Damps Hamps

Krishna Alok

Sinha

Jean Colcombet

Cécile Sözen

Heribert Hirt

YFP: Yellow Fluorescent Protein Y2H: Yeast two Hybrid

Keywords: Adenosine Tri-Phosphate Cas9: CRISPR-Associated protein 9 CDPK: Calcium-Dependent Protein Kinase Clustered Regularly Interspaced Short Palindromic Repeats Mitogen-Activated Protein Kinase MAP2K: Mitogen-Activated Protein Kinase Kinase MAP3K: Mitogen-Activated Protein Kinase Kinase Kinase MBP: Myelin Basic Protein viridiplantae, MKK3 module, phosphorylation cascade, stress responses, mitogen-activated protein kinases

Since its first description in 1995 and functional characterization 12 years later, plant MKK3-type MAP2Ks have emerged as important integrators in plant signaling. Although they have received less attention than the canonical stress-activated mitogen-activated protein kinases (MAPKs), several recent publications shed light on their important roles in plant adaptation to environmental conditions. Nevertheless, the MKK3-related literature is complicated. This review summarizes the current knowledge and discrepancies on MKK3 MAPK modules in plants and highlights the singular role of MKK3 in green plants.

In the light of the latest data, we hypothesize a general model that all clade-III MAP3Ks converge on MKK3 and C-group MAPKs, thereby defining a set of novel MAPK modules which are activated by stresses and internal signals through the transcriptional regulation of MAP3K genes.

III. IDENTIFICATION OF SIGNALING ELEMENTS

CHAPTER I: INTRODUCTION

Agricultural context

Humans started to cultivate plants more than 10.000 years ago for their alimentation.

Nowadays, 62% of crop production is intended for human food, 35% for animal feeding and 3% for bioenergy, seed and other industrial products [START_REF] Foley | Solutions for a cultivated planet[END_REF]. Thus it is clear that agriculture and food production are of great concern for people all over the world.

International organizations such as the Food and Agriculture Organization of the United Nations (FAO) or the World Health Organization have developed strategies to prevent worldwide food crises and highlighted the urgent need for research and innovation in agricultural sciences. According to the FAO, the world population will reach 9.1 billion by 2050. In order to feed that number of people, food productivity will have to increase by 60-70% (www.fao.org, 2015). The increase of food productivity is also of great importance to fight poverty in many third-world countries. To reach this objective, different levels are explored. The first one consists in increasing overall plant productivity. Breeding has been performed since crop domestication but new tools coupled to a better knowledge of plant biology are available to accelerate the process. Another possibility will consist in reducing crop losses due to non-optimal growth conditions referred as "stresses". Such stresses are generally classified in two categories. Abiotic stresses are caused by non-living entities such as drought, salt, heat, cold, whereas biotic stresses are caused by living organisms like bacteria, fungi, viruses or insects. Abiotic and biotic stresses are responsible for about 50% crop losses in the world [START_REF] Ashraf | Crop Production for Agricultural Improvement[END_REF]. It is important to stress the fact that the impact of abiotic stresses is very likely to increase in the future due to global warming issues (increasing temperatures and CO 2 emissions) (Intergovernmental Panel on Climate Change, ipcc.ch). The effects of stresses on plants are diverse: reduction of biomass, decrease of fertility or simply death. It is thus important to find a way to increase crop tolerance to environmental stresses with the aim of improving their quantity and quality. Until now, strategies used to improve crop production have relied on a massive use of chemical products and great deforestations to create surfaces devoted to cultures. Yet these practices have huge impact on human health and environment (destruction of wildlife and plants). Today, the new goal is to develop a sustainable agriculture without impacting the environment. For this reason it became critical to develop new tools based on basic science. Studying and understanding how plant respond to their environment is an important step before being able to engineer plants more resistant to environmental stresses.

Plants are confronted to mechanical stimuli of diverse nature Like many other organisms, plants are exposed to mechanical stimuli imposed by their environment that can range from a gentle breeze or the touch of neighboring objects during the growth of organs to a strong wounding caused by herbivore feeding or the accidental abscission of aged organs. The sensing of "non-wounding" mechanical stimuli have an important role in plant morphogenesis as it triggers developmental responses of the plant, the best illustrating examples being the shaping of a tree by the wind or the development of roots finding their way through obstacles in the soil (Monshausen & Gilroy, 2009). On the other hand, the wounding of plant tissues can have more detrimental effects as it can cause cell death and create entrances for pathogens thereby facilitating their development inside plant tissues. In the course of my PhD, I have been particularly interested in the mechanical stress caused by herbivorous insects.

Plants and herbivorous insects

Plants and insects have co-evolved for nearly 350 million years (Gatehouse, 2002).

Some plant-insect interactions can be beneficial for the plant as in the case of pollination. In some cases, plants can even develop at the expense of insects. An amazing example is the important source of nitrogen that carnivorous plants living on very poor soils find in insects they can catch. Yet many insects have deleterious effects on the plant as in the case of attacks by herbivorous insects that can feed themselves on the targeted plant or deposit their eggs (oviposition). Herbivorous insect species are very diverse and possess different feeding strategies. The majority is composed of beetles and caterpillars that can damage leaves with their mouthparts by chewing, tearing or snipping [START_REF] Fürstenberg-Hägg | Plant defense against insect herbivores[END_REF]. Sucking insects, like aphids, have specialized organs called stylets that can be inserted into the phloem to suck the liquid content. In this case, the wounding part of the interaction is reduced but aphids can form very large colonies which can have strong effects.

Plants have developed advanced response systems to fight and defend themselves against herbivorous insects. A first layer of defense is constitutive. Plants are naturally equipped with physical barriers like a cuticle layer, trichomes, and thorns. They also Wounding and herbivory can be perceived by the cell through the recognition of Damage-Associated Molecular Patterns (DAMPs) such as OGs, Pep1 or ATP that are self-derived molecules. Plants can also respond to Herbivore-Associated Molecular Patterns (HAMPs) found in insect oral secretions that are recognized through an unknown mechanism. Both DAMPs and HAMPs are able to trigger plant defense responses. Wounding leads to a rapid depolarization of the plasma membrane followed by an important influx of calcium and a burst of Reactive Oxygen Species (ROS). Those events lead to the de novo synthesis of phytohormones and in term to the regulation of stress-responsive genes. Plants will be able to defend themselves by activating specific defense responses in order to prevent further attacks or to repel herbivores.

Membrane depolarization

Defense responses

-physical barriers (trichomes…) -resistance to further attacks -callose deposition -production of volatile compounds accumulate secondary metabolites that can function against herbivores. But they also sense the presence of insects by detecting compounds of their oral secretions/oviposition fluids and by the mechanical damage caused by the insect feeding. In order to counteract insect effects, plants activate specific immune responses that are controlled by complex signaling pathways.

One specific response is the emission of volatile compounds to repulse herbivores, attract their predators and alert neighboring organs and plants.

Mechanical wounding of plant tissues is an unavoidable consequence of herbivory, although the intensity and the extent of damage depend on the mode of feeding of the insect (chewing, sucking…). Insects feed on leaves by continuously wounding them and swallowing small pieces of tissue, simultaneously introducing saliva and other secretions in contact of plant cells. In order to better understand plant defense responses triggered by herbivores, most studies chose to apply a single mechanical wound by crushing or puncturing leaves. Although the major part of responses is similar to the ones observed upon insect attack, a single wound is often not enough to induce the emission of volatiles [START_REF] Alborn | An Elicitor of Plant Volatiles from Beet Armyworm Oral Secretion[END_REF].

Cellular events occurring upon wounding and herbivory include membrane depolarization, increase of cytosolic calcium levels, production of Reactive Oxygen Species (ROS), activation of Mitogen-Activated Protein Kinases (MAPKs) along with phosphorylation events and phytohormone accumulation. These cascades of intracellular events finally lead to a strong transcriptional reprogramming allowing the plant to raise defenses against the perceived stress [START_REF] Savatin | Wounding in the plant tissue: the defense of a dangerous passage[END_REF][START_REF] Zebelo | Role of early signalling events in plant-insect interactions[END_REF]. Those wound-triggered cellular events are described in the first part of the introduction and additional information about insect-induced responses is given when available (figure 1.1).

Arabidopsis, a model plant to study plant defense responses

Although studies on this subject have been conducted on different plants species including tobacco (Nicotiana attenuata), tomato (Lycopersicon esculentum), Lima bean (Phaseolus lunatus) and thale cress (Arabidopsis thaliana) closer attention will be paid to literature data provided by works performed on Arabidopsis. Indeed, the latter is commonly used in the laboratory and provides a wide range of advantages. Arabidopsis thaliana was first proposed to be used as a genetic model organism about 75 years ago and started to be widely used as such during the 1980s [START_REF] Meyerowitz | Prehistory and history of Arabidopsis research[END_REF]. The study of this plant has developed our understanding of plants in general but had also an impact on some aspects of research on animals and humans. Indeed, some important processes of human biology can be more easily studied in this model plant and many discoveries made using Arabidopsis were directly useful for human health issues such as cancer or Alzheimer's disease [START_REF] Jones | The Impact of Arabidopsis on Human Health: Diversifying Our Portfolio[END_REF]. The use of Arabidopsis as a model offers advantages such as a small genome size (≈ 135 million base pairs), the convenience of crossing linked to a strict Mendelian inheritance, the large progeny of each plant (>10.000 seeds) and the short generation time (≈ 8 weeks from germination to mature seed production). Arabidopsis thaliana genome has been entirely sequenced and very well annotated (The Arabidopsis Genome Initiative, 2000). Mutations of desired target sequences have been largely facilitated by the development of various tools such as the collection of T-DNA insertion lines, the TILLING (Targeting Induced Local Lesions in Genomes) technology, the use of approaches based on RNA-silencing and more recently the CRISPR-Cas9 system based on the action of endonucleases aiming desired sites in the genome (Zhang et al., 2016). Large mutant collections are easily accessible to query using online tools.

I. Early cellular events triggered by wounding and herbivory

Sensing cell wall integrity and cell death

Wounding caused by insect feeding or other mechanical stimuli is a very complex stress. It locally induces cell death on a variable area as well as a mechanical stimulation of the neighboring living cells. The first events and their relative importance in the initiation of wounding signaling processes are poorly known. In one hand, wounding-induced cell death triggers a strong loss of compartmentalization of cellular molecules such as enzymes or ions which may be involved in the initiation of wounding signaling (ATP, Ca 2+ …). On the other hand, living cells neighboring the wounding site are abruptly disturbed and probably react by the activation of intra and intercellular signaling pathways.

Wounding and herbivore feeding affect the integrity of cuticle and cell wall.

Resulting breaches alert the plant of the presence of invading visitors. Among the warning signs are changes in mechanical properties and release of oligosaccharide fragments from the cell wall [START_REF] Nühse | Cell wall integrity signaling and innate immunity in plants[END_REF]. A loss of cell wall integrity (CWI) can be sensed by Receptor-Like Kinases (RLKs) such as THESEUS 1 but other sensors have been suggested based on studies conducted in yeast where a CWI-dependent pathway has been well characterized [START_REF] Levin | Regulation of cell wall biogenesis in Saccharomyces cerevisiae: the cell wall integrity signaling pathway[END_REF][START_REF] Hamann | The plant cell wall integrity maintenance mechanism -Concepts for organization and mode of action[END_REF]. Similar sensing systems might be involved in the detection of wounding in plants. Additionally, in the case of a pathogen attack, cell wall-degrading enzymes such as polygalacturonase, pectate lyase and pectin methylesterase, which are released by the pathogen or produced by the plant and activated upon attack, come into play [START_REF] Walton | Deconstructing the Cell Wall[END_REF]. Treatment of the plant cell wall with these enzymes can elicit defense responses [START_REF] Walton | Deconstructing the Cell Wall[END_REF]. In most cases, responses are triggered by the recognition of the enzymatic products among which oligogalacturonides have been the most studied [START_REF] Ferrari | Oligogalacturonides: plant damage-associated molecular patterns and regulators of growth and development[END_REF].

Physical signals could also be generated by stretching of the plasma membrane that could be detected by stretch-activated enzymatic activities. There is much evidence from electrophysiological studies that the plasma membrane of plant cells contain a wide diversity of mechanosensitive (MS) ion channels. A simple touch of the leaf surface can be sensed through MS channels that can trigger further signal transduction events. Thus a soft mechanical stress at the leaf surface was shown to be enough to activate the expression of touch (TCH) genes as well as other cellular responses similar to the ones induced by a stronger wounding (ROS production, Ca 2+ influx, resistance to pathogens) (Benikhlef et al., 2013). A number of MS channels activities have been functionally characterized in plant membranes and are good candidates to act as first sensors of mechanical stimuli [START_REF] Peyronnet | Mechanosensitive channels: feeling tension in a world under pressure[END_REF][START_REF] Haswell | Two MscS Homologs Provide Mechanosensitive Channel Activities in the Arabidopsis Root[END_REF]. Based on their homology to the bacterial MscS channel, the 10 MscS-Like (MSL) proteins of Arabidopsis thaliana have been hypothesized to form mechanosensitive channels in plant cell and organelle membranes. MS channels have the property to convert mechanical stimuli into electrophysiological (potential variation) or biochemical signals. Once they are activated and opened, they often lead to membrane depolarization and to the influx of second messengers such as Ca 2+ ions. The link between the activation of MS channels and Ca 2+ variation is still not clearly known [START_REF] Hamilton | United in Diversity: Mechanosensitive Ion Channels in Plants[END_REF]. Some authors suggest the involvement of a MS calcium channel that could itself generate Ca 2+ influx upon a mechanical stimulus. It seems that MSL permeability is more anionic than calcic [START_REF] Haswell | Two MscS Homologs Provide Mechanosensitive Channel Activities in the Arabidopsis Root[END_REF], but as a very low Ca 2+ permeability might be enough to induce slight increases of cytosolic Ca 2+ , a direct role for MS channels in calciuminduced activities cannot be excluded. Alternatively, it is possible that voltage-dependent Ca 2+ channels found on plasma membranes [START_REF] Miedema | Two voltage-dependent calcium channels co-exist in the apical plasma membrane of Arabidopsis thaliana root hairs[END_REF] could be activated by the depolarization triggered by the opening of MS channels.

Ion fluxes and other second messengers

As indicated earlier, the depolarization of the plasma membrane causes ion fluxes, the most important being Ca 2+ ions as they have been shown to function as second messengers in many signaling pathways [START_REF] Kudla | Calcium signals: the lead currency of plant information processing[END_REF]. Plants can discriminate between various stimuli based on calcium signatures that are defined by their amplitude, frequency, subcellular localization and duration. Peaks of cytosolic Ca 2+ are detected within 6 seconds in cells close to the injury site [START_REF] Beneloujaephajri | Production of reactive oxygen species and wound-induced resistance in Arabidopsis thaliana against Botrytis cinerea are preceded and depend on a burst of calcium[END_REF] and several studies showed that cytosolic calcium levels also increase upon herbivore attack (Arimura & Maffei, 2010). Very recently, the use of a fluorescent calcium biosensor has allowed observing elevations of Ca 2+ levels during a biotic interaction with an aphid (Myzus persicae) [START_REF] Vincent | Interplay of Plasma Membrane and Vacuolar Ion Channels, Together with BAK1, Elicits Rapid Cytosolic Calcium Elevations in Arabidopsis during Aphid Feeding[END_REF]. Using appropriate mutant lines, authors were able to show that these Ca 2+ elevations are dependent on BAK1 (Brassinosteroid insensitive-Associated Kinase 1), a well-known co-receptor of Pathogen Recognition Receptors (PRRs), as well as on GLR3.3 and 3.6 channels (GLutamate Receptor-like). In the same study, the vacuolar cation channel TPC1 (Two Pore Channel 1) was shown to importantly contribute to the observed cytosolic elevation. In another study, TPC1-dependent calcium elevations were observed upon feeding of the caterpillar Spodoptera littoralis, both locally and systemically [START_REF] Kiep | Systemic cytosolic Ca 2+ elevation is activated upon wounding and herbivory in Arabidopsis[END_REF]. Inositol trisphosphate (InsP 3 ) was shown to mediate Ca 2+ release from internal stores [START_REF] Alexandre | Effect of D-myo-Inositol 1,4,5-Trisphosphate on the Electrical Properties of the Red Beet Vacuole Membrane[END_REF]. It was recently shown, along with phosphoinositide, to act as a second messenger of wounding signal transduction and regulate the expression of wound-inducible genes [START_REF] Mosblech | Phosphoinositide and inositolpolyphosphate signalling in defense responses of Arabidopsis thaliana challenged by mechanical wounding[END_REF]. Interestingly, their production was shown to require the biosynthesis of JA as no increase of InsP 3 was observed in plants deficient in JA synthesis.

In Arabidopsis, the calcium signal is decoded by three major multigenic families of Ca 2+ sensor proteins: calmodulins or calmodulin-like proteins (CAMs), calcineurin B-like proteins (CBLs) and Calcium-Dependent Protein Kinases (CDPKs). Some of those sensors have been studied and were shown to have roles in plant immunity in response to bacterial pathogens but also herbivorous insects [START_REF] Boudsocq | CDPKs in immune and stress signaling[END_REF]Arimura & Maffei, 2010).

In Arabidopsis, some CDPKs are activated by the bacterial elicitor flg22 and were shown to be important for the transduction of defense responses [START_REF] Boudsocq | Differential innate immune signalling via Ca2+ sensor protein kinases[END_REF]. Two CDPKs, CPK3 and CPK13, were directly shown to act as positive regulators of the defense gene PDF1.2 upon exposure to Spodoptera littoralis caterpillars [START_REF] Kanchiswamy | Regulation of Arabidopsis defense responses against Spodoptera littoralis by CPK-mediated calcium signaling[END_REF]. A calmodulin-like protein, CML42 was shown to sense Ca 2+ upon application of oral secretions from S. littoralis and to connect Ca 2+ to JA signaling [START_REF] Vadassery | CML42-mediated calcium signaling coordinates responses to Spodoptera herbivory and abiotic stresses in Arabidopsis[END_REF]. cml42 plants showed enhanced resistance to the caterpillar as well as higher expression levels of the JAresponsive gene VSP2. Another calmodulin-like protein, CML37, was shown to be involved in the regulation of the Jasmonic Acid (JA) pathway and in resistance to herbivory [START_REF] Scholz | Mutation of the arabidopsis calmodulin-like protein CML37 deregulates the jasmonate pathway and enhances susceptibility to herbivory[END_REF]. Three TCH (TouCH) genes encoding calmodulin and calmodulin-like proteins were shown to be induced by mechanical stimuli such as touch or wind, supporting the important role of calcium signaling in the transduction of such environmental cues [START_REF] Braam | Rain-, wind-, and touch-induced expression of calmodulin and calmodulin-related genes in Arabidopsis[END_REF]. The role of Ca 2+ sensor proteins in a context of wounding and/or herbivory was also demonstrated in other plants species. Thus, in tomato, LeCDPK2 is involved in the wound-induced ethylene (ET) production [START_REF] Kamiyoshihara | Turnover of LeACS2, a wound-inducible 1-aminocyclopropane-1-carboxylic acid synthase in tomato, is regulated by phosphorylation/dephosphorylation[END_REF] and in tobacco (Nicotiana attenuata), NaCDPK4 and NaCDPK5 negatively regulate wounding responses and resistance to the herbivore Manduca sexta [START_REF] Yang | Silencing Nicotiana attenuata Calcium-Dependent Protein Kinases, CDPK4 and CDPK5, Strongly Up-Regulates Woundand Herbivory-Induced Jasmonic Acid Accumulations[END_REF]. Finally, in maize, ZmCPK11 is activated by wounding in a JA-dependent pathway and is involved in systemic wound responses [START_REF] Szczegielniak | Maize calcium-dependent protein kinase (ZmCPK11): Local and systemic response to wounding, regulation by touch and components of jasmonate signaling[END_REF].

In addition to calcium signaling, phospholipase D (PLD) has been suggested to play an important role in the mediation of wound-induced responses [START_REF] Wang | Involvement of phospholipase D in wound-induced accumulation of jasmonic acid in arabidopsis[END_REF]. PLD hydrolyzes phospholipids and leads to the production of phosphatidic acid (PA). PLD-induced hydrolysis was observed after wounding in bean leaves and appeared to be mediated by an increase in cytoplasmic Ca 2+ concentrations [START_REF] Ryu | Activation of phospholipase D and the possible mechanism of activation in wound-induced lipid hydrolysis in castor bean leaves[END_REF]. Interestingly, PLD was shown to be required for the production of JA and the induction of a JA-regulated gene [START_REF] Wang | Involvement of phospholipase D in wound-induced accumulation of jasmonic acid in arabidopsis[END_REF]Bargmann et al., 2009).

Production of Reactive Oxygen Species (ROS)

Reactive Oxygen Species (ROS) are normally produced in peroxisomes, chloroplasts and mitochondria due to the presence of an oxidizing activity or electron transfer chains in these organelles (Tripathy & Oelmuller, 2012). In addition, ROS production can be induced upon stress conditions and create a ROS burst that is thought to have antimicrobial effects.

Stress-induced ROS production was first described in 1983 in potato tubers infected with the oomycete Phytophthora infestans [START_REF] Doke | Generation of superoxide anion by potato tuber protoplasts during the hypersensitive response to hyphal wall components of Phytophthora infestans and specific inhibition of the reaction by suppressors of hypersensitivity[END_REF]. Since this study, many works contributed to show that ROS can also be involved in other stresses including wounding and insect feeding [START_REF] Suzuki | Reactive oxygen species-dependent wound responses in animals and plants[END_REF][START_REF] Maffei | Effects of feeding Spodoptera littoralis on lima bean leaves. III. Membrane depolarization and involvement of hydrogen peroxide[END_REF].

NADPH oxidases are well known producers of ROS [START_REF] Marino | A burst of plant NADPH oxidases[END_REF]. They transfer electrons from cytosolic NADPH (or NADH) to oxygen in the apoplast, thus leading to the production of superoxide ions (O 2 -). The latter is then converted to hydrogen peroxide (H 2 O 2 ) in the apoplast by the action of superoxide dismutase. NADPH oxidases belong to the Respiratory Burst Oxidase Homolog (RBOH) family that has 10 members (RBOHA to RBOHJ) in Arabidopsis (Torres & Dangl, 2005). In response to wounding, the observed oxidative burst is believed to be due to NADPH oxidases. Indeed, in Arabidopsis seedlings, the rapid local and systemic ROS burst is impaired in rbohd mutants [START_REF] Miller | The Plant NADPH Oxidase RBOHD Mediates Rapid Systemic Signaling in Response to Diverse Stimuli[END_REF]. In tomato (Solanum lycopersicum), the inhibition of NADPH oxidase by specific inhibitors blocks the wound-induced production of ROS and the induction of some defense genes.

NADPH oxidases are also involved in wound-induced ROS burst in other plant species such as Medicago truncatula or potato [START_REF] Soares | A proteomic analysis of the wound response in Medicago leaves reveals the early activation of a ROS-sensitive signal pathway[END_REF][START_REF] Bernards | The poly(phenolic) domain of potato suberin: a nonlignin cell wall bio-polymer[END_REF]. In addition to their oxidase domain, NADPH oxidases also possess Ca 2+ binding domains and it was demonstrated that wound-induced ROS burst is dependent on Ca 2+ peaks (Monshausen et al., 2009;[START_REF] Beneloujaephajri | Production of reactive oxygen species and wound-induced resistance in Arabidopsis thaliana against Botrytis cinerea are preceded and depend on a burst of calcium[END_REF]. Besides NADPH oxidases, ROS can also be produced through the action of apoplastic peroxidases but no evidence was found for their action in wound-induced ROS production in Arabidopsis [START_REF] Minibayeva | Roles of apoplastic peroxidases in plant response to wounding[END_REF]. Concerning herbivory, it has been shown that H 2 O 2 is released upon Spodoptera littoralis feeding on Lima bean and, to a lesser extent, upon mechanical damage [START_REF] Maffei | Effects of feeding Spodoptera littoralis on lima bean leaves. III. Membrane depolarization and involvement of hydrogen peroxide[END_REF].

Damage-Associated Molecular Patterns (DAMPs) are intercellular signaling molecules produced upon wounding

Plants are able to sense their damaged self and trigger responses very similar to those induced by pathogen infection. Endogenous molecules deriving from wounded tissue can act like Damage-Associated Molecular Patterns (DAMPs) and elicit local and systemic plant defenses. In tomato, systemin, an 18 amino-acid peptide, is released in the apoplast after wounding or insect attack [START_REF] Jacinto | Tomato prosystemin promoter confers wound-inducible, vascular bundle-specific expression of the β-glucuronidase gene in transgenic tomato plants[END_REF]. Sensing of systemin leads to the biosynthesis of JA, an important wound phytohormone, that activate defense responses locally and in the neighboring cells [START_REF] Orozco-Cardenas | Expression of an antisense prosystemin gene in tomato plants reduces resistance toward Manduca sexta larvae[END_REF]. Hydroxyproline-rich systemins have also been identified in other Solanaceae (potato, petunia, black nightshade) and are able to trigger plant immunity in response to herbivore attack. In Arabidopsis thaliana, three self-derived families of molecules have been identified so far. They are all recognized by Pattern-Recognition Receptors (PRRs) localized at the plasma membrane level. Oligogalacturonides (OGs) are released from the plant cell wall after degradation of homogalacturonan, the main component of pectin, through the action of wounding-induced hydrolytic enzymes such as polygalacturonase. The size of OGs is important and can determine their efficiency as eliciting molecules. OGs with a degree of polymerization between 10 and 15 are the most active ones [START_REF] Ferrari | Oligogalacturonides: plant damage-associated molecular patterns and regulators of growth and development[END_REF]. Wall-Associated Kinases (WAKs) are RLKs associated to the cell wall that are required for cell expansion during development but can also mediate stress responses induced by OGs released upon wounding. WAKs are encoded by 5 genes among which WAK1 and WAK2 are transcriptionally induced by wounding. In rice, OsWAK1

is also upregulated by wounding as well as salicylic acid (SA) and JA. By the use of a chimeric receptor approach, authors have shown the ability of WAK1 to bind OGs [START_REF] Brutus | A domain swap approach reveals a role of the plant wall-associated kinase 1 (WAK1) as a receptor of oligogalacturonides[END_REF]. WAK2 also seems to have a predominant role as wak2-1 plants are not able to activate defense responses induced by OGs [START_REF] Kohorn | Pectin activation of MAP kinase and gene expression is WAK2 dependent[END_REF]. Adenosine Tri Phosphate (ATP) can also act as an extracellular signaling molecule [START_REF] Tanaka | Extracellular ATP acts as a damageassociated molecular pattern (DAMP) signal in plants[END_REF]. It is released from plant cells in response to abiotic stresses [START_REF] Dark | Release of extracellular purines from plant roots and effect on ion fluxes[END_REF], fungal elicitors and mechanical stimuli [START_REF] Weerasinghe | Touch induces ATP release in Arabidopsis roots that is modulated by the heterotrimeric G-protein complex[END_REF]. Concentrations of ATP up to 40 µM were measured after wounding, probably released from neighboring cells whose plasma membrane had lost its integrity [START_REF] Jeter | Evidence of a Novel Cell Signaling Role for Extracellular Adenosine Triphosphates and Diphosphates in Arabidopsis[END_REF]. Recently the plant receptor of ATP has been identified in Arabidopsis as DORN1 (DOes not Respond to Nucleotides 1), a lectin receptor kinase [START_REF] Choi | Identification of a plant receptor for extracellular ATP[END_REF]. The characterization of dorn1 plants showed the specificity of this receptor for ATP as plants were not impaired in responses to other exogenous stimuli. Based on this observation, transcriptional responses to ATP were compared to those observed upon wounding. 60% of the genes induced by ATP were also induced by wounding and the expression of selected genes was found highly reduced in dorn1 plants after wounding, strongly suggesting that ATP is a major component of wounding response.

Pep1 is a 23 amino-acid peptide released from the C-terminal part of its precursor PROPEP1 [START_REF] Yamaguchi | Endogenous peptide elicitors in higher plants[END_REF]. The latter constitutes a family with other 7 members that are all produced in response to wounding or pathogen perception, which means that unlike OGs and systemin, whose production does not require any gene regulation, genetically encoded peptides such as Pep1 are produced in a second wave of events to reinforce responses and/or mediate intercellular signaling. Pep1 is perceived by two Receptor-Like Kinases (RLKs), PEPR1 and PEPR2 (for Pep-Receptor1 and 2), which are functionally and structurally very similar to the RLKs FLS2 and EFR (receptors of bacterial elicitors flg22 and elf18 respectively) [START_REF] Krol | Perception of the Arabidopsis danger signal peptide 1 involves the pattern recognition receptor AtPEPR1 and its close homologue AtPEPR2[END_REF]. PEPR1 and PEPR2 are transcriptionally induced by wounding and JA as well as upon herbivore attack and application of oral secretions (OS) [START_REF] Klauser | The Arabidopsis Pep-PEPR system is induced by herbivore feeding and contributes to JA-mediated plant defence against herbivory[END_REF]. Besides, the pepr1 pepr2 mutant shows increased sensitivity to Spodoptera littoralis feeding as well as a reduction of JA accumulation upon application of insect OS. Homologues of AtPep1 have been identified in maize; one of them, ZmPep3, triggers the biosynthesis of JA and ET and induces the production of volatile compounds [START_REF] Huffaker | Plant elicitor peptides are conserved signals regulating direct and indirect antiherbivore defense[END_REF].

DAMP signaling is very similar to the one induced by the recognition of PAMPs (Pathogen-Associated Molecular Patterns) that are pathogen-derived molecules, in opposition to the self-derived nature of DAMPs. First, they are recognized at the cell surface by PRRs (mostly receptor-like kinases) that are structurally very similar: a variable ectodomain potentially involved in ligand binding, a single transmembrane domain and an intracellular kinase domain, which is involved in the initiation of the intracellular signaling pathway [START_REF] Couto | Regulation of pattern recognition receptor signalling in plants[END_REF]. Secondly, the recognition of both DAMPs and PAMPs by the appropriate receptor leads to a cellular immune signaling involving the steps described earlier: rapid ion-flux changes at the plasma membrane level, increase of Ca 2+ levels, production of ROS. In a coordinated manner, activation of phosphorylation cascades conveys immune signaling to the nucleus, resulting in strong transcriptional reprogramming to establish an immune response.

JA is a central actor of plant responses to wounding and insects

Phytohormones are small molecules used in various aspects of plant life ranging from development to stress responses. Jasmonic acid, the major component of jasmine scent, and its major derivative JA-Isoleucine (JA-Ile) help to regulate various aspects of plant biology, from stress responses to development. In non-stressed plants, JA is involved in senescence and reproductive development. Stress responses in which JA is involved include some abiotic stresses (drought, UV, ozone…) and mainly biotic stresses caused by insects or other microbial pathogens [START_REF] Wasternack | Jasmonates: Biosynthesis, perception, signal transduction and action in plant stress response, growth and development. An update to the 2007 review in Annals of Botany[END_REF]Santino et al., 2013). Notably, JA has long been described as the major defense hormone against herbivores [START_REF] Howe | Plant Immunity to Insect Herbivores[END_REF].

Herbivores can activate the synthesis of JA in damaged host tissue and, in turn, JA can activate specific wound-induced defense responses [START_REF] Campos | Jasmonate-Triggered Plant Immunity[END_REF]. Most herbivore- JA is synthesized from the fatty acid α-linolenic acid (18:3) (α -LeA) released from galactolipids of chloroplast membranes. After the action of several enzymes (LOXs, AOS, AOC), OPDA is obtained and is relocated to the peroxisome. The reduction of OPDA by OPR3 followed by several cycles of β-oxidations leads to the synthesis of JA which is then translocated to the cytosol where it can be modified to JA-Ile and other derivatives.

