Obtaining deeper insights into microbiome diversity using a simple method to block host and nontargets in amplicon sequencing

Abstract Profiling diverse microbiomes is revolutionizing our understanding of biological mechanisms and ecologically relevant problems, including metaorganism (host + microbiome) assembly, functions and adaptation. Amplicon sequencing of multiple conserved, phylogenetically informative loci has therefore become an instrumental tool for many researchers. Investigations in many systems are hindered, however, since essential sequencing depth can be lost by amplification of nontarget DNA from hosts or overabundant microorganisms. Here, we introduce “blocking oligos”, a low‐cost and flexible method using standard oligonucleotides to block amplification of diverse nontargets and software to aid their design. We apply them primarily in leaves, where exceptional challenges with host amplification prevail. A . thaliana ‐specific blocking oligos applied in eight different target loci reduce undesirable host amplification by up to 90%. To expand applicability, we designed universal 16S and 18S rRNA gene plant blocking oligos for targets that are conserved in diverse plant species and demonstrate that they efficiently block five plant species from five orders spanning monocots and dicots ( Bromus erectus , Plantago lanceolata , Lotus corniculatus , Amaranth sp., Arabidopsis thaliana ). These can increase alpha diversity discovery without biasing beta diversity patterns and do not compromise microbial load information inherent to plant‐derived 16S rRNA gene amplicon sequencing data. Finally, we designed and tested blocking oligos to avoid amplification of 18S rRNA genes of a sporulating oomycete pathogen, demonstrating their effectiveness in applications well beyond plants. Using these tools, we generated a survey of the A . thaliana leaf microbiome based on eight loci targeting bacterial, fungal, oomycete and other eukaryotic microorganisms and discuss complementarity of commonly used amplicon sequencing regions for describing leaf microbiota. This approach has potential to make questions in a variety of study systems more tractable by making amplicon sequencing more targeted, leading to deeper, systems‐based insights into microbial discovery. For fast and easy design for blocking oligos for any nontarget DNA in other study systems, we developed a publicly available R package

ER body-resident myrosinases and tryptophan specialized metabolism modulate root microbiota assembly

Summary Endoplasmic reticulum (ER) bodies are ER-derived structures that contain a large amount of PYK10 myrosinase, which hydrolyzes tryptophan (Trp)-derived indole glucosinolates (IGs). Given the well-described role of IGs in root–microbe interactions, we hypothesized that ER bodies in roots are important for interaction with soil-borne microbes at the root–soil interface. We used mutants impaired in ER bodies (nai1), ER body-resident myrosinases (pyk10bglu21), IG biosynthesis (myb34/51/122), and Trp specialized metabolism (cyp79b2b3) to profile their root microbiota community in natural soil, evaluate the impact of axenically collected root exudates on soil or synthetic microbial communities, and test their response to fungal endophytes in a mono-association setup. Tested mutants exhibited altered bacterial and fungal communities in rhizoplane and endosphere, respectively. Natural soils and bacterial synthetic communities treated with mutant root exudates exhibited distinctive microbial profiles from those treated with wild-type (WT) exudates. Most tested endophytes severely restricted the growth of cyp79b2b3, a part of which also impaired the growth of pyk10bglu21. Our results suggest that root ER bodies and their resident myrosinases modulate the profile of root-secreted metabolites and thereby influence root–microbiota interactions

Microbiota-root-shoot-environment axis and stress tolerance in plants

Reminiscent to the microbiota-gut-brain axis described in animals, recent advances indicate that plants can take advantage of belowground microbial commensals to orchestrate aboveground stress responses. Integration of plant responses to microbial cues belowground and environmental cues aboveground emerges as a mechanism that promotes stress tolerance in plants. Using recent examples obtained from reductionist and community-level approaches, we discuss the extent to which perception of aboveground biotic and abiotic stresses can cascade along the shoot-root axis to sculpt root microbiota assembly and modulate the growth of root commensals that bolster aboveground stress tolerance. We propose that host modulation of microbiota-root-shoot circuits contributes to phenotypic plasticity and decision making in plants, thereby promoting adaptation to rapidly changing environmental conditions

Microbial Systems Ecology to Understand Cross-Feeding in Microbiomes

International audienceUnderstanding how microorganism-microorganism interactions shape microbial assemblages is a key to deciphering the evolution of dependencies and co-existence in complex microbiomes. Metabolic dependencies in cross-feeding exist in microbial communities and can at least partially determine microbial community composition. To parry the complexity and experimental limitations caused by the large number of possible interactions, new concepts from systems biology aim to decipher how the components of a system interact with each other. The idea that cross-feeding does impact microbiome assemblages has developed both theoretically and empirically, following a systems biology framework applied to microbial communities, formalized as microbial systems ecology (MSE) and relying on integrated-omics data. This framework merges cellular and community scales and offers new avenues to untangle microbial coexistence primarily by metabolic modeling, one of the main approaches used for mechanistic studies. In this mini-review, we first give a concise explanation of microbial cross-feeding. We then discuss how MSE can enable progress in microbial research. Finally, we provide an overview of a MSE framework mostly based on genome-scale metabolic-network reconstruction that combines top-down and bottom-up approaches to assess the molecular mechanisms of deterministic processes of microbial community assembly that is particularly suitable for use in synthetic biology and microbiome engineering

A microbiota-root-shoot circuit favours Arabidopsis growth over defence under suboptimal light

