Research Collection Doctoral Thesis Structure, culturability and adaptation cues of the Arabidopsis leaf microbiota Author(s): Müller, Daniel B. Publication Date: 2016 Permanent Link: https://doi.org/10.3929/ethz-a-010693815 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library DISS. ETH NO. 23510 Structure, culturability and adaptation cues of the Arabidopsis leaf microbiota A thesis submitted to attain the degree of DOCTOR OF SCIENCES of ETH ZURICH (Dr. sc. ETH Zurich) presented by Daniel Bastian Müller Dipl. Biol., Goethe University Frankfurt born July 10, 1984 citizen of Germany accepted on the recommendation of Prof. Dr. Julia A. Vorholt Prof. Dr. Rudolf Aebersold Prof. Dr. Martin Ackermann 2016 Contents Abstract ....................................................................................................................................................1 Zusammenfassung ....................................................................................................................................3 1. Introduction ..........................................................................................................................................5 1.1 The plant - A huge microbial habitat ............................................................................................6 1.2 Phylogenetic structure of the plant microbiota .............................................................................8 1.3 Plant microbiota establishment and driving forces .....................................................................12 1.3.1 Sources of colonizing bacteria ...........................................................................................12 1.3.2 Environmental factors .......................................................................................................14 1.3.3 Host genetics ....................................................................................................................14 1.3.4 Microbial interactions ......................................................................................................16 1.4 The plant microbiome - Adaptation to life on plants ..................................................................16 1.5 Importance of the microbiota for host fitness .............................................................................20 1.6 Scope of this thesis .....................................................................................................................22 2. Functional overlap of the Arabidopsis leaf and root microbiota ........................................................23 2.1 Abstract and Introduction ...........................................................................................................25 2.2 Results and Discussion ...............................................................................................................26 2.2.1 Bacterial culture collections from roots and leaves ...........................................................26 2.2.2 At-RSPHERE and At-LSPHERE culture collections ........................................................27 2.2.3 Comparative genome analysis of the culture collections ..................................................28 2.2.4 Synthetic community colonization of germ free plants .....................................................31 2.2.5 Niche-specific microbiota establishment with SynComs .................................................33 2.3 Conclusion ..................................................................................................................................35 2.4 References ..................................................................................................................................35 2.5 Methods ......................................................................................................................................36 2.6 Extended data figures .................................................................................................................44 3. Systems-level proteomics of two ubiquitous leaf commensals reveals complementary adaptive traits for phyllosphere colonization ................................................................................................................55 3.1 Summary ....................................................................................................................................57 3.2 Introduction ................................................................................................................................58 3.3 Experimental procedures ............................................................................................................59 3.3.1 Experimental design and statistical rational ......................................................................59 3.3.2 Bacterial strains and growth conditions ............................................................................59 3.3.3 Plant growth conditions and inoculation of phyllosphere bacteria ...................................59 3.3.4 Harvest of plants and recovery of phyllosphere bacteria ..................................................60 3.3.5 Microscopy .......................................................................................................................60 3.3.6 Preparation of protein samples for MS .............................................................................60 3.3.7 SWATH assay library generation .....................................................................................61 3.3.8 SWATH data acquisition ..................................................................................................63 3.3.9 SWATH data analysis with OpenSWATH ......................................................................63 3.3.10 Relative quantification by ANOVA ...............................................................................63 3.3.11 Protein inference by aLFQ .............................................................................................64 3.3.12 Proteome comparison of different conditions ................................................................64 3.3.13 Protein comparison with other leaf microbiota members ...............................................64 3.3.14 Data availability ..............................................................................................................65 3.4 Results ........................................................................................................................................65 3.4.1 SWATH assay library construction ..................................................................................65 3.4.2 Overview of SWATH measurements ...............................................................................66 3.4.3 Adaptation of S. melonis Fr1 to the Arabidopsis phyllosphere ........................................66 3.4.4 Adaptation of M. extorquens Fr1 to the Arabidopsis phyllosphere ..................................67 3.4.5 Metabolic specialization of Methylobacterium strains .....................................................71 3.4.6 Overlap of regulated proteins between M. extorquens PA1 and S. melonis Fr1 ..............72 3.5 Discussion ..................................................................................................................................73 3.6 References ..................................................................................................................................76 3.7 Supplementary material ..............................................................................................................80 4. Bipartite interactions and plant protective abilities of the Arabidopsis leaf microbiota ...................89 4.1 Summary ....................................................................................................................................91 4.2 Introduction ................................................................................................................................92 4.3 Methods ......................................................................................................................................93 4.3.1 Pair-wise interaction screen of phyllosphere bacteria .......................................................93 4.3.2 Analysis of natural community data .................................................................................93 4.3.3 Secondary metabolite cluster prediction ...........................................................................93 4.3.4 Plant growth and enumeration of phyllosphere bacteria ..................................................94 4.3.5 Whole genome comparison of Sphingomonas isolates ....................................................94 4.4 Results ........................................................................................................................................95 4.4.1 Binary interaction network of phyllosphere bacteria .......................................................95 4.4.2 Identification of putative gene clusters of natural product biosynthesis ..........................98 4.4.3 Screen for plant colonization efficiency of individual isolates ......................................100 4.4.4 Plant protective abilities of selected leaf microbiota