JA-Ile is then perceived by the SCF/COI1 complex and allows the expression of defense genes as well as genes encoding enzymes of its own synthesis.

induced responses are also induced by treatments with JA. Moreover, wounding is sufficient to trigger increases of JA and JA-Ile, thus demonstrating that insect-derived molecules are not required to activate JA production [START_REF] Koo | A rapid wound signal activates the systemic synthesis of bioactive jasmonates in Arabidopsis[END_REF]. Mutants defective in JA biosynthesis or perception are highly susceptible to insect attack or other pathogens, highlighting the essential role of JA in defense [START_REF] Thomma | Separate jasmonate-dependent and salicylatedependent defense-response pathways in Arabidopsis are essential for resistance to distinct microbial pathogens[END_REF][START_REF] Stotz | Evidence for regulation of resistance in Arabidopsis to Egyptian cotton worm by salicylic and jasmonic acid signaling pathways[END_REF]. Hereafter is an overview of JA functions in the context of wounding and herbivory, including its biosynthesis, signaling, as well as some indications of crosstalks with other phytohormones.

a. JA homeostasis in response to wounding and herbivory Jasmonates form a family of oxylipins deriving from enzymatic oxygenation of 18 and 16-carbon triunsaturated fatty acids [START_REF] Wasternack | Jasmonates: Structural Requirements for Lipid-Derived Signals Active in Plant Stress Responses and Development[END_REF]. JA is synthesized from the fatty acid α-linolenic acid (18:3) (α-LeA) released from galactolipids of chloroplast membranes (figure 1.2). The enzyme responsible for the release of α-LeA was identified as phospholipase 1 (PLA1). α-LeA is then oxidized by a lipoxygenase (LOX) that is responsible for the insertion of an oxygen at the C-13 position of the acid, thus forming 13-HPOT. The genome of Arabidopsis includes 4 LOXs involved in JA synthesis upon wounding (LOX2, 3, 4 and 6). LOX2 was shown to be required for JA production proximal to the wound [START_REF] Glauser | Velocity estimates for signal propagation leading to systemic jasmonic acid Characterization of a MAPK module involved in Arabidopsis response to wounding 113 accumulation in wounded Arabidopsis[END_REF]. However, JA and JA-Ile are still synthesized in the lox2-1 mutant suggesting the involvement of other LOXs. More recent studies using combinations of multiple LOXs mutants revealed that all 4 LOXs contribute to JA synthesis upon wounding [START_REF] Chauvin | Four 13-lipoxygenases contribute to rapid jasmonate synthesis in wounded Arabidopsis thaliana leaves: A role for lipoxygenase 6 in responses to long-distance wound signals[END_REF]. LOX6 was found to have a particular role in early JA synthesis in distal leaves [START_REF] Farmer | The squeeze cell hypothesis for the activation of jasmonate synthesis in response to wounding[END_REF]. The next step of the JA biosynthesis pathway leads to the formation of its precursor cis-12-oxo-phytodienoic acid (OPDA). The enzymes responsible for this step are ALLENE OXIDE CYCLASE (AOC) and ALLENE OXIDE SYNTHASE (AOS). OPDA is then transported to the peroxisome where it is reduced to OPC-8:0 by 12-oxophytodienoate reductase (OPR3). Next, (+)-7-iso-JA is produced after several rounds of β-oxidations. Upon its subsequent transport to the cytoplasm, JA is modified to methyl-JA (meJA), JA-Ile or other derivatives. JA-Ile is formed by the action of the JAR1 enzyme (Jasmonic Acid

Resistant 1) and is the most active form of JA as it seems to be the one that actually plays the major role in Arabidopsis leaves [START_REF] Staswick | The oxylipin signal jasmonic acid is activated by an enzyme that conjugates it to isoleucine in Arabidopsis[END_REF]Fonseca et al., 2009b).

At the histological level, jasmonate biosynthesis in Arabidopsis mainly takes place in the vascular bundles of the vegetative parts of the plant. However, some data suggest a production of JA in cells outside the vessels. For example, LOX2 is found strongly expressed in mesophyll cells [START_REF] Montillet | An abscisic acid-independent oxylipin pathway controls stomatal closure and immune defense in Arabidopsis[END_REF]. The use of promoter::GUS lines allowed the observation of some genes encoding enzymes of the AOC family in Arabidopsis roots [START_REF] Stenzel | ALLENE OXIDE CYCLASE (AOC) gene family members of Arabidopsis thaliana: tissueand organ-specific promoter activities and in vivo heteromerization[END_REF].

The Arabidopsis microarray datasets from various stress conditions and developmental stages revealed transcriptional regulation of all JA biosynthesis genes [START_REF] Pauwels | Jasmonate-inducible gene: what does it mean?[END_REF]van Verk et al., 2011). However, the activity of some enzymes seems to be post-translationally regulated. Thus, the activity of OPR3 seems to result from a monomer/dimer equilibrium [START_REF] Schaller | Enzymes in jasmonate biosynthesis -structure, function, regulation[END_REF]. Unstressed leaves of Arabidopsis contain very low amounts of bioactive JA, typically about 20-50 pmol/g fresh mass but this level increases within 5 minutes upon wounding (Glauser et al., 2008b;[START_REF] Glauser | Velocity estimates for signal propagation leading to systemic jasmonic acid Characterization of a MAPK module involved in Arabidopsis response to wounding 113 accumulation in wounded Arabidopsis[END_REF].

In the wounded leaf JA levels increase up to 500-fold, reaching about 10 nmol/g fresh mass whereas JA-Ile levels reach less than 10% of the level of JA [START_REF] Suza | The role of JAR1 in Jasmonoyl-l-isoleucine production during Arabidopsis wound response[END_REF]. In more details, the first significant increase of JA in the wounded leaf is observed less than 30 seconds after wounding and its level doubles every 20 seconds during the first minute.

Concerning JA-Ile, its first significant increase is observed 5 min after wounding [START_REF] Koo | A rapid wound signal activates the systemic synthesis of bioactive jasmonates in Arabidopsis[END_REF]. This remarkable speed of production suggests that all biosynthetic enzymes involved in production of JA/JA-Ile are already present in resting cells.

The molecular events linking wounding to the de novo synthesis of JA are largely unknown. Genetic and pharmacological disruption of cell wall integrity has been shown to constitutively activate the JA pathway [START_REF] Hamann | Identification of cell-wall stress as a hexose-dependent and osmosensitive regulator of plant responses[END_REF]. There is also evidence suggesting that JA/JA-Ile are produced upon perception of DAMPs or bacterial effectors [START_REF] Campos | Jasmonate-Triggered Plant Immunity[END_REF]. In Arabidopsis, the perception of AtPep1 by its receptors PEPR1 and PEPR2 was shown to have a role in JA production as pepr1 pepr2 plants accumulated less JA upon herbivore oral secretion [START_REF] Klauser | The Arabidopsis Pep-PEPR system is induced by herbivore feeding and contributes to JA-mediated plant defence against herbivory[END_REF]. In maize, ZmPEPR3 activates JA synthesis by increasing the level of transcripts encoding the biosynthetic enzymes AOC and AOS [START_REF] Huffaker | An endogenous peptide signal in Arabidopsis activates components of the innate immune response[END_REF]. The application of ATP induces the expression of genes encoding enzymes for JA biosynthesis in Arabidopsis [START_REF] Song | Extracellular ATP induces the accumulation of superoxide via NADPH oxidases in Arabidopsis[END_REF][START_REF] Choi | Identification of a plant receptor for extracellular ATP[END_REF].

The responses induced by OGs were however shown to be independent from JA signaling [START_REF] Ferrari | Resistance to Botrytis cinerea induced in Arabidopsis by elicitors is independent of salicylic acid, ethylene, or jasmonate signaling but requires PHYTOALEXIN DEFICIENT3[END_REF]. The exact mechanism by which DAMP signaling leads to JA biosynthesis still remains elusive. Among the intracellular events that could link the perception of self-derived elicitors to JA production are Ca 2+ ions, ROS, MAPK cascades and CDPKs (Arimura & Maffei, 2010;[START_REF] Zebelo | Role of early signalling events in plant-insect interactions[END_REF]. For instance, Ca 2+ fluxes and associated CDPKs exert a control during the activation of JA biosynthesis genes (Bonaventure et al., 2007). However, it is not clear whether any of the enzymes involved in the biosynthesis of JA are regulated by CDPK/MAPK-dependent phosphorylation or cellular redox changes, although JA-induced phosphorylation of JA signaling components has been observed [START_REF] Zhai | Phosphorylation-coupled proteolysis of the transcription factor MYC2 is important for jasmonate-signaled plant immunity[END_REF].

In addition to JA-Ile, diverse conjugated and oxidized derivatives of JA are produced. These forms of jasmonates include hydroxyhasmonates (HOJAs) and HOJA-Ile as well as dicarboxyjasmonate (HOOCJA-Ile). HOJAs and HOOCJA-Ile accumulate in midveins of wounded leaves [START_REF] Glauser | Spatial and temporal dynamics of jasmonate synthesis and accumulation in Arabidopsis in response to wounding[END_REF] and were shown to be products of JA catabolism (Heitz et al., 2012;Koo et al., 2012). These derivatives being less active than JA-Ile in promoting the binding of COI1 to JAZ proteins, they participate to the attenuation of JA responses. Enzymes involved in JA-Ile catabolism are also rapidly induced upon wounding, herbivory and exogenous JA treatments [START_REF] Bhosale | Predicting gene function from uncontrolled expression variation among individual wild-type Arabidopsis plants[END_REF]. This co-expression of catabolism genes with other JA-response genes reflects the tight control of JA signaling.

b. JA signaling through COI1 -JAZ-MYC signaling module

In 1994, the first JA resistant mutant coi1 (coronatine insensitive 1) was isolated and

shown to be impaired in the receptor of JA-Ile (Feys et al., 1994). In Arabidopsis, plants impaired in COI1 do not respond to external JA application. In addition, biochemical approaches showed direct binding of JA-Ile (or its structural mimic coronatine) to COI1 [START_REF] Yan | The Arabidopsis CORONATINE INSENSITIVE1 Protein Is a Jasmonate Receptor[END_REF]. COI1 is an F-box protein containing 18 leucine-rich repeat (LRR) that forms a complex with SCF (Skp1/Cullin/F-box), an E3 ubiquitin ligase which mediates protein ubiquitination for targeted degradation by the 26S proteasome [START_REF] Xie | COI1: An Arabidopsis Gene Required for Jasmonate-Regulated Defense and Fertility[END_REF]. SCF/COI1 defines a complex specifically involved in jasmonate signaling [START_REF] Xu | The SCF(COI1) ubiquitin-ligase complexes are required for jasmonate response in Arabidopsis[END_REF]. Mutants lacking a functional COI1 are compromised in all known JA-regulated processes and do not respond to exogenous JA, as shown in tobacco, tomato and Arabidopsis [START_REF] Paschold | Co(i)-ordinating defenses: NaCOI1 mediates herbivore-induced resistance in Nicotiana attenuata and reveals the role of herbivore movement in avoiding defenses[END_REF][START_REF] Li | The tomato homolog of CORONATINE-INSENSITIVE1 is required for the maternal control of seed maturation, jasmonate-signaled defense responses, and glandular trichome development[END_REF][START_REF] Xie | COI1: An Arabidopsis Gene Required for Jasmonate-Regulated Defense and Fertility[END_REF]. Interestingly, the accumulation of JA-Ile was shown to be dependent on COI1 as wound-induced JA levels were far lower in coi1 plants than in wildtype ones [START_REF] Chung | Regulation and function of Arabidopsis JASMONATE ZIM-domain genes in response to wounding and herbivory[END_REF]. Jasmonate ZIM-Domain (JAZ) proteins that form a family of 13 members in Arabidopsis are negative regulators of the transcription of JA-responsive genes [START_REF] Thines | JAZ repressor proteins are targets of the SCFCOI1 complex during jasmonate signalling[END_REF]. The absence of DNA-binding domains in JAZ proteins suggests b) When JA-Ile is produced in necessary amounts, it binds to the SCF/COI1 complex, allowing the latter to recruit JAZ repressors and promote their ubiquitination for further degradation by the proteasome. MYC factors are then set free and can activate the expression of defense genes.

that the negative regulation is probably not performed by direct binding to gene promoter sequences but rather by interfering with transcription factors. Indeed, JAZ proteins can bind to many transcription factors and repress their activity. Among them the basic helix-loophelix (bHLH) transcription factors MYC2, MYC3 and MYC4 have been extensively studied (Fernandez-Calvo et al., 2011). In the absence of JA-Ile, JAZ proteins are stable and repress the action of MYCs, and thus the expression of the downstream MYC-dependent genes, by recruiting two co-repressors TPL (TOPLESS) and NINJA (NOVEL INTERACTOR OF JAZ)

and by competing with MED25 (MEDIATOR 25) for interaction with MYCs (figure 1.3).

The myc2 myc3 myc4 triple mutant was shown to be highly susceptible to Spodoptera littoralis feeding [START_REF] Schweizer | Arabidopsis basic helix-loop-helix transcription factors MYC2, MYC3, and MYC4 regulate glucosinolate biosynthesis, insect performance, and feeding behavior[END_REF]. JA-Ile binding to COI1 promotes ubiquitination of JAZ proteins and their subsequent degradation by proteolysis [START_REF] Chini | The JAZ family of repressors is the missing link in jasmonate signalling[END_REF]. Proteinprotein interaction assays showed that the COI1-JAZ interaction is stimulated by JA-Ile but not by meJA or OPDA [START_REF] Thines | JAZ repressor proteins are targets of the SCFCOI1 complex during jasmonate signalling[END_REF]. Poly-ubiquitination of JAZ proteins has been indirectly observed using proteomic approaches [START_REF] Durand | The Ubiquitin System and Jasmonate Signaling[END_REF] and their proteasomedependent degradation was demonstrated by the use of MG132 treatments that stabilized JAZ proteins [START_REF] Thines | JAZ repressor proteins are targets of the SCFCOI1 complex during jasmonate signalling[END_REF].

Interestingly, the expression of JAZ repressors is induced upon wounding and herbivory [START_REF] Chung | Regulation and function of Arabidopsis JASMONATE ZIM-domain genes in response to wounding and herbivory[END_REF]. More particularly, the JAZ10 gene is a well-described early marker for JA signaling in wounded leaves [START_REF] Yan | A downstream mediator in the growth repression limb of the jasmonate pathway[END_REF][START_REF] Acosta | Jasmonates. The Arabidopsis Book[END_REF]. The authors showed that this induction was correlated with the accumulation of JA and JA-Ile and moreover that it was dependent on COI1. Although most of JAZ proteins were shown to be rapidly degraded upon JA-Ile perception, some studies also showed a relative stability of some JAZ proteins following fluctuating JA-Ile levels [START_REF] Chung | Alternative splicing expands the repertoire of dominant JAZ repressors of jasmonate signaling[END_REF]. This negative feedback loop probably acts to restrain the duration and amplitude of JA-triggered responses.

However, JA is also involved in a so-called positive feedback loop where it can stimulate its own synthesis by inducing MYC2-dependent expression of genes like LOX2, OPR3, AOS and AOC (Browse, 2009). The wound-induced expression of AOS and OPR3 was shown to be COI1-dependent [START_REF] Devoto | Expression profiling reveals COI1 to be a key regulator of genes involved in wound-and methyl jasmonate-induced secondary metabolism, defence, and hormone interactions[END_REF].

c. JA crosstalk with other stress related phytohormones Plant pathogens are usually divided into biotrophs and necrotrophs based on their lifestyles. Biotrophic pathogens feed on living tissue whereas necrotrophs kill the host tissue and feed on dead residues. While it is generally admitted that JA has a role in defense against necrotrophic pathogens and herbivorous insects, SA rather activates resistance against biotrophic pathogens [START_REF] Thomma | Separate jasmonate-dependent and salicylatedependent defense-response pathways in Arabidopsis are essential for resistance to distinct microbial pathogens[END_REF]. At first glance, SA and JA have antagonistic roles in plants as mutants impaired in one of them display enhanced expression of marker genes of the other [START_REF] Kloek | Resistance to Pseudomonas syringae conferred by an Arabidopsis thaliana coronatine-insensitive ( coi1 ) mutation occurs through two distinct mechanisms[END_REF][START_REF] Glazebrook | Contrasting Mechanisms of Defense Against Biotrophic and Necrotrophic Pathogens[END_REF]. However, the reality is more complicated. For instance, the hemi-biotrophic bacteria Pseudomonas syringae induces both JA and SA signaling pathways in tomato (Stout et al., 1999).

NPR1 was described as the receptor of SA. When SA levels increase, NPR1, which is present as a homodimer, is "monomerized" and goes to the nucleus where it can regulate the expression of specific genes. The JA/SA antagonism principally occurs at the transcriptional level where SA can suppress the induction of JA-dependent genes [START_REF] Spoel | NPR1 Modulates Cross-Talk between Salicylate-and Jasmonate-Dependent Defense Pathways through a Novel Function in the Cytosol[END_REF][START_REF] Caarls | How salicylic acid takes transcriptional control over jasmonic acid signaling[END_REF]. The well-known JA marker gene PDF1.2 is under the control of several transcription factors among which ORA59 from the ERF family (Ethylene Responsive Factor) that is activated by ethylene (ET) and JA. ORA59 was shown to be degraded by SA thus blocking the expression of PDF1.2. This suppression of PDF1.2 expression still occurs in coi1-1 mutants showing that the SA/JA crosstalk is independent on COI1 and is likely to occur downstream. Moreover, redox signaling was shown to be important for SA-induced suppression of JA responses. High SA levels lead to a reduced redox state. Inhibition of glutathione synthesis, a reducing agent, blocked the SA-mediated antagonism of PDF1.2 expression. However, the results of JA and SA crosstalk can vary with hormone concentrations and the type of stimuli [START_REF] Mur | The Outcomes of Concentration-Specific Interactions between Salicylate and Jasmonate Signaling Include Synergy , Antagonism , and Oxidative Stress Leading to[END_REF][START_REF] Spoel | Regulation of tradeoffs between plant defenses against pathogens with different lifestyles[END_REF].

There are evidence of a JA/SA crosstalk in response to wounding. First, external application of aspirin and SA can block JA biosynthesis in wounded tomato leaves and thus inhibit the expression of some wound-induced genes (Peña-Cortés et al., 1993). Moreover, SA was shown to inhibit the JA-induced production of proteinase inhibitor (PI) in wounded tomato leaves [START_REF] Doares | Oligogalacturonides and chitosan activate plant defensive genes through the octadecanoid pathway[END_REF]. In addition, the SA-inducing pathogen Hyaloperonospora arabidopsidis was shown to strongly suppress JA-mediated defenses that are activated by caterpillars of Pieris rapae [START_REF] Koornneef | Kinetics of salicylate-mediated suppression of jasmonate signaling reveal a role for redox modulation[END_REF]. Less is known about the effect of JA on SA. However, SA-mediated defense responses seem to be enhanced in JA mutants, as demonstrated in wounded tobacco leaves [START_REF] Niki | Antagonistic Effect of Salicylic Acid and Jasmonic Acid on the Expression of Pathogenesis-Related ( PR ) Protein Genes in Wounded Mature Tobacco Leaves[END_REF]. Very recently, JA was shown to induce the expression of EDS5, a gene involved in SA biosynthesis, upon elicitation with flg22 [START_REF] Mine | An incoherent feed-forward loop mediates robustness and tunability in a plant immune network[END_REF]. The same study showed that JA also represses the expression of In the context of herbivores, SA and JA were shown to mediate defense responses towards Spodoptera littoralis, the Egyptian cotton worm [START_REF] Stotz | Evidence for regulation of resistance in Arabidopsis to Egyptian cotton worm by salicylic and jasmonic acid signaling pathways[END_REF]. By analyzing coi1 and npr1 mutants in Arabidopsis, authors showed that SA and JA have opposite effects on plant. Moreover, exogenous application of SA was able to reduce JA amounts induced by the insect. Another study showed this inhibition of JA-induced resistance by SA in plants challenged by the herbivore Spodoptera exigua [START_REF] Cipollini | Salicylic acid inhibits jasmonic acid-induced resistance of Arabidopsis thaliana to Spodoptera exigua[END_REF]). Using a phloemfeeding insect (Bemicia tabaci) another study showed that Arabidopsis plants accumulate SAresponsive defense genes both locally and systemically (Zarate et al., 2006). On the contrary, JA-responsive genes were repressed or not affected by the insect challenge. Moreover, the insect development was boosted in plants constitutively activating SA defenses (cim10, for Constitutive IMmunity 10) and plants impaired in JA defenses (coi1). Finally a more recent study showed that different patterns of phytohormones production can be observable depending on the herbivore species [START_REF] Diezel | Different Lepidopteran Elicitors Account for Cross-Talk in Herbivory-Induced Phytohormone[END_REF].

JA-induced responses are usually described as being dependent on two different branches (figure 1.4). MYC2 is the master regulator of the MYC branch that is co-regulated by JA and abscisic acid (ABA) and leads to the activation of the downstream marker genes VSP2 and LOX2 [START_REF] Lorenzo | JASMONATE-INSENSITIVE1 encodes a MYC transcription factor essential to discriminate between different jasmonate-regulated defense responses in Arabidopsis[END_REF][START_REF] Vos | Onset of herbivore-induced resistance in systemic tissue primed for jasmonatedependent defenses is activated by abscisic acid[END_REF]. The ERF branch is co-regulated by JA and ET and involves transcription factors of EIN3, EIL1, and ERF families such as ERF1 and ORA59. This branch activates the downstream marker gene PDF1.2 [START_REF] Zhu | Derepression of ethylenestabilized transcription factors ( EIN3 / EIL1 ) mediates jasmonate and ethylene signaling synergy in Arabidopsis[END_REF].

Like JA, ET and ABA generally antagonize SA responses.

ET is produced in response to various stress conditions including herbivore attacks or mechanical injury and can modulate the expression of specific ET-regulated genes but also plant physiology (von [START_REF] Dahl | Deciphering the Role of Ethylene in Plant-Herbivore Interactions[END_REF]. In Arabidopsis thaliana, the production of ET was observed during interaction with the lepidopteran Pieris rapea and the homopteran Myzus persicae [START_REF] De Vos | Signal Signature and Transcriptome Changes of Arabidopsis During Pathogen and Insect Attack[END_REF]. Since then, several studies have elucidated the signaling pathway of ET from the sensing by its receptors to the downstream signaling components and the ethylene-responsive genes [START_REF] Ju | Mechanistic Insights in Ethylene Perception and Signal Transdcution[END_REF]. ET can act both positively and negatively on the immune response. In the case of an attack by necrotrophic pathogens, ET acts in synergy with JA for the control of the signaling branch dependent on ERF transcription factors whereas it antagonizes the MYC-dependent branch. It can also affect SA responses. Wounding of a leaf leads to the production of JA and JA-Ile in distal leaves within a restricted area of the plant. Consequently, JAZ repressors are also degraded in distal leaves and transcriptional regulations can occur. For instance, JAZ10, a marker of JA signaling is found induced in distal leaves.

For example, when plants are treated with both ET and SA, the SA-induced expression of PR1 (Pathogenesis-Related 1) is strongly amplified [START_REF] De Vos | Herbivore-induced resistance against microbial pathogens in Arabidopsis[END_REF]. On the other hand, ET-responsive transcription factors EIN3 and EIL1 were shown to repress the expression of SA biosynthesis gene SID2 leading to a reduced SA accumulation (Chen et al., 2009). Finally, ET also plays an important role in the JA/SA interaction as it was shown to be able to stop the SA-mediated suppression of JA responses (Leon-Reyes et al., 2010a). Indeed, when ET and JA are produced prior to SA, the antagonistic effect of SA is abolished. This event is no more observable when an inhibitor of ET is used, confirming its important role.

ABA is the major drought and salt hormone. However, a production of ABA was observed in potato and tomato plants after wounding (Peña-Cortés et al., 1995). In Arabidopsis thaliana, ABA was shown to affect the expression of herbivore-induced genes and JA biosynthesis [START_REF] Bodenhausen | Signaling Pathways Controlling Induced Resistance to Insect Herbivores in Arabidopsis[END_REF]. ABA acts in synergy with JA to induce MYC2-dependent gene expression during wound responses [START_REF] Anderson | Antagonistic Interaction between Abscisic Acid and Jasmonate-Ethylene Signaling Pathways Modulates Defense Gene Expression and Disease Resistance in Arabidopsis[END_REF]. It was also shown to affect JA-inducible defense responses in maize (Erb et al., 2009) and resistance to herbivores in tomato [START_REF] Thaler | Interactions between abscisic-acid-emediated responses and plant resistence to pathogens and insects[END_REF]. These data suggest that the regulation of ABA levels in response to herbivory or wounding can modulate JA-induced defense responses.

Conversely, JA signaling can have a positive effect on ABA signaling by inducing genes, such as PYL4 and PYL5 that encode proteins of the ABA receptor family as it was observed in Arabidopsis and tobacco [START_REF] Lackman | Jasmonate signaling involves the abscisic acid receptor PYL4 to regulate metabolic reprogramming in Arabidopsis and tobacco[END_REF]. Plants mutated in those genes are more sensitive to JA-induced growth inhibition but less sensitive to JA-induced anthocyanin accumulation. Thus, JA-inducible responses depend on ABA and vice versa. In Arabidopsis, ABA and SA response mechanisms also affect each other at multiple levels from biosynthesis to signal transduction pathways [START_REF] Yasuda | Antagonistic interaction between systemic acquired resistance and the abscisic acidmediated abiotic stress response in Arabidopsis[END_REF].

Long-distance signaling

The events described above start locally at the wounding site but can spread systemically throughout the plant, the aim being to alert the rest of the plant of the imminent danger. The existence of a systemic signaling molecule was first reported in tomato in 1972 [START_REF] Green | Wound-Induced Proteinase Inhibitor in Plant Leaves : A Possible Defense Mechanism against Insects[END_REF]. JA and other members of the oxylipins family related to JA were shown to be crucial components of the systemic wound signal. Indeed, wounding of Arabidopsis leaves results in a rapid accumulation of JA-Ile in distal leaves [START_REF] Koo | A rapid wound signal activates the systemic synthesis of bioactive jasmonates in Arabidopsis[END_REF] (figure 1.5). The events leading to jasmonate accumulation distal to wounds can be reduced to two principal mechanisms [START_REF] Koo | A rapid wound signal activates the systemic synthesis of bioactive jasmonates in Arabidopsis[END_REF]. One is the transport of jasmonate itself between cells or organs. Labeling experiments conducted in tobacco showed that meJA can move through xylem and phloem [START_REF] Thorpe | 11C-imaging: Methyl jasmonate moves in both phloem and xylem, promotes transport of jasmonate, and of photoassimilate even after proton transport is decoupled[END_REF]. Using jasmonate-specific antibodies, another study performed in tomato showed that jasmonates accumulate in the epidermis, the mesophyll and in parenchyma cells associated with the xylem and the phloem (Mielke et al., 2011). In all those cell types, the observation of JA accumulation was followed by the accumulation of JA-responsive transcripts, as observed by in situ hybridization. In Arabidopsis, several JA derivatives were also found enriched in the midveins of wounded leaves [START_REF] Glauser | Spatial and temporal dynamics of jasmonate synthesis and accumulation in Arabidopsis in response to wounding[END_REF].

The wound signal is estimated to move at an average of 3.4 to 4.5 cm/min through the plant to stimulate JA accumulation in distal leaves [START_REF] Glauser | Velocity estimates for signal propagation leading to systemic jasmonic acid Characterization of a MAPK module involved in Arabidopsis response to wounding 113 accumulation in wounded Arabidopsis[END_REF]. Concerning JA-Ile a speed of less than 2 cm/min is reported for the wound-induced long-distance signal leading to its production in Arabidopsis. The first significant increase of JA in distal leaves was observed 2 minutes after wounding and JA-Ile appears 3 minutes later. Regarding JAinduced genes, JAZ7 is first observed 15 minutes after wounding in distal leaves. Levels of JA in leaves distal to a wounded leaf can exceed 200 pmol/g fresh mass (Glauser et al. 2008b[START_REF] Glauser | Velocity estimates for signal propagation leading to systemic jasmonic acid Characterization of a MAPK module involved in Arabidopsis response to wounding 113 accumulation in wounded Arabidopsis[END_REF][START_REF] Koo | A rapid wound signal activates the systemic synthesis of bioactive jasmonates in Arabidopsis[END_REF]. Notably, accumulation of JA-Ile and JA-induced responses were shown to be quite transient in distal leaves compared to wounded leaves [START_REF] Koo | A rapid wound signal activates the systemic synthesis of bioactive jasmonates in Arabidopsis[END_REF]. For example, the wound-induced expression of JA-responsive genes decreases during a period when JA-Ile levels remain high [START_REF] Koo | A rapid wound signal activates the systemic synthesis of bioactive jasmonates in Arabidopsis[END_REF]. This observation suggests that injured leaves become less sensitive to the JA-Ile signal and that an efficient mechanism must operate to inactivate the signal.

The second mechanism leading to JA accumulation in leaves distal to the wound is a rapid signal able to travel through the vasculature and to act over long distances, independently of jasmonate transport. We now know that wounding of a leaf can cause electrical activity and membrane depolarization in distal leaves. Electrical signals were first proposed to activate jasmonate synthesis in 1996 [START_REF] Herde | Localized Wounding by Heat Initiates the Accumulation of Proteinase Inhibitor II in Abscisic Acid-Deficient Plants by Triggering Jasmonic Acid Biosynthesis[END_REF]. In Arabidopsis leaves, the plasma membrane depolarization induced by wounding generates Wound-Activated Surface Potentials (WASPs) that move from the tips of leaves to their center and then to a restricted number of distal leaves [START_REF] Mousavi | GLUTAMATE RECEPTOR-LIKE genes mediate leaf-to-leaf wound signalling[END_REF]. The existence of such signal was first postulated based on the observation that tomato wounded cotyledons generate an action potential in the neighboring unwounded leaves [START_REF] Wildon | Electrical signalling and systemic proteinase inhibitor induction in the wounded plant[END_REF]. WASPs were also observed in potato [START_REF] Herde | Localized Wounding by Heat Initiates the Accumulation of Proteinase Inhibitor II in Abscisic Acid-Deficient Plants by Triggering Jasmonic Acid Biosynthesis[END_REF] and avocado trees [START_REF] Oyarce | Evidence for the transmission of information through electric potentials in injured avocado trees[END_REF].

WASPs can be transmitted to leaves from a restricted area of the plant that is consistent with the expression pattern of the JA marker gene JAZ10. GLUTAMATE RECEPTOR-LIKE (GLR) family members were shown to be required for this transmission. Indeed, two mutants of these ion channel proteins, glr3.3 and glr3.6, were deficient in electrical activity in the wounded leaf and showed reduced JAZ10 expression levels in distal leaves [START_REF] Mousavi | GLUTAMATE RECEPTOR-LIKE genes mediate leaf-to-leaf wound signalling[END_REF]. Interestingly, these GLR-dependent systemic depolarization events were also triggered by caterpillar feeding [START_REF] Salvador-Recatalà | Real-time, in vivo intracellular recordings of caterpillar-induced depolarization waves in sieve elements using aphid electrodes[END_REF]. It is notable that the fact that caterpillars and mechanical wounding can induce same changes in electrical activities suggests that insect-derived molecules are not required for this response. Electrical signals being able to activate JA synthesis and lead to the expression of JA-induced responses at the transcriptional level, they might be considered as wounding-induced "elicitors" independently of wounding-induced DAMPs or insect-derived HAMPs. However, the mechanism by which this electrical signal is perceived and lead to JA production is still not known, although the involvement of the RBOHD-dependent ROS production, that is known to transmit signal to distal leaves at a speed similar to WASPs [START_REF] Miller | The Plant NADPH Oxidase RBOHD Mediates Rapid Systemic Signaling in Response to Diverse Stimuli[END_REF] was excluded [START_REF] Mousavi | GLUTAMATE RECEPTOR-LIKE genes mediate leaf-to-leaf wound signalling[END_REF]. This distal production of JA-Ile was shown to require the OPR3 and JAR1 enzymes in the unwounded leaves. Additionally, LOX6 was shown to be responsible for rapid jasmonate synthesis in tissues distal to wounds in Arabidopsis [START_REF] Chauvin | Four 13-lipoxygenases contribute to rapid jasmonate synthesis in wounded Arabidopsis thaliana leaves: A role for lipoxygenase 6 in responses to long-distance wound signals[END_REF].

How to differentiate herbivory from mechanical wounding?

Oral secretions (OS) from feeding insects can contain small molecules able to act like elicitors and thus to activate plant immunity. Such molecules called Herbivore-Associated Molecular Patterns (HAMPs) likely allow the plant to distinguish between herbivory wounding and mechanical damage [START_REF] Mithöfer | Recognition of Herbivory-Associated Molecular Patterns[END_REF]. They are believed to act like DAMPs/PAMPs whose mechanism of recognition by plants has been largely described. Some studies have shown that herbivore-induced signals can influence wound-induced transcript and phytohormone levels [START_REF] Reymond | Differential Gene Expression in Response to Mechanical Wounding and Insect Feeding in Arabidopsis[END_REF][START_REF] Reymond | A conserved transcript pattern in response to a specialist and a generalist herbivore[END_REF][START_REF] Schmelz | Phytohormone-based activity mapping of insect herbivore-produced elicitors[END_REF]. Up to now, only few HAMPs have been identified and isolated. Among them, fatty acid-amino acid conjugates (FACs) are the best studied elicitors [START_REF] Fürstenberg-Hägg | Plant defense against insect herbivores[END_REF]. The fatty acid part comes from the plant whereas the amino acid part originates from insects and the whole is synthesized in insect midguts. The first characterized FAC is volicitin, identified in oral secretions of Spodoptera exigua (beet armyworm) [START_REF] Alborn | An Elicitor of Plant Volatiles from Beet Armyworm Oral Secretion[END_REF]. Since then, FACs were found in oral secretions of many lepidopteran larvae such as Manduca sexta [START_REF] Halitschke | Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuata. III. Fatty acid-amino acid conjugates in herbivore oral secretions are necessary and sufficient for herbivore-specific plant responses[END_REF] that, together with tobacco (Nicotiana attenuata), constitutes the most studied plant-herbivore model. Application of volicitin on Zea Mays seedlings was able to trigger great volatile emission which is not observed upon wounding alone [START_REF] Alborn | An Elicitor of Plant Volatiles from Beet Armyworm Oral Secretion[END_REF]. In Arabidopsis, application of oral secretions of grasshopper was shown to activate defense signaling pathways such as increase of calcium levels, MAPKs activation and hormone production (Schäfer et al., 2011). The molecular mechanism of HAMPs perception is still not known. However, volicitin is able to bind a plasma membrane protein from Zea Mays in a reversible and saturable way (Truitt et al., 2004). This suggests the existence of a FAC-specific receptor although none was identified so far. In addition to FACs, other elicitors are found in oral secretions such as inceptins or caeliferins. Inceptins, isolated from OS of Spodoptera frugiperda, are structurally comparable to flg22 and act like elicitors by triggering hormonal production and volatile emission [START_REF] Schmelz | Cowpea chloroplastic ATP synthase is the source of multiple plant defense elicitors during insect herbivory[END_REF]. Caeliferins, isolated from regurgitate of the grasshopper Schistocerca Americana, are able to induce volatile compound emission in maize [START_REF] Alborn | Disulfooxy fatty acids from the American bird grasshopper Schistocerca americana, elicitors of plant volatiles[END_REF]. There is a huge diversity of plant perception of herbivore OS as highlighted by testing the responses of different plant species to various FACs [START_REF] Schmelz | Phytohormone-based activity mapping of insect herbivore-produced elicitors[END_REF]. None of the elicitors is able to induce responses in all tested plant species and even closely related species do not respond the same way to a given elicitor.

