LETTER doi:10.1038/nature11336 Revealing structure and assembly cues for Arabidopsis root-inhabiting bacterial microbiota Davide Bulgarelli 1 *, Matthias Rott 1 *, Klaus Schlaeppi 1 *, Emiel Ver Loren van Themaat 1 *, Nahal Ahmadinejad 1 {, Federica Assenza 1 , Philipp Rauf 1 {, Bruno Huettel 2 , Richard Reinhardt 2 , Elmon Schmelzer 3 , Joerg Peplies 4 , Frank Oliver Gloeckner 4,5 , Rudolf Amann 5 , Thilo Eickhorst 6 & Paul Schulze-Lefert 1 The plant root defines the interface between a multicellular eukaryote and soil, one of the richest microbial ecosystems on Earth 1 . Notably, soil bacteria are able to multiply inside roots as benign endophytes and modulate plant growth and development 2 , with implications ranging from enhanced crop productivity 3 to phytoremediation 4 . 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 5 . Several studies attempted to describe the structure of bacterial root endo- phytes 6 ; 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 rep- resent 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. We have grown Arabidopsis ecotypes Shakdara (Sha) and Landsberg erecta (Ler) in natural soils of contrasting geochemistry, designated Cologne (clay- and silt-rich) or Golm (sand- rich) soil, under controlled environmental conditions and at a defined planting density (Supplementary Fig. 1 and Supplementary Table 1). At early flowering stage we collected samples from three compartments: ‘unplanted soil’ (number of replicates: Cologne n C 5 13, Golm n G 5 12), ‘rhizosphere’ (n C 5 15, n G 5 12) and ‘root’ (n C 5 18, n G 5 14). The ‘rhizosphere compartment’ defines the soil particles firmly attached to roots collected by centrifugation of root washings (Supplementary Movie 1). The ‘root compartment’ is defined as root tissue depleted of soil particles and epiphytic bacteria by sequential washing and sonication treatments and is therefore enriched for root-inhabiting bacteria (Supplementary Fig. 2). We used pyrosequencing of an approximately 400 base pairs PCR amplicon of the bacterial 16S ribosomal RNA gene and analysed the variable gene segments V5–V6. To examine the taxonomic structure of the bacterial communities we performed a supervised taxonomy classification of all high quality reads using the SILVA 7 database. This classification identified a total of 43 bacterial phyla and divisions and revealed an anomalous Chloroflexi abundance in all samples (Fig. 1a). PCR-independent catalysed reporter deposition-fluorescence in situ hybridization (CARD-FISH) analysis on soil samples (Supplementary Fig. 3) and comparative PCR primer analysis indicated this is due to a PCR primer bias (Supplementary Information and Supplementary Fig. 4). After removal of reads assigned to Chloroflexi we identified Proteobacteria, Actinobacteria and Bacteroidetes as dominating phyla in root bacterial communities and significantly enriched compared to soil and *These authors contributed equally to this work. 1 Department of Plant Microbe Interactions, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany. 2 Max Planck Genome Centre, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany. 3 Central Microscopy, Max Planck Institute for Plant Breeding Research, 50829 Cologne, Germany. 4 Ribocon GmbH, 28359 Bremen, Germany. 5 Department of Molecular Ecology, Max Planck Institute for Marine Microbiology, 28359 Bremen, Germany. 6 Soil Science, Faculty of Biology and Chemistry, University of Bremen, 28359 Bremen, Germany. {Present addresses: INRES - Crop Bioinformatics, University of Bonn, 53115 Bonn, Germany (N.A.); Department of Developmental Immunology, Max Planck Institute of Immunobiology and Epigenetics, 79108 Freiburg, Germany (P.R.). Chlorolexi Acidobacteria Planctomycetes Firmicutes TM7 Bacteroidetes Actinobacteria Proteobacteria Flavobacteriaceae Streptomycetaceae Rhizobiaceae Comamonadaceae Oxalobacteraceae Pseudomonadaceae Xanthomonadaceae a b c 0 200 400 600 800 1,000 0 200 400 600 800 1,000 0 200 400 600 800 1,000 0 100 200 300 400 500 600 0 100 200 300 400 500 600 0 100 200 300 400 500 600 0 50 150 250 350 450 0 50 150 250 350 450 0 50 150 250 350 450 Relative abundance (‰) Soil Rhizosphere Root Bacteroidetes Actinobacteria Alphaproteobacteria Betaproteobacteria Gammaproteobacteria Golm soil Cologne soil Figure 1 | Taxa at high taxonomic ranks define building blocks of root- associated bacterial communities. a, Average relative abundance (% 6 s.e.m.) of the phylum Chloroflexi detected in the indicated compartments. b, Average relative abundance (% 6 s.e.m.) of the dominant phyla (. 5 %) detected in root compartments of the indicated soil types. c, Average relative abundance (% 6 s.e.m.) of families belonging to the three dominant phyla in the root compartment. In b and c average relative abundances are calculated after removal of reads assigned to Chloroflexi. Asterisks indicate significant enrichment (Benjamini–Hochberg false- discovery-rate (FDR) adjusted P value , 0.05) in the root compartment compared to soil and rhizosphere compartments. 2 AUGUST 2012 | VOL 488 | NATURE | 91 Macmillan Publishers Limited. All rights reserved ©2012