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 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.

Cofunctioning of bacterial exometabolites drives root microbiota establishment.

Soil-dwelling microbes are the principal inoculum for the root microbiota, but our understanding of microbe-microbe interactions in microbiota establishment remains fragmentary. We tested 39,204 binary interbacterial interactions for inhibitory activities in vitro, allowing us to identify taxonomic signatures in bacterial inhibition profiles. Using genetic and metabolomic approaches, we identified the antimicrobial 2,4-diacetylphloroglucinol (DAPG) and the iron chelator pyoverdine as exometabolites whose combined functions explain most of the inhibitory activity of the strongly antagonistic Pseudomonas brassicacearum R401. Microbiota reconstitution with a core of Arabidopsis thaliana root commensals in the presence of wild-type or mutant strains revealed a root niche-specific cofunction of these exometabolites as root competence determinants and drivers of predictable changes in the root-associated community. In natural environments, both the corresponding biosynthetic operons are enriched in roots, a pattern likely linked to their role as iron sinks, indicating that these cofunctioning exometabolites are adaptive traits contributing to pseudomonad pervasiveness throughout the root microbiota.

Co-evolution within the plant holobiont drives host performance.

Plants interact with a diversity of microorganisms that influence their growth and resilience, and they can therefore be considered as ecological entities, namely "plant holobionts," rather than as singular organisms. In a plant holobiont, the assembly of above- and belowground microbiota is ruled by host, microbial, and environmental factors. Upon microorganism perception, plants activate immune signaling resulting in the secretion of factors that modulate microbiota composition. Additionally, metabolic interdependencies and antagonism between microbes are driving forces for community assemblies. We argue that complex plant-microbe and intermicrobial interactions have been selected for during evolution and may promote the survival and fitness of plants and their associated microorganisms as holobionts. As part of this process, plants evolved metabolite-mediated strategies to selectively recruit beneficial microorganisms in their microbiota. Some of these microbiota members show host-adaptation, from which mutualism may rapidly arise. In the holobiont, microbiota members also co-evolved antagonistic activities that restrict proliferation of microbes with high pathogenic potential and can therefore prevent disease development. Co-evolution within holobionts thus ultimately drives plant performance.

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

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.

Tryptophan metabolism and bacterial commensals prevent fungal dysbiosis in Arabidopsis roots.

In nature, roots of healthy plants are colonized by multikingdom microbial communities that include bacteria, fungi, and oomycetes. A key question is how plants control the assembly of these diverse microbes in roots to maintain host-microbe homeostasis and health. Using microbiota reconstitution experiments with a set of immunocompromised Arabidopsis thaliana mutants and a multikingdom synthetic microbial community (SynCom) representative of the natural A. thaliana root microbiota, we observed that microbiota-mediated plant growth promotion was abolished in most of the tested immunocompromised mutants. Notably, more than 40% of between-genotype variation in these microbiota-induced growth differences was explained by fungal but not bacterial or oomycete load in roots. Extensive fungal overgrowth in roots and altered plant growth was evident at both vegetative and reproductive stages for a mutant impaired in the production of tryptophan-derived, specialized metabolites (cyp79b2/b3). Microbiota manipulation experiments with single- and multikingdom microbial SynComs further demonstrated that 1) the presence of fungi in the multikingdom SynCom was the direct cause of the dysbiotic phenotype in the cyp79b2/b3 mutant and 2) bacterial commensals and host tryptophan metabolism are both necessary to control fungal load, thereby promoting A. thaliana growth and survival. Our results indicate that protective activities of bacterial root commensals are as critical as the host tryptophan metabolic pathway in preventing fungal dysbiosis in the A. thaliana root endosphere.

Climate drives rhizosphere microbiome variation and divergent selection between geographically distant Arabidopsis populations.

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.

The Rust Fungus Melampsora larici-populina Expresses a Conserved Genetic Program and Distinct Sets of Secreted Protein Genes During Infection of Its Two Host Plants, Larch and Poplar.