Interestingly, although insect OS contain elicitors able to trigger plant immunity, a recent study showed that application of OS on Arabidopsis leaves can also suppress defense responses [START_REF] Consales | Insect oral secretions suppress wound-induced responses in Arabidopsis[END_REF]. Thus, by conducting a transcriptomic analysis, authors found that several wound-induced genes expression was suppressed by the application of OS from Pieris brassicae and Spodoptera littoralis. This transcriptional regulation was consistent with an enhanced larval growth. However this inhibition of defense responses was not dependent on JA or SA pathways. In addition to oral secretions, plants are able to perceive the deposition of eggs by some herbivorous insects (oviposition) and activate direct and indirect defenses. Two substances in oviposition fluids are known to elicit defense responses: bruchins and benzyl cyanide [START_REF] Wu | Characterization of a MAPK module involved in Arabidopsis response to wounding 126[END_REF].

Besides HAMPs, plants are able to recognize herbivory by their specific feeding pattern. The use of a programmable mechanical device called Mecworm showed that continuous wounding of Lima bean leaves can produce the same bunch of volatiles than those induced by feeding of Spodoptera littoralis larvae [START_REF] Mithöfer | Effects of feeding Spodoptera littoralis on lima bean leaves. II. Continuous mechanical wounding resembling insect feeding is sufficient to elicit herbivory-related volatile emission[END_REF].

II. Wounding and herbivory feeding induce a large set of responses

Wounding of plant tissues is a very strong stress inducing a lot of defense responses from the plant. Such responses are very diverse and include altered plant growth [START_REF] Agrawal | Overcompensation of plants in response to herbivory and the by-product benefits of mutualism[END_REF], enhanced trichome formation [START_REF] Yoshida | Jasmonic acid control of GLABRA3 links inducible defense and trichome patterning in Arabidopsis[END_REF], production of defense chemicals such as proteinase inhibitors or other proteins decreasing the performance of the feeding insect [START_REF] Fürstenberg-Hägg | Plant defense against insect herbivores[END_REF]. Interestingly, most of wounding-induced defense responses are controlled, at least in part, by JA and closer attention will be paid to those ones.

A great transcriptional reprogramming largely dependent on JA

Plants respond to mechanical wounding with the induction of numerous genes. The first wound-inducible defense genes and thus defense proteins were identified in tomato and potato and include proteinase inhibitors [START_REF] Ryan | Protease Inhibitors in Plants: Genes for Improving Defenses Against Insects and Pathogens[END_REF][START_REF] Graham | Regulation of synthesis of proteinase inhibitors I and II mRNAs in leaves of wounded tomato plants[END_REF]. In Arabidopsis many genes were found induced by mechanical wounding and interestingly many of them are also induced by JA or OPDA [START_REF] Reymond | Jasmonate and salicylate as global signals for defense gene expression[END_REF]. Studies aiming to identify more precisely genes specifically induced by wounding are not many. The most cited one reaches back to almost 20 years ago when, using a microarray technique, the expression of 150 selected defense-related genes in Arabidopsis leaves submitted to mechanical wounding was analyzed following kinetics up to 24 hours post-wounding [START_REF] Reymond | Differential Gene Expression in Response to Mechanical Wounding and Insect Feeding in Arabidopsis[END_REF]. 20 genes were already induced after 15 minutes, including pathogenesis-related genes (PR1, PR2 and PR5), some touch genes (TCH2, TCH3 and TCH4) and genes encoding MAPKs (MPK3 and MEKK1). The number of wounding-induced genes increased to 39 after 90 minutes to decrease again at later time points. Interestingly, looking at cluster of genes showing similar temporal expression, the authors identified genes involved in the biosynthesis of JA (LOX2 and AOS) as well as a JA-responsive gene JR3. Following the production of JA upon wounding, the authors observed a peak of accumulation at 2 hours post-wounding, coherently with the peak of expression observed for the JA-related genes. In order to understand the role of JA signaling in the expression of wound-induced genes, wild-type and coi1 plants were compared. 50 % of the wound-induced genes were no longer induced in coi1 plants. On the contrary, the expression of the other 50% of wound-induced genes was COI1-independent. JA is also an important mediator of defense responses toward herbivorous insects. Therefore the authors decided to compare defense responses triggered by mechanical wounding and herbivorous insects at the transcript level. For that purpose they used larvae of the cabbage butterfly Pieris rapae and compared its effect with mechanically wounded plants. Many transcripts were induced by both stresses but generally their expression level was higher upon mechanical wounding. Wounding and insect feeding led to different transcript patterns. Many genes were induced only by wounding but only a single gene was induced only by the insect.

Moreover, between 67 and 84% of insect-induced genes were shown to be dependent on JA signaling [START_REF] Reymond | A conserved transcript pattern in response to a specialist and a generalist herbivore[END_REF]. These genes mainly encode proteins involved in JA biosynthesis, components of JA signaling pathway (transcription factors), defense-related genes, etc... Insect-induced genes dependent on COI1 were typical of biotic stress responses such as PR genes and were also induced by a treatment with meJA. On the other hand, insectinduced genes that were not dependent on COI1 were mainly related to abiotic stresses (senescence, salt) and were not induced by meJA. Moreover, expression patterns observed in distal leaves were quite similar to those observed in local leaves. In order to investigate the role of insect-specific factors on gene expression, oral secretions were applied to pre-wounded leaves. Some genes expression was enhanced but never at the level of their induction by insect feeding [START_REF] Reymond | A conserved transcript pattern in response to a specialist and a generalist herbivore[END_REF]. It is usually the case as mechanical wounding is not able to fully reproduce insect-induced responses such as the emission of volatile compounds or levels of JA production that are lower after mechanical wounding.

Another following study conducted on Arabidopsis focused more particularly on genes differentially expressed at the wound site and in systemic parts of the plant [START_REF] Delessert | Spatial and temporal analysis of the local response to wounding in Arabidopsis leaves[END_REF]. A microarray containing 3500 cDNA clones was screened to enrich for genes regulated at the wound site 4 hours after the stress. Around 360 were then selected for further analysis following kinetics up to 17h post-wounding and also in response to hormonal treatments such as JA, ET and ABA. Interestingly, the genes identified at the wound site were also importantly regulated in distal sites. Coherently with the previous study, many of the systemically induced genes were also induced by JA and were mostly encoding transcription factors. On the other hand locally induced genes were mainly encoding metabolic proteins such as proteins involved in the biosynthesis of lignin. Interestingly, the authors showed that among the up-regulated genes, the majority were systemically induced whereas downregulated genes were mostly local. Combined with temporal analyses the authors concluded that the establishment of systemic responses is prioritized by the plant whereas the local response is established later. Locally, photosynthetic and drought-related genes were found induced, respectively regulated by ET and ABA. Finally, more recently, genes encoding transcription factors were shown to be importantly regulated upon wounding [START_REF] Kilian | The AtGenExpress global stress expression data set: Protocols, evaluation and model data analysis of UV-B light, drought and cold stress responses[END_REF][START_REF] Chen | Expression Profile Matrix of Arabidopsis Transcription Factor Genes Suggests Their Putative Functions in Response to Environmental Stresses[END_REF], among which members of the WRKY, ERF or Zinc finger families.

At the end, all transcriptomic studies pointed out an important overlap between genes induced by wounding and those induced by other stresses, mainly drought and pathogens [START_REF] Reymond | Differential Gene Expression in Response to Mechanical Wounding and Insect Feeding in Arabidopsis[END_REF][START_REF] Cheong | Transcriptional profiling reveals novel interactions between wounding, pathogen, abiotic stress, and hormonal responses in Arabidopsis[END_REF]. An interesting study compared transcriptomes of Arabidopsis plants grown side by side under identical conditions but submitted to different stresses (including heat cold, salt, drought, wounding and UV-B light), following a time course [START_REF] Kilian | The AtGenExpress global stress expression data set: Protocols, evaluation and model data analysis of UV-B light, drought and cold stress responses[END_REF]. Sample harvesting and RNA extraction were also performed in the exact same conditions. Overall, the study points out the existence of a common set of early-induced stress response genes, which mainly results from the fact that early signaling pathways are common to many stresses (Ca 2+ signaling, ROS production, phosphorylation events and hormone accumulation). Conversely, specific genes are induced later and are rather tissue-specific.

Various defense responses

Many defense mechanisms were described to be induced after wounding and insect feeding. I will try to list and classify them and give some information about the cellular machinery involved in their activation. It is likely that many responses are largely determined by the huge wave of genome reprogramming under the control of the rapid JA production observed after wounding.

a. Modification of metabolism to produce toxic and volatile compounds JA can regulate a wide range of wound-induced defense responses including the synthesis of toxic secondary metabolites, production of morphological structures and the control of plant growth. It was initially shown to induce the expression of proteinase inhibitor proteins, anti-digestive compounds, in tomato [START_REF] Farmer | Octadecanoid Precursors of Jasmonic Acid Activate the Synthesis of Wound-Inducible Proteinase Inhibitors[END_REF]. Another example is the wound-induced production of nicotine, a toxic alkaloid, in tobacco (Nicotiana attenuata)

that was shown to be dependent on meJA [START_REF] Baldwin | Jasmonate-induced responses are costly but benefit plants under attack in native populations[END_REF]. The observed metabolic changes were shown to be under the control of a wide genetic reprogramming, as demonstrated by the analysis of Arabidopsis cell suspension cultures or seedlings elicited with JA [START_REF] Pauwels | Jasmonate-inducible gene: what does it mean?[END_REF]. Among JA-induced metabolites are glucosinolates, anthocyanins, phenylpropanoids, isoprenoids…

In addition to toxic compounds production, attacked plants emit volatile organic compounds (VOCs) in order to repel herbivores (direct defense) or to attract predators (indirect defense) [START_REF] Kessler | Defensive Function of Herbivore-Induced Plant Volatile Emissions in Nature[END_REF]. The neighboring plants perceiving these volatile signals are also primed and can adapt in preparation for upcoming challenges [START_REF] Engelberth | Airborne signals prime plants against insect herbivore attack[END_REF]. The herbivore-induced emission of VOCs has been well described in several plant species. They mainly include terpenoids, aromatic compounds, amino acid derivatives and most importantly green leaf volatiles (GLVs) which are products of the oxylipin pathway [START_REF] Mccormick | The specificity of herbivoreinduced plant volatiles in attracting herbivore enemies[END_REF]. VOCs are usually released within 24 h after attack [START_REF] Dudareva | Plant Volatiles: Recent Advances and Future Perspectives[END_REF]. Different feeding strategies adopted by herbivores can lead to the synthesis of different volatiles. For example, leaf-eaters induce the emission of esters, monoterpenes and sesquiterpenes along with JA signaling, whereas piercing-sucking herbivores also induce SA-mediated pathways [START_REF] Walling | The myriad plant responses to herbivores[END_REF]. Phytohormones can affect VOCs emission by activating enzymes (or the expression of corresponding genes) involved in volatile synthesis [START_REF] Van Poecke | Herbivore-Induced Volatile Production by Arabidopsis thaliana Leads to Attraction of the Parasitoid Cotesia rubecula: Chemical, Behavioral, and Gene-Expression Analysis[END_REF]. It is important to note that VOCs induced by wounding and herbivory largely differ both qualitatively and quantitatively. Exogenous application of JA was shown to induce the emission of VOCs and to increase the attraction of carnivore predators [START_REF] Van Poecke | Indirect defence of plants against herbivores: Using Arabidopsis thaliana as a model plant[END_REF]. Besides, inhibition of JA signaling inhibited volatile emission in tomato [START_REF] Thaler | Antagonism Between Jasmonate-and Salicylate-Mediated Induced Plant Resistance: Effects of Concentration and Timing of Elicitation on Defense-Related Proteins, Herbivore, and Pathogen Performance in Tomato[END_REF]. Insect feeding on one leaf can also trigger volatile emission in systemic leaves as observed in several species [START_REF] Van Poecke | Indirect defence of plants against herbivores: Using Arabidopsis thaliana as a model plant[END_REF].

b. Development of physical barriers

Wounded plants were shown to develop more trichomes on new generated leaves to prevent further attacks [START_REF] Yoshida | Jasmonic acid control of GLABRA3 links inducible defense and trichome patterning in Arabidopsis[END_REF]. This increase of trichome density in newly formed leaves was abolished in mutants of JA biosynthetic or signaling pathway showing once again the involvement of JA in this wound-induced phenomenon. In addition to trichomes that function as physical barriers, plants can produce callose, lignin, suberin and other phenolic compounds that are often set up upon wounding. In Arabidopsis, the production of callose requires the callose synthase PMR4 [START_REF] Jacobs | An Arabidopsis Callose Synthase, GSL5, Is Required for Wound and Papillary Callose Formation[END_REF] but no link with JA was described to date.

c. Resistance towards attacking organisms

The main organisms whose fitness is reduced by JA-dependent immunity are necrotrophic pathogens (such as Botrytis cinerea) and herbivores such as caterpillars, aphids, beetles, spider mites… Antisense expression of LOX3 in tobacco was shown to reduce wound-induced JA accumulation and to decrease defense responses towards herbivores [START_REF] Halitschke | Antisense LOX expression increases herbivore performance by decreasing defense responses and inhibiting growth-related transcriptional reorganization in Nicotiana attenuata[END_REF]. Numerous studies also showed that the defects of JA signaling in coi1 plants lead to increased susceptibility to herbivores. For example, Spodoptera littoralis larvae grow faster on coi1 Arabidopsis plants than on wild type ones [START_REF] Stotz | Evidence for regulation of resistance in Arabidopsis to Egyptian cotton worm by salicylic and jasmonic acid signaling pathways[END_REF].

In Arabidopsis, the role of JA has also been shown in resistance towards necrotrophic fungi such as Alternaria brassicicola and Botrytis cinerea but also towards the oomycete Pythium irregulare [START_REF] Thomma | Separate jasmonate-dependent and salicylatedependent defense-response pathways in Arabidopsis are essential for resistance to distinct microbial pathogens[END_REF][START_REF] Staswick | Jasmonate signaling mutants ofArabidopsisare susceptible to the soil fungusPythium irregulare[END_REF]. The first study showed the susceptibility of coi1 mutants to necrotrophic fungi whereas the second one used different alleles of the jar1 mutant, unable to produce the bioactive form JA-Ile from JA. The involvement of JA in the mediation of defense responses was also shown against the nematode Meloidogyne graminicola in rice (Oryza sativa) using a JA biosynthesis mutant [START_REF] Nahar | The Jasmonate Pathway Is a Key Player in Systemically Induced Defense against Root Knot Nematodes in Rice[END_REF]. Overall, those studies suggest that JA is involved in defenses against herbivores and necrotrophic pathogens whereas biotrophic organisms yield to SA-dependent defenses. Wounding can also have a priming effect and protect plants from further attacks by pathogens. Thus, wounding was shown to induce a transient but strong resistance to B.cinerea [START_REF] Chassot | Wounding of Arabidopsis leaves causes a powerful but transient protection against Botrytis infection[END_REF]. This induced resistance was still occurring in mutants of JA, SA or ET signaling showing probable independence from these hormones or from the main hormonal signaling pathways. The same effect was observed after elicitation with OGs and was also independent from JA, SA and ET [START_REF] Ferrari | Resistance to Botrytis cinerea induced in Arabidopsis by elicitors is independent of salicylic acid, ethylene, or jasmonate signaling but requires PHYTOALEXIN DEFICIENT3[END_REF].

d. Growth inhibition

Plant growth is often limited by the availability of resources in the environment.

Investment in defense mechanisms to fight the continuous threat of herbivorous insects slows down the photosynthetic capacity in leaves and absorption of nutrients and water by roots. In order to reduce the metabolic cost of defense mechanisms, plants use inducible defense strategies. Genetic studies have established an important role for JA in mediating tradeoffs between growth and defense [START_REF] Havko | Control of Carbon Assimilation and Partitioning by Jasmonate: An Accounting of Growth-Defense Tradeoffs[END_REF]. The expression of defense features in response to increased JA levels is followed by a strong growth inhibition. Several studies showed that exogenous application of JA or meJA leads to root growth inhibition [START_REF] Staswick | Methyl jasmonate inhibition of root growth and induction of a leaf protein are decreased in an Arabidopsis thaliana mutant[END_REF]Feys et al., 1994). The role of the core JA signaling module in the woundinginduced growth inhibition was shown using coi1, myc2 or jar1 mutants which growth was not inhibited by wounding [START_REF] Zhang | Wound-induced endogenous jasmonates stunt plant growth by inhibiting mitosis[END_REF]. More recently, a study showed the effect of JA on growth and photosynthetic efficiency in Arabidopsis by treatments with coronatine, an agonist of the JA receptor [START_REF] Attaran | Temporal dynamics of growth and photosynthesis suppression in response to jasmonate signaling[END_REF]. Coronatine rapidly stopped plant growth and this was correlated with the expression of genes involved in defense and the repression of genes involved in growth and photosynthesis. One reason for plants to inhibit their growth can be part of their defense strategy to limit food availability for herbivores [START_REF] Havko | Control of Carbon Assimilation and Partitioning by Jasmonate: An Accounting of Growth-Defense Tradeoffs[END_REF]. These regulations are concomitant with the activation of secondary metabolism and show the ability of JA to control tradeoffs between growth and defense by reallocating energy.

Some studies showed the importance of hormonal crosstalks, especially between JA and gibberellin (GA), in this phenomenon. JAZ proteins, important actors of JA signaling, can also modulate growth responses through a direct interaction with DELLA proteins, which are repressors of the GA pathway. The production of GA leads to the degradation of DELLA proteins interacting with JAZ repressors, the consequences of which is an increased inhibition of MYC factors and thus of a greater repression of defense responses [START_REF] Hou | DELLAs Modulate Jasmonate Signaling via Competitive Binding to JAZs[END_REF].

e. Post-wounding healing

Wound healing is an essential biological process to restore functions of damaged cells and surrounding tissues. Plants must restore tissue through cellular regeneration at the damaged site and tissue reorganization. Plant regeneration has mainly been studied in roots where auxin was shown to play an important role during communication between the repair site and other parts of the plant [START_REF] Asahina | Spatially selective hormonal control of RAP2.6L and ANAC071 transcription factors involved in tissue reunion in Arabidopsis[END_REF][START_REF] Xu | A Molecular Framework for Plant Regeneration[END_REF]. Auxin accumulates on the acropetal side of the wound while lower levels are found on the basipetal side.

Extracellular ATP is known to strongly inhibit polar auxin transport (Tang et al., 2003).

Therefore ATP released upon wounding could contribute to the auxin distribution at the wound site. Besides, the addition of ATP induces the expression of genes involved in the biosynthesis of JA and ET [START_REF] Choi | Identification of a plant receptor for extracellular ATP[END_REF] and the latter have been postulated to activate specific wound responsive genes that further promote wound healing [START_REF] Asahina | Spatially selective hormonal control of RAP2.6L and ANAC071 transcription factors involved in tissue reunion in Arabidopsis[END_REF]. that phosphorylates and activates a MAPK kinase (MAP2K or MKK) which in turn phosphorylates and activates its downstream MAPK (MPK). MAP3Ks are serine/threonine kinases, thus their downstream MAP2K(s) is dually phosphorylated on serine and threonine residues following the consensus S/TXXXXXS/T motif in their activation loop. MAP2Ks are threonine/tyrosine kinases and phosphorylate their downstream MAPKs on TEY or TDY motifs of their activation loop [START_REF] Jonak | MAP kinases: universal multi-purpose signaling tools[END_REF]. Plant MAPKs share important sequence similarities with their homologues in animals and fungi (Ichimura et al., 2002). They are also found in a great amount of other plant species (rice, tobacco, grapevine …) thus indicating that MAPK cascades are much conserved among plants. In Arabidopsis thaliana, 20 genes encode MPKs that can be divided into 4 groups (A-D) based on sequence analogies. A, B and C groups are composed of MPKs whose activation loop contains a TEY motif whereas members of the group D possess a TDY motif (Ichimura et al., 2002) (figure 1.7). The 10 MAP2Ks of Arabidopsis are also divided into 4 subgroups (A-D) (figure 1.8). The fact that they are half as many as MAPKs suggests that a given MAP2K is likely to activate several MAPKs and moreover that MAP2Ks constitute the core of crosstalks between various signal transduction pathways. While MAPKs and MAP2Ks constitute quite homogeneous families, MAP3Ks form a more heterogeneous group in that its members show greater variability of primary structures and domains. The 80 MAP3K-like kinases found in Arabidopsis genome, defined by their similarities with animal MAP3Ks, cluster in 3 families named MEKK-like (20 members), Raf-like (48) and ZIK-like MAP3Ks (11). While there is lots of evidence that MEKK1-like members are indeed MAP2K activators, the last two have not been shown as such so far. The 20 MEKK-like MAP3Ks can further be divided into 3 sub-clades (figure 1.9). Finally, MAP4Ks (MAP3K kinases) can take action as a fourth level of kinases and thus act as adaptors linking upstream signaling elements to the MAPK cascades (Colcombet & Hirt, 2008). Interactions between MAPKs within a cascade can occur through docking sites that are present in the kinases or with the help of scaffolding proteins [START_REF] Tanoue | A conserved docking motif in MAP kinases common to substrates, activators and regulators[END_REF].

III. Mitogen-Activated Protein Kinase (MAPK) modules are key signaling actors of environmental perception

Activation of MAPKs can be modulated by phosphatases and among the various ones found in Arabidopsis, the 5 genes composing the MKP (MAPK phosphatase) family seem to be good candidates to control the duration of signaling events or to shut-down the pathway after signaling.

b. The iconic stress-activated MPK3, 4 and 6 reflect the complexit y of MAPK signaling

MAPKs have been extensively studied in the context of plant-pathogen interactions.

They can be activated upon the perception of Pathogen-Associated Molecular Patterns (PAMPs), which are small molecules released from pathogens betraying their presence such as flg22, EF-Tu or chitin. Most studies on MAPKs use flg22, a 22-amino acid peptide coming from Pseudomonas syringae DC3000 flagellum [START_REF] Felix | Plants have a sensitive perception system for the most conserved domain of bacterial flagellin[END_REF]. The recognition of the latter by the Pattern Recognition Receptor (PRR) FLS2 in complex with BAK1 triggers a first level of response from the plant that is called the PAMP-triggered immunity (PTI). The study of MAPKs in the context of PAMPs recognition led to the identification of MPK3, MPK6 (group A) and MPK4 (group B) that are rapidly and transiently activated by PAMPs. Their activation occurs within a minute and peaks around 15 minutes later [START_REF] Nühse | Microbial elicitors induce activation and dual phosphorylation of the Arabidopsis thaliana MAPK 6[END_REF][START_REF] Zipfel | Perception of the Bacterial PAMP EF-Tu by the Receptor EFR Restricts Agrobacterium-Mediated Transformation[END_REF]. The entire modules have been identified and are composed of MKK4/MKK5 acting upstream MPK3/MPK6 [START_REF] Asai | MAP kinase signaling cascade in Arabidopsis innate immunity[END_REF] and MEKK1-MKK1/MKK2 upstream MPK4 [START_REF] Suarez-Rodriguez | MEKK1 Is Required for flg22-Induced MPK4 Activation in Arabidopsis Plants[END_REF], Gao et al., 2008). MPK11, another member of group B very closely related to MPK4, is also rapidly activated by flg22 but the upstream MAP3Ks/MAP2Ks have not been identified yet [START_REF] Bethke | Activation of the Arabidopsis thaliana Mitogen-Activated Protein Kinase MPK11 by the Flagellin-Derived Elicitor Peptide, flg22[END_REF]

(figure 1.10A).
During the course of their evolution, pathogens have acquired the ability to counteract PTI signaling by introducing effector proteins in plant cells, thus decreasing defense responses. Such effectors are very diverse and lead to the so-called Effector-Triggered Susceptibility (ETS) corresponding to the inability of plants to respond correctly to the pathogen and thus a hypersensitive state. However some plants have also developed intracellular receptors from the NB-LRR family (Nucleotide-Binding Leucine Rich Repeat) able to recognize pathogen effectors. The recognition of effectors by such receptors leads to the Effector-Triggered Immunity (ETI) [START_REF] Jones | The plant immune system[END_REF]. One of the main features of ETI is the Hypersensitive Response (HR) which is characterized by a localized cell death inhibiting the expansion of the pathogens feeding on living cells. A recent study showed that the MKK4/5-MPK3/6 module involved in PTI was also involved in ETI signaling [START_REF] Tsuda | Dual Regulation of Gene Expression Mediated by Extended MAPK Activation and Salicylic Acid Contributes to Robust Innate Immunity in Arabidopsis thaliana[END_REF]. The difference comes mainly from their activation kinetics: MAPKs activation is quite transient during PTI (about 1 hour) whereas they are activated from 3 to 10 hours after the expression of pathogen effectors in the plant.

MAPKs are also involved in the transduction of defense responses to abiotic stresses such as cold, salt, drought, UV or ozone (Moustafa et al., 2014). Thus, for example, mkk2 plants have been shown to be less tolerant to salt and cold, suggesting a role for MKK2 and MKK2-related pathways in plant responses to salinity and temperature changes (figure 1.10B) [START_REF] Teige | The MKK2 pathway mediates cold and salt stress signaling in Arabidopsis[END_REF]. In addition to the transduction of stress signals, MAPKs were shown to be involved in developmental processes. Thus the MAP3Ks ANP1, 2 and 3 were shown to act in module with MKK6 and MPK4 in the control of cytokinesis in Arabidopsis [START_REF] Kosetsu | The MAP kinase MPK4 is required for cytokinesis in Arabidopsis thaliana[END_REF]. In stomatal cells, the MAP3K YODA (YDA) was shown to be involved in stomatal patterning [START_REF] Bergmann | Stomatal Development and Pattern Controlled by a MAPKK Kinase[END_REF]. Besides, evidence was provided that MKK4/5-MPK3/6 is the MAPK module acting downstream of YODA [START_REF] Wang | Stomatal Development and Patterning Are Regulated by Environmentally Responsive Mitogen-Activated Protein Kinases in Arabidopsis[END_REF]

(figure 1.10C).

Signaling events involving MAPKs in plants are a good way to realize the complexity of cellular mechanisms involved throughout signaling processes. It also indicates that there are still many unresolved steps to have an exhaustive insight in those events. One of the biggest gaps in our knowledge is the identification of the steps linking the perception of PAMPs to MAP3Ks. Same MAPK modules being involved in different events, it is still unknown how functional specificity is maintained between those different processes. Finally, MAPK phosphorylation cascades lead to the regulation of diverse targets (enzymes, transcription factors…). In the case of the well-studied MPK3, 4 and 6, a large number of substrates have been identified [START_REF] Popescu | MAPK target networks in Arabidopsis thaliana revealed using functional protein microarrays[END_REF][START_REF] Hoehenwarter | Identification of novel in vivo MAP kinase substrates in Arabidopsis thaliana through use of tandem metal oxide affinity chromatography[END_REF]. The diverse nature of identified targets reflects the range of cellular mechanisms that MAPK cascades can regulate.

c. MKK3 defines a new set of multi-stress-responsive MAPK modules

In this part, I will describe extensively what we know about MKK3-related modules as they largely became the topic of my PhD work. Despite having been described several decades ago (Shibata et al., 1995), our knowledge about this MAP2K and the related modules bloomed recently thanks, partly, to the work of the "Stress signaling" group, in which I realized my thesis and other groups which works are quoted thereafter.

MKK3 is an atypical MAPK2 and the only member of sub-group B (Ichimura et al., 2002) (figure 1.8). Its sequence and secondary structure are rather different from the other MAP2Ks but are very well conserved among MKK3 homologues found in other species. A major specificity of MKK3 comes from the presence of an NTF2 (for Nuclear Transport Factor 2)-like domain at its C-terminal part. The role of this domain is still mysterious. As its name suggests, it could have a role in the localization or the shuttling of MKK3 to the nucleus. However other proteins possess NTF2 domains that have been shown to be involved in other processes. For example, the C-terminal NTF2 domain of the Ca 2+ /calmodulin dependent protein kinase II (CAMKII) was shown to be involved in its oligomerization (Morris & Török et al., 2001) or, in some bacteria, NTF2-like peptides have enzymatic activities (Sultana et al., 2004). Even though MKK3 has not been studied as much as other A or C group MAP2Ks (MKK1/2, MKK4/5), data are available in the literature about its potential role in stress responses. Dóczi et al., (2007) showed the role of MKK3 during plant-pathogen interaction (figure 1.11A). They found that mkk3-1 knock-out plants were hypersensitive to P.syringae DC3000 whereas lines expressing a constitutively active MKK3 (MKK3EE) showed a slight resistance to the bacteria. Yeast-2 hybrid studies showed a specificity of interaction between C-group MAPKs (MPK1, 2, 7 and 14) and the MAP2K MKK3 (Dóczi et al., 2007;Lee et al., 2008). Consistently, immunoprecipitation assays from Arabidopsis protoplasts showed an activation of MPK7 by H 2 O 2 that was enhanced by the co-expression of MKK3. MPK1 and MPK2 were also found activated by H 2 O 2 in an independent study (Ortiz-Masia et al., 2007).

Intriguingly, an MKK3-dependent module can also work upstream Reactive Oxygen Species (ROS), as it was shown in the context of wounding (Takahashi et al., 2011). In that study, MKK3 is proposed to work upstream MPK8, a group D MAPK which is activated by wounding in a calcium/calmodulin-dependent as well as an MKK3-dependent manner (figure 1.11B). Diaminobenzidine (DAB), an organic compound that turns brown in the presence of hydrogen peroxide, was used to reveal that plants over-expressing MPK8 are impaired in wounding-induced ROS production whereas plants mutated in MPK8 have trouble to restrict ROS production to the wounding site of the leaf, suggesting a negative role for MPK8 upstream ROS production. However it is difficult to conclude about the role of MKK3 as mkk3 plants have not been tested in their ability to still produce ROS upon wounding.

MKK3 has also been claimed to act upstream MPK6 in two different contexts. A first study showed that the MKK3-MPK6 module has an important role in the JA-induced root growth inhibition in Arabidopsis (Takahashi et al., 2007) (figure 1.11C). MPK6 was rapidly activated by JA and this activation was impaired in mkk3 plants but not in mkk2 plants.

However no mock control was shown to ensure this JA-induced activation of MPK6. mpk6 as well as mkk3 plants were slightly more sensitive to an exogenous application of JA as reflected by the slightly shorter size of their roots compared to wild-type plants. These plants also showed a reduced expression of the JA marker gene PDF1.2 and increased expression of VSP2. A second study showed the involvement of the MKK3-MPK6 module in blue light signaling (Sethi et al., 2014) (figure 1.11D). MPK6 is specifically activated by blue light but not by red light. This activation is lost in mkk3 as well as in myc2 mutant plants suggesting that MKK3 and MYC2 act upstream the blue light-induced activation of MPK6. Interestingly which is a well-known actor of JA signaling.

More recently, the role of MKK3 upstream C-group MAPKs was demonstrated by the group in which I performed my thesis, in the context of the major drought hormone ABA (Danquah et al., 2015). C-group MAPKs were found activated 4 hours after ABA treatment and this activation was lost in mkk3-1 plants. The authors were able to identify the MAP3Ks acting upstream MKK3 when plants were treated with ABA. Indeed, the ABA-induced activation of MKK3-MPK7 was lost in the map3k17 map3k18 double mutant. Interestingly the study revealed that the ABA-induced activation of C-group MAPKs requires the de novo synthesis of MAP3K17 and MAP3K18 (figure 1.12). This could explain the rather slow activation kinetics of the downstream MAPKs. However the involvement of this entire module in response to ABA was also shown in an independent study (Matsuoka et al., 2015) in which a rapid activation (15 minutes) of MAP3K18 by ABA was shown using lines expressing a tagged version of the kinase. That study also demonstrated the role of MAP3K18 in the regulation of leaf senescence. Another study provided evidence that MAP3K18 is induced and activated by ABA (Mitula et al., 2015). The role of MKK3 in ABA signaling was confirmed by phenotypical studies of plants mutated in MKK3. mkk3-1 plants were more sensitive to drought as reflected by their lower ability to restrict water loss after a long-term drought experiment (Danquah et al., 2015). MKK3 was linked to ABA signaling in other plant species. Thus tobacco plants overexpressing the cotton GhMKK3 were more resistant to drought and closed their stomata more efficiently in response to ABA (Wang et al., 2016).

