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Analyzing the Viral Resistance of Tomato Plant to TYLCV, PVY, and TSWV Under Heat and Drought Conditions

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Analyzing the Viral Resistance of Tomato Plant to TYLCV, PVY, and TSWV Under Heat and Drought Conditions Analyzing the viral resistance of tomato plant to TYLCV, PVY, and TSWV under heat and drought conditions Der Naturewissenschaftlichen Fakultät der Friedrich Alexander Universität Erlangen-Nürnberg Zur Erlangung des Doktorgrades Dr. rer. nat. Vorgelegt von Wafa'a Odeh Aus Jordanien Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der Friedrich-Alexander Universität Erlangen-Nürnberg Tag der mündlichen Prüfung: 20.02.2018 Vorsitzende/r des Promotionsorgans: Prof. Dr.Georg Kreimer. Gutachter/in: Prof. Dr. Uwe Sonnewald. Prof. Dr. Wolfgang Kreis. ii To the soul of my mother & To my beloved family iii Table of Contents Summary .................................................................................................................. 1 Zusammenfassung .................................................................................................. 3 1. Introduction ......................................................................................................... 5 1.1 Tomato ............................................................................................................ 5 1.1.1 An important crop .................................................................................... 5 1.1.2 Tomato is a sensitive crop plant .............................................................. 6 1.2 Viral diseases of tomato plants ....................................................................... 6 1.2.1 Tomato yellow leaf curl virus (TYLCV) ................................................. 8 1.2.2 Tomato spotted wilt virus (TSWV) ......................................................... 9 1.2.3 Potato virus Y (PVY) ............................................................................. 10 1.3 Plants are exposed to different abiotic stress ................................................ 11 1.4 Climate change and abiotic stress ................................................................. 12 1.5 Response of plants to combined biotic and abiotic stress ............................. 12 1.6 Plant receptors: key role in pathogen recognition ........................................ 15 1.7 Host factors; an important role in viral pathogenicity .................................. 17 1.8 Development of virus resistant plants ........................................................... 20 1.9 Aims of this study ......................................................................................... 21 2. Materials and methods ..................................................................................... 23 2.1 Materials ....................................................................................................... 23 2.1.1 Chemicals, enzymes, and consumables ................................................. 23 2.1.2 Bacterial strains ...................................................................................... 23 2.1.3 Antibiotics and additives ....................................................................... 23 2.1.4 Vectors ................................................................................................... 24 2.1.5 Oligonucleotides and sequencing ......................................................... 24 2.1.6 Media ..................................................................................................... 25 2.1.7 Plant materials ........................................................................................ 26 2.1.8 Constructs for plant transformation ....................................................... 26 iv 2.2 Methods ........................................................................................................ 27 2.2.1 Growth conditions and transformation .................................................. 27 2.2.1.1 Growth conditions and transformation of Bacteria ...................... 27 .2.21.2 Growth conditions and transformation of plants ......................... 28 2.2.2 Transcriptome analysis of tomato plants exposed to triple stress (virus, heat, and drought) ................................................................................. 28 2.2.2.1 Field experiment in Jordan ........................................................... 28 2.2.2.1.1 Sites selection and time frame ............................................... 28 2.2.2.1.2 Soil analysis ........................................................................... 28 2.2.2.1.3 TYLCV transfection of tomato plants ................................... 28 2.2.2.1.4 Plants in the field ................................................................... 29 .22.2.1.5 Temperature monitoring ........................................................ 29 2.2.2.1.6 Monitoring the water content of the soil ................................ 29 2.2.2.1.7 Sample collection ................................................................... 30 2.2.2.1.8 Symptoms monitoring ........................................................... 30 2.2.2.2 Transcriptome analysis ................................................................. 30 2.2.2.2.1 RNA isolation ........................................................................ 30 2.2.2.2.2 Microarray hybridization ....................................................... 31 2.2.2.2.3 Analysis of Microarray data .................................................. 31 2.2.2.2.4 DNA extraction, PCR amplification, and sequencing analysis31 2.2.2.2.5 Prediction of the secondary structure of the LRR-RLP ......... 32 2.2.2.2.6 Quantitative real-time RT-PCR (Q-RT-PCR) of the gene receptor-like kinase (RLK) ................................................... 32 .22.2.2.7 Analyzing the conserved domain of RLK and LRR-RLP ..... 32 2.2.3 Transformation of Nicotiana tabaccum and Solanum lycopersicum with constructs having single or double genes ............................................. 32 .2.23.1 Preparation of the construct with the single gene (NSm) ............. 32 2.2.3.2 Stable transformation of N. tabaccum and S. lycopersicum plants33 2.2.3.2.1 Stable transformation of tobacco and tomato plants .............. 33 2.2.3.2.2 Analyzing regenerated plantlets ............................................ 33 2.2.3.2.3 Testing transgenic N. tabaccum plants for TSWV resistance 34 2.2.3.3 Selection of homozygous transgenic lines .................................... 34 2.2.4 Identification of host factors interacting with TYLCV ......................... 35 2.2.4.1 DNA Isolation ............................................................................... 35 v .2.24.2 Rolling circle amplification (RCA) of TYLCV DNA .................. 35 2.2.4.3 Sequencing of TYLCV isolates .................................................... 35 2.2.4.4 Cloning the open reading frames (ORFs) of TYLCV ................. 35 2.2.4.5 Subcellular Localization of TYLCV ORFs .................................. 36 2.2.4.6 Immunoprecipitation of fused protein of V1, V2, and C2 ............ 36 2.2.4.7 Tryptic digestion of the precipitated protein ................................ 37 2.2.4.8 Analysis of precipitated protein via Nano LC-Mass Spectrometry37 2.2.4.9 Data analysis ................................................................................. 38 3. Results ................................................................................................................ 39 3.1 Transcriptome analysis of tomato plants exposed to triple stress ................. 39 3.1.1 Temperature records at both sites .......................................................... 40 3.1.2 Evaluation of disease symptoms ............................................................ 41 3.1.3 Transcriptome analysis of field samples ................................................ 43 3.1.4 Differential gene regulation in resistant and susceptible lines .............. 46 3.1.5 Differentially regulated genes located on chromosome 11 ................... 48 3.1.6 Relative expression of Receptor-like kinase (RLK) .............................. 52 3.1.7 Conserved domains of RLK and LRR-RLP .......................................... 53 3.2 Evaluation of Potato virus Y (PVY) and Tomato spotted wilt virus (TSWV) resistance in transgenic Nicotiana tabacum SNN and Solanum lycopersicum M82 lines having single or double gene constructs ...................................... 54 3.2.1 Tobacco and tomato plants expressing single or double genes ............. 54 3.2.2 TSWV resistance in tobacco plants transformed with the single or double genes ..................................................................................................... 58 3.2.3 T1 transgenic tobacco and tomato lines ................................................. 60 3.3 Interaction partners of Tomato yellow leaf curl virus (TYLCV) ................. 61 3.3.1 TYLCV working isolate ........................................................................ 61 3.3.2 Subcellular localization of V1, V2, and C2 ........................................... 62 3.3.3 Candidate interaction partners of TYLCV ORFs .................................. 63 4. Discussion .......................................................................................................... 65 4.1 Gene expression does not respond to treatments .......................................... 65 4.2 LRR-RLP interacts with RLK to initiate signaling and mediates resistance response to TYLCV in resistant lines ........................................................... 66 4.3 Resistant lines are distinguished from susceptible lines via SNPs detected
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