Mechanisms required for broad-spectrum or specific host colonization of plant parasites are poorly understood. As a perfect illustration, heteroecious rust fungi require two alternate host plants to complete their life cycles. Melampsora larici-populina infects two taxonomically unrelated plants, larch, on which sexual reproduction is achieved, and poplar, on which clonal multiplication occurs, leading to severe epidemics in plantations. We applied deep RNA sequencing to three key developmental stages of M. larici-populina infection on larch: basidia, pycnia, and aecia, and we performed comparative transcriptomics of infection on poplar and larch hosts, using available expression data. Secreted protein was the only significantly overrepresented category among differentially expressed M. larici-populina genes between the basidial, the pycnial, and the aecial stages, highlighting their probable involvement in the infection process. Comparison of fungal transcriptomes in larch and poplar revealed a majority of rust genes were commonly expressed on the two hosts and a fraction exhibited host-specific expression. More particularly, gene families encoding small secreted proteins presented striking expression profiles that highlight probable candidate effectors specialized on each host. Our results bring valuable new information about the biological cycle of rust fungi and identify genes that may contribute to host specificity.

Colletotrichum higginsianum extracellular LysM proteins play dual roles in appressorial function and suppression of chitin-triggered plant immunity.

The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum, encodes a large repertoire of candidate-secreted effectors containing LysM domains, but the role of such proteins in the pathogenicity of any Colletotrichum species is unknown. Here, we characterized the function of two effectors, ChELP1 and ChELP2, which are transcriptionally activated during the initial intracellular biotrophic phase of infection. Using immunocytochemistry, we found that ChELP2 is concentrated on the surface of bulbous biotrophic hyphae at the interface with living host cells but is absent from filamentous necrotrophic hyphae. We show that recombinant ChELP1 and ChELP2 bind chitin and chitin oligomers in vitro with high affinity and specificity and that both proteins suppress the chitin-triggered activation of two immune-related plant mitogen-activated protein kinases in the host Arabidopsis. Using RNAi-mediated gene silencing, we found that ChELP1 and ChELP2 are essential for fungal virulence and appressorium-mediated penetration of both Arabidopsis epidermal cells and cellophane membranes in vitro. The findings suggest a dual role for these LysM proteins as effectors for suppressing chitin-triggered immunity and as proteins required for appressorium function.

Mosaic genome structure of the barley powdery mildew pathogen and conservation of transcriptional programs in divergent hosts.

Barley powdery mildew, Blumeria graminis f. sp. hordei (Bgh), is an obligate biotrophic ascomycete fungal pathogen that can grow and reproduce only on living cells of wild or domesticated barley (Hordeum sp.). Domestication and deployment of resistant barley cultivars by humans selected for amplification of Bgh isolates with different virulence combinations. We sequenced the genomes of two European Bgh isolates, A6 and K1, for comparative analysis with the reference genome of isolate DH14. This revealed a mosaic genome structure consisting of large isolate-specific DNA blocks with either high or low SNP densities. Some of the highly polymorphic blocks likely accumulated SNPs for over 10,000 years, well before the domestication of barley. These isolate-specific blocks of alternating monomorphic and polymorphic regions imply an exceptionally large standing genetic variation in the Bgh population and might be generated and maintained by rare outbreeding and frequent clonal reproduction. RNA-sequencing experiments with isolates A6 and K1 during four early stages of compatible and incompatible interactions on leaves of partially immunocompromised Arabidopsis mutants revealed a conserved Bgh transcriptional program during pathogenesis compared with the natural host barley despite ~200 million years of reproductive isolation of these hosts. Transcripts encoding candidate-secreted effector proteins are massively induced in successive waves. A specific decrease in candidate-secreted effector protein transcript abundance in the incompatible interaction follows extensive transcriptional reprogramming of the host transcriptome and coincides with the onset of localized host cell death, suggesting a host-inducible defense mechanism that targets fungal effector secretion or production.

Towards a holistic understanding of the beneficial interactions across the Populus microbiome.

Interactions between trees and microorganisms are tremendously complex and the multispecies networks resulting from these associations have consequences for plant growth and productivity. However, a more holistic view is needed to better understand trees as ecosystems and superorganisms, where many interacting species contribute to the overall stability of the system. While much progress has been made on microbial communities associated with individual tree niches and the molecular interactions between model symbiotic partners, there is still a lack of knowledge of the multi-component interactions necessary for holistic ecosystem-level understanding. We review recent studies in Populus to emphasize the importance of such holistic efforts across the leaf, stem and rooting zones, and discuss prospects for future research in these important ecosystems.