ABA is also a major hormone involved in the control of seed dormancy. In Arabidopsis, germination assays revealed a hypersensitivity of mkk3-1 seeds to ABA whereas MKK3EE seeds were insensitive. In barley, the identification of a recessive mutation for MKK3 revealed its role in breaking seed dormancy (Nakamura et al., 2016) and in wheat MKK3 was found to be a causal gene of seed dormancy [START_REF] Torada | A causal gene for seed dormancy on wheat chromosome 4A encodes a MAP kinase kinase[END_REF]. Additionally, one very recent study places MKK3 downstream of MAP3K20. Both map3k20 and mkk3 plants are defective in microtubule functions and the interaction of the two proteins was shown in vitro. Interestingly mpk18 plants show the same phenotype and seem to directly interact and to form a functional module with MAP3K20, without the need of an intermediate MAP2K (Benhamman et al., 2017). Finally, MKK3 and MPK1 were shown to have a role in auxin signaling as mkk3 and mpk1 plants were hypersensitive to auxin in cell expansion assays (Enders et al., 2017).

To summarize, MKK3 has been studied in different stress contexts and in modules involving different MAPKs (figure 1.11). However, while three independent studies showed the interaction of MKK3 with MPK1, 2, 7 and 14 (Dóczi et al., 2007;Lee et al., 2008;Matsuoka et al., 2015), a direct interaction between MKK3 and MPK6 or MPK8 was never proven. Besides, in protoplasts MKK3EE could not activate MPK6 or MPK8 whereas Cgroup MAPKs were activated.

Spare data suggest a complex MAPK role in wounding and herbivory interaction

Intriguingly, MAPK activation in response to wounding was largely documented in species other than Arabidopsis. The first MAPK response to wounding was reported in alfalfa (Bögre et al., 1997). In this study, wounding induces a rapid activation of the alfalfa MMK4

that is shown to be due to a post-translational regulation as it is not blocked by the addition of α-amanitin or cycloheximide, two inhibitors of the transcription and translation steps respectively. In tobacco (Nicotiana attenuata), transcript levels and kinase activities of WIPK (for Wound-Induced Protein Kinase) and SIPK (for SA-Induced Protein Kinase), which are orthologs of Arabidopsis MPK3 and MPK6 respectively, increase rapidly upon wounding (after 1 min), in both local and systemic leaves (Seo et al., 1999). This activity is amplified when oral secretions from Manduca sexta larvae are applied in addition to the wounding [START_REF] Wu | Herbivory Rapidly Activates MAPK Signaling in Attacked and Unattacked Leaf Regions but Not between Leaves of Nicotiana attenuata[END_REF]. SIPK and WIPK are involved in the regulation of JA and SA levels in tobacco plants submitted to wounding or herbivory [START_REF] Seo | The mitogen-activated protein kinases WIPK and SIPK regulate the levels of jasmonic and salicylic acids in wounded tobacco plants[END_REF][START_REF] Wu | Herbivory Rapidly Activates MAPK Signaling in Attacked and Unattacked Leaf Regions but Not between Leaves of Nicotiana attenuata[END_REF]. Moreover, NaMEK2 was shown to be the potential MAP3K acting upstream SIPK and WIPK as it was required for the phosphorylation of the latter upon herbivory simulation (wounding + oral secretion). NaMEK2 was also shown to regulate wounding-and herbivory-induced JA and ET levels [START_REF] Heinrich | Two mitogen-activated protein kinase kinases, MKK1 and MEK2, are involved in wounding-and specialist lepidopteran herbivore Manduca sexta-induced responses in Nicotiana attenuata[END_REF]. Concerning JA, the regulation occurs at the level of enzymes involved in the early steps of its biosynthesis [START_REF] Kallenbach | Nicotiana attenuata SIPK, WIPK, NPR1 and Fatty Acid-Amino Acid Conjugates Participate in the Induction of Jasmonic Acid Biosynthesis by Affecting Early Enzymatic Steps in the Pathway[END_REF]. Very similarly in tomato (Solanum lycopersicum), LeMPK1 and LeMPK2, which are orthologs of SIPK, were activated by herbivory and their silencing led to the reduction of JA levels and the expression of JA biosynthesis genes [START_REF] Kandoth | Tomato MAPKs LeMPK1, LeMPK2, and LeMPK3 function in the systeminmediated defense response against herbivorous insects[END_REF]. Tobacco MPK4 was also found activated by wounding and was shown to have an important role in the expression of JAdependent genes [START_REF] Gomi | A mitogen-activated protein kinase NtMPK4 activated by SIPKK is required for jasmonic acid signaling and involved in ozone tolerance via stomatal movement in tobacco[END_REF].

In Arabidopsis, wounding was identified among various stresses activating MPK4 and MPK6 [START_REF] Ichimura | Various abiotic stresses rapidly activate Arabidopsis MAP kinases ATMPK4 and ATMPK6[END_REF]. More recently, MPK3 and MPK6 were shown to be involved in wounding-induced ET biosynthesis [START_REF] Li | AIK1, A Mitogen-Activated Protein Kinase, Modulates Abscisic Acid Responses through the MKK5-MPK6 Kinase Cascade[END_REF] through the transcriptional regulation of ACS (1-aminocyclopropane-1-carboxylic acid synthase) genes. They were rapidly activated by wounding and their activation was strongly reduced in the mkk4 mkk5 double mutant, suggesting that MKK4 and MKK5 act upstream of MPK3 and MPK6 also in a wounding context. Coherently, a PP2C-type phosphatase that negatively regulates MPK4 and MPK6 was shown to modulate JA and ET levels upon wounding and have a role in defense responses against necrotrophic pathogens [START_REF] Schweighofer | The PP2C-type phosphatase AP2C1, which negatively regulates MPK4 and MPK6, modulates innate immunity, jasmonic acid, and ethylene levels in Arabidopsis[END_REF]. Besides, the MAP2K

MKK3 was shown to work upstream MPK8 and to regulate Reactive Oxygen Species (ROS) homeostasis upon wounding (Takahashi et al., 2011). To date, no activation of MAPKs was

shown in response to a direct insect attack. However, application of grasshopper oral secretion on Arabidopsis leaves triggered the activation of MPK3/MPK6 (Schäfer et al., 2011).

In addition to wounding, DAMPs, ROS, as well as wounding-induced phytohormones are also able to activate MAPKs. Thus, a treatment with 1 µM of AtPeps is able to rapidly activate MPK3 and MPK6 [START_REF] Bartels | The family of peps and their precursors in arabidopsis: Differential expression and localization but similar induction of pattern-Triggered immune responses[END_REF]. Their activation by AtPeps requires the expression of both PEPR1 and PEPR2 as shown by the absence of activation in the pepr1 pepr2 double mutant. Pectin fragments and OGs are also able to activate MPK3 and MPK6 [START_REF] Kohorn | Pectin activation of MAP kinase and gene expression is WAK2 dependent[END_REF][START_REF] Denoux | Activation of defense response pathways by OGs and Flg22 elicitors in Arabidopsis seedlings[END_REF], as well as a treatment with 100 µM ATP.

MPK3 and MPK6 were not activated in dorn1-1 plants thus showing the involvement of DORN1 for the ATP-induced activation of MPK3 and MPK6 [START_REF] Choi | Identification of a plant receptor for extracellular ATP[END_REF]. In addition to the highly studied MAPKs 3, 4 and 6, MPK1 and MPK2 (group C) were found activated by wounding and JA in Arabidopsis thaliana (Ortiz-Masia et al., 2007). Altogether, these data suggest an important role for MAPKs in the transduction of the wounding signal and a likely role in the regulation of hormone biosynthesis and in particular of JA and SA.

IV. Objectives of the PhD work: unveiling the function of MKK3 modules in wounding signaling

MAPKs and signal transduction before this work, a state of knowledge

As discussed earlier, many studies focused on the well-known MPK3, 4 and 6, pointing out the essential role of MAPKs, activated rapidly upon a stress perception, in signal transduction. This choice may be explained by the facts that those kinases are intrinsically strongly activated upon stresses and their activities are simple enough to monitor. In particular, "in gel" kinase assays are very efficient techniques for MPK3/4/6. Moreover, antibodies risen against the phosphorylated (=active) forms of the mammalian ERK2 is also able to recognize the phosphorylated form of Arabidopsis MPK3, 4 and 6, providing an easy way to screen for MAPK activation using western blots. Clearly, such tools created a bias in studies and drew the attention on small kinases that are highly expressed and show strong enzymatic activities at the expense of other kinases lacking such features. Some kinases have a larger size (therefore more difficult to refold in in-gel kinase assays) and are weakly expressed or less processive once activated, making their functional characterization difficult without the use of specific antibodies. Our knowledge is therefore reduced concerning other kinases from the different families that are found in plant genomes. Among the 4 sub-groups of MAPKs in Arabidopsis (A, B, C and D), not much is known about members of groups C and D, which are actually the largest ones.

The "Stress Signaling" group is working on MAPK and CDPK modules. Its strategy largely consists in developing original tools to study the classical signaling modules defined by MPK3/4/6. For instance, in the past, the group has developed the Tandem Affinity Purification (TAP) system to identify putative interacting proteins to candidate kinases along with other phosphoproteomic approaches and the use of mutations triggering constitutively active MAPKs as a genetic tool in addition to the classical knock-out mutants. Another important objective of the group is to better understand the role of MAPKs other than the most studied MPK3, 4 and 6 in Arabidopsis. The study conducted in the laboratory on the MKK3 module activated by ABA is quite novel in that it deciphered a whole new MAPK module and demonstrated that some MAPK modules are under the control of transcriptional regulation and are therefore late responsive signaling pathways (Danquah et al., 2015;Boudsocq et al,. 2015). That rather slow activation suggests a regulation of late events that are quite difficult to apprehend.

Objectives: characterizing the activation of a MAPK module upon wounding stress

The objective of my PhD thesis was to further characterize MKK3-dependent modules. The role of such modules has been largely studied by the "Stress Signaling" group, as well as by other groups, in the context of its activation by ABA (Danquah et al. 2015;Matsuoka et al., 2015;Mitula et al., 2015). However we had arguments suggesting that MKK3-dependent modules could be activated by other cues and be involved in other processes. Firstly based on the numerous studies carried out on MPK3, 4 and 6, it is now well admitted that a given MAPK module can be solicited in different contexts and can thus be

CHAPTER II: RESULTS & DISCUSSIONS

I. Toward a general model for the activation of MKK3-related modules

Previous studies carried out in our laboratory identified a complete MKK3dependent MAPK module activated by ABA. This module includes C-group MAPKs (MPK1, 2, 7 and 14) downstream of MKK3 and its activation requires the transcriptional regulation of MAP3K17 and 18, both sub-clade III MAP3Ks. From this observation, we wondered if those characteristics could be extended to all MKK3-dependent modules and built a model that led to the writing of a review [START_REF] Colcombet | Convergence of Multiple MAP3Ks on MKK3 Identifies a Set of Novel Stress MAPK Modules[END_REF].

Identification of MAP3Ks able to interact with MKK3 using the yeast 2-hybrid system

In order to identify which MAP3Ks could interact with MKK3 and thus could potentially act as activators of MKK3-dependent modules, a pair-wise yeast 2-hybrid (Y2H) experiment using the collection of kinase ORFs available in the laboratory was carried out. MAP3Ks belonging to the sub-clade III and that the latter could act upstream MKK3 to define functional modules in the context of their activation by environmental cues (Ichimura et al., 2002;[START_REF] Colcombet | Convergence of Multiple MAP3Ks on MKK3 Identifies a Set of Novel Stress MAPK Modules[END_REF].

Functional validation of MAP3Ks using a transient expression system

The fact that some sub-clade III MAP3Ks and MKK3 interact in Y2H assays does not demonstrate their functionality as phosphorylation cascades in planta. In order to test whether it is indeed the case, I used the transient expression system of Arabidopsis mesophyll protoplasts [START_REF] Yoo | Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis[END_REF]. Protoplast production and transformation is a rapid technique, commonly used in our laboratory to (co-)express proteins which then may be detected by western blot and tested for their activity after immuno-precipitation. This tool has been previously successful to characterize an MKK3-dependent module activated by ABA. It allowed demonstrating the functionality of the MAP3K18-MKK3-C-group MAPKs module in planta as well as the need for MAP3K18 production for MPK7 activation by ABA (Danquah et al., 2015).

The ability of sub-clade III MAP3Ks to act upstream MKK3 in a functional module was tested by co-expressing the latter and testing the activity of downstream MAPKs. Several works identified MPK1, 2, 7 and 14, all members of the C-group MAPKs, as being the MAPKs acting downstream MKK3 [START_REF] Schweighofer | The PP2C-type phosphatase AP2C1, which negatively regulates MPK4 and MPK6, modulates innate immunity, jasmonic acid, and ethylene levels in Arabidopsis[END_REF]Danquah et al., 2015). For technical reasons, MPK2 was used as a representative of C-group MAPKs. Plasmids containing gene ORFs of sub-clade III MAP3Ks, MKK3 and MPK2 under a 35S promoter and a specific tag at their C-terminal tail were created. MPK2-HA was co-expressed in mkk3-1 mesophyll protoplasts in combination with each sub-clade III MAP3K-YFP the in the absence or presence of MKK3-Myc. MPK2-HA was then immuno-precipitated using α-HA antibodies and its activity was assayed through the phosphorylation of Myelin Basic Protein (MBP).

MPK2-HA showed a strong activity only when both MKK3-Myc and MAP3K-YFP were coexpressed (figure 2.2A). Western blot analyses using α-YFP, α-Myc or α-HA antibodies allowed me to check that the proteins were correctly and equally expressed in all conditions.

Concerning MAP3Ks, only MAP3K16, 17, 18, 19 and 20 were detected on the western blots.

The other MAP3Ks proteins were not detectable even though their co-transformation was sufficient to increase MPK2 activity. We first hypothesized a very rapid degradation/synthesis "turn-over" of the proteins. Some data in the literature discuss this aspect of MAP3Ks regulation. Thus PP2C phosphatase ABI1 was shown to control the proteasome-mediated degradation of MAP3K18 (Ludwikow et al., 2015). In that work MAP3K18 degradation was blocked by the proteasome inhibitor MG132. To test my hypothesis, I decided to also use MG132 and see if MAP3Ks were detectable on western blots. Actors of the module were coexpressed as described above, with the exception that MG132 or DMSO (as mock) was added in the expression medium along with transformed protoplasts (see "Materials & Methods for details). Consistently, MPK2-HA was activated when both MKK3-Myc and one of the MAP3K-YFP fusions were co-expressed, regardless of the presence of MG132. Looking at protein accumulation, I was only able to get a signal for MAP3K18 in absence and presence of MG132. The other MAP3Ks (13, 14, 15 and 16) were still not detectable even in presence of MG132 (figure2.2B). As no positive control for the action of MG132 was in our possession it is difficult to ensure that it was indeed "perceived" by the cells and therefore to conclude on the reason why some MAP3Ks proteins are not visible. Overall, these results

suggest that sub-clade III MAP3Ks, MKK3 and MPK2 form a module that is functional in planta.

Sub-clade III MAP3Ks are strongly transcriptionally regulated by stresses

With the noticeable exception of MAP3K17 and MAP3K18, not much is known about sub-clade III MAP3Ks. Strikingly, the laboratory showed that the activation of MKK3-MPK7 by ABA and drought required the de novo synthesis of those two MAP3Ks.

Consequently, MAP3K17 and MAP3K18 genes are strongly transcriptionally regulated by these signals (Danquah et al., 2015). Interestingly, two other members of sub-clade III, MAP3K13 and MAP3K14, were shown to be strongly induced upon nitrate (Marchive et al., 2013;[START_REF] Chardin | Review: Mitogen-Activated Protein kinases in nutritional signaling in Arabidopsis[END_REF] [START_REF] Zimmermann | GENEVESTIGATOR. Arabidopsis microarray database and analysis toolbox[END_REF]. This database helps finding out rapidly under which conditions one gene is expressed. We got useful information about transcriptional regulations of MEKK-like MAP3Ks that were indeed found to be regulated under a broad range of both biotic and abiotic stresses. Interestingly, sub-clade III MAP3Ks were found induced by the largest number of stresses (figure 2.3). For each MEKKlike MAP3K, the principal environmental perturbations responsible for their transcriptional

Discussion

Previous studies have shown that MKK3 forms a functional module with C-group MAPKs (MPK1, 2, 7 and 14). However, little data is provided about the potential upstream MAP3K(s). A previous study of the laboratory showed the role of MAP3Ks 17 and 18 upstream MKK3 in an ABA and drought context (Danquah et al., 2015). Another study showed the interaction of MAP3K20 with MKK3 in a Y2H screen (Benhamman et al., 2017).

Interestingly, those three MAP3Ks belong to the sub-clade III and were found interacting with MKK3 along with three other MAP3Ks (15, 16 and 19) in our Y2H assay. The functionality of all members of sub-clade III as activators of MKK3 was then confirmed by transient expression in protoplasts. These include MAP3K13 and 14 which did not interact with MKK3 in Y2H. Looking closer to the structure of MAP3K13 and 14, we realized that transmembrane domains at their C-terminal tail were predicted by several software [START_REF] Schwacke | ARAMEMNON, a novel database for Arabidopsis integral membrane proteins[END_REF].

This turned out to be a specificity of those two MAP3Ks. The presence of such a domain could explain the absence of interaction between MKK3 and MAP3K13 and 14 in yeast. To These experiments were performed at least 3 times (n>3) with identical results.

Characterization of a MAPK module involved in Arabidopsis response to wounding 50 MPK2 were shown to be activated by H 2 O 2 , JA and wounding (Ortiz-Masia et al., 2007). Two independent studies also showed their activation by ABA (Umezawa et al., 2013;Danquah et al., 2015). As, H 2 O 2 , JA and ABA are all signaling molecules produced upon wounding, we decided to focus on the study of the MKK3-C-group MAPKs module in a context where plants are submitted to mechanical damage.

I. Identification and characterization of new MAPK modules activated by wounding

In this second part, I show results that led me to the identification of an almost complete MKK3-dependent MAPK module activated by wounding. The objectives were to (1) reproduce and characterize the activation of MPK1 and MPK2 by wounding which has been previously

shown (Ortiz-Masia et al., 2007) and extend this information to other C-group MAPKs, (2) test whether MKK3 could act upstream those MAPKs as shown in the context of ABA and proposed in the first part of results and (3) identify the upstream MAP3K(s) to complete the module.

Wounding activates an MKK3 module in Arabidopsis thaliana

In order to confirm the results previously obtained by Ortiz-Masia and coworkers The same tagged versions were also created in the mkk3-1 background for MPK2 and MPK7.

For technical reasons, we did not succeed to produce the corresponding MPK14 line. The activity of MPK1, 2 and 7 was tested after IP with α-HA antibodies in those transgenic lines submitted to wounding. All three MAPKs were found to be activated 30 minutes to 2 hours after wounding, with a peak of activity at 1 hour (figure 2.7 -first lines). The kinetics was similar to the one of MPK2 immuno-precipitated from Col-0 plants using α-MPK2 antibodies.

In the case of MPK2-HA and MPK7-HA lines, no signal could be observed in mkk3-1 background, confirming that their activation is dependent on MKK3. MAPKs protein amounts were analyzed by western blot after incubation with α-HA antibodies (figure 2.7second lines). MAPK amounts remained stable all along the time course and the proteins were already present at resting conditions (time 0, without wounding). Besides, protein levels were similar in Col-0 and mkk3-1 plants. Taken together, these results suggest that the MKK3-dependent increase of MAPKs activity observed upon wounding is not a consequence However, it would be necessary to ensure that the touch stress was indeed correctly perceived by the plant by testing, for instance, the activation of MPK3 and 6 that is known to happen upon a touch stress.

The wounding-induced activation of the iconic MPK3 and 6 is not dependent on MKK3

MKK3 and C-group MAPKs are obviously not the only MAPKs that are activated upon environmental stresses and more particularly upon wounding. Indeed, MPK3, 4 and 6 which are A/B-group MAPKs, are rapidly activated upon wounding of Arabidopsis leaves [START_REF] Ichimura | Various abiotic stresses rapidly activate Arabidopsis MAP kinases ATMPK4 and ATMPK6[END_REF][START_REF] Li | AIK1, A Mitogen-Activated Protein Kinase, Modulates Abscisic Acid Responses through the MKK5-MPK6 Kinase Cascade[END_REF]. Based on these data I wondered if MPK3, 4 and 6

were activated by wounding in our laboratory conditions and moreover if they showed the same characteristics as C-group MAPKs (activation kinetics, dependence on MKK3). I therefore performed western blot analyses using an antibody raised against the pTEpY motif of MAPK activation loop (and referred as anti-pTpY). This TEY motif, which is largely conserved within MAPKs of groups A, B and C, is known to be phosphorylated by upstream et al., 2015). To note, the MAP3K18locus-YFP construct includes the endogenous terminator of MAP3K18 whereas the Tnos terminator was used for all the other MAP3Klocus-YFP lines.

Plants were wounded and proteins were analyzed by western blots using α-YFP antibodies.

Unfortunately, none of the MAP3K-YFP proteins was detectable, except MAP3K18-YFP which appeared 2 hours after the wounding and was still present at 4 hours post-wounding For each mutant background, the table shows the number of experiments performed (testing MPK2 activity upon wounding) and whether the results were reproducible or not. higher in map3k14-1 plants. Studying more carefully the localization of the T-DNA insertion in the MAP3K14 locus, we realized that the mutant could still allow the production of a truncated protein including its whole kinase domain and a part of its C-terminal end (figure 2.19A). To test whether truncated MAP3K14 was able to activate the downstream kinases MKK3 and MPK2, I took advantage of the protoplast expression system. We created a vector containing the coding sequence predicted to be expressed and transcribed in the map3k14-1 mutant (named MAP3K14-1) fused to YFP in it C-terminal tail. When MAP3K14-1-YFP was co-expressed with MKK3-Myc and MPK2-HA in mkk3-1 protoplasts, I observed the activation of MPK2 (figure 2.19B -above dotted line). Interestingly, the truncated MAP3K14-1 protein was detectable whereas the wild-type MAP3K14 protein was still not as previously stated (figure 2.19B -below dotted line). This result goes in the direction of our hypothesis supporting the idea of a higher stability for the truncated form of MAP3K14 that is expressed in the map3k14-1 line and is responsible for an increased MPK2 activity after wounding. Curiously, a weak activation of MPK2 was still visible when MAP3K14-1-YFP was expressed in the absence of MKK3-Myc, which suggests the activation of MPK2 circumventing MKK3. Altogether, these results suggest a role for MAP3K14 upstream the MKK3-MPK2 module at early time-points of its activation by wounding. To strengthen this idea we wanted to have a real knock-out mutant and therefore decided to rely on the recently emerged CRISPR-Cas9 system to create it. Two independent lines including a 1bp insertion in the MAP3K14 gene leading to a frame shift were created. They were both homozygous for the mutant allele. MPK2 activity was tested in both lines and compared to Col-0.

(
Unfortunately results were quite variable (figure 2.20). Although not concluding, this result does not invalidate our hypothesis according to which MAP3K14 acts upstream MKK3-MPK2 in response to wounding. Nevertheless, it suggests a high functional redundancy with other sub-clade III MAP3Ks that are still expressed when MAP3K14 is knocked-out. This late activation could reflect the requirement for a de novo protein synthesis which, according to our model, could occur at the MAP3K level.

Discussion

D

thus its visualization by biochemical approaches. Since MAP3K14 did not interact with MKK3 in yeast it would also be interesting to test whether its truncated form can. We also observed an activation of MPK2 in the presence of the truncated MAP3K14 despite the absence of MKK3 in protoplasts. One possible explanation is that sub-clade III MAP3Ks can interact directly with C-group MAPKs, which has never been tested. However a direct interaction of a sub-clade III MAP3K with a MAPK was recently observed with the example of MAP3K20 interacting with MPK18 (Benhamman et al., 2018). Another explanation can be the involvement of other MAP2Ks which endogenous form is still expressed in mkk3-1 protoplasts. We can imagine that the missing carboxyl-terminal part of the truncated form part plays a role in the interaction specificities of MAP3K14 with downstream MAP2Ks.

III. Identification of signaling elements acting upstream MKK3 upon wounding

The large number of cellular events occurring when plants are subjected to wounding is listed in the introduction part. However, the way the different signaling elements coordinate still needs to be deciphered. To better understand the place of the MKK3-C-group MAPKs module in the response of Arabidopsis to wounding, I was interested in the identification of second messengers that are responsible for the signal transmission from the perception of the wounding stress to the activation of the MAPK cascade. These second messengers would thus act upstream our MKK3-dependent module and play an important role in its activation. In order to identify those second messengers, I used two different approaches. The first one consisted in testing whether an exogenous application of candidate molecules on the plants could trigger the activation of our MAPK module. Obviously, that method could not give a direct answer to our question as the molecule's effect was tested independently from wounding. Therefore the second approach consisted in the use of plants mutated in either genes involved in the biosynthetic pathway of candidate molecules or in their known perception systems/receptors.

Exogenous application of some candidate second messengers can activate an MKK3-dependent module

As explained in the introduction, production of second messengers, such as products of cell wall degradation, H 2 O 2 or phytohormones is one of the early events occurring upon C). To be sure that the treatment was nonetheless sensed by the plants α-pTpY western blots were performed to test MPK3 and MPK6 activation that is already known to occur upon those tested stresses [START_REF] Kovtun | Functional analysis of oxidative stressactivated mitogen-activated protein kinase cascade in plants[END_REF][START_REF] Choi | Identification of a plant receptor for extracellular ATP[END_REF]Galletti et al., 2011). Indeed MPK3 and MPK6 were found activated by H 2 O 2 and OGs. The test was not performed upon treatment with ATP but further gene expression analyses showed that their presence in plants environment was indeed correctly perceived (not shown). In the introduction part, similarities between DAMP-and PAMP-induced responses were discussed. In order to know if our module could also be activated by PAMPs, I used flg22 on Col-0 and mkk3-1 plants. ABA was also used as a positive control known to activate MPK2. Therefore MPK3 and MPK6 are not activated by an exogenous treatment with JA.

JA is perceived by the SCF/COI1 complex. In coi1 plants, JA is not perceived anymore and is therefore not able to regulate downstream JA-dependent responses. B: Col-0, map3k14-1 and map3k17 map3k18 were treated with 50 µM JA following kinetics from 15 minutes to 4 hours.

I focused only on the ones whose expression was previously found to be induced by JA.

MPK2 activity was reduced in map3k17 map3k18 plants at 30 minutes and 2 hours posttreatment. No difference was observed in map3k19 and map3k14-1 backgrounds. These results suggest a role for MAP3K17 and 18 upstream MKK3-MPK2 in the context of an external treatment with JA. Nonetheless this experiment was performed only once and does not allow drawing any definite conclusion (figure 2.28A and B).

Wounding-induced activation of the MKK3-dependent module is disrupted in mutants of the JA signaling pathway

As the growth conditions were quite different between plants intended for the wounding stress (4 week-old grown on soil) and those used for exogenous treatment with signaling molecules (10 day-old grown on ½ MS medium), I decided to test mutants of biosynthesis or perception of our candidate molecules even though they were not able to activate MPK2 in vitro. The aim was to identify which of our candidate molecules could act upstream the MKK3-MPK2 module in its activation by wounding. Concerning H 2 O 2 , I used the rbohd rbohf double mutant impaired in two members of Arabidopsis NADPH oxidase family, which is responsible for the production of O 2 -(superoxide) and H 2 O 2 (hydrogen peroxide) at the plasma membrane level. RBOHD, the most highly expressed RBOH in plants was shown to be involved in the control of cell death upon both biotic and abiotic stresses (Torres et al., 2005). Most importantly rbohd plants are impaired in wound-induced ROS burst. RBOHF is known to work redundantly with RBOHD as the rbohd rbohf double mutant shows stronger responses than the single rbohd in defense responses towards pathogens (Torres et al., 2005). However, NADPH oxidases are not the only producers of ROS in plant cells [START_REF] Minibayeva | Wound-induced apoplastic peroxidase activities: Their roles in the production and detoxification of reactive oxygen species[END_REF]. Unfortunately, results were highly heterogeneous (figure 2.29A) and do not suggest a major role for RBOHD/F upstream wounding-induced activation of MPK2. In order to test if ATP is an intermediate signal between the wounding and the activation of our MAPK module, dorn1-1 plants mutated in the recently identified DORN1 receptor were used [START_REF] Choi | Identification of a plant receptor for extracellular ATP[END_REF]. MPK2 activation by wounding was unchanged in dorn1 plants (figure 2.29B). Similarly, wak2-1 and wak2-2 plants unable to respond to oligogalacturonides (OGs) were used [START_REF] Brutus | A domain swap approach reveals a role of the plant wall-associated kinase 1 (WAK1) as a receptor of oligogalacturonides[END_REF]. No difference could be observed between those mutants and Col-0 plants in the activation of MPK2 by wounding (figure

2.29C).

ABA is an important phytohormone that was found produced in potato and tomato plants after wounding (Peña-Cortés et al., 1995). We already know that MPK2 is activated by an exogenous treatment with ABA (Danquah et al., 2015). Under the assumption that wounding-induced ABA production is conserved in Arabidopsis I used two independent lines mutated in the HAB1 PP2C gene [START_REF] Robert | A hypermorphic mutation in the protein phosphatase 2C HAB1 strongly affects ABA signaling in Arabidopsis[END_REF] that are known to be ABA-insensitive.

No striking difference was observed between Col-0 and hab1 plants when testing the activation of MPK2 by wounding (figure 2.30A). In addition, several plant lines impaired in genes of JA biosynthesis pathway or perception system were used.

MKK3-MPK2 is not activated in systemic leaves

As described in the introduction part, wounding induces local and systemic responses in the plant. JA has been shown to be an important element involved in the transmission of the stress signal from the wounded site to a restricted area of distal leaves [START_REF] Glauser | Velocity estimates for signal propagation leading to systemic jasmonic acid Characterization of a MAPK module involved in Arabidopsis response to wounding 113 accumulation in wounded Arabidopsis[END_REF].

For example, when a single leaf (n°8) is wounded, JA levels increase in this leaf but also in a distal leaf (n°13). However it accumulates at a far lesser extent in leaf n°9, which can then be used as a negative control. In the same way, the JAZ10 gene, a well-known marker of JA, is induced in the wounded leaf n°8 as well as in the distal leaf n°13 but not in the distal leaf n°9 [START_REF] Mousavi | GLUTAMATE RECEPTOR-LIKE genes mediate leaf-to-leaf wound signalling[END_REF]. To note, the level of JAZ10 expression in leaf n°13 is similar to the one observed in the leaf n°8. As JA seems to have an important role upstream our MKK3dependent MAPK module, I wondered if a JA-dependent systemic activation of this module could occur upon wounding. Leaf n°8 of Col-0 and mkk3-1 plants was wounded and MPK2 activity was tested in leaves n°8 (local), n°9 (negative control) and n°13 (systemic). As usual, MPK2 was activated in the wounded leaf n°8 in Col-0 but not in mkk3-1 plants (figure 2.34A). As expected, no activation was observed in the negative control leaf n°9. Sadly MPK2 was not activated in the distal leaf n°13 where the JA signal is supposed to be transmitted. This experiment was performed three times increasing the percentage of wounded leaf surface each time (40%, 50% and then 100%) but no activation of MPK2 could be observed in the distal leaf n°13. It was important to check that JA indeed accumulated in the distal leaf n°13 in our hands. Therefore, I studied the expression of the JAZ10 marker in leaves n°8, 9 and 13 upon wounding of leaf n°8. Leaves were harvested 1 hour after wounding leaf n°8 and JAZ10 expression levels were compared to leaves harvested on nonwounded plants. In Col-0 plants, JAZ10 expression was highly increased in leaf n°8 after wounding (figure 2.34B). However no increase was noticed in leaf n°13. I decided to also add the mkk3-1 line to see if the expression of this JA marker could be dependent on MKK3. As Intriguingly, JAZ10 was importantly more expressed in leaves n°13 of mkk3-1 plants after wounding. This observation suggests a negative control of JAZ10 expression by MKK3 in distal leaves and thus a negative regulation of JA-dependent responses. Moreover, as JAZ10 is a marker of JA signaling, this result could also suggest a negative regulation of JA signaling in distal leaves by MKK3. Wondering if the two waves of MAPK activation observed upon wounding were also occurring in response to JA, we tested the activation of MPK3 and MPK6. According to Takahashi and coworkers (Takahashi et al., 2007), MPK6 is activated by JA and this activation is impaired in mkk3-1 plants. However no mock control was shown to ensure this JA-induced activation of MPK6, which was necessary since MPK6 is activated by many stresses, including touch. In our conditions, adding a mock control as well as a positive control (flg22), neither MPK6 nor MPK3 was activated by JA.

Discussion

In addition, JA was able to trigger the expression of several candidate MAP3Ks.

However, their expression pattern was quite different from what was observed after wounding and their expression levels were also quite low. These differences can easily be explained by the difference of plants developmental stage at the moment when the stress is applied as well as their growth conditions (4 week-old plants grown on soil for wounding and 10 day-old plants grown on ½MS for JA treatment). Besides, the concentration of externally applied JA is 5 times higher than the physiological concentrations measured after wounding (up to 10 nmol/g FW, corresponding roughly to 10 µM) [START_REF] Glauser | Spatial and temporal dynamics of jasmonate synthesis and accumulation in Arabidopsis in response to wounding[END_REF]. To be able to compare expression patterns of MAP3Ks between plants treated with JA and wounded plants it would be wiser to apply JA by spraying it on 4 week-old plants grown on soil in the same conditions as the plants aimed for wounding.

b. Wounding-induced activation of MKK3 -dependent modules is largel y dependent on JA Application of exogenous molecules was tested independently from wounding.

