Drought Stress Results in a Compartment-Specific Restructuring of the Rice Root-Associated Microbiomes.

Plant roots support complex microbial communities that can influence plant growth, nutrition, and health. While extensive characterizations of the composition and spatial compartmentalization of these communities have been performed in different plant species, there is relatively little known about the impact of abiotic stresses on the root microbiota. Here, we have used rice as a model to explore the responses of root microbiomes to drought stress. Using four distinct genotypes, grown in soils from three different fields, we tracked the drought-induced changes in microbial composition in the rhizosphere (the soil immediately surrounding the root), the endosphere (the root interior), and unplanted soils. Drought significantly altered the overall bacterial and fungal compositions of all three communities, with the endosphere and rhizosphere compartments showing the greatest divergence from well-watered controls. The overall response of the bacterial microbiota to drought stress was taxonomically consistent across soils and cultivars and was primarily driven by an enrichment of multiple Actinobacteria and Chloroflexi, as well as a depletion of several Acidobacteria and Deltaproteobacteria While there was some overlap in the changes observed in the rhizosphere and endosphere communities, several drought-responsive taxa were compartment specific, a pattern likely arising from preexisting compositional differences, as well as plant-mediated processes affecting individual compartments. These results reveal that drought stress, in addition to its well-characterized effects on plant physiology, also results in restructuring of root microbial communities and suggest the possibility that constituents of the altered plant microbiota might contribute to plant survival under extreme environmental conditions.IMPORTANCE With the likelihood that changes in global climate will adversely affect crop yields, the potential role of microbial communities in enhancing plant performance makes it important to elucidate the responses of plant microbiomes to environmental variation. By detailed characterization of the effect of drought stress on the root-associated microbiota of the crop plant rice, we show that the rhizosphere and endosphere communities undergo major compositional changes that involve shifts in the relative abundances of a taxonomically diverse set of bacteria in response to drought. These drought-responsive microbes, in particular those enriched under water deficit conditions, could potentially benefit the plant as they could contribute to tolerance to drought and other abiotic stresses, as well as provide protection from opportunistic infection by pathogenic microbes. The identification and future isolation of microbes that promote plant tolerance to drought could potentially be used to mitigate crop losses arising from adverse shifts in climate

Lignin engineering in field-grown poplar trees affects the endosphere bacterial microbiome

Cinnamoyl-CoA reductase (CCR), an enzyme central to the lignin bio-synthetic pathway, represents a promising biotechnological target to reduce lignin levels and to improve the commercial viability of lignocellulosic biomass. However, silencing of the CCR gene results in considerable flux changes of the general and monolignol-specific lignin pathways, ultimately leading to the accumulation of various extractable phenolic compounds in the xylem. Here, we evaluated host genotype-dependent effects of field-grown, CCR-down-regulated poplar trees (Populus tremula x Populus alba) on the bacterial rhizosphere microbiome and the endosphere microbiome, namely the microbiota present in roots, stems, and leaves. Plant-associated bacteria were isolated from all plant compartments by selective isolation and enrichment techniques with specific phenolic carbon sources (such as ferulic acid) that are up-regulated in CCR-deficient poplar trees. The bacterial microbiomes present in the endosphere were highly responsive to the CCR-deficient poplar genotype with remarkably different metabolic capacities and associated community structures compared with the WT trees. In contrast, the rhizosphere microbiome of CCR-deficient and WT poplar trees featured highly overlapping bacterial community structures and metabolic capacities. We demonstrate the host genotype modulation of the plant microbiome by minute genetic variations in the plant genome. Hence, these interactions need to be taken into consideration to understand the full consequences of plant metabolic pathway engineering and its relation with the environment and the intended genetic improvement

The Genetic Architecture of Adaptation to Leaf and Root Bacterial Microbiota in Arabidopsis thaliana