A synthetic root microbial community rescues weak growth under low light and enhances immunity in Arabidopsis. Transcription factor MYC2 regulates both this coordination between rhizosphere and shoots and the growth/defence trade-off under low light conditions. Bidirectional root-shoot signalling is probably key in orchestrating stress responses and ensuring plant survival. Here, we show that Arabidopsis thaliana responses to microbial root commensals and light are interconnected along a microbiota-root-shoot axis. Microbiota and light manipulation experiments in a gnotobiotic plant system reveal that low photosynthetically active radiation perceived by leaves induces long-distance modulation of root bacterial communities but not fungal or oomycete communities. Reciprocally, microbial commensals alleviate plant growth deficiency under low photosynthetically active radiation. This growth rescue was associated with reduced microbiota-induced aboveground defence responses and altered resistance to foliar pathogens compared with the control light condition. Inspection of a set of A. thaliana mutants reveals that this microbiota- and light-dependent growth-defence trade-off is directly explained by belowground bacterial community composition and requires the host transcriptional regulator MYC2. Our work indicates that aboveground stress responses in plants can be modulated by signals from microbial root commensals

Microbial Interkingdom Interactions in Roots Promote Arabidopsis Survival

Roots of healthy plants are inhabited by soil-derived bacteria, fungi, and oomycetes that have evolved independently in distinct kingdoms of life. How these microorganisms interact and to what extent those interactions affect plant health are poorly understood. We examined root-associated microbial communities from three Arabidopsis thaliana populations and detected mostly negative correlations between bacteria and filamentous microbial eukaryotes. We established microbial culture collections for reconstitution experiments using germ-free A. thaliana. In plants inoculated with mono- or multi-kingdom synthetic microbial consortia, we observed a profound impact of the bacterial root microbiota on fungal and oomycetal community structure and diversity. We demonstrate that the bacterial microbiota is essential for plant survival and protection against root-derived filamentous eukaryotes. Deconvolution of 2,862 binary bacterial-fungal interactions ex situ, combined with community perturbation experiments in planta, indicate that biocontrol activity of bacterial root commensals is a redundant trait that maintains microbial interkingdom balance for plant health

Root-Associated Bacterial and Fungal Community Profiles of Arabidopsis thaliana Are Robust Across Contrasting Soil P Levels

Plant survival depends on the ability of roots to sense and acquire nutrients in soils, which harbor a rich diversity of microbes. A subset of this microcosm interacts with plant roots and collectively forms root-associated microbial communities, termed the root microbiota. Under phosphorus-limiting conditions, some plants can engage in mutualistic interactions, for example with arbuscular mycorrhizal fungi. Here, we describe how Arabidopsis thaliana, which lacks the genetic capacity for establishing the aforementioned symbiosis, interacts with soil-resident bacteria and fungi in soil from a long-term phosphorus fertilization trial. Long-term, contrasting fertilization regimes resulted in an similar to 6-fold and similar to 2.4-fold disparity in bioavailable and total phosphorous, respectively, which may explain differences in biomass of A. thaliana plants. Sequencing of marker genes enabled us to characterize bacterial and fungal communities present in the bulk soil, rhizosphere, and root compartments. Phosphorus had little effect on alpha- or beta-diversity indices, but more strongly influences bacterial and fungal community shifts in plant-associated compartments compared with bulk soil. The significant impact of soil P abundance could only be resolved at operational taxonomic unit level, and these subtle differences are more pronounced in the root compartment. We conclude that despite decades of different fertilization, both bacterial and fungal soil communities remained unexpectedly stable in soils tested, suggesting that the soil biota is resilient over time to nutrient supplementation. Conversely, low-abundance, root-associated microbes, which collectively represent 2 to 3% of the relative abundance of bacteria and fungi in the roots, exhibited a subtle, yet significant shift between the two soils

Root microbiota assembly and adaptive differentiation among European Arabidopsis populations

Across large spatial scales, climate is more important than soil conditions for plant adaptation and variation in root-associated filamentous eukaryotic communities. Factors that drive continental-scale variation in root microbiota and plant adaptation are poorly understood. We monitored root-associated microbial communities in Arabidopsis thaliana and co-occurring grasses at 17 European sites across 3 years. We observed strong geographic structuring of the soil biome, but not of the root microbiota. A few phylogenetically diverse and geographically widespread bacteria consistently colonized plant roots. Among-site and across-year similarity in microbial community composition was stronger for the bacterial root microbiota than for filamentous eukaryotes. In a reciprocal transplant between two A. thaliana populations in Sweden and Italy, we uncoupled soil from location effects and tested their contributions to root microbiota variation and plant adaptation. Community differentiation in plant roots was explained primarily by location for filamentous eukaryotes and by soil origin for bacteria, whereas host genotype effects were marginal. Strong local adaptation between the two A. thaliana populations was observed, with differences in soil properties and microbes of little importance for the observed magnitude of adaptive differentiation. Our results suggest that, across large spatial scales, climate is more important than soil conditions for plant adaptation and variation in root-associated filamentous eukaryotic communities, whereas soil properties are primary drivers of bacterial community differentiation in roots

Root Endophyte Colletotrichum tofieldiae Confers Plant Fitness Benefits that Are Phosphate Status Dependent

A staggering diversity of endophytic fungi associate with healthy plants in nature, but it is usually unclear whether these represent stochastic encounters or provide host fitness benefits. Although most characterized species of the fungal genus Colletotrichum are destructive pathogens, we show here that C. tofieldiae (Ct) is an endemic endophyte in natural Arabidopsis thaliana populations in central Spain. Colonization by Ct initiates in roots but can also spread systemically into shoots. Ct transfers the macronutrient phosphorus to shoots, promotes plant growth, and increases fertility only under phosphorus-deficient conditions, a nutrient status that might have facilitated the transition from pathogenic to beneficial lifestyles. The host's phosphate starvation response (PSR) system controls Ct root colonization and is needed for plant growth promotion (PGP). PGP also requires PEN2-dependent indole glucosinolate metabolism, a component of innate immune responses, indicating a functional link between innate immunity and the PSR system during beneficial interactions with Ct