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Identification, Cloning and Functional Characterisation of Related Genes On Isabelle Vogt Institut für Züchtungsforschung an Obst Fire blight: identifi cation, cloning and functional characterisation of related genes on Malus ×robusta Dissertationen aus dem Julius Kühn-Institut Julius Kühn-Institut Bundesforschungsinstitut für Kulturpfl anzen Kontakt/Contact: Isabelle Vogt Kamenzerstr. 42b 01099 Dresden Die Schriftenreihe ,,Dissertationen aus dem Julius Kühn-lnstitut" veröffentlicht Doktorarbeiten, die in enger Zusammenarbeit mit Universitäten an lnstituten des Julius Kühn-lnstituts entstanden sind. The publication series „Dissertationen aus dem Julius Kühn-lnstitut" publishes doctoral dissertations originating from research doctorates and completed at the Julius Kühn-Institut (JKI) either in close collaboration with universities or as an outstanding independent work in the JKI research fields. Der Vertrieb dieser Monographien erfolgt über den Buchhandel (Nachweis im Verzeichnis lieferbarer Bücher - VLB) und OPEN ACCESS im lnternetangebot www.julius-kuehn.de Bereich Veröffentlichungen. The monographs are distributed through the book trade (listed in German Books in Print - VLB) and OPEN ACCESS through the JKI website www.julius-kuehn.de (see Publications). Wir unterstützen den offenen Zugang zu wissenschaftlichem Wissen. Die Dissertationen aus dem Julius Kühn-lnstitut erscheinen daher OPEN ACCESS. Alle Ausgaben stehen kostenfrei im lnternet zur Verfügung: http://www.julius-kuehn.de Bereich Veröffentlichungen. We advocate open access to scientific knowledge. Dissertations from the Julius Kühn-lnstitut are therefore published open access. All issues are available free of charge under http://www.julius-kuehn.de (see Publications). Bibliografische Information der Deutschen Nationalbibliothek Die Deutsche Nationalbibliothek verzeichnet diese Publikation In der Deutschen Nationalbibliografie: detaillierte bibliografische Daten sind im lnternet über http://dnb.d-nb.de abrufbar. Bibliographic information published by the Deutsche Nationalbibliothek (German National Library) The Deutsche Nationalbibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data are available in the Internet at http://dnb.dnb.de. ISBN 978-3-95547-057-9 DOI 10.5073/dissjki.2018.001 Herausgeber l Editor Julius Kühn-lnstitut, Bundesforschungsinstitut für Kulturpflanzen, Quedlinburg, Deutschland Julius Kühn-lnstitut, Federal Research Centre for Cultivated Plants, Quedlinburg, Germany Dieses Werk ist lizenziert unter einer Creative Commons – Namensnennung – Weitergabe unter gleichen Bedingungen – 4.0 Lizenz. This work is licensed under a Creative Commons – Attribution – ShareAlike – 4.0 license. Fire blight: identification, cloning and functional characterisation of related genes on Malus ×robusta DISSERTATION zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) vorgelegt der Fakultät Mathematik und Naturwissenschaften der Technischen Universität Dresden von Diplom-Chemikerin Isabelle Vogt geboren am 24.09.1984 in Zittau Eingereicht am 06.10.2017 Die Dissertation wurde in der Zeit von August 2010 bis Dezember 2013 am Julius Kühn-Institut (JKI), Institut für Züchtungsforschung an Obst, in Dresden-Pillnitz angefertigt. Contents List of Abbreviations7 List of Figures9 List of Tables 11 1. Introduction 13 1.1. Description and history of the plant disease fire blight . 13 1.2. Conventional control methods of fire blight . 14 1.3. Genetic engineering to improve host resistance against fire blight . 16 1.4. General aspects of plant-pathogen interactions . 18 1.5. Genetics of fire blight . 19 1.6. The E. amylovora effector AvrRpt2EA ................... 20 1.7. Aim of the study . 21 2. Materials and methods 23 2.1. Materials . 23 2.1.1. Plant material . 23 2.1.2. Bacteria and yeast strains . 23 2.1.3. Vectors and plasmids . 24 2.2. Methods . 25 2.2.1. Inoculation of apple shoots . 25 2.2.2. Cultivation of bacteria . 25 2.2.3. Cultivation of yeast . 25 2.2.4. DNA extraction . 26 2.2.5. RNA extraction . 26 2.2.6. cDNA synthesis . 27 2.2.7. PCR based methods . 27 3 Contents 2.2.8. Cloning of E. coli ........................... 30 2.3. Sequencing of the avrRpt2EA gene of different E. amylovora strains . 31 2.4. Establishment of complemented E. amylovora strains . 31 2.5. Yeast two-hybrid assay . 32 2.5.1. Cloning . 32 2.5.2. Mating . 34 2.6. Western blot . 35 2.6.1. Protein extraction . 35 2.6.2. Electrophoresis of proteins . 35 2.6.3. Membrane transfer and detection . 36 2.7. RNA-seq . 37 2.7.1. Sample preparation and RNA extraction . 37 2.7.2. cDNA library construction, sequencing and mapping . 37 2.7.3. Data analysis and bioinformatics . 37 2.8. Gene expression analysis with the BioMark™ HD system . 38 2.8.1. Primer development and optimisation . 38 2.8.2. Differential gene expression analysis . 39 2.8.3. Statistical analysis . 40 3. Results 41 3.1. Analysis of the AvrRpt2EA effector . 41 3.1.1. Sequencing of the avrRpt2EA gene . 