Understanding the role of the host genome in modulating microbiota variation is a need to shed light on the holobiont theory and overcome the current limits on the description of host-microbiota interactions at the genomic and molecular levels. However, the host genetic architecture structuring microbiota is only partly described in plants. In addition, most association genetic studies on microbiota are often carried out outside the native habitats where the host evolves and the identification of signatures of local adaptation on the candidate genes has been overlooked. To fill these gaps and dissect the genetic architecture driving adaptive plant-microbiota interactions, we adopted a genome-environment association (GEA) analysis on 141 whole-genome sequenced natural populations of Arabidopsis thaliana characterized in situ for their leaf and root bacterial communities in fall and spring, and a large range of nonmicrobial ecological factors (i.e., climate, soil, and plant communities). A much higher fraction of among-population microbiota variance was explained by the host genetics than by nonmicrobial ecological factors. Importantly, the relative importance of host genetics and nonmicrobial ecological factors in explaining the presence of particular operational taxonomic units (OTUs) differs between bacterial families and genera. In addition, the polygenic architecture of adaptation to bacterial communities was highly flexible between plant compartments and seasons. Relatedly, signatures of local adaptation were stronger on quantitative trait loci (QTLs) of the root microbiota in spring. Finally, plant immunity appears as a major source of adaptive genetic variation structuring bacterial assemblages in A. thaliana

Computational analyses of the plant-associated microbiota

Plants harbor phylogenetically diverse microbes on the exterior and interior of all organs and they form intimate relationships with the colonized microbiota. Multi-omics dramatically facilitates and expands our knowledge in plant-microbiota interactions and associations. To establish causalities, manipulation of microbiota populating plants under strictly controlled conditions is a necessity, which forged the development of reductionist approaches for studying plant-microbiota interactions, including the process of deconstruction and reconstruction of the plant microbiota. Deconstruction of the plant microbiota requires the establishment of genome-indexed microbial culture collections representing the plant microbiota of interests. The reconstruction step is to design synthetic microbial communities (SynComs) by mixing the strains from the culture collections and inoculate onto the plants. In this dissertation, I introduced a software named Rbec that is developed to exclusively characterize the accurate microbial composition in SynComs subject to amplicon sequencing by both correcting PCR/sequencing errors and identifying maker gene paralogues within the same strain. Rbec also provides a novel feature for contamination identification in the SynCom experiments, which has been overlooked in previous studies but is a necessity to verify the robustness of the readouts from SynCom experimentations. Further, with the established pipelines for analyzing amplicon sequencing data from either natural or synthetic communities, I analyzed the microbial compositions from different studies including the study of the host preference of Arabidopsis thaliana and Lotus Japonicus commensals, the phycosphere microbiota, the effects of plant metabolites on soil microbiota and how bacterial antibiotics shape root microbiota. Genome-indexed microbial culture collections allow us to study the functional capacities of microbiota. We systematically analyzed the biosynthetic gene clusters and the spread of antimicrobial 2,4-diacetylphloroglucinol synthetic gene clusters in Pseudomonas in established culture collections. Moreover, I studied the recent horizontal gene transfer (HGT) in bacteria from different culture collections assembled from different host plants and sites. This provides an atlas of the active taxa involved in HGT and the frequently transferred functional orthologues in plant-associated niches. In addition, it reveals the selection forces exerted on different taxa in the relevant environments. In summary, our work tried to move the reductionist approaches forward in the aspect of computational analyses. We not only introduced a new computational method for accurately profiling microbial compositions in SynComs, but also digged deeper into the genome- indexed culture collections by making full use of genome sequences. With the valuable integrated genome information of the plant microbiota, it’ll provide the opportunity to study the functional diversities, evolutionary trajectories, genomic contents related to adaptations to hosts. However, with the increased volume of available genomes, novel methodology will be required to fast processing large datasets in a computational-efficient way

Antimicrobial peptide expression in a wild tobacco plant reveals the limits of host-microbe-manipulations in the field

Plant-microbe associations are thought to be beneficial for plant growth and resistance against biotic or abiotic stresses, but for natural ecosystems, the ecological analysis of microbiome function remains in its infancy. We used transformed wild tobacco plants (Nicotiana attenuata) which constitutively express an antimicrobial peptide (Mc-AMP1) of the common ice plant, to establish an ecological tool for plant-microbe studies in the field. Transgenic plants showed in planta activity against plant-beneficial bacteria and were phenotyped within the plants´ natural habitat regarding growth, fitness and the resistance against herbivores. Multiple field experiments, conducted over 3 years, indicated no differences compared to isogenic controls. Pyrosequencing analysis of the root-associated microbial communities showed no major alterations but marginal effects at the genus level. Experimental infiltrations revealed a high heterogeneity in peptide tolerance among native isolates and suggests that the diversity of natural microbial communities can be a major obstacle for microbiome manipulations in nature