Sequential delivery of host-induced virulence effectors by appressoria and intracellular hyphae of the phytopathogen Colletotrichum higginsianum.

Phytopathogens secrete effector proteins to manipulate their hosts for effective colonization. Hemibiotrophic fungi must maintain host viability during initial biotrophic growth and elicit host death for subsequent necrotrophic growth. To identify effectors mediating these opposing processes, we deeply sequenced the transcriptome of Colletotrichum higginsianum infecting Arabidopsis. Most effector genes are host-induced and expressed in consecutive waves associated with pathogenic transitions, indicating distinct effector suites are deployed at each stage. Using fluorescent protein tagging and transmission electron microscopy-immunogold labelling, we found effectors localised to stage-specific compartments at the host-pathogen interface. In particular, we show effectors are focally secreted from appressorial penetration pores before host invasion, revealing new levels of functional complexity for this fungal organ. Furthermore, we demonstrate that antagonistic effectors either induce or suppress plant cell death. Based on these results we conclude that hemibiotrophy in Colletotrichum is orchestrated through the coordinated expression of antagonistic effectors supporting either cell viability or cell death.

Obligate biotrophy features unraveled by the genomic analysis of rust fungi.

Rust fungi are some of the most devastating pathogens of crop plants. They are obligate biotrophs, which extract nutrients only from living plant tissues and cannot grow apart from their hosts. Their lifestyle has slowed the dissection of molecular mechanisms underlying host invasion and avoidance or suppression of plant innate immunity. We sequenced the 101-Mb genome of Melampsora larici-populina, the causal agent of poplar leaf rust, and the 89-Mb genome of Puccinia graminis f. sp. tritici, the causal agent of wheat and barley stem rust. We then compared the 16,399 predicted proteins of M. larici-populina with the 17,773 predicted proteins of P. graminis f. sp tritici. Genomic features related to their obligate biotrophic lifestyle include expanded lineage-specific gene families, a large repertoire of effector-like small secreted proteins, impaired nitrogen and sulfur assimilation pathways, and expanded families of amino acid and oligopeptide membrane transporters. The dramatic up-regulation of transcripts coding for small secreted proteins, secreted hydrolytic enzymes, and transporters in planta suggests that they play a role in host infection and nutrient acquisition. Some of these genomic hallmarks are mirrored in the genomes of other microbial eukaryotes that have independently evolved to infect plants, indicating convergent adaptation to a biotrophic existence inside plant cells.

Physiochemical interaction between osmotic stress and a bacterial exometabolite promotes plant disease.

Various microbes isolated from healthy plants are detrimental under laboratory conditions, indicating the existence of molecular mechanisms preventing disease in nature. Here, we demonstrated that application of sodium chloride (NaCl) in natural and gnotobiotic soil systems is sufficient to induce plant disease caused by an otherwise non-pathogenic root-derived Pseudomonas brassicacearum isolate (R401). Disease caused by combinatorial treatment of NaCl and R401 triggered extensive, root-specific transcriptional reprogramming that did not involve down-regulation of host innate immune genes, nor dampening of ROS-mediated immunity. Instead, we identified and structurally characterized the R401 lipopeptide brassicapeptin A as necessary and sufficient to promote disease on salt-treated plants. Brassicapeptin A production is salt-inducible, promotes root colonization and transitions R401 from being beneficial to being detrimental on salt-treated plants by disturbing host ion homeostasis, thereby bolstering susceptibility to osmolytes. We conclude that the interaction between a global change stressor and a single exometabolite from a member of the root microbiome promotes plant disease in complex soil systems.

Laser microdissection and microarray analysis of Tuber melanosporum ectomycorrhizas reveal functional heterogeneity between mantle and Hartig net compartments.

