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Characterization of a MAPK Module Involved in Arabidopsis Response to Wounding Cecile Sözen

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Characterization of a MAPK Module Involved in Arabidopsis Response to Wounding Cecile Sözen Characterization of a MAPK module involved in Arabidopsis response to wounding Cecile Sözen To cite this version: Cecile Sözen. Characterization of a MAPK module involved in Arabidopsis response to wounding. Vegetal Biology. Université Paris Saclay (COmUE), 2017. English. NNT : 2017SACLS472. tel- 02388086 HAL Id: tel-02388086 https://tel.archives-ouvertes.fr/tel-02388086 Submitted on 1 Dec 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. NNT : 2017SACLS472 THESE DE DOCTORAT DE L’UNIVERSITE PARIS-SACLAY PREPAREE A L’UNIVERSITE PARIS-SUD ECOLE DOCTORALE N°567 Sciences du végétal : du Gène à l’Ecosystème Spécialité de doctorat : Biologie Par Mlle Cécile SÖZEN Characterization of a MAPK module involved in Arabidopsis response to wounding Thèse présentée et soutenue à Orsay (IPS2) le 29 novembre 2017 Composition du Jury : Dr. Christian MEYER Directeur de Recherche - IJPB, Versailles Président du jury Dr. Claudia JONAK Directrice de Recherche - AIT, Vienne Rapportrice Dr. Debora GASPERINI Directrice de Recherche - LIPB, Halle Rapportrice Dr. Heribert HIRT Directeur de Recherche - KAUST,Thuwal Examinateur Dr. Jean COLCOMBET Chargé de Recherche - IPS2, Orsay Directeur de thèse Dr. Axel de ZELICOURT Maître de Conférences - IPS2, Orsay Invité LIST OF ABBREVIATIONS ABA: Abscisic Acid APS: Ammonium PerSulfate ATP: Adenosine Tri-Phosphate Cas9: CRISPR-Associated protein 9 CDPK: Calcium-Dependent Protein Kinase CHX: CycloHeXimide CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats C-terminal: Carboxyl-terminal DMSO: DiMethylSulfOxide DTT: DiThioThreitol ET: Ethylene EtOH: Ethanol GLR: Glutamate Receptor-like HA: HemAgglutinin hpi: hours post-inoculation H2O2: Hydrogen peroxide JA: Jasmonic Acid kDa: KiloDalton MAPK: Mitogen-Activated Protein Kinase MAP2K: Mitogen-Activated Protein Kinase Kinase MAP3K: Mitogen-Activated Protein Kinase Kinase Kinase MBP: Myelin Basic Protein meJA: methyl-Jasmonate NaF: Sodium Fluoride OG: OligoGalacturonide PRR: Pathogen Recognition Receptor ROS: Reactive Oxygen Species RT-qPCR: Reverse Transcription - quantitative Polymerase Chain Reaction SA: Salicylic Acid SDS-PAGE: Sodium Dodecyl Sulfate - PolyAcrylamide Gel Electrophoresis TBS: Tris-Buffered Saline TEMED: TEtraMEthyleneDiamine T-DNA: Transfer DNA YFP: Yellow Fluorescent Protein Y2H: Yeast two Hybrid Characterization of a MAPK module involved in Arabidopsis response to wounding 2 LIST OF FIGURES & TABLES CHAPTER I: INTRODUCTION Figure 1.1: Simplified model depicting cellular events triggered by wounding and herbivory – p.11 Figure 1.2: Biosynthetic pathway of Jasmonic Acid (JA) – p.19 Figure 1.3: Perception and signaling of Jasmonic Acid – p.22 Figure 1.4: Relationship of JA with other phytohormones – p.24 Figure 1.5: JA and systemic signaling – p.25 Figure 1.6: MAPK cascades are key elements of the stress signal transduction in plants – p.35 Figure 1.7: 20 MAPKs in Arabidopsis divided into 4 subgroups – p.36 Figure 1.8: MKK3, the sole member of group B – p.36 Figure 1.9: 20 MEKK-like MAP3Ks divided into 3 clades – p.37 Figure 1.10: PAMP-activated MAPK cascades illustrate the complexity of MAPK signaling – p.37 Figure 1.11: Biotic and abiotic stresses activating MKK3-dependent pathways – p.39 Figure 1.12: Identification of a complete MKK3-dependent module activated by ABA – p.40 CHAPTER II: RESULTS & DISCUSSIONS Figure 2.1: MKK3 and sub-clade III MAP3Ks interact in yeast – p.46 Figure 2.2: Sub-clade III MAP3Ks, MKK3 and MPK2 constitute a functional module in protoplasts –p.47 Figure 2.3: Transcriptional regulation of MEKK-like MAP3Ks in Arabidopsis thaliana – p.48 Table 2.1: Main conditions under which sub-clade III MAP3Ks are regulated – p.48 Figure 2.4: General model based on the conserved transcriptional regulation of sub-clade III MAP3Ks – p.49 Figure 2.5: MPK2 is activated by wounding in an MKK3-dependent way – p.50 Figure 2.6: MPK1, MPK2 and MPK7 are not transcriptionally regulated by wounding – p.51 Figure 2.7: MPK1, MPK2 and MPK7 are activated by wounding in an MKK3-dependent way – p.51 Figure 2.8: MPK2 is not activated by gentle touch – p.52 Figure 2.9: MPK3 and MPK6 activation by wounding is not dependent on MKK3 – p.52 Figure 2.10: MPK3 and MPK6 have no effect on the activation of MPK2 by wounding – p.53 Figure 2.11: MPK1, MPK2 and MPK7 activation by wounding requires a de novo protein synthesis– p.53 Figure 2.12: CHX induces a constitutive activation of MPK3 and MPK6 – p.54 Figure 2.13: Expression of sub-clade III MAP3Ks upon wounding – p.55 Figure 2.14: Sub-clade III MAP3Ks protein accumulation