Klaus Schlaeppi

Revealing structure and assembly cues for Arabidopsis root-inhabiting bacterial microbiota

The plant root defines the interface between a multicellular eukaryote and soil, one of the richest microbial ecosystems on Earth. Notably, soil bacteria are able to multiply inside roots as benign endophytes and modulate plant growth and development, with implications ranging from enhanced crop productivity to phytoremediation. Endophytic colonization represents an apparent paradox of plant innate immunity because plant cells can detect an array of microbe-associated molecular patterns (also known as MAMPs) to initiate immune responses to terminate microbial multiplication. Several studies attempted to describe the structure of bacterial root endophytes; however, different sampling protocols and low-resolution profiling methods make it difficult to infer general principles. Here we describe methodology to characterize and compare soil- and root-inhabiting bacterial communities, which reveals not only a function for metabolically active plant cells but also for inert cell-wall features in the selection of soil bacteria for host colonization. We show that the roots of Arabidopsis thaliana, grown in different natural soils under controlled environmental conditions, are preferentially colonized by Proteobacteria, Bacteroidetes and Actinobacteria, and each bacterial phylum is represented by a dominating class or family. Soil type defines the composition of root-inhabiting bacterial communities and host genotype determines their ribotype profiles to a limited extent. The identification of soil-type-specific members within the root-inhabiting assemblies supports our conclusion that these represent soil-derived root endophytes. Surprisingly, plant cell-wall features of other tested plant species seem to provide a sufficient cue for the assembly of approximately 40% of the Arabidopsis bacterial root-inhabiting microbiota, with a bias for Betaproteobacteria. Thus, this root sub-community may not be Arabidopsis-specific but saprophytic bacteria that would naturally be found on any plant root or plant debris in the tested soils. By contrast, colonization of Arabidopsis roots by members of the Actinobacteria depends on other cues from metabolically active host cells.

Structure and Functions of the Bacterial Microbiota of Plants

Plants host distinct bacterial communities on and inside various plant organs, of which those associated with roots and the leaf surface are best characterized. The phylogenetic composition of these communities is defined by relatively few bacterial phyla, including Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria. A synthesis of available data suggests a two-step selection process by which the bacterial microbiota of roots is differentiated from the surrounding soil biome. Rhizodeposition appears to fuel an initial substrate-driven community shift in the rhizosphere, which converges with host genotype-dependent fine-tuning of microbiota profiles in the selection of root endophyte assemblages. Substrate-driven selection also underlies the establishment of phyllosphere communities but takes place solely at the immediate leaf surface. Both the leaf and root microbiota contain bacteria that provide indirect pathogen protection, but root microbiota members appear to serve additional host functions through the acquisition of nutrients from soil for plant growth. Thus, the plant microbiota emerges as a fundamental trait that includes mutualism enabled through diverse biochemical mechanisms, as revealed by studies on plant growth-promoting and plant health-promoting bacteria.

Quantitative divergence of the bacterial root microbiota in Arabidopsis thaliana relatives

Significance All plants carry distinctive bacterial communities on and inside organs such as roots and leaves, collectively called the plant microbiota. How this microbiota diversifies in related plant species is unknown. We investigated the diversity of the bacterial root microbiota in the Brassicaceae family, including three Arabidopsis thaliana ecotypes, its sister species Arabidopsis halleri and Arabidopsis lyrata, and Cardamine hirsuta. We show that differences in root microbiota profiles between these hosts are largely quantitative and that host phylogenetic distance alone cannot explain the observed microbiota diversification. Our work also reveals a largely conserved and taxonomically narrow root microbiota, which comprises stable community members belonging to the Actinomycetales, Burkholderiales, and Flavobacteriales. Plants host at the contact zone with soil a distinctive root-associated bacterial microbiota believed to function in plant nutrition and health. We investigated the diversity of the root microbiota within a phylogenetic framework of hosts: three Arabidopsis thaliana ecotypes along with its sister species Arabidopsis halleri and Arabidopsis lyrata, as well as Cardamine hirsuta, which diverged from the former ∼35 Mya. We surveyed their microbiota under controlled environmental conditions and of A. thaliana and C. hirsuta in two natural habitats. Deep 16S rRNA gene profiling of root and corresponding soil samples identified a total of 237 quantifiable bacterial ribotypes, of which an average of 73 community members were enriched in roots. The composition of this root microbiota depends more on interactions with the environment than with host species. Interhost species microbiota diversity is largely quantitative and is greater between the three Arabidopsis species than the three A. thaliana ecotypes. Host species-specific microbiota were identified at the levels of individual community members, taxonomic groups, and whole root communities. Most of these signatures were observed in the phylogenetically distant C. hirsuta. However, the branching order of host phylogeny is incongruent with interspecies root microbiota diversity, indicating that host phylogenetic distance alone cannot explain root microbiota diversification. Our work reveals within 35 My of host divergence a largely conserved and taxonomically narrow root microbiota, which comprises stable community members belonging to the Actinomycetales, Burkholderiales, and Flavobacteriales.

