„Transcriptional regulation of defence gene expression by a VQ-motif containing protein” Dissertation Zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) der Naturwissenschaftlichen Fakultät I – Biowissenschaften – der Martin-Luther-Universität vorgelegt von Herrn Martin Weyhe geb. am 14.03.1987 in Merseburg Gutachter: Prof. Dr. Dierk Scheel | Leibniz Institut für Pflanzenbiochmie, Halle Prof. Dr. Ulla Bonas | Martin-Luther-Universität Halle-Wittenberg Prof. Dr. Thorsten Nürnberger | Zentrum für Molekularbiologie der Pflanzen, Tübingen Verteidigung: 03.07.2019 Index Index List of abbreviations ................................................................................................................... VI 1. Introduction ........................................................................................................................ 1 1.1. Plant immunity ....................................................................................................................... 1 1.1.1 Pattern recognition........................................................................................................ 1 1.1.2. Early MTI responses ....................................................................................................... 3 1.1.3. Late MTI responses ........................................................................................................ 4 1.1.4 Pathogen effectors and effector-triggered immunity (ETI) ........................................... 5 1.2. MAPK cascades ....................................................................................................................... 6 1.2.1. MAMP-induced MAPKs ................................................................................................. 7 1.2.2. MAPK targets involved in immunity .............................................................................. 8 1.3. WRKY transcription factors .................................................................................................. 10 1.3.1 The WRKY domain ....................................................................................................... 10 1.3.2 WRKY function and regulation .................................................................................... 10 1.4. VQ-motif containing proteins ............................................................................................... 12 1.4.1. VQs are transcriptional co-regulators ......................................................................... 12 1.4.2. MPK3/6-targeted VQs (MVQs) .................................................................................... 14 1.5. Aim of the present work ....................................................................................................... 16 2. Materials and methods ...................................................................................................... 17 2.1. Materials ............................................................................................................................... 17 2.1.1. Chemicals ..................................................................................................................... 17 2.1.2. Media ........................................................................................................................... 17 2.1.3. Bacteria ........................................................................................................................ 17 2.1.4. Plant material and growth conditions ......................................................................... 18 2.2. Methods ............................................................................................................................... 18 2.2.1 Molecular cloning ........................................................................................................ 18 2.2.2 Transformation of bacteria.......................................................................................... 19 2.2.3 Generation of MVQ phospho-site mutants ................................................................. 19 2.2.4 Genotyping T-DNA insertion lines ............................................................................... 19 2.2.5 Generation of CRISPR-Cas9 constructs ........................................................................ 20 2.2.6 Agrobacterium-mediated plant transformation ......................................................... 20 2.2.7 Quantitative Real-time PCR ......................................................................................... 20 2.2.8 Isolation of genomic DNA ............................................................................................ 20 III Index 2.2.9 Southern Blot ............................................................................................................... 21 2.2.10 Preparation and transfection of A. thaliana mesophyll protoplasts ........................... 21 2.2.11 Microscopy .................................................................................................................. 21 2.2.12 Promoter activation assay ........................................................................................... 22 2.2.13 Microarray analysis ...................................................................................................... 22 2.2.15 Expression of recombinant proteins in Escherichia coli .............................................. 23 2.2.16 Protein purification ...................................................................................................... 24 2.2.17 SDS-PAGE and immunoblot analysis ........................................................................... 24 2.2.18 Electrophoretic mobility shift assay (EMSA)................................................................ 25 2.2.19 DNA-protein interaction assay (DPI-ELISA).................................................................. 25 2.2.20 Chromatin Immunoprecipitation followed by sequencing (ChIP-seq) ........................ 26 2.2.21 Infection of Arabidopsis thaliana with Botrytis cinerea .............................................. 26 3. Results .............................................................................................................................. 28 3.1 Subcellular localisation of MVQs ..........................................................................................28 3.1.1 MVQs display two distinct subcellular localisation patterns ....................................... 28 3.1.2 Mutation of phosphorylation sites affects localisation of some MVQs ...................... 31 3.2 Effects of MVQ1 and other MVQs on defence gene promoter activity ...............................33 3.2.1 MVQ1 dampens MAMP-induced activation of pNHL10 via its VQ-motif .................... 33 3.2.2 MVQs differentially modulate MAMP-induced activation of pNHL10 ........................ 35 3.2.3 MVQ1 suppresses MAMP-induced activation of additional defence-related genes .. 38 3.2.4 MVQ1 antagonises WRKY-mediated activation of pNHL10 ........................................ 39 3.3 Confirmation of MVQ1 interactions with WRKY transcription factors ................................41 3.4 Transcriptome analysis of MVQ1 misexpression lines .........................................................44 3.4.1 Characterisation of MVQ1 misexpressing plant lines for transcriptome analysis ....... 44 3.4.2 Microarray analysis reveals differentially expressed genes in MVQ1 misexpression lines .............................................................................................................................. 45 3.4.3. Effect of altered MVQ1 levels on the transcriptome in control conditions ................ 46 3.4.4. Impact of MVQ1 on the transcriptome after MAMP-treatment................................. 49 3.4.5. Validation of potential MVQ1-suppressed defence genes by qRT-PCR ...................... 53 3.5 Influence of MVQ1 on DNA-binding of WRKYs ....................................................................55 3.5.1. EMSAs reveal interactions of MVQ1 with DNA-bound WRKY33 ................................. 55 3.5.2. MVQ1 stimulates binding of some WRKY-domains to DNA ........................................ 57 3.5.3. MVQ2-6 can also stimulate binding of WRKY33 cDBD to DNA ................................... 59 3.6 Potential MVQ1 targets identified by Chromatin Immunoprecipitation (ChIP) ...................61 IV Index 3.7 The role of MVQ1 in resistance against Botrytis cinerea ..................................................... 65 4. Discussion ......................................................................................................................... 67 4.1 MVQs are potential transcriptional co-regulators of defence genes ................................... 67 4.2 MVQ1 is a negative regulator of defence gene expression ................................................. 68 4.3 The molecular mode of action for MVQ1............................................................................. 70 4.3.1 Does MVQ1 affect DNA-binding or transcriptional activity of WRKYs? ...................... 70 4.3.2 MVQ1 is associated with target gene promoters via WRKYs ...................................... 73 4.3.3 Regulation of MVQ1 activity by MAPKs ...................................................................... 74 4.3.4 Integration of MVQ1 into the WRKY network ............................................................. 75 5. Summary ..........................................................................................................................