However, the best way to identify signaling elements acting upstream of an MKK3-dependent module was to use plants impaired in biosynthesis or perception of candidate molecules.

Wounding-induced activation of MPK2 was impaired only in plants unable to synthesize or to perceive JA (opr3,. In addition, some MAP3Ks expression induced by wounding was impaired in plants not perceiving JA correctly (coi1-16 and coi1-34). The role of JA in the activation of MKK3-MPK2 by wounding seems to be preeminent and probably goes through the transcriptional regulation of MAP3K14, 15, 17 and 18 genes (figure 2.35).

Many signaling molecules are produced upon wounding and it is possible that several participate to MAPK activation by wounding but because of high redundancy between signaling pathways, the study of mutants impaired in only one of them do not show significant results. That is one explanation for the absence of MPK2 activity decrease when other signaling molecules mutants were used (dorn1, wak2 …) and the rather strong but not total decrease of MPK2 activity and MAP3Ks induction observed in coi1 plants. To address this issue it would be necessary to have a multiple mutant unable to produce most of those second messengers. However it is to note that none of the known DAMPs in Arabidopsis could activate MKK3 modules when applied exogenously.

IV. Activation of an MKK3-dependent module by cell walldamaging pathogens

Wounding is an important signal triggered by cell wall-damaging pathogens such as necrotrophic fungi and herbivorous insects. The results presented in the previous part showed that JA has an important role upstream the activation of MKK3-C-group MAPKs module in

B

the context of wounding. Interestingly, in Arabidopsis thaliana, JA has been shown to be an important mediator of defense responses against necrotrophic pathogens as well as herbivores.

Indeed, plants mutated in COI1 are more sensitive to an inoculation with spores of the necrotroph B. cinerea [START_REF] Thomma | Separate jasmonate-dependent and salicylatedependent defense-response pathways in Arabidopsis are essential for resistance to distinct microbial pathogens[END_REF]. The importance of the jasmonate pathway in resistance towards generalist herbivores was also shown using coi1 plants [START_REF] Reymond | A conserved transcript pattern in response to a specialist and a generalist herbivore[END_REF]. Therefore, we wondered if our MKK3-dependent module would be activated by pathogens causing wounding of the leaves. We chose to work with Botrytis cinerea as a representative of necrotrophic fungi, which has commonly been used in our institute and Spodoptera littoralis as a representative of herbivorous insects, in the frame of collaboration with Axel Mithofer (MPI Jena, Germany).

Botrytis cinerea activates an MKK3-dependent module in Arabidopsis thaliana

If mechanical wounding is partly mimicking the effect of necrotrophic fungi, it is possible that the MKK3-MPK2 module is also activated when plants are challenged with Botrytis cinerea. In their work, Chassot and coworkers [START_REF] Chassot | Wounding of Arabidopsis leaves causes a powerful but transient protection against Botrytis infection[END_REF] Previous works showed that wounding of Arabidopsis leaves causes a transient resistance against the necrotrophic fungus Botrytis cinerea [START_REF] Chassot | Wounding of Arabidopsis leaves causes a powerful but transient protection against Botrytis infection[END_REF]

Spodoptera littoralis activates an MKK3-dependent module in Arabidopsis thaliana

In the same way, we wondered if MKK3-MPK2 could be activated when plants were challenged with an herbivorous insect. For this purpose, we collaborated with Axel Mithöfer (Max Planck Institute in Jena, Germany). He kindly performed some insect feeding assays on

Col-0, mkk3-1 and mkk3/MKK3locus-YFP plants. Larvae are left to starve for some time and are then put on leaves to feed for 1 hour or 3 hours. Larvae and leaves which have been chewed are then collected to be weighed or used to characterize plant responses respectively (see Scholz et al., 2012;[START_REF] Vadassery | CML42-mediated calcium signaling coordinates responses to Spodoptera herbivory and abiotic stresses in Arabidopsis[END_REF]. After submitting plants to the generalist S. (pictures from Williamson et al., 2007) replicates to test MPK2 activation and found it activated in Col-0 plants 1 hour after the feeding start (figure 2.38). This activity was lost in mkk3-1 plants and restored in the complemented line, therefore showing that MPK2 activation by S. littoralis is dependent on MKK3. To complete the module it is necessary to study the expression of MAP3Ks upon S. littoralis feeding and test the corresponding mutant lines in their ability to still activate MKK3-MPK2 as it was done in the case of mechanical wounding.

Discussion

In this part I showed the activation of the MKK3-MPK2 module by two cell walldamaging pathogens: Botrytis cinerea and Spodoptera littoralis. MPK2 is activated 48 hours after inoculation with Botrytis spores in an MKK3-dependent way. Making a parallel with the wounding-induced activation of MKK3-MPK2, such a late activation is probably due to the time going on between the inoculation and the beginning of tissue maceration causing the actual cell damage. Notably, it is actually difficult to know exactly how much time after the tissue lesion our module is activated. This also may explain the difference observed in MAP3Ks induction between wounding and fungal inoculation. Indeed, only MAP3K19 was found strongly induced 48 hours post-inoculation (although the analysis was performed only once). Another explanation is that obviously the two stresses are not exactly the same and despite triggering similar cellular events, the response specificity of each could be reflected at the transcriptional level. That observation is also consistent with our model (see figure 2.4) according to which different stresses can trigger the MKK3-MPK2 module through the transcriptional regulation of different MAP3Ks. MPK2 was less active when the inoculation was preceded by wounding which is known to cause a protection against the fungus and therefore weaker lesions caused by the latter.

MPK2 was also activated in an MKK3-dependent way by the generalist herbivore

Spodoptera littoralis 1 hour after the feeding start. Interestingly the peak of MPK2 activation observed at the 1h time-point is consistent with the one observed in a wounding context.

Obviously, more time-points would be necessary to conclude about a real consistency regarding the kinetics of activation.

In addition to degrading the cell wall, these two pathogens trigger JA production in the plant cell, which mediates defense responses (figure 2.39). It could be interesting to check

Experiment n°

Appearence of first lesions

Effect of mkk3-1 on resistance to B. cinerea

Effect of mkk3-1 on wounding-induced resistance

Significance

Behavior of complemented line

1 48h YES (mkk3-1 more resistant) NO YES X 2 48h NO NO NO X 3 72h YES (mkk3-1 more resistant) NO YES different from Col-0 4 72h NO X NO X

Table 2.3: Summary of results obtained with B. cinerea inoculations

This table summarizes the results obtained concerning an effect of the mkk3-1 mutation on the resistance of plants to B. cinerea. The resistance was based on the size of outgrowing lesions and was tested in the context of plants wounded or not prior to the inoculation.

whether JA signaling has an important role upstream our MKK3-dependent module, as in the context of "lab-performed" mechanical wounding, using coi1 plants.

V. Study of the role of an MKK3-dependent module in woundingrelated responses

After showing that an MKK3-dependent module is strongly activated by mechanical wounding and cell wall-damaging pathogens, we assumed that this central element has an important role in the transduction of wounding-induced defense responses. As MKK3 acts as a "hub" upstream C-group MAPKs, plants knocked-out in MKK3 constitute a powerful tool to study the role of the MKK3-dependent module in the context of wounding. Therefore, we phenotyped mkk3-1 mutant plants and compared them to Col-0 plants on several levels, focusing particularly on its role in the context of an interaction with cell-wall damaging pathogens. the more important as the fungus is applied shortly after the wounding [START_REF] Chassot | Wounding of Arabidopsis leaves causes a powerful but transient protection against Botrytis infection[END_REF].

Plants impaired in MKK3 signaling do not show clear detectable phenotype upon Botrytis infection

Nevertheless, this acquired resistance could still be observed up to 48 hours after the wounding. I first wanted to confirm the priming effect of wounding on B. cinerea First of all, I was able to show that the chosen genes were indeed up-regulated in the presence of B. cinerea, from 24 hours after the inoculation, increasing even more at 48 hours. Some of them were apparently misregulated in mkk3-1 plants. But this experiment having been performed only once, it is difficult to draw any conclusion. Nevertheless, it could be interesting to go further in this direction once the experiment is correctly set up to reduce variability, as well as including other genetic material (complemented line, mkk3-2 and map3k19 mutants).

development

Plants impaired in MKK3 signaling do not properly respond to herbivorous insects a. Resistance towards Spodoptera littoralis

Since MKK3-MPK2 is activated upon S. littoralis feeding, it was interesting to look for a role for MKK3 in the resistance towards this generalist herbivore. All insect resistance assays were performed by Axel Mithöfer, in his laboratory in Jena. His feeding assays were performed on Col-0 and mkk3-1 plants and larvae were collected and weighed 8 days after the feeding start. The sensitivity of the plant is reflected by larvae weights at the end of the feeding period. Indeed, the more the plant is sensitive the more the larva will feed on it and become heavy. Three biologically independent experiments were performed with around 25 measurements for each genotype and each replicate. Only one out of the three showed a statistical difference between larvae grown on Col-0 and mkk3-1 plants (figure 2.42). The difference is suggesting a higher sensitivity for mkk3-1 plants and would consequently suggest that MKK3 is a positive regulator of plant defenses towards S. littoralis.

Unfortunately, this trend is not strong enough to be significant in all replicates. The same experiment was repeated later including the mkk3/MKK3locus-YFP line but unfortunately no differences could be observed (not shown). I therefore conclude that MKK3 is not involved in resistance towards S. littoralis. Another possibility is that the phenotype is too weak and requires a higher number of replicates to be detected out of the experimental variability.

b. Role of MKK3 in the regulation of phytohormone levels upon wounding and Spodoptera littoralis feeding After performing insect feeding assays, Axel Mithöfer kindly offered to measure phytohormone levels in the different genetic backgrounds we provided him with. As mentioned earlier in the introduction, the few studies mentioning MAPKs in the context of mechanical wounding and herbivory suggested their role in the regulation of phytohormone production [START_REF] Seo | The mitogen-activated protein kinases WIPK and SIPK regulate the levels of jasmonic and salicylic acids in wounded tobacco plants[END_REF][START_REF] Heinrich | Two mitogen-activated protein kinase kinases, MKK1 and MEK2, are involved in wounding-and specialist lepidopteran herbivore Manduca sexta-induced responses in Nicotiana attenuata[END_REF][START_REF] Li | AIK1, A Mitogen-Activated Protein Kinase, Modulates Abscisic Acid Responses through the MKK5-MPK6 Kinase Cascade[END_REF]. Therefore, our module being activated upon insect feeding, we could also expect its role in the regulation of phytohormone production. Col-0, mkk3-1 and mkk3/MKK3locus-YFP plants were submitted to insect feeding and leaves were harvested, as before, 1 hour and 3 hours after the feeding start. The experiment was performed on four biologically independent samples. Levels of JA, JA-Ile, SA and ABA are shown in figure 2.43. As expected, JA and JA-Ile levels were increased by the insect challenge. We measured a peak at 1 hour (among the tested timepoints) decreasing back at 3 hours to a level that was still higher than at resting conditions.

Interestingly these levels are increased in mkk3-1 plants 1 hour and 3 hours after the feeding start. The complemented line behaves like the Col-0, thus confirming that the increase observed in mkk3-1 lines is indeed due to the mutation. The result was even more interesting concerning SA levels. No increase of SA could be observed in Col-0 and mkk3/MKK3locus-YFP plants whereas a high production of SA occurred in mkk3-1 plants. The peak was observed at 1 hour after the feeding start, as for JA. ABA levels were unchanged in all genotypes. These data suggest that MKK3 is involved in the negative regulation of JA, JA-Ile and SA levels upon S. littoralis feeding. In the case of SA, MKK3 is even totally inhibiting the production of SA upon insect feeding. MKK3-MPK2 being activated 1h after the feeding start, and probably already earlier, a role for MKK3 in the regulation of SA and JA levels is not absurd.

After these results, we wondered if the same effect of MKK3 on JA and SA production could be observed in response to mechanical wounding. Col-0, mkk3-1 and mkk3/MKK3locus-YFP plants were wounded with a forceps and wounded leaves were collected 1 hour and 3 hours later. Samples from three biologically independent replicates were sent to Axel Mithöfer's laboratory for phytohormone measurements. Results are shown on figure 2.44. As observed after the insect attack, JA and JA-Ile levels were found increased upon wounding of Col-0 plants, peaking at 1 hour post-wounding. A slight increase of ABA levels could also be observed upon wounding. SA levels did not vary. Because mkk3-1 plants behaved like Col-0 plants, I concluded that MKK3 is not involved in hormonal regulation upon wounding. That would mean that the MKK3-dependent variations observed when plants were submitted to S.littoralis are specific of the insect, independently of wounding. Indeed, in addition to the mechanical wounding resulting from the insect feeding on the leaf, some chemical substances from the insect saliva also come into play. Therefore, it would be interesting to perform an experiment where insect oral secretions are applied to previously wounded leaves and check if the hormonal phenotype is restored. Another explanation is that the mechanistic of wounding performed with forceps is quite different from the one of insectinduced wounds. Indeed, Mithöfer and his collaborators showed that S. littoralis has a specific feeding pattern [START_REF] Mithöfer | Effects of feeding Spodoptera littoralis on lima bean leaves. II. Continuous mechanical wounding resembling insect feeding is sufficient to elicit herbivory-related volatile emission[END_REF]. To be able to reproduce it, they developed a robot named "Mecworm" that is able to mimic the strength and the frequency of S. littoralis feeding A: Expression levels of SA biosynthetis genes PAL1, PAL2, PAL4 and SID2

B: Expression levels of JA biosynthesis genes LOX2, LOX3, LOX4, LOX6 and AOS

A B

on lima bean leaves. We could also submit our different genotypes to that robot. Finally, a third hypothesis which could explain the observed differences between wounding and insect feeding is the likely difference in plant growth conditions between our two laboratories. At this moment, samples are in progress of preparation to test those different hypotheses and the results might be presented during the defense.

c. Role of MKK3 module in the regulation of SA -and JA-related genes upon insect feeding

In tobacco, MAPKs were shown to regulate JA levels after wounding by regulating the activity of enzymes involved in its biosynthesis [START_REF] Kallenbach | Nicotiana attenuata SIPK, WIPK, NPR1 and Fatty Acid-Amino Acid Conjugates Participate in the Induction of Jasmonic Acid Biosynthesis by Affecting Early Enzymatic Steps in the Pathway[END_REF] and in wounded Arabidopsis, ethylene levels were shown to be regulated by some MAPKs through the transcriptional regulation of ACS genes [START_REF] Li | AIK1, A Mitogen-Activated Protein Kinase, Modulates Abscisic Acid Responses through the MKK5-MPK6 Kinase Cascade[END_REF]. Therefore, in order to have a deeper understanding of the role of MKK3 on the previously observed hormonal regulations, I decided to focus on the enzymes involved in their biosynthesis. Since the tools required to measure the activity of enzymes involved in SA and JA biosynthesis pathways were not available in our vicinity, I decided to analyze the expression levels of genes encoding these enzymes. The idea was to show that the negative regulation of SA and JA levels by MKK3 could occur by the negative regulation of genes involved in their biosynthesis. Some genes were selected and their expression was analyzed by RT-qPCR with specific primers.

Concerning SA, two synthesis pathways are known. One of them allows the production of SA from its precursor chorismate. The main enzyme involved in that pathway is isochorismate synthase (ISC), which is encoded by two genes in Arabidopsis (ICS1 and ICS2). ics1 ics2 double mutant showed around 96% of SA reduction after UV treatment, suggesting the existence of an alternative pathway [START_REF] Garcion | Characterization and biological function of the ISOCHORISMATE SYNTHASE2 gene of Arabidopsis[END_REF]. The alternative pathway leads to the synthesis of SA from cinnamate via the action of phenylalanine ammonia lyase (PAL). In Arabidopsis 4 PAL isoenzymes are found (PAL1, 2, 3 and 4). Chemical inhibition of PAL genes in Arabidopsis led to a reduced pathogen-induced SA accumulation [START_REF] Mauch-Mani | Production of Salicylic Acid Precursors Is a Major Function of Phenylalanine Ammonia-Lyase in the Resistance of Arabidopsis to Peronospora parasitica[END_REF]. Two other genes, PBS3 and EPS1, operating downstream of PALs or ICS1, have been shown to play an important role in pathogen-induced SA accumulation [START_REF] Jagadeeswaran | Arabidopsis GH3-LIKE DEFENSE GENE 1 is required for accumulation of salicylic acid, activation of defense responses and resistance to Pseudomonas syringae[END_REF], Zheng et al., 2009). To this day, the exact way SA is produced in plants is not fully understood. Thus, based on these pieces of information, I decided to study the expression of PALs and ICS1 (also called SID2). PAL1 and PAL2 expression were found induced upon S. littoralis feeding on Col-0 plants (figure 2.45A). This observation is quite puzzling as no SA production was observed in Col-0 plants. In mkk3-1 plants, these two genes levels were higher than in Col-0 at time 0 but largely decreased after 1 and 3 hours. More disturbingly, mkk3/MKK3locus-YFP plants did not behave at all like Col-0 plants. PAL4 and SID2 were not induced by insect feeding in any of the three genotypes. Concerning JA, most of the genes participating in its synthesis have been characterized by molecular, genetic and biochemical approaches [START_REF] Kombrink | Chemical and genetic exploration of jasmonate biosynthesis and signaling paths[END_REF]. I decided to study the expression of genes operating at the early steps of JA synthesis: all four LOX genes and AOS. All tested genes except LOX6 were induced by S. littoralis feeding on Col-0 plants, following the same pattern as JA production (peaking at 1 hour and decreasing at 3 hours post-feeding). Their expression pattern was quite intriguing in mkk3-1 plants. LOX1, LOX2 and AOS expression was higher after 1 hour but lower after 3 hours, in comparison with Col-0 plants. LOX3 expression was lower in mkk3-1 plants at all time-points. In any case, mkk3/MKK3lcous-YFP plants never behaved like Col-0 plants (figure 2.45B).

In a second part I was interested in the study of SA-and JA-responsive genes. I chose some that were well described in the literature. JAZ10, VSP2, PDF1.2 are well-known markers of JA signaling and insect responses. PR1 is known to be highly induced upon pathogen challenge and SA accumulation. JAZ10 and VSP2 were indeed induced in Col-0 plants upon insect feeding (figure 2.46). Their expression peaked at 1 hour and decreased back at 3 hours post-feeding. PDF1.2 and PR1 were not induced by insect feeding on Col-0 plants. JAZ10 and VSP2 expression was decreased in mkk3-1 plants 1 and 3 hours postfeeding. On the contrary PR1 expression was induced in mkk3-1 plants. PR1 was actually the only gene for which a consistency was found with the previously observed SA levels.

Unfortunately, once again, the complemented line did not fully play its part.

All the qPCR analyses of this part have been performed only once. Additionally, Axel Mithöfer provided us with very few single leaves on which insects had fed. It is known that stress responses strongly depend on leaf age as well as rosette phylotaxy and the fact that the leaves provided by Axel for each genotype and time-point are not of the same age may induce a strong variability hiding a transcriptional phenotype. On another hand, it was confusing for us to note that the complemented line was not able to complement gene expression disturbances whereas it was nicely complementing hormonal deregulations observed in mkk3-1 plants upon insect feeding as well as MPK2 activity decrease observed in mkk3-1 plants upon wounding, B. cinerea and S. littoralis. The most direct conclusion would

B

be that the differences observed in genes expression in mkk3-1 plants are not due to the mutation of MKK3 but other variations in the genome. As this non-complementation of gene expressions was observed several times in other experiments when mkk3/MKK3locus-YFP plants were used, we favored the assumption that something could be wrong with this line.

We formulated the hypothesis that somehow the C-terminal YFP tag could be disturbing for gene regulations. Nevertheless this hypothesis brings another one: as MPK2 activity is restored in the complemented line, the potential effect of MKK3 on genes expression would be direct (MKK3 directly interacts with some transcription factors or gene promoters) rather than indirect (the regulation goes through the downstream MAPKs). We are currently in the process of creating untagged mkk3/MKK3locus-YFP lines.

Wounding-induced trichome formation

One of the documented JA-dependent wound responses is the formation of trichomes, specialized defense cells. Herbivore-or wound-induced damage on old leaves leads to an increased trichome density in newly forming leaves. This wounding-induced trichome formation is abolished in mutants of JA biosynthetic or signaling pathway [START_REF] Yoshida | Jasmonic acid control of GLABRA3 links inducible defense and trichome patterning in Arabidopsis[END_REF]. As the wounding-induced activation of our MKK3-dependent module depends also largely on JA signaling, I decided to investigate a putative role of MKK3 in trichome formation. For this purpose, I used the published protocol [START_REF] Poudel | Mutations in jasmonoyl-L-isoleucine-12-hydroxylases suppress multiple JA-dependent wound responses in Arabidopsis thaliana[END_REF] which consists in wounding specific leaves of Arabidopsis plants with intervals over the course of 12 days.

At the end, new formed leaves are observed under a stereomicroscope and trichomes are counted. In my case, two different tools were used for the wounding. On one hand, leaves were squeezed through their midvein using a forceps. On the other hand, leaves were punctured once on each side of the midvein with a needle. Wounding was performed on both cotyledons and the two first true leaves of 2 week-old Col-0 and mkk3-1 plants grown on soil.

Trichomes were observed on leaf n°5. Measurements were made regardless of the leaf surface which obviously created a bias since wounded plants had a retarded development and were thus far smaller than control plants at day 12. The result is shown on figure 2.47A. The wounding-induced trichome formation was not visible in our conditions: since wounded leaves were smaller they also had less trichome. However, although the trichome density was not measured, no increase was notable by eye (figure 2.47B).

Figure 2.48: Transcriptomic analysis of mkk3-1 plants submitted to wounding did not highlight any MKK3-regulated genes A: Pair comparisons used for the microarray. For comparisons among the same genotype, expression ratios of genes differentially regulated in wounded plants compared to non-wounded plants were calculated. For comparisons among the same condition, expression ratios of genes differentially regulated in mkk3-1 plants compared to Col-0 plants were calculated. Numbers of statistically up-and downregulated genes (with ratios ≥1 or ≤-1) for each comparison are highlighted in respectively red and green.

B:

The graph shows the average Log2 ratio of wounded Col-0 plants on non-wounded Col-0 plants compared to the average Log2 ratio of wounded mkk3-1 plants on non-wounded mkk3-1 plants . The correlation coefficient (R²=0,9659) reflects the absence of major differences between the two genotypes. Differentially expressed genes are highlighted in red.

ATG number Ratio

Copia-like retrotransposon family AT5G35935 -2,4636

MKK3 AT5G40440 -1,7031 BETA-AMYLASE 5 AT4G15210 -0,9606 CALMODULIN-LIKE 41 AT3G50770 -0,9525 RECEPTOR LIKE PROTEIN 23 AT2G32680 -0,9013 WRKY30 AT5G24110 -0,8715 GLYCINE RICH PROTEIN 9 AT2G05440 -0,8674
Table 2.4: Few MKK3-regulated genes were found

The table displays names, ATG numbers and expression ratios of genes that were found misregulated in mkk3-1 plants compared to Col-0 plants upon wounding. A positive ratio reflects a higher expression of the corresponding gene in mkk3-1 plants whereas a negative ratio indicates a lower expression.

Transcriptomic analysis of mkk3-1 plants submitted to wounding did not highlight any MKK3-regulated genes

An easy way to gain clues about the role of a protein is to identify misregulated genes in the corresponding mutant. Our laboratory has performed a microarray to compare Col-0 and mkk3-1 plants treated or not with ABA. The analysis had been performed on plants harvested 4 hours after the ABA treatment and thus after the peak of MPK2 activity (Danquah et al., 2015). In the case of wounding, the peak of MPK2 activity is observed at 1 hour postwounding. Thus we decided to carry out the microarray on samples harvested 2 hours postwounding. Non-wounded samples were also used as controls. Col-0 and mkk3-1 plants were compared as shown in figure 2.48A. Each condition was tested three times from biologically independent samples. Many genes were regulated by wounding and with rather high log change values (figure 2.48B). For example, 2347 and 1929 genes were found to be statistically up-and down-regulated respectively in Col-0 plants. On the other hand, we were very astonished when we realized that Col-0 and plants impaired in MKK3 virtually do not show any strong differences in term of gene expression. Only a handful of genes were found statistically deregulated when wounded Col-0 and wounded mkk3-1 samples were compared, including XTR3, QQS and SDE3 which are up-regulated in mkk3-1 and BAM5, CL41 or RLP23 that are down-regulated in mkk3-1 (figure 2.48B (in red) and table 2.4). None of the well-known wound-induced genes were found. In conclusion, according to statistical analysis, the wounding stress was indeed reflected at the transcriptional level but no role for MKK3 was highlighted.

It is surprising that this approach did not identify "interesting" genes whose expression could depend on MKK3 upon wounding. Several explanations are possible. First of all, it is possible that the chosen time-point, 2 hours after wounding, is not very relevant.

As no MKK3-dependent wounding-induced (or JA-induced) genes were described in the literature, we had no reference beside the microarray performed in an ABA context. Another possibility is that the wounding I applied, which was very strong, triggered redundant signaling pathways, rending MKK3 module not necessary. This is supported by the fact that the number of wound-regulated genes we identified is four times higher than other published transcriptomic studies [START_REF] Kilian | The AtGenExpress global stress expression data set: Protocols, evaluation and model data analysis of UV-B light, drought and cold stress responses[END_REF]. Last, it is possible that MKK3 pathway does not regulate gene reprogramming but other cellular processes such as enzymatic activities involved in wounding responses. It would be interesting to test this hypothesis by performing

Discussion

In this part were presented phenotyping experiments I performed to identify the potential roles of MKK3 in stress responses. I was not able to show any important role for MKK3 in the resistance towards B. cinerea and S. littoralis, the two tested pathogens, which are known to largely induce wounding-related stresses. However, I showed that MKK3 might play a role in phytohormone regulations upon S. littoralis feeding by negatively regulating JA and SA levels. Interestingly this hormonal regulation defect has not been observed using mechanical wounding, suggesting a role for MKK3 specifically under herbivore-induced wounds. A possibility is that insect saliva contains molecules necessary for the complete hormonal response or, alternatively, that wounding performed in the laboratory with forceps does not mimic the insect-induced wounding. Such hypothesis is currently under investigation in the laboratory of our collaborator Axel Mithöfer. Interestingly, JA, which seems to be negatively regulated by MKK3-dependent modules based on mkk3-1 phenotype upon insect interaction, is also a potent activator of the module. This suggests a negative feedback loop in which MKK3 module acts to restrain the duration and the amplitude of the downstream defense-responses it triggers (figure 2.49). Surprisingly, mkk3-1 plants produced SA upon insect feeding whereas Col-0 plants did not. This suggests that one of MKK3 functions is to inhibit SA production upon stresses.

The way MKK3 exerts a control on hormonal levels remains to be determined. A possible regulation at the transcriptional level of enzymes involved in their biosynthesis was briefly tested but would need to be pushed further. The variation of hormone levels upon insect feeding was actually the only phenotype for mkk3-1 plants that we obtained. Based on our knowledge on the SA/JA antagonism that SA inhibits JA-induced responses at the transcriptional level, we can expect that JA-dependent wounding-induced (or herbivoreinduced) defense responses are reduced in mkk3-1 plants which, unlike Col-0 plants, produce a high amount of SA upon insect feeding. One study assesses the impact of SA and JA on the resistance of Arabidopsis to S. littoralis [START_REF] Stotz | Evidence for regulation of resistance in Arabidopsis to Egyptian cotton worm by salicylic and jasmonic acid signaling pathways[END_REF] using coi1 and npr1 mutants. The authors showed that SA and JA have opposite effects on plant resistance as, compared to wild type plants, coi1 plants showed enhanced susceptibility to larvae whereas npr1 plants were more resistant. The fact that npr1 plants, which are impaired in the perception of SA, show an increased resistance to the insect suggests that SA is engaged and mediates susceptibility towards S. littoralis. The authors also claim to have measured SA production upon S. littoralis but do not show any data regarding that point. The authors also tested the relationship between the two hormones. Exogenous application of SA was able to reduce JA amounts induced by the insect. We could not draw any conclusion from our resistance study on the role of MKK3. However among the three independent experiments, the one showing a significant difference between Col-0 and mkk3-1 plants highlighted a higher susceptibility of mkk3-1 plants. If such a difference was to be confirmed by the increase of experimental repetitions, it would be consistent with the pattern of phytohormone production. Indeed, the SA produced in mkk3-1 plants would negatively regulate the JA-mediated resistance towards the insect compared to Col-0 plants and therefore decrease plant resistance.

Intriguingly, the regulation of phytohormone levels was not reflected by the large microarray analysis carried out on mkk3-1 mutant. We could have expected differential expression of JA-and SA-related genes. This discrepancy supports the hypothesis that MKK3 rather acts at the post-translational level by regulating the activity or the localization of its targets.

Wounding-induced trichome formation was tested as one of the many JA-induced responses without any conclusive outcome. Many others could still be tested such as the production of metabolic and volatile compounds or growth inhibition. Concerning the latter, a study showed a role for MKK3 in the JA-induced root growth inhibition (Takahashi et al., 2007). Since wounding also leads to root growth inhibition through the production of JA (Gasperini et al., 2015), it would be interesting to monitor the growth of mkk3-1 roots upon wounding.

CHAPTER III: CONCLUSIONS & PERSPECTIVES

I. A MAPK signaling network activated in response to wounding

Toward a sequential and interdependent activation of two MAPK modules

Before I started my PhD project, our knowledge about MAPK involvement in response to wounding in plants was reduced and was mainly concerning A-group MAPKs. In tobacco, WIPK and SIPK, orthologs of Arabidopsis MPK3 and MPK6 respectively, were

shown to be rapidly activated by wounding. WIPK was shown to regulate JA levels as transgenic plants in which WIPK (homologue of AtMPK3) expression was suppressed were not able to accumulate JA upon wounding (Seo et al., 1999). In addition, a constitutive expression of the gene led to higher levels of JA compared to wild-type plants (Seo et al., 1999). Silencing of SIPK (homologue of AtMPK6) also led to decreased JA levels [START_REF] Seo | The mitogen-activated protein kinases WIPK and SIPK regulate the levels of jasmonic and salicylic acids in wounded tobacco plants[END_REF]. NaMEK2, identified as the MAP2K acting upstream of WIPK and SIPK upon wounding, was also shown to regulate JA and ET levels [START_REF] Heinrich | Two mitogen-activated protein kinase kinases, MKK1 and MEK2, are involved in wounding-and specialist lepidopteran herbivore Manduca sexta-induced responses in Nicotiana attenuata[END_REF]. Very similarly in tomato (Solanum lycopersicum), LeMPK1 and LeMPK2, which are orthologs of SIPK, were activated by herbivory and their silencing led to the reduction of JA levels and the expression of JA biosynthesis genes [START_REF] Kandoth | Tomato MAPKs LeMPK1, LeMPK2, and LeMPK3 function in the systeminmediated defense response against herbivorous insects[END_REF]. These observations suggest that in Arabidopsis, MAPKs activated by wounding could also be involved in the regulation of JA levels. In Arabidopsis, the classical MPK3 and MPK6 are also rapidly activated by wounding [START_REF] Ichimura | Various abiotic stresses rapidly activate Arabidopsis MAP kinases ATMPK4 and ATMPK6[END_REF][START_REF] Li | AIK1, A Mitogen-Activated Protein Kinase, Modulates Abscisic Acid Responses through the MKK5-MPK6 Kinase Cascade[END_REF]. Beside these iconic stress-activated MAPKs, another study has shown the activation of the C-group MAPKs MPK1 and MPK2 by wounding (Ortiz-Masia et al., 2007), which has been the starting point of my work.

The results I obtained throughout this work gave a more precise view of MAPK cascades in a wounding context. I was able to show that two apparently independent modules are activated with distinct kinetics. First, the well-known MPK3 and MPK6 are activated few minutes after wounding and their activity fades after 30 minutes and I showed that their activation is not dependent on MKK3. Besides MPK3/MPK6, I was able to show that MKK3 and C-group MAPKs (MPK1, 2, 7 and 14) form a functional module activated by wounding. My experiments also suggest that their activation, which occurs quite belatedly, from 30 minutes post-wounding and lasts a couple of hours, is likely dependent on the transcriptional regulation of sub-clade III MAP3Ks and partially under the control of JA. This observation of two waves of MAPK activation is intriguing and raises the question of whether they are dependent on each other. In mpk3 and mpk6 mutants, I was not able to see any decrease of wounding-induced activation of MPK2 but a functional redundancy between MPK3 and MPK6, which has been shown in other contexts, cannot be excluded. Sadly, available mpk3 mpk6 double mutants are lethal because of a redundant role in development [START_REF] Wang | Stomatal Development and Patterning Are Regulated by Environmentally Responsive Mitogen-Activated Protein Kinases in Arabidopsis[END_REF]. A recent study showed that MPK3/MPK6 activation by wounding is impaired in the mkk4 mkk5 double mutant [START_REF] Li | AIK1, A Mitogen-Activated Protein Kinase, Modulates Abscisic Acid Responses through the MKK5-MPK6 Kinase Cascade[END_REF]

Spatial organization of MAPK activation upon wounding

As stated in the introduction, wounding triggers many cellular events and is therefore very complex to study. An important aspect which deserves some attention is the spatial organization of those events. It is obvious that there is a strong gradient of responses between the cells which have been physically wounded -and are meant to die-and the ones surrounding this central area, which are likely to adapt and survive. Closest cells to the wound site feel the wounding through mechanical repercussion whereas distant cells, sometimes located in other tissues or organs, are alerted by electrical or chemical signals. Neighboring plants may in some extend also react without being injured themselves. This theoretical problematic is complex to tackle but very important if we want to understand the coordination of stress signaling and describe local responses. One possibility is that the MKK4/5-MPK3/6 module is activated very locally at the wounded side and that a systemic signal, JA for instance, allows the activation of MKK3-MPK1/2/7/14 in a surrounding area. In this condition, finding a phenotype controlled by MKK3-related modules would become more complicated without a first characterization of the spatial distribution of MAPK modules.