41 3.1.2. Development and virulence analysis of the complemented E. amyl- ovora strains . 42 3.1.3. Real-Time qRT-PCR with the complemented E. amylovora strains 44 3.2. RIN4 of Malus ×robusta 5.......................... 47 3.2.1. Sequence analysis of RIN4 from Mr5 . 47 3.2.2. Western blot with the RIN4 antibody . 50 3.3. Yeast two-hybrid assay to prove protein-protein interactions . 51 3.4. RNA-seq and gene expression analysis with the BioMark™ HD system . 54 3.4.1. Differential transcriptome analysis by RNA-seq . 54 3.4.2. Gene expression analysis with the BioMark™ HD system . 60 3.4.3. Comparison of the results obtained by RNA-seq and the Bio- Mark™ HD system . 67 4 Contents 3.4.4. Comparison between prediction, sequencing and RNA-seq data in case of RIN4 from Mr5 . 68 3.5. Fire blight resistance gene FB MR5 of Mr5 . 70 4. Discussion 73 4.1. Role of the E. amylovora effector AvrRpt2EA ................ 73 4.2. Identification and characterisation of RIN4 of Mr5 . 76 4.3. Protein-protein interaction in the system Mr5 and E. amylovora ..... 79 4.4. Differential transcriptome analysis of Mr5 by RNA-seq . 81 4.5. Gene expression analysis of Mr5 with the BioMark™ HD system . 87 4.6. Fire blight resistance gene FB MR5 of Mr5 . 90 5. Conclusion 92 Bibliography 95 Appendix 108 A. Materials . 108 A.1. Used chemicals . 108 A.2. Composition of buffers and media . 109 A.3. Laboratory equipment . 110 A.4. Consumables . 113 B. Erwinia amylovora strains . 114 C. Tables of primers . 116 C.1. General primers . 116 C.2. Primers for the synthesis of the complemented strains . 117 C.3. Primers used in Y2H assay . 118 C.4. Primers for RACE-PCR of FB MR5 and RIN4 .......... 119 C.5. Primers used in gene expression analysis with the BioMark™ HD system . 120 D. RT-PCR of the complemented E. amylovora strains . 124 E. Analysis of Real-Time q-RT data of the complemented E. amylovora strains125 F. Sequence of the avrRpt2EA-eu gene . 126 G. Alignment of the sequences received by 5’ RACE-PCR of RIN4 from Mr5 127 H. Protein sequence alignment of RIN4 . 131 5 Contents I. Protein sequence identity matrix of RIN4 . 134 J. RNA Integrity Number of the samples used for RNA-seq . 135 J.1. RIN value of the sample Ea1189 2 hpi . 135 J.2. RIN value of the sample Ea1189 48 hpi . 136 J.3. RIN value of the sample ZYRKD3-1 2 hpi . 137 J.4. RIN value of the sample ZYRKD3-1 48 hpi . 138 K. FastQC results of the transcriptome analysis . 139 L. Significant DEGs obtained from RNA-seq . 140 L.1. DEGs at 2 or 48 hpi . 140 L.2. DEGs at 2 and 48 hpi . 144 M. Assignment of RNA-seq data via MapMan . 145 N. CNRQ values of the genes analysed by BioMark™ HD system . 147 O. Genes analysed by BioMark™ HD system . 155 P. Statistical analysis by SAS of the CNRQ values of the genes analysed by BioMark™ HD system . 156 P.1. Shapiro-Wilk, Kruskal-Wallis and Levene’s test . 156 P.2. ANOVA and Tukey’s HSD test . 157 P.3. Mann-Whitney U test . 159 Q. Sequences of the FB MR5 gene obtained by 3’ and 5’ RACE-PCR . 160 Q.1. 5’ RACE-PCR of the FB MR5 gene . 160 Q.2. 3’ RACE-PCR of the FB MR5 gene . 162 Acknowledgements 164 Declaration of Authorship 165 List of publications 166 Curriculum vitae 168 6 List of Abbreviations ABA Abscisic acid ANOVA Analysis of variance APS Ammonium persulfate bp Base pair bZIP Basic leucine zipper cDNA Complementary DNA CDS Coding sequence cfu Colony-forming unit Chr Chromosome CNRQ Calibrated normalised relative quantities Ct Cycle threshold cv Cultivar ddH2O Double-distilled water DEG Differential expressed gene DNA Deoxyribonucleic acid dNTPs Deoxynucleotide triphosphate dpi Days post inoculation DTT Dithiothreitol ERF Ethylene responsive factor ETI Effector-triggered immunity FDR False discovery rate GDR Genome database for Rosaceae GO Gene Ontology hpi Hours post inoculation HR Hypersensitive response HSD Honest significant difference ID Identification 7 Contents JA Jasmonic acid kb Kilo base LG Linkage group MAB Marker-assisted breeding MAPK Mitogen-activated protein kinase mRNA Messenger RNA Mr5 Malus ×robusta 5 OD Optical density PAGE Polyacrylamide gel electrophoresis PCR Polymerase chain reaction PTI Pattern-triggered immunity PVDF Polyvinylidene difluoride PVE Phenotypic variation explained qRT-PCR Quantitative reverse transcription-PCR QTL Quantitative trait locus RACE Rapid amplification of cDNA ends RCS RIN4 cleavage sites RNA-seq RNA sequencing rpm Revolutions per minute RT Room temperature SA Salicylic acid SAS Statistical analysis system SDS Sodium dodecyl sulfate SNP Single nucleotide polymorphism T3SS Type III secretion system TA Annealing temperature TEMED Tetramethylethylenediamine Tris Tris(hydroxymethyl)aminomethane U Unit UTR Untranslated region Y2H Yeast two-hybrid 8 List of Figures 1.1. Shoots of Mr5 infected with the wild type strain Ea1189 or the avrRpt2EA deletion mutant ZYRKD3-1. 20 2.1. Assembly of the western blot apparatus. 36 3.1. Amino acid sequence and SNP analysis of the avrRpt2EA gene.
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