
Structural and functional analysis of the bacterial root microbiota of Arabidopsis thaliana and relative species Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Universität zu Köln vorgelegt von Nina Dombrowski aus Wyk auf Föhr Köln, 2015 Die vorliegende Arbeit wurde am Max-Planck-Institut für Pflanzenzüchtungsforschung in Köln in der Abteilung für Pflanze-Mikroben Interaktionen (Direktor: Prof. Dr. Paul Schulze-Lefert) angefertigt. Berichterstatter: Prof. Dr. Paul Schulze-Lefert Prof. Dr. Alga Zuccaro Prof. Dr. George Coupland Prüfungsvorsitzender: Prof. Dr. Gunther Döhlemann Tag der mündlichen Prüfung: 15.04.2015 `The most exciting phrase to hear in science, the one that heralds new discoveries, is not ‘eureka!’ but ‘that’s funny…’ Isaac Asimov Publication Publication Schlaeppi, K., Dombrowski, N., Oter, R.G., Themaat, E.V.L. van, and Schulze-Lefert, P. (2014). Quantitative divergence of the bacterial root microbiota in Arabidopsis thaliana relatives. PNAS 111, 585–592. I Table of contents Table of contents Publication ................................................................................................................................................ I Table of contents ................................................................................................................................... III Abstract ................................................................................................................................................ VII Zusammenfassung ................................................................................................................................. IX Chapter 1 Structural and functional characterization of the bacterial microbiota of Arabidopsis thaliana and relative species 1. Introduction ....................................................................................................................... 1 1.1. Structure and functions of the bacterial microbiota ....................................................... 1 1.1.1. Soil bacterial communities ............................................................................................ 1 1.1.2. Rhizosphere bacterial communities ............................................................................... 2 1.2. Root bacterial communities ........................................................................................... 4 1.2.1. Structure of the bacterial root microbiota ...................................................................... 6 1.2.2. The bacterial root microbiota of A. thaliana .................................................................. 7 1.3. The arctic-alpine perennial Arabis alpina ..................................................................... 9 1.4. Techniques to study bacterial communities ................................................................. 10 1.4.1. Amplicon-based techniques ......................................................................................... 10 1.4.2. Techniques to dissect functional capacities of bacterial communities ........................ 12 1.5. Thesis aim .................................................................................................................... 13 2. Results............................................................................................................................... 15 2.1. The structure of the bacterial root microbiota of A. thaliana and its relatives ............ 15 2.2. Comparing 454 and Illumina sequencing platforms .................................................... 23 2.3. The bacterial microbiota of perennial A. alpina .......................................................... 25 2.3.1. Influence of time and development on the bacterial microbiota of A. alpina .............. 25 2.3.2. Influence of environmental conditions on the bacterial microbiota of A. alpina ........ 32 2.4. Isolation and functional characterization of bacteria of the root microbiota of Brassicaceae plant species ........................................................................................... 40 2.4.1. Isolation of bacteria from roots of three Brassicaceae plant species ........................... 40 2.4.2. Functional characterization of bacteria of the shared microbiota of A. thaliana ......... 42 2.4.3. Functional role of root-associated bacteria on A. thaliana relatives ............................ 46 3. Discussion ......................................................................................................................... 49 3.1. Compartment, soil type, residence time and environmental conditions are major determinants of community structure .......................................................................... 49 III Table of contents 3.2. Plant species and plant development influence community structure only to a minor extent ........................................................................................................................... 53 3.3. Defining a shared Brassicaceae root microbiota .......................................................... 55 3.4. Members of the bacterial root microbiota are culturable ............................................. 57 3.5. Bacteria of the shared root microbiota alter root architecture of A. thaliana .............. 59 3.6. The effect of bacteria of the shared microbiota cannot be predicted for other Brassicaceae than A. thaliana ...................................................................................... 61 3.7. Conclusions and future perspectives ........................................................................... 63 4. Materials and Methods ................................................................................................... 65 4.1. Materials ...................................................................................................................... 65 4.1.1. Storage and preparation of soil material ...................................................................... 65 4.1.2. Plant material ............................................................................................................... 66 4.1.3. Oligonucleotides .......................................................................................................... 66 4.1.4. Enzymes ....................................................................................................................... 67 4.1.5. Chemicals and antibiotics ............................................................................................ 67 4.1.6. Buffers and solutions ................................................................................................... 67 4.2. Methods ....................................................................................................................... 67 4.2.1. Agarose gel electrophoresis ......................................................................................... 67 4.2.2. Seed sterilization .......................................................................................................... 68 4.2.3. Plant growth conditions in the greenhouse .................................................................. 68 4.2.4. Plant growth of A. alpina under natural conditions ..................................................... 68 4.2.5. Sampling of the root, soil and rhizosphere compartment ............................................ 69 4.2.6. Preparation of root samples for scanning electron microscopy (SEM) ....................... 69 4.2.7. DNA isolation from the root, soil and rhizosphere compartments .............................. 70 4.2.8. Generation of 16S rRNA gene amplicon libraries for pyrosequencing (454) ............. 70 4.2.9. Generation of 16S rRNA gene amplicon libraries for Illumina sequencing ................ 71 4.2.10. Bioinformatic analysis of amplicon libraries ............................................................... 72 4.2.11. Isolation and identification of root-associated bacteria ............................................... 75 4.2.12. Purification of root-associated bacteria ....................................................................... 76 4.2.13. Plant growth-promotion assays .................................................................................... 76 4.2.14. Statistical analysis for plant growth-promotion assays ................................................ 77 IV Table of contents Chapter II Root-associated rhizobial members: Plant growth-promoting properties on A. thaliana 1. Introduction ..................................................................................................................... 79 1.1. Colonization of plant roots by bacteria ........................................................................ 79 1.2. The role of the bacterial root microbiota on plant health ............................................ 81 1.2.1. Nutrient availability ..................................................................................................... 81 1.2.2. Interference with plant hormones ................................................................................ 84 1.2.3. Biocontrol activities ..................................................................................................... 85 1.3. Rhizobia as plant growth-promoting rhizobacteria ..................................................... 86 1.3.1. Rhizobia and their interactions with legume plants ....................................................
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