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Early Responses of Medicago Truncatula Roots After Contact with the Symbiont Rhizophagus Irregularis Or the Pathogen Aphanomyces Euteiches

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Early Responses of Medicago Truncatula Roots After Contact with the Symbiont Rhizophagus Irregularis Or the Pathogen Aphanomyces Euteiches Early responses of Medicago truncatula roots after contact with the symbiont Rhizophagus irregularis or the pathogen Aphanomyces euteiches Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) der Naturwissenschaftlichen Fakultat¨ I - Biowissenschaften der Martin-Luther-Universitat¨ Halle-Wittenberg, vorgelegt von Frau Dorothee´ Klemann geb. am 27. Mai 1983 in Kempten (Allgau)¨ Gutachter: 1. Prof. Bettina Hause 2.Prof. Edgar Peiter 3. Prof. Helge Kuster¨ Halle (Saale), 29. Februar 2015 Contents 1 Introduction1 1.1 Medicago truncatula ...............................1 1.2 Mycorrhiza: a symbiosis between plants and fungi...............2 1.2.1 The Arbuscular Mycorrhiza.......................2 1.2.2 Structures and life cycle of AMF.....................3 1.2.3 Plant benefits from AMF.........................5 1.3 Aphanomyces euteiches - an oomycete pathogen of legumes..........6 1.4 Communication between plant and rhizosphere microorganisms - friend or foe.8 1.4.1 The role of plant derived volatile organic compounds..........8 1.4.2 The role of root exudates on rhizosphere microorganisms........ 10 1.4.3 Communication between plants and AMF................ 11 1.4.4 Induced plant defense responses..................... 13 1.5 Aim of the study................................. 14 2 Material and methods 15 2.1 Material...................................... 15 2.1.1 Chemicals and supplies.......................... 15 2.1.2 Plants................................... 15 2.1.3 Microorganisms............................. 15 2.1.4 Oligonucleotides and Plasmids...................... 15 2.2 Biological methods................................ 16 2.2.1 Fertilizer and media for cultivation of plants and microorganisms... 16 2.2.2 Axenic cultivation of AM fungal material................ 18 2.2.3 Sterile cultivation of A. euteiches ..................... 18 2.2.4 Cultivation systems for M. truncatula .................. 19 2.3 Microscopic works................................ 20 2.3.1 Ink staining of AMF in root tissue.................... 20 2.3.2 Evaluation of mycorrhization rates via the Gridline intersection method“ 20 ” 2.3.3 Staining with WGA-AlexaFluor488................... 20 2.3.4 Localization of GFP-fused proteins................... 21 2.4 Molecular biological methods.......................... 21 2.4.1 Isolation of genomic DNA........................ 21 2.4.2 Isolation of Plasmid DNA........................ 21 2.4.3 Isolation of RNA............................. 21 2.4.4 Synthesis of cDNA............................ 21 2.4.5 Agarose Gel Electrophoresis....................... 21 2.4.6 Polymerase Chain Reaction (PCR).................... 22 2.4.7 Quantitative Real-Time PCR (qRT-PCR)................ 23 i 2.4.8 Affymetrix whole genome array of M. truncatula ............ 23 2.4.9 Genotyping of Tnt1 insertion lines.................... 24 2.4.10 Cloning techniques............................ 25 2.5 Microbiological Methods............................. 27 2.5.1 Production of chemically competent E. coli cells............ 27 2.5.2 Transformation of E. coli cells...................... 27 2.5.3 Production of chemically competent Agr. rhizogenes cells....... 28 2.5.4 Transformation of Agr. rhizogenes cells................. 28 2.5.5 M. truncatula transient root transformation mediated by Agr. rhizogenes 28 2.5.6 Transformation of N. benthamiana mediated by Agr. tumefaciens ... 29 2.6 Metabolite analysis................................ 30 2.6.1 Gas chromatography–mass spectrometry (GC-MS) for volatile analysis 30 2.6.2 Non-targeted metabolite profiling of root exudates with LC/MS.... 31 2.7 Different sample processing of root exudates for LC-MS analysis........ 32 3 Results 33 3.1 Evaluation of 33 M. truncatula accessions for mycorrhization capacity..... 33 3.2 Timing of earliest transcriptional events after AMF contact........... 37 3.2.1 Gene expression of known early induced genes............. 37 3.2.2 Test array at 3 different time points after inoculation.......... 39 3.3 Transcriptional changes 2 hrs after microbial challenge............. 40 3.3.1 Microarray analysis............................ 40 3.3.2 Validation of some interesting genes via qRT-PCR........... 43 3.4 Functional characterization of some candidate genes, early regulated after mi- crobial contact with R. irregularis or A. euteiches ................ 47 3.4.1 Screen for Tnt1 insertion lines...................... 47 3.4.2 Tnt1 insertion for TS ........................... 47 3.4.3 Functional characterization of putative terpene synthase in M. truncat- ula roots................................. 