upon wounding – p.55 Figure 2.15: Subcellular localization of MAP3K18-YFP upon ABA and wounding – p.56 Table 2.2: Testing the wounding-induced activation of MPK2 in map3k mutants led to variable results – p.56 Figure 2.16: MPK2 activity upon wounding is increased in map3k14-1 plants compared to Col-0 – p.56 Figure 2.17: MPK3 and MPK6 activity upon wounding is unchanged in map3k14-1 plants – p.56 Figure 2.18: Some MAP3Ks expression is increased in map3k14-1 plants– p.56 Characterization of a MAPK module involved in Arabidopsis response to wounding 3 Figure 2.19: Truncated MAP3K14 is functional in vivo and leads to a more stable protein – p.57 Figure 2.20: MPK2 activity upon wounding is unchanged in CRISPR map3k14 plants compared to Col-0 p.57 Figure 2.21: Theoretical model depicting the two MAPK groups activated upon wounding – p.58 Figure 2.22: Theoretical model depicting the involvement of specific sub-clade III MAP3Ks depending on the wounding-induced component – p.59 Figure 2.23: MPK2 is not activated by an exogenous treatment with H2O2, OGs, ATP or flg22 – p.60 Figure 2.24: MPK1, 2 and 7 are activated by JA in an MKK3-dependent manner – p.61 Figure 2.25: MPK3 and MPK6 are not activated by an exogenous treatment with JA – p.62 Figure 2.26: MPK2 activation by JA requires its perception by COI1 – p.62 Figure 2.27: MAP3Ks expression upon exogenous treatment with JA – p.63 Figure 2.28: JA-induced activation of MPK2 is reduced in some map3k mutants – p.63 Figure 2.29: MPK2 activation by wounding is not highly dependent on ATP, OGs or H2O2 signaling – p.64 Figure 2.30: JA, but not ABA, has a major role upstream of MKK3-MPK2 in response to wounding – p.64 Figure 2.31: JA does not have a major role upstream of MPK3/MPK6 upon wounding – p.64 Figure 2.32: Expression of candidate MAP3Ks upon wounding is reduced in coi1-16 plants – p.64 Figure 2.33: Expression of candidate MAP3Ks upon wounding is reduced in coi1-34 plants – p.65 Figure 2.34: MKK3-MPK2 is not activated in systemic leaves – p.66 Figure 2.35: Model depicting the JA-dependent MAPK module activated by wounding – p.67 Figure 2.36: MKK3-MPK2 is activated by B. cinerea – p.68 Figure 2.37: MAP3K19 is highly induced 48 hours after inoculation with B. cinerea – p.69 Figure 2.38: MKK3-MPK2 is activated by Spodoptera littoralis – p.70 Figure 2.39: MKK3-MPK2 is activated by S. littoralis and B. cinerea, two JA-producing pathogens – p.70 Table 2.3: Summary of results obtained with B. cinerea inoculations – p.71 Figure 2.40: Lesion outgrowth after inoculation with B. cinerea spores – p.71 Figure 2.41: Some Botrytis-induced genes expression is misregulated in mkk3-1 plants – p.72 Figure 2.42: MKK3 is not involved in resistance towards Spodoptera littoralis – p.73 Figure 2.43: MKK3 negatively regulates SA and JA accumulation after insect feeding – p.73 Figure 2.44: MKK3 is not involved in hormonal regulation upon wounding – p.74 Figure 2.45: Role of MKK3 in the regulation of genes involved in SA and JA biosynthesis pathways – p.75 Figure 2.46: Role of MKK3 in the regulation of SA- and JA-responsive genes – p.76 Figure 2.47: Wounding-induced trichome formation – p.77 Figure 2.48: Transcriptomic analysis of mkk3-1 plants submitted to wounding did not highlight any MKK3- regulated genes – p.78 Table 2.4: Few MKK3-regulated genes were found – p.78 Figure 2.49: Theoretical model on the role of MKK3 in phytohormone regulation upon S. littoralis feeding - p.79 Characterization of a MAPK module involved in Arabidopsis response to wounding 4 CHAPTER III: CONCLUSIONS & PERSPECTIVES Figure 3.1: Hypothetical model on the interdependent activation of two MAPK modules upon wounding – p.82 Figure 3.2: MPK7 is activated by nitrate in an MKK3- and NLP7- dependent manner – p.84 Figure 3.3: mkk3-1 plants show increased dormancy – p.90 CHAPTER IV: MATERIALS & METHODS Table 4.1: Mutant lines mentioned in this manuscript – p.92 Table 4.2: Exogenous treatments applied on Arabidopsis plantlets – p.97 Table 4.3: Preparation of the enzymatic solution – p.98 Table 4.4: Composition of W5 and WI solutions – p.98 Table 4.5: Composition of the MMg solution – p.99 Table 4.6: Composition of the PEG solution – p.99 Table 4.7: Primers used to amplify loci for the creation of lines expressing tagged versions of MAP3Ks, MKK3 and MAPKs – p.102 Table 4.8: Composition of the RT mix – p.103 Table 4.9: Genes used for qPCR experiments and corresponding primer couples – p.104 Table 4.10: Description of samples sent for micro-array analyses – p.104 Table 4.11: Dilutions of antibodies used for western blots – p.106 Characterization of a MAPK module involved in Arabidopsis response to wounding 5 TABLE OF CONTENT CHAPTER I: INTRODUCTION ..........................................................................................
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