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Disentangling the contribution of climatic and edaphic factors to microbiome variation and local adaptation in plants requires an experimental approach to uncouple their effects and test for causality. We used microbial inocula, soil matrices and plant genotypes derived from two natural Arabidopsis thaliana populations in northern and southern Europe in an experiment conducted in climatic chambers mimicking seasonal changes in temperature, day length and light intensity of the home sites of the two genotypes. The southern A. thaliana genotype outperformed the northern genotype in the southern climate chamber, whereas the opposite was true in the northern climate chamber. Recipient soil matrix, but not microbial composition, affected plant fitness, and effects did not differ between genotypes. Differences between chambers significantly affected rhizosphere microbiome assembly, although these effects were small in comparison with the shifts induced by physicochemical differences between soil matrices. The results suggest that differences in seasonal changes in temperature, day length and light intensity between northern and southern Europe have strongly influenced adaptive differentiation between the two A. thaliana populations, whereas effects of differences in soil factors have been weak. By contrast, below-ground differences in soil characteristics were more important than differences in climate for rhizosphere microbiome differentiation.

Evaluating metagenomic software is key for optimizing metagenome interpretation and focus of the Initiative for the Critical Assessment of Metagenome Interpretation (CAMI). The CAMI II challenge engaged the community to assess methods on realistic and complex datasets with long- and short-read sequences, created computationally from around 1,700 new and known genomes, as well as 600 new plasmids and viruses. Here we analyze 5,002 results by 76 program versions. Substantial improvements were seen in assembly, some due to long-read data. Related strains still were challenging for assembly and genome recovery through binning, as was assembly quality for the latter. Profilers markedly matured, with taxon profilers and binners excelling at higher bacterial ranks, but underperforming for viruses and Archaea. Clinical pathogen detection results revealed a need to improve reproducibility. Runtime and memory usage analyses identified efficient programs, including top performers with other metrics. The results identify challenges and guide researchers in selecting methods for analyses.

Plant roots host diverse fungal communities that affect plant health. Here, Mesny et al. use comparative genomics and transcriptomics of fungal isolates from the Arabidopsis thaliana root mycobiota, together with root colonization assays, to identify genetic determinants of endophytism. The roots of Arabidopsis thaliana host diverse fungal communities that affect plant health and disease states. Here, we sequence the genomes of 41 fungal isolates representative of the A. thaliana root mycobiota for comparative analysis with other 79 plant-associated fungi. Our analyses indicate that root mycobiota members evolved from ancestors with diverse lifestyles and retain large repertoires of plant cell wall-degrading enzymes (PCWDEs) and effector-like small secreted proteins. We identify a set of 84 gene families associated with endophytism, including genes encoding PCWDEs acting on xylan (family GH10) and cellulose (family AA9). Transcripts encoding these enzymes are also part of a conserved transcriptional program activated by phylogenetically-distant mycobiota members upon host contact. Recolonization experiments with individual fungi indicate that strains with detrimental effects in mono-association with the host colonize roots more aggressively than those with beneficial activities, and dominate in natural root samples. Furthermore, we show that the pectin-degrading enzyme family PL1_7 links aggressiveness of endophytic colonization to plant health.

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.

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.

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.

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.

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.

Recent field and laboratory experiments with perennial Boechera stricta and annual Arabidopsis thaliana suggest that the root microbiota influences flowering time. Here we examined in long-term time-course experiments the bacterial root microbiota of the arctic-alpine perennial Arabis alpina in natural and controlled environments by 16S rRNA gene profiling. We identified soil type and residence time of plants in soil as major determinants explaining up to 15% of root microbiota variation, whereas environmental conditions and host genotype explain maximally 11% of variation. When grown in the same soil, the root microbiota composition of perennial A. alpina is largely similar to those of its annual relatives A. thaliana and Cardamine hirsuta. Non-flowering wild-type A. alpina and flowering pep1 mutant plants assemble an essentially indistinguishable root microbiota, thereby uncoupling flowering time from plant residence time-dependent microbiota changes. This reveals the robustness of the root microbiota against the onset and perpetual flowering of A. alpina. Together with previous studies, this implies a model in which parts of the root microbiota modulate flowering time, whereas, after microbiota acquisition during vegetative growth, the established root-associated bacterial assemblage is structurally robust to perturbations caused by flowering and drastic changes in plant stature.

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.

The sessile nature of plants forced them to evolve mechanisms to prioritize their responses to simultaneous stresses, including colonization by microbes or nutrient starvation. Here, we compare the genomes of a beneficial root endophyte, Colletotrichum tofieldiae and its pathogenic relative C. incanum, and examine the transcriptomes of both fungi and their plant host Arabidopsis during phosphate starvation. Although the two species diverged only 8.8 million years ago and have similar gene arsenals, we identify genomic signatures indicative of an evolutionary transition from pathogenic to beneficial lifestyles, including a narrowed repertoire of secreted effector proteins, expanded families of chitin-binding and secondary metabolism-related proteins, and limited activation of pathogenicity-related genes in planta. We show that beneficial responses are prioritized in C. tofieldiae-colonized roots under phosphate-deficient conditions, whereas defense responses are activated under phosphate-sufficient conditions. These immune responses are retained in phosphate-starved roots colonized by pathogenic C. incanum, illustrating the ability of plants to maximize survival in response to conflicting stresses.