Smart fluorescent sensors have been developed in animals [START_REF] Harvey | A genetically encoded fluorescent sensor of ERK activity[END_REF][START_REF] Lindenburg | Engineering genetically encoded FRET sensors[END_REF][START_REF] De La Cova | A Real-Time Biosensor for ERK Activity Reveals Signaling Dynamics during C. elegans Cell Fate Specification[END_REF] but will deserve some adaptation for plant system (with the problem of MAPK specificity). An alternative for MKK3-related modules will consist in studying the spatial expression of MAP3Ks. We showed that sub-clade III MAP3Ks are strongly transcriptionally regulated in time and we may assume that they also are in space.

Strikingly a preliminary experiment showed that it might be complex: MAP3K18 is expressed specifically in guard cells upon ABA treatment whereas it is expressed in epidermal cells upon wounding. Promoter::GUS lines would be more informative at the tissue and organ levels. Overall, this suggests that it would be useful to resolve the spatial expression of each sub-clade III MAP3Ks in response to stresses upon which they are found induced.

II. A novel MKK3-dependent module activated by environmental constraints through transcriptional regulation of MAP3K genes

1. An emerging general working model…

In the literature, not much was known about the involvement of MKK3/C-group modules in stress responses and even less in the context of wounding and herbivory. Some studies have shown the activation of C-group MAPKs by wounding and JA (Ortiz-Masia et al., 2007) and some even placed MKK3 upstream them in response to H 2 O 2 , pathogens or ABA (Dóczi et al., 2007;Danquah et al., 2015). Studies previously carried out by Agyemang Danquah, Marie Boudsocq and Axel de Zelicourt in the "Stress Signaling" group unveiled an atypical mode of activation of MKK3-dependent modules in response to ABA, occurring through an important step of transcriptional regulation of upstream MAP3K genes. This protein synthesis step was responsible for a delay in the activation of the module (Boudsocq et al., 2015;Danquah et al., 2015). This finding was breaking our idea according to which MAPKs are rapidly activated upon stress perception. My work, as well as the work of Camille A: Col-0 and mkk3-1 plants expressing MPK7-HA were supplied with NO 3 after 3 days of starvation. MPK7 activity was assayed over a time course following resupply. KCl was used as a control.

B: Col-0 and nlp7 plants expressing MPK7-HAwere supplied with NO 3 , MPK7 activity was assayed 15 minutes after resupply. ) and not by ammonium or glutamine, and this activation was strictly dependent on MKK3 and partially dependent on NLP7 (which belong to a multigenic family). The laboratory is currently testing the role of MAP3K13 and 14 upstream of MKK3-MPK7.

Interestingly, MAP3K13 has also been found recently in a transcriptomic experiment among target genes of NLP8, a very close homologue of NLP7, which is involved in the nitratedependent control of seed dormancy [START_REF] Yan | NIN-like protein 8 is a master regulator of nitrate-promoted seed germination in Arabidopsis[END_REF].

I was able to show that all sub-clade III MAP3Ks interact with MKK3 and activate MKK3-MPK2 in mesophyll protoplasts. Based on the observations made in the context of ABA and nitrate and on Genevestigator data, we realized that sub-clade III MAP3Ks are generally transcriptionally regulated [START_REF] Colcombet | Convergence of Multiple MAP3Ks on MKK3 Identifies a Set of Novel Stress MAPK Modules[END_REF]. A recent study showed that Arabidopsis lines over-expressing MAP3K14,15,16,17 or 18 share a same phenotype of insensitivity to ABA during seed germination (Choi et al., 2017), indicating a functional redundancy among them. Besides, such a result also suggests that strategies of overexpression can lead to wrong conclusions. Overall, these studies reinforced our model according to which transcriptional regulation is a conserved feature of sub-clade III MAP3Ks

and that different association of the latter are induced by different stresses.

…coexisting with other models?

Several studies showed the involvement of MKK3 in module with others MAPKs. MAPKs functioning in alternative modules. For example, MKK3 was shown to act upstream MPK6 in response to JA (Takahashi et al., 2007) and to blue light (Sethi et al., 2014).

Another study placed MKK3 upstream of MPK8 in response to wounding to control ROS homeostasis (Takahashi et al., 2011). I showed the regulation of MKK3 by JA but was unable to show any JA-induced regulation of MPK6. Furthermore, the laboratory has not been able to show that MPK6 is under the control of MKK3. Indeed, expression of constitutive MKK3 (MKK3EE) in protoplasts does not activate MPK6 or MPK8 (Danquah et al., 2015). It is important also to note that authors often did not include careful controls and therefore the observed MPK6 activation by JA could be due to mechanical stresses related to manipulation.

At last, none of those studies could show a direct interaction of MKK3 with MPK6 or MPK8, whereas MKK3 is able to interact with C-group MAPKs and MPK6 with MKK4/5 in Y2H assays [START_REF] Schweighofer | The PP2C-type phosphatase AP2C1, which negatively regulates MPK4 and MPK6, modulates innate immunity, jasmonic acid, and ethylene levels in Arabidopsis[END_REF]Lee et al., 2008).

The Y2H assay that we carried out pointed out that MKK3 interacts only with subclade III MAP3Ks. However, the reciprocal relationship has never been tested, therefore leaving the possibility that sub-clade III MAP3Ks interact and activate other MAP2Ks. One study from the literature suggests that MAP3K20 could act upstream MKK5-MPK6 in response to ABA [START_REF] Li | AIK1, A Mitogen-Activated Protein Kinase, Modulates Abscisic Acid Responses through the MKK5-MPK6 Kinase Cascade[END_REF].The building of a multiple map3k mutant would help to shut down any potential parallel module which activation is also dependent on sub-clade III MAP3Ks. MAP3K20 has also been proposed to interact directly with MAPK18 (Benhamman et al., 2017).

Open questions about the functioning of sub-clade III MAP3Ks-MKK3-C-group MAPKs modules

a. Functional redundancy within the module at the MAP3K and MAPK levels Upon wounding, I was able to show that many sub-clade III MAP3Ks are transcriptionally induced. Among them, I was able to highlight an important role for MAP3K14 in the activation of MKK3-MPK2 by wounding. However the corresponding single knock-out mutants did not completely shut down the activation of MKK3-MPK2, probably because of high functional redundancy with other MAP3Ks. Similarly, the knockout mutations of MAP3K17 and MAP3K18 do not totally impair the ABA-dependent MPK7 activation (Danquah et al., 2015) Preliminary experiments performed by Jean Colcombet in the laboratory suggested that wildtype MKK3 localizes in the cytosol and the nucleus at resting conditions. However, we were not able to see any relocation upon ABA treatment. It is possible that MKK3 nucleocytoplasmic turnover increases upon stresses without modifying the relative quantity of nuclear and cytosolic MKK3s. This is difficult to bring to light without using complex microscopy tools such as FRAP (Fluorescence Recovery After Photobleaching). Another possibility is that C-group MAPKs are localized in the nucleus and require the presence of MKK3 there to be activated. It would be interesting to know where MPK1, 2, 7 and 14 are located in resting conditions. Alternatively, the C-terminal NTF2 domain of the Ca 2+ /calmodulin dependent protein kinase II (CAMKII) was shown to be involved in its oligomerization (Morris & Török et al., 2001). We could imagine the same mechanism occurring for MKK3, in which case NTF2 would have a structural function necessary for the stability and the activity of the protein. To test the hypothesis of MKK3 dimerization, interaction assays could be performed between two MKK3 proteins. Another possibility is to perform western blot analyses to detect MKK3 after protein extraction in non-denaturant conditions. Concerning the effect of the NTF2 domain on MKK3 activity, mkk3-1 plants could be complemented with a version of MKK3 lacking its NTF2 domain and the activity of downstream MAPKs tested.

III. A role for MKK3 in stress responses?

A well-conserved MAPK through evolution

Although the activation of MKK3-dependent modules upon wounding and diverse conditions was not very complicated to demonstrate, I did not succeed to find and understand the responses controlled by MKK3-dependent signaling pathways in response to mechanical wounding. It is possible that the role of the protein is quite weak and technically difficult to reveal and that appropriate conditions were not fulfilled to bring it to light. However, the protein could still have an important evolutionary role. MKK3 is an atypical MAP2K and the only member of sub-group B (Ichimura et al., 2002). Its sequence and secondary structure are rather different from the other MAP2Ks but are very well conserved among MKK3 homologues found in other plant species [START_REF] Colcombet | Convergence of Multiple MAP3Ks on MKK3 Identifies a Set of Novel Stress MAPK Modules[END_REF]. It is highly conserved in almost all monocots and dicots as well as in the primitive angiosperm Amborella trichopoda.

MKK3-like kinases are also found in the moss Physcomitrella patens. In addition, a MAP2K possessing an NTF2-like domain in its C-terminal end is also found in the unicellular green algae Chlamydomonas reinhardtii. These observations suggest that MKK3-related pathways could also be conserved among all photosynthetic eukaryotes and probably have a very important role.

A role in herbivory signaling?

Throughout my work, the only phenotype obtained for mkk3-1 plants was the increase of JA and SA levels observed upon insect feeding. This hormonal regulation seems to be specific of insect feeding since it was not observed upon mechanical wounding. It would be interesting to better understand such specificity: is it due to insect oral secretions or to their specific feeding pattern? And moreover, is it specific of that particular insect (S. littoralis) or could the observation be extended to other species? To address those questions, we could take advantage of tools developed by our collaborators to test the effects of oral secretions applied on previously mechanically wounded plants or used the Mecworm robot able to mimic insect feeding pattern [START_REF] Mithöfer | Effects of feeding Spodoptera littoralis on lima bean leaves. II. Continuous mechanical wounding resembling insect feeding is sufficient to elicit herbivory-related volatile emission[END_REF]. Assays could also be performed with a specialist herbivore such as the commonly used Pieris rapae.

In addition to mechanical wounding and insect feeding, MKK3 was found activated by the necrotroph B.cinerea. Broadly speaking, MKK3 seems to be activated by stresses upon which JA is known to have a prevailing role in the mediation of defense responses. It could be interesting to also measure phytohormone levels upon interaction with B. cinerea comparing Col-0 and mkk3-1 plants. Biotrophic pathogens, in response to which SA is the major hormone activating defense responses and JA has a minor role, were not tested in their ability to activate MKK3-dependent modules. However, flg22, which can mimic the interaction with the biotrophic bacterium P. syringae, did not activate MKK3-dependent modules. Moreover, the wounding-induced resistance to B. cinerea was not affected by the mkk3-1 mutation.

Since an important production of SA was observed in mkk3-1 plants upon caterpillar feeding, it would be interesting to see if a pre-interaction of mkk3-1 plants with S. littoralis could lead to an increased resistance toward biotrophic pathogens. The theoretical experiment would consist in submitting Col-0 and mkk3-1 plants to S. littoralis feeding prior to an infection with P.syringae and compare bacterial growth in both genotypes.

Characterized phenotypes in other stress and developmental contexts

On the other hand, MKK3 has been studied in other stress contexts and some phenotypes have been characterized. mkk3-1 plants were studied in a context of plantpathogen interaction and were shown to be hypersensitive to Pseudomonas syringae DC3000 (DÓczi et al., 2007). MKK3 was also studied in a JA context together with MPK6 and was shown to have a role in JA-induced growth inhibition. Indeed, mkk3-1 plants were more sensitive to exogenous JA as they displayed shorter roots. mkk3-1 plants also showed a reduced expression of the JA marker gene PDF1.2 (Takahashi et al., 2007). Since wounding was shown to trigger root growth inhibition through the production of JA (Gasperini et al., 2015), it would be interesting to monitor the growth of mkk3-1 roots upon wounding. Other studies showed the hypersensitivity of mkk3-1 plants to auxin (Enders et al., 2017) and gibberellin as well as insensitivity to red light [START_REF] Lee | Mitogen-activated protein kinase kinase 3 is required for regulation during dark-light transition[END_REF]. Arabidopsis plants impaired in MKK3 were studied in a drought context and appeared to be less efficient to restrict water loss upon long-term mild drought conditions (Danquah et al., 2015). Similarly in cotton, plants over-expressing GhMKK3 were shown to be more resistant to drought and more efficient to close their stomata in response to ABA (Wang et al., 2016). In addition, mkk3-1 plants were shown to be hypersensitive to ABA in root elongation and germination. Indeed, mkk3-1 seeds show a higher decrease of germination rate upon ABA treatment compared to wild-type seeds as well as greater ABA-induced root growth inhibition (Danquah et al., 2015). In both wheat and barley, recessive mutations of MKK3 orthologs revealed a role for MKK3 in the control of dormancy, a phenomenon in which ABA is largely involved (Nakamura et al., 2016;[START_REF] Torada | A causal gene for seed dormancy on wheat chromosome 4A encodes a MAP kinase kinase[END_REF]. Recently, some germination assays were performed in the "Stress Signaling" group, comparing Col-0, mkk3-1 and mkk3/MKK3locus-YFP plants. mkk3-1 seeds germinated significantly more belatedly compared to the two other genotypes (figure 3.3). Overall, these data clearly suggest a role for MKK3 in germination/dormancy. Therefore it would be interesting to go further in that direction and better characterize MKK3 and its partners during germination. First it would be necessary to study the expression and the activation of MKK3 in seeds at different stages. Then it would be interesting to check if candidate partners of MKK3 share its dormancy phenotype. Thus, germination assays could be performed on mpk1/2/7/14 quadruple mutants and map3k mutant combinations.

The hunt of MPK1/2/7/14 substrates

As many other signaling components, MKK3-related modules are also shared between different stresses (ABA treatment, JA, wounding, pathogens…) thus the specificity of responses should occur downstream the activation of the module and likely at the transcriptional level of stress-responsive genes, which constitutes an output of signaling processes. Unfortunately, our microarray analysis did not point out any MKK3-related wounding-induced genes. Another possibility would be to identify direct substrates of MKK3 and MKK3-related modules. We could take advantage of the TAP-tag technique that is wellmastered in the "Stress Signaling" group that, couple to mass spectrometry, could help identifying interactants of candidate MAPKs. Those could later be validated by Y2H or in planta BiFC (Bimolecular Fluorescent Complementation) assays. Alternatively, phosphoproteomic approaches comparing plants impaired in MKK3-related modules with Col-0 in response to stresses could point candidate substrates that could also be confirmed by in vitro kinase assays. A first hint comes from the work of Umezawa and coworkers which identified MAPK phosphosites in response to ABA (Umezawa et al., 2013). The nature of identified substrates could give some clue about the nature of cellular mechanisms regulated by MKK3 and related modules. So far only one substrate of MKK3-related modules was published. MPK1 was shown to interact with and phosphorylate RBK1 (Rop Binding Protein Kinase 1) in vitro (Enders et al., 2017). Besides, rbk1 mutants shared the auxin hypersensitivity phenotype of mkk3-1 and mpk1 plants but the activation of the whole pathway by auxin was not tested. RBK1 is a predicted S/T protein kinase that was shown to interact with and phosphorylate ROP proteins. These are small GTPases involved in diverse aspects of plant development but also in response to environmental cues [START_REF] Nibau | RAC/ROP GTPases: "hubs" for signal integration and diversification in plants[END_REF].

It could be interesting to test if RBK1 is a substrate of MKK3-dependent modules in the context of wounding.

Overall, my work helped better understanding stress signaling events occurring upon wounding and paved the way to new hypotheses that would be worth to work on in the near future.

CHAPTER IV: MATERIALS & METHODS

I. Materials

Plant material

The Columbia (Col-0) and Wassilewskija-0 (Ws-0) ecotypes of Arabidopsis thaliana were used. Mutant lines are listed below.  YPD medium (complex medium): 1% yeast extract, 2% peptone, 2% dextrose; for solid medium 1.2% of agar was added before autoclaving.

 YNB medium (minimal medium): 0.17% yeast nitrogen base without amino acids, 2% dextrose, 0.5% (NH 4 )2SO 4 , 0.2% Drop out mix. pH was adjusted to 5.6 with 5 M NaOH.

For solid medium, 1.4% of bacto agar was added before autoclaving.

Antibodies

a. Primary antibodies:

 α-pTpY: Rabbit monoclonal antibody (Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204)) (Cell Signaling 4370S).

 α-Myc: Rabbit polyclonal antibody (SIGMA C3956).

 α-HA: Rat monoclonal antibody (SIGMA A2095).

 α-GFP: Mouse monoclonal antibody (Roche).

 α-MPK6: Rabbit polyclonal antibody (SIGMA A7104).

 α-MPK3: Rabbit polyclonal antibody (Davids Biotechnology, Regensburg, Germany).

 α-MPK1 and α-MPK2: Rabbit polyclonal antibody kindly provided by M.J. Marcote (Ortiz et al., 2007)  α-MPK7: Rabbit polyclonal antibody developed in Heribert Hirt's laboratory in Vienna (Dóczi et al., 2007) b. Secondary antibodies:

 α-rabbit: Goat polyclonal antibody conjugated to horseradish peroxidase (SIGMA A6154).

 α-mouse: Goat polyclonal antibody conjugated to horseradish peroxidase (SIGMA A5906).

 α-rat: Goat polyclonal antibody conjugated to horseradish peroxidase (SIGMA A9037).

Vector backbones

 p2GWF7: Destination Gateway expression vector with C-terminal GFP tag for expression of fusion protein in protoplasts.

 pDEST22: Destination Gateway vector for yeast 2 hybrid. Allow a constitutive expression of proteins N-terminal fused to GALD4 AD under the control of ADH1 promoter.

 pDEST32 modified: Destination Gateway vector for constitutive expression of proteins fused to GAL4 BD under control of ADH1 promoter. This vector has been modified to have a Kanamycin resistance gene.

 pDONR207: donor Gateway vector (Invitrogen) used in cloning for generating entry clones.

 pGREEN0229: binary vector for the generation of transgenic plants (Halens).

 pGREEN0229-HA-Tnos: home-made binary vectors derived from pGREEN0229 for the generation of transgenic plants allowing carrying a MCS-HAx2-Tnos to allow an easy fusion of promotor-ORF or locus with HA in C-terminal.

 pGREEN0229-YFP-Tnos: home-made binary vectors derived from pGREEN0229 for the generation of transgenic plants allowing carrying a MCS-YFP-Tnos to allow an easy fusion of promotor-ORF or locus with YFP in C-terminal. was prepared and sprayed equally to the plant tray. For mock control, only DMSO was diluted in water.

Buffers and solutions

c. Touch stress

For gentle touch stress, 4 week-old Arabidopsis leaves were bent 10 times back and forward as described previously [START_REF] Chehab | Arabidopsis touch-induced morphogenesis is jasmonate mediated and protects against pests[END_REF]. Touched leaves were harvested following a time course. Samples were stressed differentially and harvested at the same time.  Digestion of Arabidopsis mesophyll cells: Arabidopsis leaves are cut into very small slices with a razor and put in a Petri dish containing the enzymatic solution previously filter through a 0.45 µm filter. The whole is then submitted to vacuum for 30 minutes and then covered with aluminium fold and left to incubate for 2h30 in Percival chambers.

 Collection of protoplasts: 10 mL of buffer W5 is added in the Petri dish. The solution is then collected with a pipette and put in a rounded 30mL tube through a mesh (35-75µm).

The tube is then centrifuged at 800 rpm break 1 for 1 minute. The supernatant is removed and protoplasts are resuspended in 10mL of buffer W5. After a second centrifugation of 2 minutes at 800 rpm break 1, protoplasts are resuspended in 3mL of buffer W5 and kept on ice.

Molecular biology methods

a. Yeast 2-hybrid assay  Vector preparation: Entry clone containing ORFs of interest in a STOP version were recombined using LR enzyme mix in pDEST22 and pDEST32-modified using the Gateway technology.

 Yeast preparation: an overnight 10 mL culture of MaV203 yeast in YPD medium was diluted in 50 mL fresh YPD medium to reach an OD 600 of 0.4. The culture was grown for an additional 4 hours at 28 °C. Yeast cells were then collected after centrifugation at 2500 rpm for 5 minutes and the supernatant was discarded. The pellet was re-suspended in 40 mL of 1X TE (10 mM Tris, pH 7.5, 1 mM EDTA) buffer and centrifuged again for 5 minutes at 2500 rpm. The pellet was re-suspended in 7 mL of 1X LiAc/0.5X TE solution (100 mM Lithium Acetate pH 7.5, 5 mM Tris-HCl, pH 7.5, 0.5 mM EDTA) and incubated at room temperature for 10 minutes.

 Yeast co-transformation: 1 µg of plasmid DNA (for both prey and bait) was mixed with 100 µg of denatured sheared salmon sperm DNA and 100 µL of yeast suspension. A 700 µL solution of 1X LiAc/40% PEG-3350/1X TE (100 mM Lithium Acetate, pH 7.5, 40%

PEG-3350, 10 mM Tris-HCl pH 7.5, 1 mM EDTA) was added to the suspension and mixed thoroughly. The mixture was incubated at 30 °C in water bath for 30 minutes.

After adding 80 µL of DMSO, the mixture was heat-shocked at 42 °C for 7 minutes. Ligation products were transformed in DH5alpha bacteria. Few colonies were selected to prepare DNA from an overnight 10 mL culture and sequenced for the CRISPR guide. The vector was introduced into thermo-competent Agrobacterium tumefaciens C58C1 strain and used to transform Arabidopsis Col-0 plants using a classical floral dipping method [START_REF] Clough | Floral dip: a simplified method forAgrobacterium-mediated transformation ofArabidopsis thaliana[END_REF]. About 20 T0-Basta R plants were then selected on pots. The number of inserts was then assessed using the BastaR segregation in T1 seeds in plate (½MS+ 1% sucrose).

Basta sensitive T1 plants from lines carrying a single insert were transferred carefully on soil.

About 50% survived and were genotyped for mutations in MAP3K13 and MAP3K14 genes.

For MAP3K13, genomic DNA was amplified using Pr2664

(CACGTGGGAAATTTTACAAAGATGAG) and Pr51 (CTAACTTCCACCCAACCATCTG) and the 1.5 kb PCR product was sequenced using Pr51. For MAP3K14, genomic DNA was amplified using Pr2661 (CACTTCTTCTTCTTCGTCTTCTTG) and Pr1591 (GAAGAGGCTGACAACTTCACG) and PCR products were sequenced using Pr3212 (GAGTTCCGATAACCCCACCG). Several lines were identified carrying a single mutation in one of the two targeted loci or on both. If necessary, homozygous plants were selected in T2. In parallel, pGREEN-HA-Tnos and pGREEN-YFP-Tnos were also linearized using compatible enzymes in presence of CIP (NEB). PCR fragments and vectors were run on electrophoresis gel, DNA was extracted from gel and both partners were ligated with a ratio 1:1 using T4 ligase (NEB) during 1 hour at room temperature. Ligation products were then transformed in home-made thermo-competent DH5aplha bacteria and next day colonies were screened by PCR. 3-5 positive colonies were then grown overnight in 10 mL LB culture at 37 °C. Plasmid DNA was extracted and construction checked by restriction pattern. Finally, the whole insert was sequenced. The vector was then introduced into thermocompetent Agrobacterium tumefaciens C58C1 strain and used to transform Arabidopsis Col-0 plants using a classical floral dipping method [START_REF] Clough | Floral dip: a simplified method forAgrobacterium-mediated transformation ofArabidopsis thaliana[END_REF]. About 20 T0-Basta R plants were then selected on pots. The number of inserts was assessed using the BastaR segregation in T1 seeds in plate (½MS + 1 % sucrose). Homozygous plants were selected in T2. In the case of MAP3K14locus-YFP construct, we decided in a second step to change the Tnos terminator for the MAP3K14 endogenous terminator. for 90 minutes following manufacturer instruction (BioRAD). Membranes are then blocked in TBS-T + 5% BSA (for further α-pTpY probing) or in TBS-T + 5% milk (for other antibodies) for 1h. Primary antibody diluted in 5% BSA (or milk) was added to membrane and left to incubate over-night at 4°C (or 2-3h at room temperature) under gentle shaking. Membranes were washed 3 times 10 minutes with TBS-T and incubated with secondary antibody coupled to horseradish peroxidase (HRP) diluted in TBS-T 5% BSA (or 5% milk) for 1h. After 3 more washes of 10 minutes with TBS-T, HRP activity on the membrane is revealed using enhanced chemiluminescence reagent (Clarity Western ECL Substrate -Biorad). The subsequent chemiluminescent signal is detected using a ChemiDoc imaging system (Biorad).

ACTIN2 AT3G18780 CGTTTCTATGATGCACTTGTGTG GGGAACAAAAGGAATAAAGAGG MPK1 AT1G10210 TGGTCACTTATCACCGAGGG GCTCCACGACCAATAGGCTT MPK2 AT1G59580 AGAGAGTCAACGGTCATAGCG CGCCATTCTTCTACCTCCCG MPK7 AT2G18170 CAACAACAACAACGACTCGGG TGGCTCAACTAACATCGCCA MAP3K13 AT1G07150 CGCGGTGAGATGAGATCGAG ACAACTATTTGGCTGTAAACGAGA MAP3K14 AT2G30040 ACCAGCTTGGGAAGATCACG GAGTTCCGATAACCCCACCG MAP3K15 AT5G55090 GTCGAGGCTCAACAGCTACT CGATGAAGAAAACTCGGCGG MAP3K16 AT4G26890 CGGCTGATCGGATCGAGAAA ATCCATCCGCCATCGTCTTC MAP3K17 AT2G32510 TACTCGGAGAGGATCGGACG TGTTCCTTCACACCTCGCTC MAP3K18 AT1G05100 TTCACCGGTCGGAGTTCTTG TGTGGAAGGGCTCTCTCGTA MAP3K19 AT5G67080 CGCCGTTAAGATTGCGGATT TTAACAGATTCCGGCGCCAT MAP3K20 AT3G50310 TATCGCAGTGTGCAAACCCT GAAACTCCGGCGACTTTCCT JAZ10 AT5G13220 ATCCCGATTTCTCCGGTCCA ACTTTCTCCTTGCGATGGGAAGA ERF1 AT3G23240 TCTAATCGAGCAGTCCACGC CTCTTATCTCCGCCGCGAAT ERF4 AT3G15210 CTCCGACGTTAGTTGTGCCT CGTTACCGATCCCCATCAGG ERF5 AT5G47230 GGTGGAGAGACGTTTCCGTT ACCAAACGGTGGATGAGGAG PR4 AT3G04720 CGGCAAGTGTTTAAGGGTGA CAAATCCAAGCCTCCGTTGC RHL41 AT5G59820 ATGGGACAAGCTTTGGGAGG TTCAACGTAGTCACCGTGGG STZ AT1G27730 GTAGCGTGTCCAACTCCGAA TCAGGGATCGGAGGGATGTT WRKY15 AT2G23320 GATCTCCACATCCAAGAGGATATTA GCGGCGGAGAGAGAATGATTA MYB51 AT1G18570 CCCTTCACGGCAACAAATGG CCGGAGGTTATGCCCTTGTG PAL1 AT2G37040 ACTTATTAGATTCCTTAACGCCGGA CCGTTGGGACCAGTAGCTTT PAL2 AT3G53260 CTCATTAGATTTTTGAACGCCGGA CGGAGAGGTAGTGACGGAGA PAL4 AT3G10340 CCATCCCGGTCAGATCGAAG ACGTACGTAAAGCGTACCGAT SID2 AT1G74710 AGCTGGAAGTGACCCATCTT TGGTGAACTGCAAAAACAACA LOX2 AT3G45140 GCTAGTTGAAGAGTGGCCGT TAGCATCATAGCCTGGCGTG LOX3 AT1G17420 ACGACCTTGGAAATCCCGAC GCTCTCCGCGTCTTTATCAGA LOX4 AT1G72520 TGATCTCATCCGAAGGGGGA GTACCAGGCTTGGAGCTCAG LOX6 AT1G67560 AGAAAAGGCGCTTGAGGAGAA TCTCGGATTCGGCTGTAGGA AOS AT5G42650 TTATCAACCGTTGGCGACGA TTCGACCACAACACATGCCT VSP2 AT5G24770 TCAGTGACCGTTGGAAGTTGTG GTTCGAACCATTAGGCTTCAATATG PDF1.2 AT5G44420 TTTGCTGCTTTCGACGCAC CGCAAACCCCTGACCATG PR1 AT2G14610 GATCCTCGTGGGAATTATGTG TTCTCGTAATCTCAGCTCTTATTTG

INTRODUCTION

Mitogen-activated protein kinase (MAPK) modules are important signaling actors found in all eukaryotic cells. They are constituted of the MAPK per se, a serine/threonine kinase which is phosphorylated and activated by a MAPK Kinase (MAP2K), itself activated by phosphorylation by a MAP2K Kinase (MAP3K). These modules are encoded in plants by large multigenic kinase families. For example, the Arabidopsis genome codes for 20 MAPKs, 10 MAP2Ks, and about 80 MAP3Ks (Ichimura et al., 2002). The MAPK and MAP2K families are both organized in four well-defined sub-clades (denoted as clades A-D). Under the generic name, MAP3Ks gather several distinct kinase families which were shown in animals to activate MAP2Ks. These include the MEKK-like kinases (20 members in Arabidopsis) which are unambiguously involved in the activation of MAP2Ks/MAPKs in plants, the large 48 membered Raf family for which functional data are scarce and the 11 ZIK kinases which have not been reported to act upstream MAP2Ks in plants so far. Three modules defined by the three iconic MAPKs, MPK3, MPK4, and MPK6, were the focus of the vast majority of MAPK studies (Colcombet and Hirt, 2008;[START_REF] Suarez-Rodriguez | Mitogen-activated protein kinase signaling in plants[END_REF].

MKK3 Modules in Plant Signalling

Although some reports exist on the majority of MAPKs and MAP2Ks, plant MKK3s have been the subject of less attention.

MKK3, AN ATYPICAL MAP2K, FOUND IN ALL PLANT SPECIES

The first MKK3-like MAP2K sequence, NtNPK2, was identified from a tobacco cell suspension cDNA library twenty years ago and proved to code for an active kinase (Shibata et al., 1995). Classical MAP2Ks are usually rather small (about 350 amino acids) and are globular proteins with only a single kinase domain. In contrast, NtNPK2 and all MKK3 homologs are considerably longer (>500 amino acids) and contain, besides the protein kinase domain, a NUCLEAR TRANSPORT FACTOR2 (NTF2)-like domain in their C-terminal regions. The MAP2K N-terminal tail, which contains a docking site (D-site) for MAPKs, is also well conserved within MKK3 homologues but divergent from those of other MAP2Ks. Additionally, MKK3 N-terminal tails have secondary structure motifs which are usually not found in other MAP2Ks.

In the Arabidopsis genome, there is a single MKK3 gene (At5g40440) which defines the B subclade of plant MAP2Ks (Ichimura et al., 2002). In the published plant genomes, B-type MAP2Ks are found in almost all dicots and monocots as well as in the primitive angiosperm Amborella trichopoda as a single copy gene. The two gymnosperm sequenced genomes (Pinus taeda and Picea abies) do not seem to contain any MKK3 but the explanation could also be due to their low sequence coverage. Alternatively, a loss of MKK3 in gymnosperms could be possible. The moss Physcomitrella patens possesses two welldefined MKK3-like kinases (PpMKK3-1 and PpMKK3-2). The unicellular green algae Chlamydomonas reinhardtii codes for very few genes having homology to MAP2Ks. Singularly, the best MAP2K candidate [referred as MAP Kinase Kinase 1 in NCBI (XP_001696437) and CrMKK3 in this article] possesses an NTF2-like domain in its C-terminus. Overall, the CrMKK3 sequence is not very similar to other MKK3s but the CrMKK3 gene shares with AtMKK3 several conserved introns which are not found in other Arabidopsis MAP2Ks, suggesting that CrMKK3 and AtMKK3 have a common ancestor containing an NTF2 domain and that the primary gene structure is highly conserved during evolution. This also suggests that the MKK3-related pathways are conserved among all photosynthetic eukaryotes.