Root microbiota functions in mitigating abiotic and biotic stresses in Arabidopsis

In nature, plants face both biotic and abiotic stresses while at the same time engaging in complex interactions with a vast diversity of commensal microorganisms comprising bacteria, fungi, and oomycetes. This so-called plant microbiota is thought to promote resistance to pathogens and tolerance to specific environmental constraints, likely driving local adaptation in natural plant populations. Reductionist approaches with synthetic microbial communities assembled from microbial culture collections and gnotobiotic plant systems now allow detailed dissection of microbiota-plant-stress interactions under strictly controlled laboratory conditions. Mechanistic understanding into how the root microbiota promotes mineral nutrition and pathogen protection in plants is now emerging. However, whether belowground response to microbial root commensals and aboveground response to abiotic stresses are connected remains largely unexplored. By reconstituting a synthetic, multi-kingdom root microbiota with different microbial input ratios in two gnotobiotic systems (the calcined-clay system and the FlowPot system) (Chapter I), I first showed that distinct input ratios of bacteria, fungi, and oomycetes converge into a similar output community composition, with stable effects on Arabidopsis growth. By testing different abiotic and biotic stresses in three gnotobiotic plant systems (the FlowPot system, the calcined-clay system, and the white sand system) (Chapter I), I provided evidence that salt, drought, and shade stresses negatively affected plant growth across all three systems, whereas nutritional stress affected on plant performance in a system-dependent manner. Moreover, I demonstrated that a synthetic multi-kingdom root microbiota rescued Arabidopsis growth under salt, drought and light limitation stresses in the FlowPot system and the white sand system (Chapter I). Given the importance of light for plant growth, in chapter II, I further dissected the extent to which response to the synthetic root microbiota and light are interconnected. By manipulating light conditions (low photosynthetically active radiation, LP; end of day far red-light treatment, EODFR) in the FlowPot system, I demonstrated that microbial root commensals confer Arabidopsis tolerance to light limitation stresses and that reciprocally, modification in aboveground light condition shifts the composition of root microbial communities. Notably, this shift in the structure of root bacterial community significantly explains the microbiota-induced growth rescue under LP. Arabidopsis transcriptome analysis revealed that immune responses in root and systemic defense responses in shoot were induced in the presence of the root microbiota under normal light conditions. These host responses were largely shut down under light limiting conditions and were correlated with increased susceptibility to unrelated leaf pathogens, implying that root microbiota-induced systemic defense responses were modulated by light. Through an extensive Arabidopsis mutant screen, I demonstrated that root microbiota-mediated plant survival under LP depends on jasmonic acid biosynthesis and signaling, cryptochromes and brassinosteroids. Furthermore, I present genetic evidence that orchestration of this light-dependent growth-defense trade-off requires the transcriptional regulator MYC2. The data suggest that plants can take advantage of root commensals to activate either growth or defense depending on aboveground light conditions

Host genotype and age shape the leaf and root microbiomes of a wild perennial plant

Bacteria living on and in leaves and roots influence many aspects of plant health, so the extent of a plant's genetic control over its microbiota is of great interest to crop breeders and evolutionary biologists. Laboratory-based studies, because they poorly simulate true environmental heterogeneity, may misestimate or totally miss the influence of certain host genes on the microbiome. Here we report a large-scale field experiment to disentangle the effects of genotype, environment, age and year of harvest on bacterial communities associated with leaves and roots of Boechera stricta (Brassicaceae), a perennial wild mustard. Host genetic control of the microbiome is evident in leaves but not roots, and varies substantially among sites. Microbiome composition also shifts as plants age. Furthermore, a large proportion of leaf bacterial groups are shared with roots, suggesting inoculation from soil. Our results demonstrate how genotype-by-environment interactions contribute to the complexity of microbiome assembly in natural environments