The ectomycorrhizal (ECM) symbiosis, a mutualistic plant-fungus association, plays a fundamental role in forest ecosystems by enhancing plant growth and by providing host protection from root diseases. The cellular complexity of the symbiotic organ, characterized by the differentiation of structurally specialized tissues (i.e. the fungal mantle and the Hartig net), is the major limitation to study fungal gene expression in such specific compartments. We investigated the transcriptional landscape of the ECM fungus Tuber melanosporum during the major stages of its life cycle and we particularly focused on the complex symbiotic stage by combining the use of laser capture microdissection and microarray gene expression analysis. We isolated the fungal/soil (i.e. the mantle) and the fungal/plant (i.e. the Hartig net) interfaces from transverse sections of T. melanosporum/Corylus avellana ectomycorrhizas and identified the distinct genetic programmes associated with each compartment. Particularly, nitrogen and water acquisition from soil, synthesis of secondary metabolites and detoxification mechanisms appear to be important processes in the fungal mantle. In contrast, transport activity is enhanced in the Hartig net and we identified carbohydrate and nitrogen-derived transporters that might play a key role in the reciprocal resources' transfer between the host and the symbiont.

Genome-resolved metatranscriptomics reveals conserved root colonization determinants in a synthetic microbiota.

The identification of processes activated by specific microbes during microbiota colonization of plant roots has been hampered by technical constraints in metatranscriptomics. These include lack of reference genomes, high representation of host or microbial rRNA sequences in datasets, or difficulty to experimentally validate gene functions. Here, we recolonized germ-free Arabidopsis thaliana with a synthetic, yet representative root microbiota comprising 106 genome-sequenced bacterial and fungal isolates. We used multi-kingdom rRNA depletion, deep RNA-sequencing and read mapping against reference microbial genomes to analyse the in planta metatranscriptome of abundant colonizers. We identified over 3,000 microbial genes that were differentially regulated at the soil-root interface. Translation and energy production processes were consistently activated in planta, and their induction correlated with bacterial strains' abundance in roots. Finally, we used targeted mutagenesis to show that several genes consistently induced by multiple bacteria are required for root colonization in one of the abundant bacterial strains (a genetically tractable Rhodanobacter). Our results indicate that microbiota members activate strain-specific processes but also common gene sets to colonize plant roots.

A comprehensive analysis of genes encoding small secreted proteins identifies candidate effectors in Melampsora larici-populina (poplar leaf rust).

The obligate biotrophic rust fungus Melampsora larici-populina is the most devastating and widespread pathogen of poplars. Studies over recent years have identified various small secreted proteins (SSP) from plant biotrophic filamentous pathogens and have highlighted their role as effectors in host-pathogen interactions. The recent analysis of the M. larici-populina genome sequence has revealed the presence of 1,184 SSP-encoding genes in this rust fungus. In the present study, the expression and evolutionary dynamics of these SSP were investigated to pinpoint the arsenal of putative effectors that could be involved in the interaction between the rust fungus and poplar. Similarity with effectors previously described in Melampsora spp., richness in cysteines, and organization in large families were extensively detailed and discussed. Positive selection analyses conducted over clusters of paralogous genes revealed fast-evolving candidate effectors. Transcript profiling of selected M. laricipopulina SSP showed a timely coordinated expression during leaf infection, and the accumulation of four candidate effectors in distinct rust infection structures was demonstrated by immunolocalization. This integrated and multifaceted approach helps to prioritize candidate effector genes for functional studies.

High-Throughput Profiling of Root-Associated Microbial Communities.

Roots of healthy plants are colonized by a great diversity of bacteria and fungi but also other microorganisms that are collectively referred to as the root microbiota. Root microbiota composition is shaped by environmental cues, by host genetics, but also by microbe-microbe interactions, and recent evidence indicates that a direct link exists between root microbiota assembly and host health. In order to characterize the root microbiota or to study the complex interplay between plants and their associated microbes, the assessment of microbial community structure via marker gene amplicon sequencing has become a key tool. Herein, we present detailed methods for the preparation of 16S rRNA gene and internal transcribed spacer (ITS) amplicon libraries to characterize Arabidopsis thaliana-associated bacterial and fungal communities along the soil-root continuum. The protocols can be easily adapted for different host organs or plant species.