At present, a role of the NTF2 domain in plant MKK3s has not been reported. A NUCLEAR TRANSPORT FACTOR2 domain suggests a function in the localization or shuttling of the protein kinase. In eukaryotes, NTF2s are small proteins which have indeed been involved in protein import into the nucleus [START_REF] Moore | Purification of a Ran-interacting protein that is required for protein import into the nucleus[END_REF] and play an important role in the β-importin-dependent macromolecular trafficking by loading the nucleus with RanGTP [START_REF] Stewart | Structural basis for molecular recognition between nuclear transport factor 2 (NTF2) and the GDP-bound form of the Ras-family GTPase Ran[END_REF]. But other proteins containing an NTF2-like domain have distinct roles. For example, metazoan p15, through interaction with NTF2s is an important actor of nuclear mRNA export [START_REF] Fribourg | Structural basis for the recognition of a nucleoporin FG repeat by the NTF2-like domain of the TAP/p15 mRNA nuclear export factor[END_REF][START_REF] Stutz | The interplay of nuclear mRNP assembly, mRNA surveillance and export[END_REF]. These data indeed suggest that MKK3 signaling could require specific shuttling mechanisms between cellular compartments. However, Ca 2+ /calmodulin dependent protein kinase II (CAMKII) also possesses in its C-terminal tail an NTF2-like domain which has been shown to be involved in its assembly of dodecamers (Morris and Török, 2001;[START_REF] Rellos | Structure of the CaMKII??/calmodulin complex reveals the molecular mechanism of CamKII kinase activation[END_REF]. Therefore, the NTF2 domain might also function in homo-or oligomerization of MKK3 proteins. But some NTF2like domains may also have catalytic activities. For example, the NTF2-like Streptomyces SnoaL codes for a small polyketide cyclase involved in antibiotic synthesis and the bacterial δ5-3ketosteroid isomerase catalyzes the stereospecific isomerization of steroids [START_REF] Kim | High-resolution crystal structures of delta5-3-ketosteroid isomerase with and without a reaction intermediate analogue[END_REF]Sultana et al., 2004), opening up the possibility that the NTF2-like domain of plant MKK3s might also have an enzymatic activity which would be coupled to MAPK signaling. Structure-function investigations would be necessary to elucidate the exact role of this mysterious domain.

IN PLANTS, MKK3 HAS BEEN INVOLVED IN SEVERAL MAPK MODULES AND RESPONSES TO ENVIRONMENT

In plants and mainly in Arabidopsis, several functional studies placed MKK3 within MAPK modules highlighting its function in several distinct physiological signaling processes. Figure 1 gives an overview of our actual knowledge about MKK3 modules in physiological processes.

MKK3 in ROS Signaling and Homeostasis

MKK3 was first reported to play a role during plant-pathogen interaction (Dóczi et al., 2007) (Figure 1A): mkk3 knockout mutants were shown to be hypersensitive to infection by Pseudomonas syringae DC3000 whereas Arabidopsis lines expressing a constitutively active MKK3 (MKK3EE) showed mild resistance to the pathogen. Using yeast two hybrid and co-immunoprecipitation assays from protoplasts, this study also suggested that MKK3 and the four C-group MAPKs MPK1/2/7/14 interact together to define a functional MAPK module, that can be activated by H 2 O 2 . In support of this notion, MPK1/2 activation by H 2 O 2 has been confirmed in vivo by an independent study (Ortiz-Masia et al., 2007).

Interestingly, an MKK3-based MAPK module has also been reported to act upstream of reactive oxygen species (ROS) production in response to wounding (Takahashi et al., 2011) (Figure 1B). In this study, the authors showed that plants mutated in MPK8, which codes for a MAPK of the poorly characterized D-clade, fail to locally restrict ROS production in response to leaf wounding. Coherently, MPK8 over-expressing plants do not produce ROS anymore in response to wounding as revealed by DAB staining. The authors also showed that MPK8 activation upon wounding depends on calcium/calmodulin as well as on MKK3. This indirectly suggests that MKK3, which is activated by H 2 O 2 , is also involved in ROS production upon stress conditions such as wounding. To test whether MKK3-MPK8 forms a functional module, it could be relevant to test whether mkk3 plants are impaired in wound-induced ROS production.

MAP3K17/18-MKK3-MPK1/2/7/14 in Response to ABA, Senescence, and Dormancy

In Arabidopsis, MPK1 and MPK2 were shown to be activated by abscisic acid (ABA) (Ortiz-Masia et al., 2007;Umezawa et al., 2013;Danquah et al., 2015). Recently, two independent studies demonstrated that MKK3, together with MAP3K17 and MAP3K18, functions upstream of MPK1, MPK2, MPK7, and MPK14 in an ABA-activated MAPK module (Danquah et al., 2015;Matsuoka et al., 2015) (Figure 1E). Apparent discrepancies between these studies also suggest a dual mode of activation of the ABA-related MKK3 module. Indeed, Danquah et al. (2015) showed that, under their experimental conditions, activation of C-group MAPKs depends on MAP3K18 synthesis, explaining their activation with a rather slow kinetics. Other studies have shown a more rapid activation of the C-group MAPKs which is compatible with a post-translational activation of MAP3Ks (Ortiz-Masia et al., 2007;Umezawa et al., 2013). Interestingly, using lines constitutively expressing tagged MAP3K18, Matsuoka et al. (2015) showed that ABA is also able to directly activate the kinase within 15 min and that MAP3K18 activity peaks at 30 min. Taken together, this suggests that the pathway has two modes of functioning, which may be dependent on unidentified experimental differences between the laboratories working on this ABA-dependent MAPK pathway. Finally, a direct interaction between MAP3K18 and the PP2C phosphatase ABI1, but not with ABI2, has been described (Mitula et al., 2015). The authors hypothesized that besides being one of the main actors of the ABA core signaling module, ABI1 is also able to dephosphorylate MAP3K18 in the absence of ABA, resulting in the degradation of MAP3K18 by the proteasome. When ABA is perceived by PYR/PYL receptors, ABI1 is dissociated from MAP3K18 and MAP3K18 is stabilized and able to activate downstream factors of the signaling module.

Phenotypical characterization in plants with impaired MKK3 module function confirmed the role of MKK3 in ABA signaling and/or ABA-dependent processes. Danquah et al. (2015) characterized mutant plants in a drought and abiotic stress context and showed that plants impaired in the MKK3 module are less able to restrict water loss when submitted to a long-term mild drought experiment. More directly, the stomata of map3K18 plants are compromised to close in response to ABA (Mitula et al., 2015) whereas MAP3K18 over-expressors show accelerated leaf senescence (Matsuoka et al., 2015), a process known to be under control of ABA [START_REF] Song | Abscisic acid as an internal integrator of multiple physiological processes modulates leaf senescence onset in Arabidopsis thaliana[END_REF]. A very recent study similarly reported that Nicotiana benthamiana plants overexpressing the cotton (Gossypium hirsutum) GhMKK3 were more resistant to drought and closed more efficiently their stomata in response to ABA (Wang et al., 2016). Additionally, by QTL mapping for seed dormancy, two recessive mutations in MKK3 orthologs have been identified in both wheat and barley (Nakamura et al., 2016;[START_REF] Torada | A causal gene for seed dormancy on wheat chromosome 4A encodes a MAP kinase kinase[END_REF]. In plants, dormancy is under control of seed hormonal content during maturation and desiccation, with one of the main hormones being ABA [START_REF] Shu | Two faces of one seed: hormonal regulation of dormancy and germination[END_REF]. In the case of the barley mutant cultivar "Azumamugi, " the recessive mutation decreases MKK3 activity and thereby increases seed dormancy, suggesting that the activation of the MKK3 module releases seed dormancy (Nakamura et al., 2016).

MKK3-MPK6-MYC2 Is Involved in JA and BL Signaling MKK3 has also been reported to work upstream of MPK6 in the context of two pathways (Figures 1C,D). A first study showed that the MKK3-MPK6 module plays a central role in jasmonic acid (JA)-induced root growth inhibition (Takahashi et al., 2007). MPK6 is activated by JA within 10 min and this activation is impaired in mkk3 but not in mkk2 mutant plants. Since MPK6 is very efficiently activated by many stresses, including touch, it is unfortunate that mock samples were not tested in these JA-dependent MPK6 activation assays. Coherently, the authors showed that both mkk3 and mpk6 mutants are slightly more sensitive to exogenous JA at the root elongation level as well as show reduced activation of the JA-marker gene PDF1.

MKK3-MPK6 has also been implicated in blue light (BL) signaling. MPK6 is activated specifically by BL but not red light. Using knock-out mutants, the authors showed that this activation is MKK3-and MYC2-dependent. Lee also reported that an mkk3 KO mutant shows insensitivity to red light and hypersensitivity to gibberellin [START_REF] Lee | Mitogen-activated protein kinase kinase 3 is required for regulation during dark-light transition[END_REF]. Interestingly, it has also recently been shown that MPK6 was able to both interact with the MYC2 promoter to induce its transcription and to phosphorylate MYC2 (Sethi et al., 2014). Interestingly, this module was shown to also negatively regulate the expression of the MYC2 transcription factor which is a well-known actor of JA signaling.

How to Conciliate Apparently Opposite Data

Taking advantage of the yeast 2 hybrid interaction assay, several groups reported on the interactions between MKK3 and the MAPKs encoded by the Arabidopsis genome (Dóczi et al., 2007;Lee et al., 2008;Matsuoka et al., 2015). In these studies, MKK3 interacted with C-group MAPKs but not with MPK6 or MPK8. The absence of an MKK3-MPK6 interaction was not due to technical problems as MPK6 was otherwise able to interact with its native MAP2Ks MKK4, and MKK5 (Lee et al., 2008). In protoplast activation assays, a constitutively active MKK3 was also not able to activate MPK6 or MPK8 (Danquah et al., 2015) and no direct interaction between MKK3 and MPK6 could be shown in the studies reporting that MKK3 and MPK6 constitute a functional MAPK module (Takahashi et al., 2007;Sethi et al., 2014). The only suggestion that MKK3 could activate directly MPK6 came from an in vitro kinase assay in which a constitutively active MKK3 was able to trigger MPK6-dependent phosphorylation of MBP (Takahashi et al., 2007).

From these results, it is tempting to hypothesize that MKK3 does not interact with MPK6 or MPK8, suggesting that the MKK3-dependent activation of MPK6 may be indirect. The fact that MPK1/2, which have been shown to function downstream of MKK3 in response to ROS and ABA, are also activated by JA (Ortiz-Masia et al., 2007) would promote a model in which the MKK3-MPK1/2/7/14 module is necessary for the proper functioning of the upstream players of JA-or BL-dependent MPK6 activation. The investigation of JA-dependent MPK6 activation in mutants impaired in MAPK genes of the C-clade would help to resolve this issue.

ARE MAP3Ks OF SUBCLADE III ACTIVATORS OF MKK3 MODULES?

MAP3K13-20 Define a Subclade of MEKK-Like Kinases Which Are Strongly Transcribed in Response to Stresses ZIK-, Raf-, and MEKK-like kinases have been shown to act as MAP2K Kinases in animal cells. In plants, ZIKlike kinases (11 members in Arabidopsis) have not been connected to MAPK signaling so far and Raf-like kinases (about 50 members), such as CTR1 and EDR1, seem to act rather as negative regulators of MAPK modules [START_REF] Yoo | Dual control of nuclear EIN3 by bifurcate MAPK cascades in C2H4 signalling[END_REF][START_REF] Zhao | EDR1 physically interacts with MKK4/MKK5 and negatively regulates a MAP kinase cascade to modulate plant innate immunity[END_REF]. In contrast, several MEKK1like kinases have been shown to be activators of MAP2Ks (Colcombet and Hirt, 2008). The 20 members of this family are organized in three subclades (Figure 2A). Genes of subclades I and II have many introns and code for kinases carrying an additional long C-and N-terminus, respectively. Interestingly, MEKK-like kinases of subclade III do not have any introns and code for kinases with rather short tails.

MAP3Ks of subclade III are largely uncharacterized, with the notable exception of MAP3K18 which has been shown to be an important player of ABA signaling (Danquah et al., 2015). This work also showed that in contrary to other MAP3Ks which are thought to be mainly regulated by phosphorylation, MAP3K18 is strongly transcriptionally regulated in response to ABA and the consequent kinase production triggers the phosphorylation cascade, explaining the delayed activation kinetics of its downstream target MAPKs MPK1, 2, 7, and 14 (Boudsocq et al., 2015;Danquah et al., 2015). Interestingly, we noticed that other members of subclade III are also transcriptionally regulated (Danquah et al., 2015). For example, MAP3K13 and MAP3K14 have been shown to be induced upon nitrate feeding after a starvation period (Marchive et al., 2013). Analysis of the Genevestigator database, which integrates many Affymetrixbased transcriptome experiments, confirms that MAP3K genes from clade III are under transcriptional regulation by various stresses (Figure 2B). This suggests that a transcriptional regulation, as shown in the case of ABA regulation of MAP3K18, might be a general feature of the regulation of clade III MAP3Ks.

Toward a General Model for a Slow Activation of MKK3 Modules by Stresses

In yeast 2 hybrid assays, MKK3 was shown to interact with six of the eight MAP3Ks belonging to clade III but none of the MAP3Ks of clades I and II (Figure 2A) [START_REF] Danquah | Identification and Validation of Key Factors of Stress Tolerance in Arabidopsis thaliana[END_REF]Danquah et al., 2015). Interestingly, clade-III MAP3Ks as well as type-C MAPKs were found in all species containing MKK3. In Arabidopsis, MPK1 and MPK2 have been shown to be activated by several stresses, such as wounding, ABA, JA, and H 2 O 2 .. We propose a general model in which clade-III MAP3Ks, MKK3,

RESUME EN FRANÇAIS

  ACTING UPSTREAM MKK3 UPON WOUNDING .................................................................................................................................... 1. Exogenous application of some candidate second messengers can activate an MKK3-dependent module................................................................................... 2. Wounding-induced activation of the MKK3-dependent module is disrupted in mutants of the JA signaling pathway .................................................................. 3. MKK3-MPK2 is not activated in systemic leaves ................................................ 4. Discussion ............................................................................................................. IV. ACTIVATION OF AN MKK3-DEPENDENT MODULE BY CELL WALL-DAMAGING PATHOGENS ................................................................................................................. 1. Botrytis cinerea activates an MKK3-dependent module in Arabidopsis thaliana 2. Spodoptera littoralis activates an MKK3-dependent module in Arabidopsis thaliana ............................................................................................................... 3. Discussion ............................................................................................................. V. STUDY OF THE ROLE OF AN MKK3-DEPENDENT MODULE IN WOUNDING-RELATED RESPONSES .................................................................................................................. 1. Plants impaired in MKK3 signaling do not show clear detectable phenotype upon Botrytis infection ................................................................................................. 2. Plants impaired in MKK3 signalingdo not properly respond to herbivorous insects .................................................................................................................. 3. Wounding-induced trichome formation ................................................................ 4. Transcriptomic analysis of mkk3-1 plants submitted to wounding did not highlight any MKK3-regulated genes ................................................................................ 5. Discussion ............................................................................................................. CHAPTER III: CONCLUSIONS & PERSPECTIVES ..................................................... I. A MAPK SIGNALING NETWORK ACTIVATED IN RESPONSE TO WOUNDING ..................... 1. Toward a sequential and interdependent activation of two MAPK modules ........ 2. Spatial organization of MAPK activation upon wounding ................................... II. A NOVEL MKK3-DEPENDENT MODULE ACTIVATED BY ENVIRONMENTAL CONSTRAINTS THROUGH TRANSCRIPTIONAL REGULATION OF MAP3K GENES ................................... 1. An emerging general working model… ................................................................ 2. …coexisting with other models? ........................................................................... 3. Open questions about the functioning of sub-clade III MAP3Ks-MKK3-C-group MAPKs modules ................................................................................................. III. A ROLE FOR MKK3 IN STRESS RESPONSES? ................................................................ 1. A well-conserved MAPK through evolution ......................................................... 2. A role in herbivory signaling? ............................................................................... 3. Characterized phenotypes in other stress and developmental contexts ................. 4. The hunt of MPK1/2/7/14 substrates .....................................................................

Figure 1 . 1 :

 11 Figure 1.1: Simplified model depicting cellular events triggered by wounding and herbivory

Figure 1 . 2 :

 12 Figure 1.2: Biosynthetic pathway of Jasmonic Acid (JA) (from Acosta & Farmer, 2010)

Figure 1 . 3 :

 13 Figure 1.3: Perception and signaling of Jasmonic Acid (from Acosta & Farmer , 2010) a) In the absence of JA-Ile, the repressor JAZ is bound to transcription factors of the MYC family, thus restraining their action.

Figure 1 . 4 :

 14 Figure 1.4: Relationship of JA with other phytohormonesJA acts in synergy with ABA for the activation of the MYC branch and with ET for the activation of the ERF branch. The antagonism of SA and JA occurs at the transcriptional level where SA inhibits the action of the ORA59 transcription factor from the ERF family thus inhibiting the expression of PDF1.2.

Figure 1 . 5 :

 15 Figure 1.5: JA and systemic signaling (Modified from Nature Protocols -Farmer lab)

Figure 1 . 6 :

 16 Figure 1.6: MAPK cascades are key elements of the stress signal transduction in plantsMAPK cascades can be activated upon the perception of a stress by the plant. They are composed of three levels of kinases activating each other by phosphorylation. The module usually starts with the activation of a MAP3K that phosphorylates a MAP2K on two serine and threonine residues following the consensus S/TxxxxxS/T motif. The activated MAP2K will then in turn activate a MAPK by phosphorylating threonine and tyrosine residues on TEY or TDY motif. At the end, MAPKs will be able to phosphorylate and regulate specific substrates in the cytosol or the nucleus such as enzymes or transcription factors. Those regulations will help the plant to trigger specific defense responses toward the perceived stress.

Figure

  Figure 1.7: 20 MAPKs in Arabidopsis divided into 4 subgroups In Arabidopsis, MAPKs can be divided into 4 groups based on their sequence analogy. A, B and C groups contain TEY phosphorylation motifs whereas members of group D are phosphorylated on TDY motifs.

Figure 1 Figure 1

 11 Figure 1.9: 20 MEKK-like MAP3Ks are divided into 3 clades (from Colcombet et al., 2016) In Arabidopsis, there are 20 MEKK-like MAP3Ks separated into 3 clades. Members of the clade III display a sequence specificity as they do not possess any intron unlike members of clades I and II.

Figure 1 .

 1 Figure 1.11: Biotic and abiotic stresses activating MKK3-dependent pathways A: MKK3 is involved in pathogen signaling. It is transcriptionally induced by P. syringae DC3000 and regulates pathogen-associated genes such as PR1. MKK3 interacts with and activates C-group MAPKs in vitro. It was also shown to activate MPK7 upon H 2 0 2. B: MPK8 is activated by wounding in an MKK3-dependent manner. This activation is also dependent on calcium. The module seems to play a role in ROS homeostasis by regulating the expression of RBOHD and OXI1. C: MKK3 acts upstream MPK6 in its activation by JA and controls the expression of PDF1.2 and VSP2. The MKK3-MPK6 module negatively regulates the root growth inhibition induced by JA. D: MKK3 acts upstream of MPK6 in its activation by blue light. The whole module controls the expression of the MYC2 gene.

Figure 1 .

 1 Figure 1.12: Identification of a complete MKK3-dependent module activated by ABA (Danquah et al., 2015)ABA can activate a module composed of MAP3K17 and 18, the MAP2K MKK3 and the MAPKs of group C, MPK1, 2, 7 and 14. The activation of the module requires a de novo synthesis of the two MAP3Ks. This MKK3-dependent module transduces the ABA signal to regulate drought responses.

  activated by different signals to mediate different responses depending on the context. More directly, an old study had shown the activation of MPK1 and MPK2, which at that time had not been associated with MKK3, by other stresses than ABA such as JA, H 2 O 2 and wounding(Ortiz-Masia et al., 2007). Finally, based on Genevestigator data, we noticed that MAP3Ks belonging to the same group as MAP3K17/18 found upstream MKK3 in the ABA context were subjected to a high transcriptional regulation by diverse stresses. Such an observation suggests a shared regulation mechanism for MAP3Ks of that group.During the first year of my PhD I took advantage of the model plant Arabidopsis thaliana as well as biochemical tools developed in the "Stress Signaling" group to identify stresses able to activate some C-group MAPKs and in particular MPK2. Among the signals that gave a positive response were wounding and jasmonic acid (JA) thus confirming previous results obtained in other groups(Ortiz-Masia et al., 2007). MKK3 being expected to act upstream MPK2 and the other C-group MAPKs, I decided to further characterize MKK3related modules in a context where plants are submitted to wounding. As the majority of studies dealing with wounding, I chose to apply a single (not continuous) mechanical wound by crushing Arabidopsis leaves with a forceps. Taking advantage of several MAPK mutants that were already present in our group as well as newly created lines, my first aim was to identify the complete module (including the MAP3K(s), MKK3 and the downstream MAPKs) that is activated by wounding. All the results that helped me to address that question are gathered in the two first parts of the chapter "Results & Discussions" (I and II). One of the major gaps in our knowledge in plant transduction events is the sequence in which they occur. Thus, it is not quite clear which components induced by wounding are responsible for the activation of MAPKs. Based on data collected in the literature and presented earlier in the introduction, I was able to test different hypotheses. The obtained results pointed out an important role for JA signaling and clarified our vision of signaling pathways induced by wounding; they are presented in the third part of the chapter "Results & Discussions" (III).Finally, after deciphering the signaling pathway leading to the activation of an MKK3-related module by wounding, I was interested in the role of such a module in the activation of defense responses that are induced by wounding. To address that question I mostly relied on plants knocked-out in the MKK3 gene since the latter can be considered as a hub for the modules we studied. In addition to testing the role of MKK3 in the transduction of defense responses to mechanical wounding, I had the chance to study MKK3-related modules in a more "natural" context with the help of colleagues as well as tools that were already set up in our institute. Indeed, as mentioned earlier, wounding is a way to mimic wounds caused by pathogens and particularly herbivorous insects. Thus, our collaboration with Axel Mithöfer from the Max Planck Institute in Jena (Germany) provided new insights on the role of MKK3 in plants attacked by the generalist herbivore Spodoptera littoralis. In addition, since JA turned out to have a key role in the activation of our module, I was also interested in the role of MKK3 in the context of an attack by a necrotrophic pathogen that is known to trigger JA-dependent defense responses. I chose to work with the necrotrophic fungus Botrytis cinerea as it was routinely used by my colleagues. The results obtained regarding the hunt of a role for MKK3related modules are gathered in the two last parts of the chapter "Results & Discussions" (IV and V).

Figure 2 . 1 :

 21 Figure 2.1: MKK3 and sub-clade III MAP3Ks interact in yeast Yeast 2-hybrid experiment showing the interaction of MEKK-like MAP3Ks with MKK3. 16 of the 20 MEKK-like kinases of Arabidopsis and MKK3 were respectively fused to the activation domain (AD) and the DNA binding domain (BD) of a transcription factor allowing the growth of yeast on selective media. L: Leucine; W: Tryptophane; H: Histidine; 3-AT: 3-amino-1,2,4-triazole

  This technique enables to test direct protein-protein interactions in vivo by taking advantage of the simplicity of Saccharomyces cerevisiae transformation and the rapid growth of the latter. It is based on the interaction-dependent reconstruction of a functional transcription factor that will allow the subsequent expression of reporter genes necessary for yeast growth on selective media. 16 of the 20 MEKK-like kinases of Arabidopsis and MKK3 were respectively fused to the activation domain (AD fusion) and the DNA binding domain (BD fusion) of the transcription factor and expressed in yeast cells. Yeast growth was observed only when AD-MKK3 construct was co-transformed with 6 of the tested BD-MEKK constructs (figure 2.1). Interestingly, these MAP3Ks all belong to the sub-clade III of the MEKK-like family (figure 1.9). This result suggests an interaction specificity of MKK3 with

  Figure 2.2: Sub-clade III MAP3Ks, MKK3 and MPK2 constitute a functional module in protoplasts Constructs containing the gene locus of sub-clade III MAP3Ks, MKK3 or MPK2 under a 35S promoter and a specific tag at their C-terminal end (YFP for the MAP3Ks, Myc for MKK3 and HA for MPK2) were created. MPK2-HA, MKK3-Myc and MAP3K-YFP were co-expressed in Arabidopsis mkk3-1 mesophyll protoplasts. Autoradiographs (above dotted lines) show MPK2 activity after immunoprecipitation with α-HA antibodies. Western blots (below dotted lines) show protein amounts of MAP3K-YFP, MKK3-Myc and MPK2-HA after incubation with respectively α-YFP, α-Myc and α-HA antibodies. Coomassie stainings of western blot membranes show total protein amounts of each sample. A: MAP3Ks-YFP and MPK2-HA were co-expressed in the presence or absence of MKK3-Myc. B: All partners were co-expressed in the presence or absence of MG132.

Figure 2

 2 Figure 2.3: Transcriptional regulation of MEKK-like MAP3Ks in Arabidopsis thalianaThe graph shows the distribution of all MEKK-like MAP3Ks according to the number of stresses inducing a strong transcriptional regulation (fold-change > 3, P-value < 0.001). It was designed based on data obtained from Genevestigator. Sub-clade III MAP3Ks are highlighted in gray.

Figure 2 . 4 :

 24 Figure 2.4: General model based on the conserved transcriptional regulation of sub-clade III MAP3KsSub-clade III MAP3Ks, MKK3 and C-group MAPKs (MPK1, 2, 7 and 14) define a functional module activated by various stresses. The activation of the module starts with the transcriptional regulation of the upstream MAP3Ks.

  Figure 2.5: MPK2 is activated by wounding in an MKK3-dependent way Autoradiographs show MPK2 kinase activity through MBP phosphorylation after immunoprecipitation with specific antibodies from plants submitted to wounding. All protein levels were normalized by Bradford assay. A: Col-0 and mkk3-1 plants were wounded following kinetics from 0 to 4 hours. B: mpk1 mpk2 plants were used to confirm the identity of the observed signals. Plants were wounded and MPK2 activity was assayed 1 hour post-wounding. C: Col-0, mkk3-1 and mkk3-2 plants were wounded following kinetics from 0 to 2 hours. D: Col-0, mkk3-1 and mkk3/MKK3locus-YFP plants were used to confirm the role of MKK3. Plants were wounded and MPK2 activity was assayed 1 hour post-wounding.

(

  (Ortiz-Masia et al., 2007), i.e. the activation of MPK2 by wounding, plants were wounded by squeezing their leaves with a forceps (3 leaves per plant) and wounded leaves were harvested at indicated time-points (see "Materials & Methods" for details). MPK2 was then immunoprecipitated using a specific antibody produced by collaborators (referred as α-MPK2)(Ortiz- Masia et al., 2007) and its activity was tested in a classical kinase assay using MBP as a heterologous substrate. I found that MPK2 was indeed activated by wounding in Col-0 plants.MPK2 is transiently activated from 30 minutes to 2 hours after wounding, showing a peak at 1 hour post-wounding (figure 2.5A -first line). The signal was lost in mpk1 mpk2 plants, showing that the observed activity is indeed specific of MPK2 (and MPK1) (figure 2.5B). According to my working model (described in the previous part), MKK3 should be the MAP2K acting upstream MPK1 and MPK2 in the context of an activation by wounding. In order to confirm this hypothesis, Col-0, mkk3-1 and mkk3-2 plants were wounded and MPK2 activity was assayed.Wounding-induced activation of MPK2 was completely lost in both mkk3 mutants (figures 2.5A and 5C). Finally we created mkk3-1 plants complemented with

Figure 2 . 6 :

 26 Figure 2.6: MPK1, MPK2 and MPK7 are not transcriptionally regulated by wounding MPK1, 2 and 7 expression levels were analyzed by RT-qPCR after total RNA extraction from Col-0 and mkk3-1 plants submitted to wounding. Actin2 (AT3G18780) was used as a housekeeping gene. The experiment was performed only once. 0 0,05 0,1 0,15 0,2

Figure 2 . 8 :

 28 Figure 2.8: MPK2 is not activated by gentle touch Autoradiographs show MPK2 activity through MBP phosphorylation after IP α-MPK2 from Col-0, mkk3-1 and mkk3/MKK3locus-YFP plants submitted to a gentle touch stress.

  MAP2Ks to activate MAPKs. Western blots were performed on soluble protein extracts from Col-0 and mkk3 plants submitted to wounding. Two bands (around 44 kDa and 42 kDa) appeared very rapidly upon wounding (from 5 to 30 minutes, with a peak at 15 minutes) (figure 2.9A -second line). Unlike MPK2 activity, the intensity of these two bands was unchanged in mkk3 loss-of-function mutants (figures 2.9A and 2.9B) indicating that the phosphorylation of the MAPKs corresponding to the observed signals is not dependent on

Figure 2 .Figure 2

 22 Figure 2.10: MPK3 and MPK6 have no effect on the activation of MPK2 by woundingAutoradiographs show MPK2 kinase activity through MBP phosphorylation in mpk3-1 and mpk6-2 plants submitted to wounding. Three biologically independent experiments were performed showing highly variable results.

Figure 2 . 13 :Figure 2

 2132 Figure 2.13: Expression of sub-clade III MAP3Ks upon woundingMAP3Ks expression levels were measured in Col-0 plants submitted to wounding by RT-qPCR using specific primers. Actin2 (AT3G18780) was used as a housekeeping gene. This result shows an average of three biologically independent experiments (n=3).

  Figure 2.15: Subcellular localization of MAP3K18-YFP upon ABA and wounding Cotyledons of map3k17/18 MAP3K18locus-YFP plantlets treated with ABA or wounding were observed under a confocal microscope.A: Plantlets were treated with 25 µM ABA and observation was made 4 hours after treatment.B:Cotyledons were wounded with a needle and observation was made 4 hours post-wounding.

Figure 2 Figure 2

 22 Figure 2.17: MPK3 and MPK6 activity upon wounding is unchanged in map3k14-1 plants Western blot after incubation with α-pTpY antibodies shows MPK3 and MPK6 phosphorylation upon wounding in Col-0 and map3k14-1 plants. Coomassie staining shows total protein amount. All protein levels were normalized by Bradford assay.

  expressing mutant versions of MAP3K14 show different levels of MPK2 activit y compared to Col -0 plants After having identified which MAP3Ks were induced by wounding, I wanted to identify which of them act(s) upstream MKK3-C-group MAPKs using a genetic approach. In this purpose, I used map3k mutant plants impaired in the expression of sub-clade III MAP3Ks and looked if MPK2 was still activated by wounding in those plants. To prove that one of our candidate MAP3Ks acts upstream MKK3-MPK2, we expected MPK2 activity to be decreased or totally abolished in the corresponding map3k mutant background(s). I took advantage of lines that were already available in our group. Mutant lines had been ordered through the SIGnAL database and all lines were carrying a T-DNA insertion. The insertion was in the region encoding the kinase domain of MAP3K16 and MAP3K18. Concerning all the others, the insertion was located on the C-terminal part of the gene, after the kinase domain. The intensity of MPK2 activity in tested mutants was compared to the one observed in Col-0 plants. This experiment was repeated several times on biologically independent replicates, giving highly heterogeneous results that are summarized in table 2.2. Interestingly, wounding-induced activation of MPK2 was increased in the map3k14-1 background whereas the opposite effect would have been expected if MAP3K14 acts positively upstream of our module of interest (figure 2.16). I was also interested in the activation of MPK3 and MPK6 in map3k14-1 plants after wounding. Comparing Col-0 and map3k14-1 plants, both MPK3 and MPK6 activation was unchanged in both genetic backgrounds (figure 2.17). However I could observe the appearance of some bands below MPK3. We hypothesize that these bands very likely correspond to MPK2 and other C-group MAPKs which would be consistent with the increase of MPK2 activity observed in the map3k14-1 background.It was curious to see a decrease of MPK2 activity in the map3k14-1 mutant background. Therefore I wondered if this could be due to an up-regulation of other members of sub-clade III to compensate the absence of MAP3K14. To test this hypothesis, I performed RT-qPCR analyses on material from Col-0 and map3k14-1 plants submitted to wounding. Among all MAP3Ks that are induced upon wounding, MAP3K17 is the only one which expression was unchanged in map3k14-1 plants. Indeed, MAP3Ks 15, 18 and 19 expression was higher in map3k14-1 plants compared to Col-0 (figure 2.18). This experiment was performed only once but could suggest a compensation of the mutation of MAP3K14 by the over-expression of other sub-clade III MAP3Ks. Surprisingly, MAP3K14 expression was also

Figure 2 .Figure 2

 22 Figure 2.19: Truncated MAP3K14 is functional in planta and leads to a more stable protein A: Schematic representation of MAP3K14 locus including the position of the T-DNA insertion. The latter is inserted after the region coding for the kinase domain of the MAP3K14 protein. B: Constructs containing the gene locus of one MAP3K (14, 14-1 or 18), MKK3 or MPK2 under a 35S promoter and a specific tag at their C-terminal end (YFP for the MAP3Ks, Myc for MKK3 and HA for MPK2) were created. MPK2-HA, MKK3-Myc and MAP3K-YFP were co-expressed in Arabidopsis mkk3-1 mesophyll protoplasts. Autoradiographs (above dotted lines) show MPK2 activity after immunoprecipitation with HA-specific antibodies. Western blots (below dotted lines) show protein amounts of MAP3Ks, MKK3 and MPK2 after incubation with respectively α-YFP, α-Myc and α-HA antibodies. Coomassie stainings of western blot membranes show total protein amounts of each sample.

  a. Two waves of MAPK activationIn this second part, I identified an almost complete MAPK module activated by wounding in Arabidopsis thaliana. First, I was able to confirm the results previously obtained by Ortiz-Masia and coworkers(Ortiz-Masia et al., 2007) that is the activation of MPK1 and MPK2 by wounding. Thanks to our HA-tagged lines I was also able to extend this observation

Figure 2 .