The glutathione‐deficient mutant pad2‐1 accumulates lower amounts of glucosinolates and is more susceptible to the insect herbivore Spodoptera littoralis

Summary Plants often respond to pathogen or insect attack by inducing the synthesis of toxic compounds such as phytoalexins and glucosinolates (GS). The Arabidopsis mutant pad2-1 has reduced levels of the phytoalexin camalexin and is known for its increased susceptibility to fungal and bacterial pathogens. We found that pad2-1 is also more susceptible to the generalist insect Spodoptera littoralis but not to the specialist Pieris brassicae. The PAD2 gene encodes a gamma-glutamylcysteine synthetase that is involved in glutathione (GSH) synthesis, and consequently the pad2-1 mutant contains about 20% of the GSH found in wild-type plants. Lower GSH levels of pad2-1 were correlated with reduced accumulation of the two major indole and aliphatic GSs of Arabidopsis, indolyl-3-methyl-GS and 4-methylsulfinylbutyl-GS, in response to insect feeding. This effect was specific to GSH, was not complemented by treatment of pad2-1 with the strong reducing agent dithiothreitol, and was not observed with the ascorbate-deficient mutant vtc1-1. In contrast to the jasmonate-insensitive mutant coi1-1, expression of insect-regulated and GS biosynthesis genes was not affected in pad2-1. Our data suggest a crucial role for GSH in GS biosynthesis and insect resistance.

The plant microbiome at work.

Plants host distinct microbial communities on and inside their tissues designated the plant microbiota. Microbial community profiling enabled the description of the phylogenetic structure of the plant microbiota to an unprecedented depth, whereas functional insights are largely derived from experiments using individual microorganisms. The binary interplay between isolated members of the plant microbiota and host plants ranges from mutualistic to commensalistic and pathogenic relationships. However, how entire microbial communities capable of executing both growth-promoting and growth-compromising activities interfere with plant fitness remains largely unknown. Ultimately, unravelling the net result of microbial activities encoded in the extended plant genome-the plant microbiome-will be key to understanding and exploiting the full yield potential of a crop plant. In this perspective, we summarize first achievements of plant-microbiome research, we discuss future research directions, and we provide ideas for the translation of basic science to application to capitalize on the plant microbiome at work.

Disease resistance of Arabidopsis to Phytophthora brassicae is established by the sequential action of indole glucosinolates and camalexin.

