Aus dem Institut für Phytopathologie der Christian-Albrechts-Universität zu Kiel Investigation on the role of plant defensin proteins in regulating plant-Verticillium longisporum interactions in Arabidopsis thaliana Dissertation zur Erlangung des Doktorgrades der Agrar- und Ernährungswissenschaftlichen Fakultät der Christian-Albrechts-Universität zu Kiel vorgelegt von M.Sc. Shailja Singh aus Lucknow, India Kiel, 2020 Dekan/in: Professor Dr. Dr. Christian Henning 1. Berichterstatter: Prof. Dr. Daguang Cai 2. Berichterstatter: Prof. Dr. Professor Joseph-Alexander Verreet Tag der mündlichen Prüfung: 17.06.2020 Schriftenreihe des Instituts für Phytopathologie der Christian-Albrechts-Universität zu Kiel; Heft 7, 2020 ISSN: 2197-554X Gedruckt mit Genehmigung der Agrar- und Ernährungswissenschaftlichen Fakultät der Christian-Albrechts-Universität zu Kiel Table of Contents Table of Contents Table of Contents ........................................................................................................... I List of Figures ................................................................................................................ V List of Tables ............................................................................................................... VII List of Abbreviations .................................................................................................. VIII Chapter I: General Introduction .................................................................................... 1 1 Induced plant defense responses .......................................................................... 2 1.1 Phytohormones - general role in plants and special role in plant defense .... 4 1.2 JA/ET-mediated defense against pathogens and signaling pathways .......... 5 1.3 Crosstalk between the phytohormones ET, JA, and SA ............................... 9 2 Role of plant defensins in immunity ...................................................................... 11 2.2 Structure of plant defensins ........................................................................ 12 2.3 Modes of action of plant defensins ............................................................. 14 2.4 Role of defensins in plant defense .............................................................. 17 2.5 Biotechnological applications ..................................................................... 18 3 The plant pathogen Verticillium longisporum ....................................................... 19 3.1 Taxonomy and morphology of Verticillium longisporum ............................. 19 3.2 Symptom development and life cycle of Verticillium longisporum ............... 20 3.3 Plant response to V. longisporum infection ................................................ 22 3.4 Disease Management ................................................................................. 24 4 The model plant Arabidopsis thaliana .................................................................. 26 5 Aim of the thesis................................................................................................... 27 Chapter II: The fungus Verticillium longisporum suppresses plant-defensin AtPDF2.2 to achieve its colonization on its host Arabidopsis ................................ 29 1 Abstract ................................................................................................................ 30 2 Introduction .......................................................................................................... 31 3 Materials and methods ......................................................................................... 36 3.1 Plant material and growth conditions ................................................................ 36 4 Results ................................................................................................................. 43 4.1 Trancript analysis indicates suppression of AtPDF2.2 in Arabidopsis roots at the early infection stage .................................................................................... 43 I Table of Contents 4.2 AtPDF2.2 is constitutively expressed in plants and suppressed by the V. longisporum infection at the early infection stage ............................................. 45 4.3 Knockdown and overexpression of AtPDF2.2 alter the plant phenotype and flowering time ................................................................................................... 47 4.4 Knockdown and overexpression of AtPDF2.2 in transgenic Arabidopsis differentially affects the expressions of JA, SA and ET-associated genes ....... 49 4.5 AtPDF2.2 is involved in plant-Verticillium interactions ................................ 51 4.6 AtPDF2.2 is involved in plant-bacterial interactions .................................... 54 4.7 AtPDF2.2 is involved in plant-S. sclerotiorum interactions ......................... 55 5 Discussion ........................................................................................................... 57 6 Acknowledgments ................................................................................................ 70 7 Supporting Information ......................................................................................... 71 Chapter III: Overexpression of AtPDF1.2a confers resistance to fungal pathogens in Arabidopsis thaliana ............................................................................................... 77 1 Abstract ................................................................................................................ 78 2 Introduction .......................................................................................................... 79 3 Materials and methods ......................................................................................... 82 4 Results ................................................................................................................. 89 4.1 Upregulation of AtPDF1.2a gene in Arabidopsis roots at the early infection stage ................................................................................................................. 89 4.2 In-silico analysis of expression of Class- I AtPDF genes ............................ 91 4.3 Knockdown and overexpression of the AtPDF1.2 gene alters the phenotype and rosette size ................................................................................................ 94 4.4 Knockout of AtPDF1.2a prompts differential expressions of defense related genes ................................................................................................................ 95 4.5 SA-signaling pathway defecient mutants show impeded AtPDF1.2a expression ........................................................................................................ 96 4.6 V. longisporum (Vl43) infection inhibits the primary root length of KO-pdf1.2a ......................................................................................................................... 97 4.7 Overexpression of AtPDF1.2a enhanced plant resistance to V. longisporum infection ............................................................................................................ 98 II Table of Contents 4.8 Pseudomonas syringae (Pst DC3000) induces the expression of AtPDF1.2a ....................................................................................................................... 100 4.9 OE-PDF1.2a plants displayed improved resistance to S. sclerotiorum infection .......................................................................................................... 101 5 Discussion .......................................................................................................... 103 6 Acknowledgments .............................................................................................. 116 7 Supporting Information ....................................................................................... 117 Chapter IV: General discussion ............................................................................... 119 2 V. longisporum suppresses the expression of AtPDF2.2 and induces the expression of AtPDF1.2a in the early phase of infection ....................................... 121 3 In-silico promoter analysis of AtPDF2.2 and AtPDF1.2 indicates complex regulatory mechanisms of AtPDFs ........................................................................ 123 4 Promoter analysis revealed a gradual suppression of AtPDF2.2 from roots to leaves .................................................................................................................... 124 5 Plant defensins are functionally involved in plant growth and development ....... 125 6 Knockdown and overexpression of AtPDF2.2 and AtPDF1.2a lead to altered phenotype in transgenic plants ............................................................................. 127 7 Digital transcript profiles of AtPDF2.2 and AtPDF1.2a under biotic and abiotic stresses ................................................................................................................. 128 8 Knockdown/out of AtPDF2.2 and AtPDF1.2a prompt the differential expressions of AtPDF genes and JA, SA or ET-associated genes in transgenic plants ............... 130 9 Translational regulation of AtPDF2.2 and AtPDF1.2a ........................................ 132 10 Overexpression of AtPDF2.2 and AtPDF1.2a enhances plant resistance to V. longisporum infection in transgenic plants ...........................................................