Structure and functions of the bacterial root microbiota in wild and domesticated barley

Transcription

1 Structure and functions of the bacterial root microbiota in wild and domesticated barley Ruben Garrido Oter Structure, Function and Dynamics in Microbial Communities 30 / 11 / 2014

2 Introduction Othersources Atmosphere ~ m 3 endophytic microbiome rhizosphere microbiome Leafarea ~ cm 2 Soil ~ g 1 Rhizosphere ~ g 1 Rootendosphere ~ g 1 Bacterial phyla Actinobacteria Bacteroidetesand Firmicutes Proteobacteria Bulgarelli et al. Annu. Rev. Plant Biol. (2012) Hirsch, Mauchline, et al. Nature Biotech. (2012)

3 Outline introduction part I structure of the barley root and rhizosphere bacterial communities effect of domestication in the barley root microbial diversity part II the barley rhizosphere metagenome: taxonomic characterization of the rhizosphere metagenome functions enriched in root-associated taxa w.r.t. the surrounding soil biota signatures of possitive selection and microevolution future work & conclusions

4 Three different accessions represent three stages in the domestication process of barley Hv ssp. spontaneum Hv ssp. vulgare Landrace Hv ssp. vulgare Modern

5 Three different accessions represent three stages in the domestication process of barley ~ years ~ 60 years (advent of agriculture) (green revolution)

6 Experimental setup unplanted soil controls (3 replicates) (x 2 soil batches) H. vulgare ssp. spontaneum H. vulgare ssp. vulgare Landrace H. vulgare ssp. vulgare Modern rhizosphere (3 replicates) root (3 replicates) rhizosphere (3 replicates) root (3 replicates) rhizosphere (3 replicates) root (3 replicates) 16S rrna: - 42 samples - 691,822 reads rhizosphere (shotgun metagenome) rhizosphere (shotgun metagenome) rhizosphere (shotgun metagenome) metagenome: - 6 samples ~ 255 M paired-end reads

7 Factors influencing the diversity of the barley root-associated bacterial communities Microhabitat 22% of variance; P < 0.005, CI = 17%, 30% Genotype 5.7% of variance; P < 0.005, CI = 5%, 6.4%

8 Marked rhizosphere effect across barley accessions barley Soil Arabidopsis Soil Root Rhizosphere Root Rhizosphere Root OTUs Rhizosphere OTUs Root and Rhizosphere OTUs Bulgarelli et al., Nature, 2012

9 Outline introduction part I structure of the barley root and rhizosphere bacterial communities effect of domestication in the barley root microbial diversity part II the barley rhizosphere metagenome: taxonomic characterization of the rhizosphere metagenome functions enriched in root-associated taxa w.r.t. the surrounding soil biota signatures of possitive selection and microevolution future work & conclusions

10 Taxonomic abundances in the barley rhizosphere metagenome Bacteroidetes Acidobacteria Firmicutes δ - proteobacteria α - proteobacteria γ - proteobacteria Ignavibacteriales Mycoplasmatales Acholeplasmatales Bdellovibrionales Desulfobacterales Syntrophobacterales Desulfarculales Desulfovibrionales Myxococcales Desulfuromonadales Campylobacterales Nautiliales Sphingomonadales Magnetococcales Rhizobiales Caulobacterales Rickettsiales Rhodospirillales Parvularculales Rhodobacterales Legionellales Pasteurellales Xanthomonadales Acidithiobacillales Cardiobacteriales Alteromonadales Enterobacteriales Pseudomonadales Vibrionales Oceanospirillales Salinisphaerales Chromatiales Thiotrichales Methylococcales β - proteobacteria Nitrospirales Aeromonadales Gallionellales Sphingobacteriales Flavobacteriales Chlorobiales Rhodocyclales Nitrosomonadales Methylophilales Hydrogenophilales Bacteroidetes Inc. sedis Bacteroidales Neisseriales Burkholderiales Cytophagales Mariprofundales Elusimicrobiales Phycisphaerales Deferribacterales Haloplasmatales Planctomycetales Poribacteria Coriobacteriales Thermotogales Actinomycetales Dictyoglomales Bifidobacteriales Aquificales Gaiellales Fusobacteriales Rubrobacterales Fibrobacterales Solirubrobacterales Holophagales Acidimicrobiales Solibacterales Nostocales Stigonematales Acidobacteriales Lactobacillales Prochlorales Bacillales Halanaerobiales Oscillatoriales Chroococcales Actinobacteria Thermoanaerobacterales Clostridiales Caldilineales Gloeobacterales Erysipelotrichales Selenomonadales Spirochaetales Synergistales Sulfolobales Sphaerobacterales Chloroflexales Herpetosiphonales Desulfurococcales Thermoproteales Thermoplasmatales Halobacteriales Thermococcales Methanosarcinales Methanocellales Chlamydiales Anaerolineales Ktedonobacterales Thermomicrobiales Methanomicrobiales Dehalococcoidales Methanobacteriales Methanococcales Archaeoglobales Nitrosopumilales Cenarchaeales Armatimonadetes Thermales Deinococcales Gemmatimonadales Spartobacteria Verrucomicrobiales Methylacidiphilales Puniceicoccales Lentisphaerales Cyanobacteria Chloroflexi Archaea Chlamydiales / Verrucomicrobia metagenome abundances 16S amplicon abundances overlap

