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Characterization of Erwinia Amylovora - Host Plant and - Biocontrol Agent Interactions

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Characterization of Erwinia Amylovora - Host Plant and - Biocontrol Agent Interactions Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2013 Characterization of Erwinia amylovora - host plant and - biocontrol agent interactions Kamber, Tim Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-89948 Dissertation Originally published at: Kamber, Tim. Characterization of Erwinia amylovora - host plant and - biocontrol agent interactions. 2013, University of Zurich, Faculty of Science. Characterization of Erwinia amylovora - Host Plant and - Biocontrol Agent Interactions Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von Tim Kamber von Hauenstein-Ifenthal (SO) Promotionskomitee: Prof. Dr. Beat Keller (Vorsitz) Prof. Dr. Robert Dudler Dr. Brion Duffy (Leitung der Dissertation) Zürich, 2013 Table of contents Table of contents 3 Summary 6 Zusammenfassung 8 Chapter 1. General Introduction 11 Erwinia amylovora – causal agent of the fire blight disease 12 Pantoea vagans C9-1 – a biocontrol agent of E. amylovora 16 Malus x domestica – host plant of E. amylovora 17 Objectives 18 Chapter 2. Genomics and current genetic understanding of Erwinia amylovora and the fire blight antagonist Pantoea vagans 19 Abstract 20 Introduction 20 Erwinia amylovora genomics 21 Erwinia inter-species genomics 23 Selected features clarified using comparative genomics 28 Type III secretion systems 28 Exopolysaccharides 32 Siderophores 34 Phylogenomic applications 34 Erwinia billingiae 35 Pantoea biocontrol agent genomics 35 Pantoea comparative genomics 36 Perspectives 37 Acknowledgements 37 Chapter 3. Identification of the type VI secretion regulome in Erwinia amylovora using RNA-seq 39 Abstract 40 Introduction 40 Material and Methods 42 Bacterial strains, media and growth conditions 42 Construction of T6SS mutants 42 RNA isolation and reverse transcription RT-PCR 43 RNA-seq data analysis 44 Immature pears assay 44 3 Shoot inoculation assay 44 Flower assay 44 Motility assay 45 Results 46 Characterization of T6SS mutants 46 In vitro transcriptome WT versus ∆T6SS-C1C3 46 Immature pear assay 48 Shoot inoculation 48 Flower assay 49 In planta transcriptome WT versus T6SS double mutant 50 Motility assay 50 Discussion 51 Chapter 4. Characterization of the antibacterial peptide herbicolin I biosynthetic operon in Pantoea vagans biocontrol strain C9-1 and incidence in Pantoea species 55 Abstract 56 Introduction 56 Materials and Methods 58 Bacterial strains and plasmids 58 Antimicrobial production and activity assays 58 Mutational analysis 58 Immature pear fruit assay 59 Sequence analysis 59 Antibiotic purification 59 Sensitivity of orchard isolates of E. amylovora to antibiotics of P. vagans C9-1 60 Results 61 Chemical identification of antibiotic compound 61 Identification of the herbicolin I gene cluster 66 Sequence analysis 68 Screening for dapdiamide biosynthetic genes in Pantoea species 70 Disscussion 70 Acknowledgements 73 Chapter 5. The apple flower reactome in response to fire blight infection 75 Abstract 76 Introduction 76 Materials and Methods 78 4 Bacterial strains, media and growth conditions 78 Flower inoculation 78 RNA extraction 78 RNA-sequencing 79 Data analysis 79 cDNA construction and RT-PCR 80 Results 80 Apple transcriptome analysis in response to E. amylovora 80 Differentially expressed genes 84 Reverse transcription PCR (RT-PCR) 86 Discovery of novel potential ORF and CNS 87 Discussion 91 Chapter 6. General conclusion and outlook 95 Conclusion 96 Outlook 96 Chapter 7. References 99 Chapter 8. Appendix 113 Table 1 All significantly differentially expressed transcripts from in vitro experiments comparing ∆T6SS-C1C3 to the E. amylovora wild type strain 114 Table 2 All significantly differentially expressed transcripts from in planta experiments comparing ∆T6SS-C1C3 to the E. amylovora wild type strain 126 Table 3 All significantly differentially expressed M. x domestica transcripts in response to E. amylovora two days post inoculation 128 Table 4 Annotation of previously unassigned significantly differential expressed M. x domestica transcripts in response to E. amylovora two days post inoculation 156 Table 5 Annotation of previously unassigned expressed M. x domestica transcripts two days post inoculation 159 Acknowledgements 183 Curriculum vitae 184 5 Summary Fire blight is caused by the phytopathogenic Gram-negative enterobacterium Erwinia amylovora and infests a wide range of commercially, ornamentally and ecologically important members of the Rosaceae family. Fire blight is a devastating necrogenic disease and the damage to apple and pear production is a major concern. The pathogen