Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2014 Functional Analysis of Syrbactin Synthetase-Like Gene Clusters in Photorhabdus luminescens and Rhizobium sp. and the Role of Syrbactins in Pseudomonas syringae Strains Colonizing Grass Species Dudnik, Alexey Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-101438 Dissertation Originally published at: Dudnik, Alexey. Functional Analysis of Syrbactin Synthetase-Like Gene Clusters in Photorhabdus lu- minescens and Rhizobium sp. and the Role of Syrbactins in Pseudomonas syringae Strains Colonizing Grass Species. 2014, University of Zurich, Faculty of Science. Functional Analysis of Syrbactin Synthetase-Like Gene Clusters in Photorhabdus luminescens and Rhizobium sp. and the Role of Syrbactins in Pseudomonas syringae Strains Colonizing Grass Species Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Alexey Dudnik aus Kasachstan Promotionskomitee Prof. Dr. Robert Dudler (Vorsitz und Leitung der Dissertation) Prof. Dr. Leo Eberl Prof. Dr. Julia Vorholt Zürich, 2014 Table of Contents Table of Contents 1. Summary ................................................................................................................................................ 5 2. Zusammenfassung .................................................................................................................................. 8 3. General Introduction ............................................................................................................................ 11 3.1. Plant-microbe interactions ...................................................................................................... 11 3.2. The plant immune system ....................................................................................................... 11 3.3. Pseudomonas syringae ............................................................................................................ 14 3.4. Pathogenicity and virulence factors of P. syringae ................................................................. 14 3.5. Syringolin A ............................................................................................................................ 18 3.6. Syrbactins ................................................................................................................................ 21 3.7. Aims of the thesis .................................................................................................................... 22 4. Heterologous Expression of a Photorhabdus luminescens Syrbactin-Like Gene Cluster Results in Production of the Potent Proteasome Inhibitor Glidobactin A .......................................................................... 25 4.1. Abstract ................................................................................................................................... 26 4.2. Introduction ............................................................................................................................. 26 4.3. Materials and Methods ............................................................................................................ 28 4.4. Results ..................................................................................................................................... 30 4.5. Discussion ............................................................................................................................... 32 5. The Endophytic Strain Rhizobium sp. AP16 Produces the Proteasome Inhibitor Syringolin A .......... 34 5.1. Abstract ................................................................................................................................... 35 5.2. Introduction ............................................................................................................................. 35 5.3. Materials and methods ............................................................................................................ 37 5.4. Results ..................................................................................................................................... 40 5.4.1. The syl-like gene cluster of Rhizobium sp. AP16 ....................................................... 40 5.4.2. Rhizobium sp. AP16 produces syringolin A and related variants .............................. 42 5.4.3. The AP16 SylC homolog PMI03_05100 exhibits tandem thiolation domains ........... 45 5.4.4. Rhizobium sp. AP16 is closely related Rhizobium rhizogenes K84 ........................... 45 5.5. Discussion ............................................................................................................................... 46 5.6. Supplementary figures and tables ........................................................................................... 48 6. Virulence Determinants of Pseudomonas syringae Strains in the Context of a Small Type III Effector Repertoire .......................................................................................................................................................... 52 6.1. Abstract ................................................................................................................................... 53 6.2. Introduction ............................................................................................................................. 53 6.3. Materials and methods ............................................................................................................ 56 6.4. Results ..................................................................................................................................... 59 2 Table of Contents 6.4.1. Syringolin production shows strain-specific effects on symptom development in wheat…………………………………………………………………………………………59 6.4.2. The T3SS is the key pathogenicity determinant .......................................................... 61 6.4.3. Importance of individual T3Es ................................................................................... 63 Contribution of exopolysaccharides to virulence of Psy SM on wheat ................................... 65 6.4.4. The type VI secretion system is involved in inter-species competition, but not in virulence .................................................................................................................................. 66 6.5. Discussion ............................................................................................................................... 68 6.6. Supplementary figures and tables ........................................................................................... 71 7. Genomics-Based Exploration of Virulence Determinants and Host-Specific Adaptations of Pseudomonas syringae Strains Isolated from Grasses ...................................................................................... 77 7.1. Abstract ................................................................................................................................... 78 7.2. Introduction ............................................................................................................................. 78 7.3. Results and Discussion ............................................................................................................ 81 7.3.1. Phylogenetic assessment of the strains ...................................................................... 81 7.3.2. Genome comparison and identification of Poaceae-specific genes ........................... 83 7.3.3. The type III secretion system and effector repertoire................................................. 85 7.3.4. Other virulence factors .............................................................................................. 88 7.3.5. Mobile and Extrachromosomal Elements .................................................................. 95 7.3.6. Other notable genome components ............................................................................ 98 7.4. Experimental section ............................................................................................................. 100 7.5. Conclusions ........................................................................................................................... 101 8. General Discussion ............................................................................................................................. 102 9. Literature ............................................................................................................................................ 105 Appendix 1: High-Quality Draft Genome Sequence of Pseudomonas syringae Pathovar syringae Strain SM Isolated from Wheat ........................................................................................................................................ 134 A1.1. Abstract .................................................................................................................................... 135 A1.2. Genome announcement ............................................................................................................ 135 A1.3. Nucleotide sequence accession numbers .................................................................................. 136