally validated candidates from two sets of plant-associated genes: one involved in plant colonization, and the other serving in microbe-microbe competition between plant-associated bacteria. We also identified 64 plant-associated protein domains that potentially mimic plant domains; some are shared with plant-associated fungi and oomycetes. This work expands the genome-based understanding of plant-microbe interactions and provides potential leads for efficient and sustainable agriculture through microbiome engineering.

PLANT MICROBIOME. Salicylic acid modulates colonization of the root microbiome by specific bacterial taxa.

Immune systems distinguish "self" from "nonself" to maintain homeostasis and must differentially gate access to allow colonization by potentially beneficial, nonpathogenic microbes. Plant roots grow within extremely diverse soil microbial communities but assemble a taxonomically limited root-associated microbiome. We grew isogenic Arabidopsis thaliana mutants with altered immune systems in a wild soil and also in recolonization experiments with a synthetic bacterial community. We established that biosynthesis of, and signaling dependent on, the foliar defense phytohormone salicylic acid is required to assemble a normal root microbiome. Salicylic acid modulates colonization of the root by specific bacterial families. Thus, plant immune signaling drives selection from the available microbial communities to sculpt the root microbiome.

Exercising influence: distinct biotic interactions shape root microbiomes.

Root microbiomes are formed from diverse microbial soil settings with extraordinary consistency, suggesting both defined mechanisms of assembly and specific microbial activity. Recent improvements in sequencing technologies, data analysis techniques, and study design, allow definition of the microbiota within these intimate and important relationships with increasing accuracy. Comparing datasets provides powerful insights into the overlap of plant microbiomes, as well as the impacts of surrounding plants and microbes on root microbiomes and long-term soil conditioning. Here we address how recent studies tease apart the impact of various biotic interactions, including: plant-plant, plant-microbe, and microbe-microbe on root microbiome composition.

Microbiota and Host Nutrition across Plant and Animal Kingdoms.

Plants and animals each have evolved specialized organs dedicated to nutrient acquisition, and these harbor specific bacterial communities that extend the host's metabolic repertoire. Similar forces driving microbial community establishment in the gut and plant roots include diet/soil-type, host genotype, and immune system as well as microbe-microbe interactions. Here we show that there is no overlap of abundant bacterial taxa between the microbiotas of the mammalian gut and plant roots, whereas taxa overlap does exist between fish gut and plant root communities. A comparison of root and gut microbiota composition in multiple host species belonging to the same evolutionary lineage reveals host phylogenetic signals in both eukaryotic kingdoms. The reasons underlying striking differences in microbiota composition in independently evolved, yet functionally related, organs in plants and animals remain unclear but might include differences in start inoculum and niche-specific factors such as oxygen levels, temperature, pH, and organic carbon availability.

Greater than the sum of their parts: characterizing plant microbiomes at the community-level.

Specific subsets of microbes are capable of assembly into plant-associated communities that influence the fitness of both the host and the microbes. While there is a large spectrum of plant phenotypes cause by microbes, the microbial community members benefit from living in protected and nutrient rich plant-associated environments. Recent advances in '-omics' technologies have provided researchers with the ability to identify and assign functions to even unculturable microbes inhabiting both above-ground and below-ground plant tissues. Thus, we are beginning to unravel the molecular mechanisms of microbiome assembly and activities that contribute to overall plant health, not only for individuals, but also at the community-level.