50 3.4.4 TS- overexpression............................ 51 3.4.5 RNAi for some candidate genes..................... 51 3.4.6 Subcellular localization of proteins of interest.............. 55 3.5 Analysis of root exudates and emitted volatiles of aeroponically grown plants. 56 3.5.1 Microscopic sample analysis....................... 57 3.5.2 Root exudates 12 hrs after microbial contact............... 57 3.5.3 Volatiles exuded 12 hrs after microbial contact............. 65 3.5.4 Bioassay to test biological function of limonene............. 68 3.5.5 Bioassay to test biological function of Nerolidol............ 69 3.6 Transcriptional changes 12 hrs after microbial challenge............ 70 ii 4 Discussion 73 4.1 Transcriptomics in M. truncatula roots 2 hrs after contact to different microor- ganisms reveals a mainly quantitative difference in gene expression....... 73 4.1.1 Genes differentially regulated in roots 2 hrs after microbial contact... 75 4.1.2 Characterization of selected candidate genes putatively involved in plant- pathogen interaction........................... 76 4.1.3 Characterization of candidate genes putatively involved in plant-AMF interaction................................ 79 4.1.4 Concluding remarks on transcriptomics 2 hrs after microbial contact.. 82 4.2 Transcriptomics 12 hrs past microbial contact to M. truncatula roots reveal an important role of TFs for a microbial-specific plant reaction........... 83 4.3 M. truncatula root exudates and volatiles emitted 12 hrs after microbial contact contain specific secondary metabolites depending on the microorganism.... 87 4.3.1 Root exudates identified by untargeted LC-MS............. 88 4.3.2 Volatiles.................................. 92 4.4 Model about early interaction of M. truncatula with different types of microor- ganisms and outlook............................... 93 5 Summary 94 6 Zusammenfassung 95 7 Appendix 110 8 Acknowledgments 124 9 Eidesstattliche Erklarung¨ 125 iii List of Figures 1 Phylogenetic classification of R. irregularis .....................2 2 Life cycle of AMF................................4 3 A: Phylogenetic classification of A.euteiches; B: Phylogenetic tree illustrating relationships within Oomycota..........................6 4 Life cycle of Aphanomyces euteiches .......................7 5 Classification of organic rhizodeposition..................... 10 6 Schematic summary of pre-symbiotic communication of plant roots towards AMF 11 7 Common symbiotic signaling pathway for AM and root-nodule symbiosis... 12 8 AMF colonized harry carrot roots on two partitioned plates........... 18 9 M. truncatula plants at 8 days or 6 weeks after seedling transfer to aeroponics.. 19 10 Scheme for primer design of Tnt1 insertion lines................. 25 11 Scheme of the RNAi cassette in the pRedRoot vector.............. 26 12 Scheme of the RNAi cassette in pAGM11978 adapted from the pRedRoot vec- tor for a golden gate cloning system....................... 27 13 Different sample processing of root exudates for LC-MS analysis........ 32 14 Root mycorrhization of 32 accessions grown on G. intraradices inoculum.... 33 15 FW of roots and shoots of 32 accessions grown on G. intraradices inoculum.. 34 16 A: Root mycorrhization B: Fresh weight of roots and shoots........... 35 17 Root mycorrhization of 32 accessions grown on sandy soil from GB for 35 days. 36 18 Cultivation and treatment of M. truncatula with spores /zoospres on plate... 37 19 Relative transcript accumulation of 5 AM induced early marker genes of M. truncatula ..................................... 38 20 Venn diagram showing number of genes differentially expressed in roots after AMF inoculation at 3 incubation times...................... 39 21 Venn diagram of transcriptional data from M. truncatula plants grown on plate and inoculated for 2 hrs with R. irregularis or A. euteiches ........... 40 22 Classification of regulated genes......................... 41 23 Expression-pattern of regulated genes by microbial treatments for 2 hrs..... 42 24 RNA quality control................................ 42 25 Expression-pattern of candidate genes after microbial challenge according to transcriptomics, validated by qRT-PCR...................... 44 26 Expression-pattern of candidate genes after microbial challenge according to transcriptomics, validated by qRT-PCR...................... 45 27 Mean shoot biomass and number of flowering plants of ts plants after treatment with A. euteiches zoospores............................ 48 28 Root and shoot biomass in ts vs Tnt1-BG plants................. 49 29 Transcript accumulation of Myc/ Ae marker genes and of MtTS ......... 49 30 Volatile collection of roots after A. euteiches treatment.............. 50 iv 31 Peak area of volatiles collected from six week old M. truncatula roots, 12 h after inoculation with zoospores of A. euteiches ................. 50 32 Molecule structure of three sesquiterpenes, synthesised
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