spreads systemically in host plants after entering through natural openings (nectarthodes, stomata) or wounds. Dependent on the plant part affected, disease symptoms develop as blossom blight, shoot blight or rootstock blight. Until now, no systemic cure for infected plants is known, and diseased tissues have to be removed by pruning. E. amylovora overwinters in cankers and following warming temperature emerges as ooze in spring. The ooze consists of bacterial cells and exopolysaccharides produced by the pathogen. The viscous texture of the ooze protects bacterial cells from abiotic stress factors and facilitates dispersal from plant to plant. Pathogenicity of E. amylovora to host plants is strictly dependent on a functional type III secretion system and the exopolysaccharide amylovoran. Factors contributing to virulence and ecological fitness of E. amylovora are reviewed in Chapter 2. Availability of genome sequences in the genus Erwinia provides the basis to analyze the full arsenal of critical determinants for virulence, host range and ecological fitness of E. amylovora. Preventive disease control measures include the application of the antibiotics streptomycin and oxytetracycline to protect flowers. The emerging resistance of E. amylovora to the most effective antibiotic streptomycin raises the need for novel management measures. The application of biocontrol agents can significantly reduce the epiphytic growth of the pathogen. The effectiveness of these agents is based on combined modes of action, e.g.site exclusion, nutrient competition, and antibiotic production. Beside this strategy, apple breeding programs led to the development of cultivars with improved resistance to E. amylovora. Nevertheless, the main apple cultivars produced and consumed (e.g., ‘Golden Delicious’, ‘Gala’) are highly susceptible to the fire blight disease. The underlying processes leading to resistance or susceptibility of apple to E. amylovora are largely unknown and knowledge of these might lead to the development of novel control measures of the disease. The first aim of this thesis was to elucidate additional virulence factors of E. amylovora (Chapter 3). Using a comparative genomic approach, three type VI secretion systems (T6SS) of unknown function were identified in E. amylovora and characterized. Plant assays showed a moderate contribution of T6SSs to E. amylovora virulence. Analysis of transcriptomic data generated by RNA-sequencing from E. amylovora in vitro cultures and in 6 planta experiments indicated that the T6SSs influence metabolic processes, chemotaxis and motility of the bacteria. The second aim was to identify and characterize the antibiotic biosynthesis operon for herbicolin I in Pantoea vagans biocontrol strain C9-1 described in Chapter 4. Analysis of mutants defective in herbicolin I biosynthesis revealed a gene cluster consisting of ten open reading frames located on the plasmid pPag2. Deficiency in herbicolin I production reduced the biocontrol efficacy of P. vagans C9-1 on E. amylovora. Prevalence screening of 45 Pantoea spp. from biocontrol, environmental, and clinical origins for herbicolin I biosynthetic genes revealed this to be a rare trait among the tested strains. Chapter 5 addresses the third aim of the presented thesis, the transcriptional response of susceptible apple (Malus x domestica variety ‘Golden Delicious’) to E.amylovora in order to identify genes and pathways affected in the host plant. The data revealed differential expression of genes involved in phytohormone biosynthesis and phenylpropanoid pathways, as well as putative resistance genes, transcription factors and pathogenesis-related genes. In addition, previously unrecognized genes in the apple genome sequence and potential new open reading frames in apple and peach (Prunus persica) were identified. The data presented in this thesis provides a comprehensive knowledge of the T6SSs in E. amylovora, focusing on their effects on virulence and the identification of a potential T6SS regulome. The identified gene cluster for the antibiotic herbicolin I in P. vagans C9-1 will facilitate the screening process for novel biocontrol agents producing this effective antibiotic. The transcriptional responses revealed by RNA-seq of susceptible apple to E. amylovora provide a profound knowledge of this host-pathogen interaction and a basis for the development of novel fire blight control measures. 7 Zusammenfassung Feuerbrand wird durch das Gram-negative Bakterium Erwinia amylovora verursacht und befällt ein breites Spektrum an Zierpflanzen, der kommerziell und ökologisch wichtigen Mitgliedern der Rosaceae Familie. Feuerbrand ist eine verheerende nekrogene Krankheit deren Schäden ein zentrales Problem für die Apfel- und Birnenproduktion darstellen.
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