11 Biases in abundance estimates 16S rrna in shotgun metagenome reads primer bias ρ = 0.89 (P = 1.55E-14) 16S rrna amplicon survey (excluding Cyanobacteria) primer bias rrna operon copy number ρ = 0.86 (P = 1.75E-12) metagenome bins (excluding Eukaryotes, Archaea and Cyanobacteria)

12 Functions enriched in root and rhizosphere-associated bacterial taxa with respect to the surrounding soil biota Soil Root Rhizosphere Root OTUs Rhizosphere OTUs Root and Rhizosphere OTUs * Wilcoxon test, FDR correction

13 Functions enriched in root and rhizosphere-associated bacterial taxa with respect to the surrounding soil biota Root Soil Rhizosphere Root OTUs Rhizosphere OTUs Root and Rhizosphere OTUs Functional Category P value * Protein secretion system Type III Adhesion Regulation of virulence Siderophores Secretion Transposable elements Periplasmic Stress Sugar Phosphotransferase Systems Bacteriophage integration excision lysogeny Invasion and intracellular resistance Detoxification Protein secretion system Type VI Acid stress * Wilcoxon test, FDR correction

14 Outline introduction part I structure of the barley root and rhizosphere bacterial communities effect of domestication in the barley root microbial diversity part II the barley rhizosphere metagenome: taxonomic characterization of the rhizosphere metagenome functions enriched in root-associated taxa w.r.t. the surrounding soil biota signatures of possitive selection and microevolution future work & conclusions

15 Protein families related to host-microbe AND microbe-microbe interactions exhibit strong signatures of positive selection density mean pathogenesis / secretion ~dn/ds pathogenesis / secretion phages ~dn/ds Philipp Muench

16 Microbial elicitors of plant immunity under positive selection in the barley rhizosphere ~dn/ds 7.5 Gap proportion TIGR00377: anti-anti-sigma factor N /ds 5 TIGR01573: CRISPR-associated endoribonuclease Cas TIGR02241: conserved hypothetical phage tail region protein ~dn/ds Sequence Position (nt) clusters of residues with high dn / ds ratios in well conserved genes (putative epitopes) Philipp Muench

17 Conclusions wild and cultivated barley accessions host a taxonomically coherent microbiota whose structure is determined by the host microhabitat and fine-tuned by the host genotype small but statistically significant effect of domestication in the assembly of root-associated microbiota manifested by combinations of bacterial taxa protein families and functional processes relating to pathogenicity, phages and secretion, are significantly enriched in root-associated bacterial taxa evidence that protein families related to the same traits are affected by co-evolutionary arms races with the host as well as with other microbes the combined effect of both, microbe-microbe and host-microbe interactions shapes the differentiation of root-associated microbiota from the surrounding soil

18 Acknowledgements MPIPZ Paul Schulze-Lefert PSL - root group HHUD Alice McHardy Algorithmic bioinformatics group Yang Bai Davide Bulgarelli Nina Dombrowski Stéphane Hacquard Haruhiko Inoue Ryohei Thomas Nakano Girish Srinivas Spaepen Stijn Rafal Zgadzaj Barbara Kracher Marc André Daxer Johannes Dröge Ivan Gregor Thorsten Klingen Andreas Klötgen Sebastian Konietzny David Lähnemann Philipp Münch Yao Pan Aaron Weimann

19 Thank you!

20 Root-enriched bacterial taxa in barley Barley root OTUs Arabidopsis root OTUs Actinobacteria Actinomycetales Streptomycetaceae Microbacteriaceae 0.1% 1% 10% relative abundance (log transformed) β Burkholderiales Thermomonosporaceae Comamonadaceae Oxalobacteraceae γ Pseudomonadaceae Bacteria Proteobacteria Xanthomonadaceae α δ Rhizobiales Myxococcales Bacteroidetes Flavobacteriaceae kingdom phylum class order family

21 The genotype-effect is manifested by combinations of a large number of bacterial OTUs ns 5.5 P value E 2 * % of variance 5.0 E 4 ** number of OTUs permutated number of OTUs permutated Permutation-based analysis of the significance of the observed genotype-effect in barley

22 The barley root-inhabiting bacterial microbiota Rhizosphere Root Cholorflexi Firmicutes Soil Hv ssp. spontaneum Hv ssp. vulgare Landrace Hv ssp vulgare Modern Bacteroidetes Actinobacteria Proteobacteria Phyllobacteriaceae Nocardioidaceae Hyphomicrobiaceae Alteromonadaceae Xanthomonadaceae Streptomycetaceae Oxalobacteraceae Rhizobiaceae Flavobacteriaceae Comamonadaceae

23 SnapShot: Plant Immune Response Pathways Ralph Panstruga, J ane E. Parker, and Paul Schulze-Lefert