We have analysed the role of tryptophan-derived secondary metabolites in disease resistance of Arabidopsis to the oomycete pathogen Phytophthora brassicae. Transcript analysis revealed that genes encoding enzymes involved in tryptophan, camalexin and indole glucosinolate (iGS) biosynthesis are coordinately induced in response to P. brassicae. However, a deficiency in either camalexin or iGS accumulation has only a minor effect on the disease resistance of Arabidopsis mutants. In contrast, the double mutant cyp79B2 cyp79B3, which has a blockage in the production of indole-3-aldoxime (IAOx), the common precursor of tryptophan-derived metabolites including camalexin and iGS, is highly susceptible to P. brassicae. Because cyp79B2 cyp79B3 shows no deficiencies in other tested disease resistance responses, we concluded that the lack of IAOx-derived compounds renders Arabidopsis susceptible despite wild-type-like pathogen-induced hypersensitive cell death, stress hormone signaling and callose deposition. The susceptibility of the double mutant pen2-1 pad3-1, which has a combined defect in camalexin synthesis and PEN2-catalysed hydrolysis of iGS compounds, demonstrates that both camalexin and products of iGS hydrolysis are important for disease resistance to P. brassicae. Products of iGS hydrolysis play an early defensive role, as indicated by enhanced epidermal penetration rates of Arabidopsis mutants affected in iGS synthesis or degradation. Our results show that disease resistance of Arabidopsis to P. brassicae is established by the sequential activity of the phytoanticipin iGS and the phytoalexin camalexin.

A widespread plant-fungal-bacterial symbiosis promotes plant biodiversity, plant nutrition and seedling recruitment

Highly diverse microbial assemblages colonize plant roots. It is still poorly understood whether different members of this root microbiome act synergistically by supplying different services (for example, different limiting nutrients) to plants and plant communities. In order to test this, we manipulated the presence of two widespread plant root symbionts, arbuscular mycorrhizal fungi and nitrogen-fixing rhizobia bacteria in model grassland communities established in axenic microcosms. Here, we demonstrate that both symbionts complement each other resulting in increased plant diversity, enhanced seedling recruitment and improved nutrient acquisition compared with a single symbiont situation. Legume seedlings obtained up to 15-fold higher productivity if they formed an association with both symbionts, opposed to productivity they reached with only one symbiont. Our results reveal the importance of functional diversity of symbionts and demonstrate that different members of the root microbiome can complement each other in acquiring different limiting nutrients and in driving important ecosystem functions.

High‐resolution community profiling of arbuscular mycorrhizal fungi

Community analyses of arbuscular mycorrhizal fungi (AMF) using ribosomal small subunit (SSU) or internal transcribed spacer (ITS) DNA sequences often suffer from low resolution or coverage. We developed a novel sequencing based approach for a highly resolving and specific profiling of AMF communities. We took advantage of previously established AMF-specific PCR primers that amplify a c. 1.5-kb long fragment covering parts of SSU, ITS and parts of the large ribosomal subunit (LSU), and we sequenced the resulting amplicons with single molecule real-time (SMRT) sequencing. The method was applicable to soil and root samples, detected all major AMF families and successfully discriminated closely related AMF species, which would not be discernible using SSU sequences. In inoculation tests we could trace the introduced AMF inoculum at the molecular level. One of the introduced strains almost replaced the local strain(s), revealing that AMF inoculation can have a profound impact on the native community. The methodology presented offers researchers a powerful new tool for AMF community analysis because it unifies improved specificity and enhanced resolution, whereas the drawback of medium sequencing throughput appears of lesser importance for low-diversity groups such as AMF.

Root surface as a frontier for plant microbiome research

Plants associate—analogous to animals or us humans—with a multitude of microorganisms, which collectively function as a microbiome. A major discovery of the last decade is that numerous organisms of a microbiome (aka microbiota) are not unpretentious background actors. Instead, some microbiota members influence host processes including behavior, appetite, and health in animals (1) and contribute to nutrition and health of plants (2–4). Recently, the compositions of the plant root-associated microbiota from numerous plant species, including major crops, were revealed using high-throughput DNA sequencing. Factors such as soil type or host genotype influence the root-associated microbiota. However, the processes that determine the acquisition of the root microbiota, its resistance to stress, and its ecological function remain poorly understood. Edwards et al. (5) present the third publication in a recent series of PNAS articles about the bacterial microbiota associated with plant roots of maize (6), related Brassicaceae (7), and now Oryza sativa (rice). It comprises a comprehensive characterization of three microbial habitats that are in the proximity of, on, and inside plant roots, which are named rhizosphere, rhizoplane, and root endosphere (Fig. 1).

Root microbiota dynamics of perennial Arabis alpina are dependent on soil residence time but independent of flowering time

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.