 2 Figure 2.21: Theoretical model depicting the two MAPK groups activated upon wounding A: Schematic graph showing activation kinetics and peaks of MPK3, MPK6 and MPK2 activities in plants submitted to wounding. Two waves of MAPK activation are visible. B: Theoretical model: the rapid and transient activation of MPK3 and MPK6 is not dependent on MKK3, whereas the rather late activation of MPK1, 2 and 7 is dependent on MKK3.

  another member of C-group MAPKs. My results also confirmed our model according to which MKK3 is the MAP2K acting upstream C-group MAPKs in a wounding context. I was also interested in two other A-group MAPKs, MPK3 and MPK6, that were already described to be activated by wounding and a plethora of other stresses[START_REF] Ichimura | Various abiotic stresses rapidly activate Arabidopsis MAP kinases ATMPK4 and ATMPK6[END_REF][START_REF] Li | AIK1, A Mitogen-Activated Protein Kinase, Modulates Abscisic Acid Responses through the MKK5-MPK6 Kinase Cascade[END_REF]. As expected, their activation by wounding was not dependent on MKK3. To summarize, two waves of MAPKs activation occur upon wounding (figure 2.21A). Firstly, two A-group MAPKs (MPK3 and MPK6) that are already activated after 5 minutes and whose activation is not dependent on MKK3. Secondly, three C-group MAPKs (MPK1, 2 and 7) activated later (from 30 minutes after the stress) and whose activation is dependent on MKK3. Testing the effect of the first wave on the other, no major decrease of MPK2 activity was observed. However, these results do not strictly exclude the possibility for MPK3 and MPK6 to act upstream MKK3-MPK2. Indeed, MPK3 and MPK6 being considered as functionally redundant, it would have been necessary to use plants impaired in both MAPK genes. It has been shown that such plants are lethal[START_REF] Wang | Stomatal Development and Patterning Are Regulated by Environmentally Responsive Mitogen-Activated Protein Kinases in Arabidopsis[END_REF] but alternative genetic tools exist which could help to address this question(Aoyama & Chua, 1997). Recently, mpk3 mpk6 double mutant rescued with mutant versions of MPK3 or MPK6 sensitive to a specific inhibitor were generated and constitute an interesting tool to compensate the lethality of the double mutant(Su et al., 2017). Moreover, the wounding-induced activation of MPK3 and MPK6 was shown to be abolished in mkk4 mkk5 plants, placing those two MAP2Ks upstream MPK3 and MPK6 in a wounding context[START_REF] Li | AIK1, A Mitogen-Activated Protein Kinase, Modulates Abscisic Acid Responses through the MKK5-MPK6 Kinase Cascade[END_REF]. Such a mutant brings out a new tool to compensate the difficulty to obtain plants where both MPK3 and MPK6 are inactive. b. Sub-clade III MAP3Ks are transcriptionall y regulated by wounding Compared to the classical activation of MPK3 and 6 that is rapid and transient, the activation of C-group MAPKs by wounding occurs quite belatedly. Such slow activation of C-group MAPKs was also observed in the context of a treatment by ABA(Danquah et al., 2015) where it was explained by the requirement for a de novo synthesis of the identified upstream MAP3Ks. Therefore we postulated that the same mechanism is taking place in a wounding context (figure 2.21B). The requirement of a de novo synthesis for the activation of the MKK3-MPK2 module by wounding was shown using the inhibitor of translation cycloheximide. In addition, some of the sub-clade III MAP3Ks were indeed found induced by wounding. To prove that the activation of MPK2 is dependent on the transcriptional induction of the candidate MAP3Ks we used plants mutated in those candidate genes. Although no

Figure 2 .

 2 Figure 2.22: Theoretical model depicting the involvement of specific sub-clade III MAP3Ks depending on the wounding-induced component A functional redundancy among sub-clade III MAP3Ks upon wounding is very likely. Such a redundancy could be due to the plethora of signals that are induced by wounding and are each responsible for the activation of specific sub-clade III MAP3Ks.

  Figure 2.24: MPK1, 2 and 7 are activated by JA in a MKK3-dependent manner Autoradiographs show MPK2 kinase activity through MBP phosphorylation after IP with specific antibodies. Western blots show MAPKs protein levels after incubation with α-HA antibodies. Coomassie stainings show total protein amounts. All protein levels were normalized by Bradford assay. A: Col-0 and mkk3-1 plants were treated with ethanol (EtOH) or 50 µM JA. Plant material was harvested up to 4 hours post-treatment. MPK2 activity was assayed after IP with α-MPK2 antibodies. B: Col-0 and mkk3-1 plants expressing MPK2-HA were treated with ethanol or 50 µM JA. MPK2 activity was assayed after IP with α-HA antibodies. Col-0 plants expressing the wild-type version of MPK2 were used as control. C: Col-0 and mkk3-1 plants expressing MPK1-HA or MPK7-HA were treated with ethanol or 50 µM JA.

  flg22 was not able to activate MPK2 (figure 2.23D). As previously, α-pTpY western blot was performed to ensure that the peptide was sensed by the plants. MPK3 and MPK6 were indeed rapidly activated by the flg22 treatment. In contrast, MPK2 was activated 1 hour after treatment with JA and this activation was abolished in mkk3-1 plants therefore demonstrating that MPK2 is activated by JA in an MKK3-dependent way (figure 2.24A). No activation was observed when plants were treated with ethanol 96° (mock control), thus confirming that the observed signal was indeed triggered by JA and not by a mechanical stress caused by the application itself. As among the tested molecules JA was the only one to give a positive result, I went further on the study of its effect on C-group MAPKs activation. As done previously in the context of wounding, I used plants expressing HA-tagged versions of MPK1, 2 and 7 in order to be able to 1) test those two other C-group MAPKs activation by JA and 2) follow MAPKs protein amounts all along the treatment time course. I first tested the activity of MPK2 in Col-0 and mkk3-1 plants expressing MPK2-HA treated with ethanol (mock control) or 50 µM of JA. MPK2-HA was activated by JA from 30 minutes to 2 hours after the treatment and this activation was lost in the mkk3-1 background (figure 2.24B). No activation was observed when ethanol was

Figure 2 .

 2 Figure 2.25: MPK3 and MPK6 are not activated by an exogenous treatment with JA Col-0 and mkk3-1 plants were subjected to treatment with JA, flg22 or EtOH (mock control). Western blot analyses show MPK3 and MPK6 phosphorylation after incubation with α-pTpY antibodies. Coomassie stainings show total protein amounts.

Figure 2 . 27 :Figure 2

 2272 Figure 2.27: MAP3Ks expression upon exogenous treatment with JACol-0 plants were submitted to wounding and samples were harvested following a kinetics up to 4 hours post-treatment. Sub-clade III MAP3Ks expression was quantified by RT-qPCR using specific primers. Actin2 (AT3G18780) was used as a housekeeping gene. The graph shows an average of expression levels obtained from 2 biologically independent replicates. MAP3Ks expression levels are presented as a ratio between their levels in plants treated with JA and those treated with ethanol.

  Figure 2.29: MPK2 activation by wounding is not highly dependent on ATP, OGs or H 2 O 2 signaling Autoradiographs show MPK2 activity through MBP phosphorylation after IP with α-MPK2 antibodies from plants submitted to wounding. A: Col-0 and rbohd rbohf were wounded and leaves were collected 1 hour post-stress. Two independent experiments are shown. B: Col-0 and dorn1-1 plants were submitted to wounding. Leaves were harvested 1 hour post-stress.C: Wild-type (Col-0 and WS) and wak2 (wak2-1 and wak2-2) plants were wounded and leaves were harvested following kinetics up to 1 hour post-wounding.

Figure 2 Figure 2

 22 Figure 2.31: JA does not have a major role upstream of MPK3/MPK6 upon wounding Col-0 and coi1-34 plants were subjected to wounding and samples were harvested following a time course up to 1 hour post-wounding. Western blot shows MPK3/MPK6 activity upon wounding after incubation with α-pTpY antibodies. Commassie staining shows total protein amounts.

Figure 2 . 33 :

 233 Figure 2.33: Expression of candidate MAP3Ks upon wounding is reduced in coi1-34 plants Expression of sub-clade III MAP3Ks in Col-0 and coi1-34 plants submitted to wounding was analyzed by RT-qPCR with specific primers. Actin2 (AT3G18780) was used as a housekeeping gene. Results are displayed as the percentage of expression in coi1-34 compared to Col-0 plants. Average of three biologically independent experiments is shown. Dotted lines indicate the expression level in Col-0 plants, which was brought to 100%.

Figure

  Figure 2.34: MKK3-MPK2 is not activated in systemic leaves A: Autoradiographs show MPK2 activity through MBP phosphorylation in leaves n°8, 9 and 13 of Col-0 and mkk3-1 plants wounded on their leaf n°8. B: Levels of JAZ10 expression were measured by RT-qPCR in leaves n°8, 9 and 13 of Arabidopsis plants. Samples were harvested 1 hour after wounding leaf n°8 of Col-0 plants and also from nonwounded Col-0 plants as controls. C: JAZ10 expression was also measured in mkk3-1 plants. Results are displayed as a ratio of JAZ10 expression between mkk3-1 and Col-0 plants 1 hour after wounding leaf n°8. Dotted lines indicate the level of JAZ10 expression in Col-0 plants, which was brought to 100%. Results of three biologically independent experiments are shown .

  were quite different from one experiment to another, results are displayed as a ratio of JAZ10 expression between mkk3-1 and Col-0 plants. JAZ10 levels were a little higher in leaf n°8 of mkk3-1 plants compared to Col-0 plants (figure 2.34C).

  a. C-group MAPKs, but not MPK3/MPK6, are activated by an exogenous treatment with JA The aim of this third part was to identify wounding-induced second messengers that could link the perception of wounding to the activation of the MKK3-C-group MAPKs module. None of the exogenously applied molecules was able to activate MPK2 except JA, which confirmed the results previously obtained by Ortiz-Masia and coworkers (Ortiz-Masia et al., 2007). Using HA-tagged lines I also showed the JA-induced activation of MPK1 and MPK7. Most importantly my results confirmed that MKK3 is the MAP2K acting upstream Cgroup MAPKs in the context of their activation by JA. Notably, the observed kinetics of Cgroup MAPKs activation by JA was similar to the one observed upon wounding.

Figure 2 .

 2 Figure 2.35: Model depicting the JA-dependent MAPK module activated by wounding MKK3-MPK2 activation is largely dependent on JA signaling. This activation probably goes through the transcriptional regulation of MAP3K14, 15, 17 and 18.

Figure 2 .

 2 Figure 2.36: MKK3-MPK2 is activated by B. cinerea. Autoradiographs show MPK2 kinase activity on MBP after immuno-precipitation with α-MPK2 antibodies from plants with different genetical backgrounds inoculated with B. cinerea spores or PDA. A: MPK2 activity was tested 24 and 48 hours after inoculation with B. cinerea or PDA in Col-0, mkk3-1 and mkk3/MKK3locus-YFP plants. B: MPK2 activity was tested 24 and 48 hours after inoculation with B. cinerea or PDA in Col-0 and mkk3-1 plants. Plants were wounded or not 1 hour prior to the inoculation.

  observed an activation of MPK3 and MPK6, 24 hours after inoculation with B. cinerea. Based on this data I chose to test the activity of MPK2 at late time-points: 24 and 48 hours after inoculation. 4 week-old Col-0, mkk3-1 and mkk3/MKK3locus-YFP plants were inoculated with B. cinerea spores or PDA (mock control) and MPK2 activity was monitored as described previously. In Col-0 plants, while inoculation with PDA could not activate MPK2, the latter was found active 48 hours after inoculation with the fungus whereas no activation could be observed in mkk3-1 background (figure 2.36A). The complementation with a tagged version of MKK3locus restored Botrytis-triggered MPK2 activation. This result which has been replicated three times on independent plants, leads to the conclusion that MPK2 is activated by B. cinerea in an MKK3-dependent way.

Figure 2 Figure 2

 22 Figure 2.38: MKK3-MPK2 is activated by Spodoptera littoralis feeding Autoradiograph shows MPK2 kinase activity on MBP after immuno-precipitation with MPK2specific antibodies in Col-0, mkk3-1 and mkk3/MKK3-YFP plants submitted to S. littoralis feeding. Leaves were harvested 1 hour and 3 hours after the feeding has started.

  Figure 2.40: Lesion outgrowth after inoculation with B. cinerea spores Pictures show lesions caused by spores of B. cinerea on Col-0, mkk3-1, mkk3/MKK3locus-YFP and coi1-16 leaves. Pictures were taken 48 hours after the inoculation.

  Before my arrival in the team, colleagues had tested the role of MKK3 in resistance towards B. cinerea by comparing lesions outgrowth on Col-0 and mkk3-1 plants (Axel De Zelicourt -personal communication). No significant difference could be observed. As stated above, wounding can induce a transient resistance towards B. cinerea and this resistance is all

Figure 2 .

 2 Figure 2.41: Some Botrytis-induced genes expression is misregulated in mkk3-1 plants The expression of several Botrytis-induced genes was quantified by RT-qPCR in Col-0 and mkk3-1 plants. Results are presented as a ratio between gene expression levels in plants inoculated with B. cinerea and those inoculated with PDA only. Actin2 (AT3G18780) was used as a housekeeping gene. The experiment was performed only once (n=1).

Figure 2 .

 2 Figure 2.42: MKK3 is not involved in resistance towards Spodoptera littoralis Weights of larvae fed on Col-0 and mkk3-1 plants were measured 8 days after larvae were put on leaves to feed. Three biologically independent experiments were performed with around 25 measurements for each genotype and each replicate. *: p-value =0,05 (M&W)

Figure 2 .

 2 Figure 2.44: MKK3 is not involved in hormonal regulation upon wounding Col-0, mkk3-1 and mkk3/MKK3locus-YFP plants were submitted to mechanical wounding. Leaves were harvested 1 hour and 3 hours later. SA, JA, JA-Ile and ABA levels were measured. Each bar represents an average of 3 independent samples.

Figure 2 .

 2 Figure 2.45: Role of MKK3 in the regulation of genes involved in SA and JA biosynthesis pathways Expression levels were measured by RT-qPCR in Col-0, mkk3-1 and mkk3/MKK3locus-YFP plants submitted to insect feeding. Actin2 (AT3G18780) was used as a housekeeping gene. The experiment was performed only once (n=1).

Figure 2 . 46 :

 246 Figure 2.46: Role of MKK3 in the regulation of SA-and JA-responsive genes JAZ10, VSP2, PDF1.2 and PR1 genes expression levels were measured by RT-qPCR in Col-0, mkk3-1 and mkk3/MKK3locus-YFP plants submitted to insect feeding. Actin2 (AT3G18780) was used as a housekeeping gene. The experiment was performed only once (n=1).

Figure 2 .

 2 Figure 2.47: Wounding-induced trichome formationCol-0 and mkk3-1 plants were regularly wounded on their cotyledons and two first leaves and trichomes were counted on leaf n°5 after 12 days.A:The graph shows trichome numbers on leaf n°5 of Col-0 and mkk3-1 plants. Each bar represents an average of around 10 measurements.B: Pictures of trichomes on leaf n°5 of Col-0 plants.

Figure 2 . 49 :

 249 Figure2.49: Theoretical model on the role of MKK3 in phytohormone regulation upon S. littoralis feeding MKK3 negatively regulates JA and SA levels upon insect feeding. Assuming that JA could also have a key role upstream MKK3-MPK2 in the activation upon S. littoralis feeding, a feedback loop would exist that acts to restrain the duration of MKK3-dependent responses.

Figure 3 . 1 :

 31 Figure 3.1: Hypothetical model on the interdependent activation of two MAPK modules upon woundingUpon wounding, the MKK4/MKK5-MPK3/MPK6 module is rapidly activated and leads to the synthesis of JA. The production of JA activates MKK3-dependent modules through the transcriptional regulation of MAP3Ks. The whole cascade controls ethylene synthesis.

  Figure 3.2: MPK7 is activated by nitrate in an MKK3-and NLP7-dependent manner Autoradiographs show MPK7 activity through MBP phodphorylation after IP with α-HA antibodies. Western blot show MPK7 protein amount after incubation with α-HA antibodies. Coomassie stainings show total protein amount.

Figure 1 .

 1 Figure 1.11 tries to summarize literature about sub-clade-III MAP3Ks, MKK3 and C-group

Figure 3 . 3 :

 33 Figure 3.3: mkk3-1 plants show increased dormancyCol-0, mkk3-1 and mkk3/MKK3locus-YFP seeds were harvested differentially and sown on a new medium. Germination rate was calculated 4 days later.



  8) c. Soluble proteins extraction from A. thaliana plant material  Laccus Buffer (10X): 250 mM Tris (pH 7.5), 50 mM EGTA (pH 7.5), 50 mM EDTA (pH 7.5), 750 mM NaCl and 0.5% Triton X-100 Extraction buffer: 10% Laccus Buffer 10X, 20 mM NaF, 20 mM β-glycerophosphate, 5 mM DTT and EDTA-free protease inhibitors cocktail (cOmplete ™ , Mini, EDTA-free Protease Inhibitor Cocktail -Roche) d. Western blot  Transfer Buffer (10X): 0.25 M Tris and 1.92 M glycine  Transfer Buffer (1X): 70% H 2 0, 10% Transfer Buffer 10X and 20% ethanol 96°.  TBS Buffer (10X): 1 M Tris and 1.5 M NaCl  TBS-Tween (TBS-T) (1X): 90% H 2 0, 10% TBS 10X and 0.1% Tween-20 of Arabidopsis leaves For all the wounding experiments, 4 week-old Arabidopsis leaves were squeezed with a forceps through their midveins. Leaves were wounded three times, thus covering approximately 90% of their surface. For each time point and genotype, three leaves from three different plants (9 in total) were wounded. For time courses, leaves were wounded at appropriate time during the day and all samples were harvested at the same time, usually around 1:00 pm. Samples were put in 1.5 mL Eppendorf tubes, frozen in liquid nitrogen and stored at -80°C for further experiments. Wounding usually started at 9:00 am. Here is a timeline of a typical wounding experiment. b. Cycloheximide (CHX) treatment CHX (Sigma-C4859) stock solution (100mg/mL) was kept at 4 °C. When CHX was applied prior to wounding, plants were all sprayed at the same time, 1h30 before first plants were wounded. A solution containing 100 µM CHX in DMSO diluted in water (14 µL for 50 mL)

  Yeast cells were collected by centrifugation at 2500 rpm for 10 seconds. The pellet was washed with 1 mL of 1X TE buffer and re-suspended in 100µL of 1X TE buffer. The transformed yeast cells were grown on a selective media (-L-T) for 2/3 days at 28 °C. Interaction test: single colonies of transformed yeasts were grown in 500µL of liquid selective media (-L-T) overnight at 30 °C. Cells were then dilutes 20 times in water and 5 µL droplets were spotted on selective media -L-T-H + 3AT 60 mM. Yeast growth was monitored at 28 °C for 3 days b. Construction of CRISPR-generated mutants Two primers (Cs9-3K13&14-F: ATTGTCCGCTTTGGATGGCTCCGG; Cs9-3K13&14-R: AAACCCGGAGCCATCCAAAGCGGA) to create a CRISPR guide targeting a 100% conserved sequence shared by both MAP3K13 and MAP3K14 were designed following manufacturer instructions. pDGE65 was digested (5 µL DNA 200 ng/µL, 1 µL buffer for T4DNA Ligase (NEB), 3 µL H20, 1 µL Bbs1 Thermo, 37 for 2 hours) and primers were ligated (4.5 µL of the previous digestion mix, 3 µL primers mix (50 pmole/µL each), 1µL buffer for T4DNA Ligase (NEB), 0.5µL T4 Ligase NEB, incubation overnight 16°C).

FIGURE 1 |

 1 FIGURE 1 | Overview of MKK3 implication in MAPK modules in Arabidopsis thaliana. MKK3 functions upstream of MPK1/2/7/17 in response to H 2 O 2 (A) and ABA (E), of MPK8 in response to wounding (B) and with MPK6 in response to jasmonic acid (JA) (C) and red light (D).

FIGURE 2 |

 2 FIGURE 2 | Are MAP3K of clade III building a functional module with MKK3-MPK1/2/7/14? (A) MEKK-like MAP3Ks are organized in three subclades with distinct locus organization (adapted from Danquah et al., 2015). Only members of clade III are interacting with MKK3 (determined using yeast 2 hybrid assay). n.d., not determined. (B) Number of conditions reported in Genevestigator as able to transcriptionally regulate MAP3K genes (fold-change > 3, P-value < 0.001). MAP3Ks of clade-III are shown in gray. (C) General working model for the activation of MKK3-MPK1/2/7/14 module by the stress-dependent transcriptional regulation of MAP3Ks of clade-III.

  Caractérisation d'un module MAPK impliqué dans la réponse à la blessure chez Arabidopsis thaliana Chapitre I : Vers un modèle général d'activation de modules MKK3-dépendants Des études ont montré que MKK3 interagit seulement avec les MAPKs du groupe C (MPK1, 2, 7 et 14). Dans le contexte d'une activation par l'ABA, les MAP3Ks 17 et 18 ont été identifiées comme agissant en amont de MKK3 et les MAPKs du groupe C. Une régulation transcriptionnelle de ces MAP3Ks a été montrée comme étant nécessaire à l'activation du module. En se basant sur ces données, nous nous sommes demandés si ce mode de régulation pourrait être généralisé à tous les modules MKK3-dépendants et avons construit un modèle qui a conduit à la publication d'une revue. Dans un premier temps nous avons réalisé une expérience de double-hybride pour tester l'interaction en levure de MKK3 avec les 20 MAP3Ks fonctionnelles chez Arabidopsis thaliana. Parmi ces MAP3Ks, MKK3 interagit seulement avec 6 des 8 MAP3Ks du sousclade III (MAP3K15 à MAP3K20). Par la suite et afin de valider la fonctionalité d'un module contenant les MAP3Ks du sous-clade III, MKK3 et les MAPKs du groupe C, les différents partenaires du module ont été exprimés transitoirement en protoplastes. L'activité de MPK2 a été testée par kinase assay en présence des différentes MAP3Ks avec ou sans MKK3. MPK2 est activée lorsque les MAP3Ks du sous-clade III sont co-exprimées, confirmant ainsi la fonctionalité in planta d'un tel module. De plus, les données Genevestigator ont fait apparaître que les MAP3Ks du sous-clade III sont particulièrement régulées par divers stress en comparaison avec les autres MAP3Ks. En nous basant sur ces données nous avons donc construit un modèle selon lequel différentes MAP3Ks du sous-clade III sont recrutées en fonction du stress perçu et conduisent à l'activation de modules MKK3-dépendants.Chapitre II : Identification de nouveaux modules MAPK activés par la blessure Dans cette partie, mes résultats ont permis d'identifier un module presque complet activé par la blessure et impliquant la MAP2K MKK3. Mes essais kinase ont pu montrer que les MPK1, 2 et 7 sont activées par la blessure. Leur activation commence 30 minutes après la blessure et dure jusqu'à 2h post-stress avec un pic à 1h. Les niveaux de protéine ne sont pas affectés par la blessure. L'utilisation de mutants mkk3 a pu montrer que cette activation est dépendante de MKK3, donc que MKK3 est la MAP2K en amont des MAPKs du groupe C dans le contexte de la blessure. En revanche ces MAPKs ne sont pas activées par un simple toucher. En parallèle nous avons étudié l'activation des MAPKs 3, 4 et 6 connus pour être rapidement activées par de nombreux stress. Par western-blot en utilisant des anticorps reconnaissant spécifiquement les MAPKs phosphorylées, j'ai pu montrer que les MAPK 3 et 6 sont effectivement rapidement activées par la blessure (dès 5 minutes). En revanche leur activation est indépendante de MKK3 comme l'atteste la présence de leur activation dans le mutant mkk3. En résumé j'ai pu observer deux vagues d'activation des MAPKs. D'abord les MAPKs 3 et 6 activées dès 5 minutes après la blessure indépendamment de MKK3. Puis les MAPKs du groupe C activées bien plus tardivement et dépendamment de MKK3. Cette observation nous a conduits à émettre l'hypothèse selon laquelle la première vague d'activation influencerait la seconde. Pour tester cette idée, j'ai testé l'activation de MPK2 par la blessure chez les simples mutants mpk3 et mpk6. Les résultats obtenus sont très variables et ne nous ont par permis de tirer une conclusion définitive. Dans un second temps nous avons voulu savoir si l'activation des MAPKs du groupe C nécessite une régulation transcriptionelle. Pour tester cette idée, j'ai utilisé un inhibiteur de la traduction, le cycloheximide (CHX). Lorsque les plantes sont traitées par le CHX avant la blessure, aucune activation de MAPK du groupe C n'est observée. La production des protéines de MAPKs ou de MKK3 n'est en revanche pas affectée. Ce résultat suggère que l'activation des MAPKs du groupe C par la blessure requiert la régulation transcriptionelle d'une protéine agissant en amont de MKK3. L'effet du CHX a également été testé sur l'activation des MAPKs 3 et 6. Le CHX conduit à une activation constitutive de ces MAPKs, indépendamment de la blessure. L'étape d'après consistait à identifier les MAP3Ks en amont de MKK3 dans le contexte de blessure. Afin de savoir si, comme dans le contexte d'une activation par l'ABA, l'activation des MAPKs nécessite une régulation transcriptionelle des MAP3Ks, j'ai testé l'expression des MAP3Ks du sous-clade III en réponse à la blessure. J'ai pu quantifier cette expression par une approche de RT-qPCR. L'expression de la MAP3K14 est rapidement et fortement induite par la blessure. Plus tardivement les MAP3Ks 15, 17, 18 et 19 sont également induites mais moins fortement. En revanche pour des raisons techniques les niveaux de protéines des MAP3Ks n'ont pas pu être quantifiés. Nous avons pu étudier la localisation subcellulaire de la MAP3K18 en réponse aux deux stress en réponse auxquels elle est induite : l'ABA et la blessure. Après un traitement ABA, la MAP3K18 est exprimée dans les cellules de garde. En revanche en réponse à la blessure la MAP3K18 est exprimée dans les cellules épidermiques. Pour aller plus loin dans l'identification des MAP3Ks en amont de MKK3, j'ai testé l'activation de MPK2 dans divers mutants map3k. Etant donné que la MAP3K14 était fortement et rapidement induite par la blessure, elle est une bonne candidate pour agir en amont de MKK3 et des MAPKs du groupe C dans les points précoces de leur activation par la blessure. Etonnamment, l'activation de MPK2 par la blessure est plus forte et un peu plus précoce dans le mutant map3k14-1. Après avoir quantifié l'expression du gène MAP3K14 dans ce mutant nous avons réalisé que le mutant permet l'expression d'une version tronquée de la MAP3K14 qui est surexprimée par rapport à la version sauvage dans des plantes sauvages. Afin de savoir si une telle forme tronquée est toujours fonctionelle et peut effectivement permettre l'activation des MAPKs en aval, j'ai exprimé cette forme tronquée en protoplastes en présence de MKK3 et MPK2. MPK2 est activée même quand la forme tronquée de la MAP3K14 est co-exprimée. De plus, cette protéine tronquée est visible en western-blot contrairement à la forme sauvage. Cela suggère que la forme tronquée est plus stable que la forme sauvage, ce qui pourrait expliquer la sur-activation de MPK2 dans le mutant exprimant cette forme tronquée. Nous avons par la suite crée des mutants perte de fonction par la méthode CRISPR-Cas 9 mais les expériences ont donné des résultats très variables. Chapitre III : Identification d'éléments de signalisation en amont de l'activation des MAPKs en réponse à la blessure Afin d'identifier les acteurs de la signalisation agissant en amont de l'activation des MAPKs par la blessure, nous avons d'abord tester certaines molécules pour leur capacité à activer les MAPKs du groupe C lorsqu'elles sont appliquées de manière exogène. Plusieurs molécules ont été testées dont l'H2O2, l'acide jasmonique (JA), les oligogalacturonides et l'ATP. Seul le JA est capable d'activer les MAPKs du groupe C. Comme dans le cas de la blessure, cette activation est dépendante de MKK3. Les niveaux de protéines correspondantes ne sont pas affectés par le traitement. Par comparaison les MAPKs 3 et 6 ne sont pas activées par le JA alors qu'elles sont rapidement activées par la blessure.Afin de savoir si le JA a un rôle important en amont de l'activation des modules MKK3-dépendants en réponse à la blessure, j'ai utilisé divers mutants de biosynthèse et de perception du JA. Dans tous les cas, une forte diminution de l'activité de MPK2 a pu être observée, suggérant ainsi que le JA a un rôle prépondérant en amont de MKK3. J'ai également pu étudier l'expression des MAP3Ks induites par la blessure dans les mutants JA.Cette expression est diminuée de manière importante dans ces mutants en comparaison avec les plantes sauvages.Le JA est une phytohormone impliquée dans la signalisation longue distance dans le contexte de blessure ou d'attaque herbivore. Pour cette raison je me suis intéressée à l'activation de MKK3/MPK2 dans des feuilles situées à distance du site de blessure. Aucune activation de MPK2 n'a pu être observée dans ces feuilles.Chapitre IV : Activation de modules MKK3-dépendants par des pathogènes dégradant la paroi cellulaire La blessure est un important signal qui peut être déclenché par des facteurs abiotiques tels que la pluie ou le vent mais surtout par des agents biotiques tels que les champignons, les bactéries ou les insectes herbivores. ¨Parmi les pathogènes de plantes, le JA a été montré comme ayant un rôle important en réponse aux champignons nécrotrophes ainsi qu'aux insectes herbivores. Nous avons donc décidé d'étudier le module MKK3/MPK2 en réponse au champignon nécrotrophe Botrytis cinerea et à l'herbivore Spodoptera littoralis. MPK2 est activée 48h après une inoculation avec des spores de B. cinerea. Cette activation est dépendante de MKK3. De la même façon, MPK2 est activée 1h après une attaque par S. littoralis, dépendamment de MKK3. Chapitre V : Etude du role de MKK3 en réponse à la blessure Dans cette dernière partie, nous avons étudié le rôle de MKK3 et des modules MKK3dépendants en réponse à la blessure et à des stress liés. Dans un premier temps nous avons étudié la résistance des plantes mkk3 aux deux pathogènes B. cinerea et S. littoralis précédemment montrés comme activateurs du module. Aucune différence n'a pu être observée entre les plantes sauvages et les plantes mutantes en réponse à ces pathogènes. Nous avons également effectué une étude transcriptomique comparant des plantes sauvages et mkk3-1 2h après une blessure. 5 gènes ont été identifiés comme étant différemment exprimés dans les deux fonds génétiques. Cependant aucun gène marqueur e la blessure ou du JA n'a été identifié.ConclusionEn conclusion, mon travail de thèse a permis d'identifier un module MAPK comprenant MKK3 et les MAPKs du groupe C et activé par la blessure ainsi que par des pathogènes induisant de la blessure. Dans ce contexte, la MAP3K14 a un rôle important en amont de MKK3. La signalisation JA semble également avoir un rôle prépondérant en amont de l'activation du module. En ce qui concerne le rôle du module, aucun phénotype n'a pus être trouvé pour le moment pour le mutant mkk3-1. Un point intéressant est la présence de 2 vagues d'activation de MAPKs. Il serait intéressant d'aller plus loin dans cette direction et de tester l'activation de MAPKs du groupe C dans le double mutant mkk4 mkk5. En effet une étude récente a montré que MKK4 et MKK5 sont les deux MAPKs en amont de MPK3 et 6 en réponse à la blessure.

1. MAPKs are important stress responsive modules in plants

	Among all cellular actors found in plants cells are kinases whose function is to
	phosphorylate and thus regulate specific substrates. Phosphorylation events are part of the
	most widespread post-translational modifications that proteins undergo. In Arabidopsis, 5% of
	the genome encodes protein kinases (The Arabidopsis Genome Initiative, 2000). Among
	them, nearly 10% are involved in MAPK signaling pathways. Coherently, a majority of
	cellular proteins are thought to be phosphorylated.
	Due to the rather large amount of articles published on MAPKs and their role in
	stress signal transduction, it is difficult to describe them exhaustively here. I decided to briefly
	present the most well established module involving the MAPKs 3, 4 and 6 and to focus on the
	state of art concerning the module that is at the heart of my project. Thereafter I present the
	current knowledge about the involvement of MAPKs in the context of wounding and/or
	herbivory. Presented data mostly come from studies carried out on Arabidopsis and tobacco.
	a. MAPK modules are abundant in plant genomes
	Mitogen-Activated Protein Kinases (MAPKs) are very well conserved proteins
	among all eukaryotes, from yeast to vertebrates. They were first described in mammals and
	are now largely studied in plants. They act in signaling cascades activated upon a stress
	perception, transducing environmental cues into intracellular responses (figure 1.6). The
	activation of the module commonly starts with a MAPK kinase kinase (MAP3K or MEKK)

Table 2

 2

	Kinase	Conditions
	MAP3K13	Inhibited during germination
	MAP3K14	IAA, High light, NO 3 , drought , hypoxia
	MAP3K15	PAMPs, ABA, salt
	MAP3K16	Iron and sulfure deficiency, salt, auxin
	MAP3K17	ABA, drought
	MAP3K18	ABA, drought, high light, salt, Pst DC3000, MeJA

.1: Main conditions under which sub-clade III MAP3Ks expression are regulated.

  regulation were identified and are summarized in table 2.1. In addition, we noticed that subclade III MAP3Ks were the ones showing the highest levels of transcriptional changes under the different perturbations, suggesting that they undergo more important regulations than the other MEKK-like MAP3K genes. These observations support our idea of a conserved transcriptional regulation of sub-clade III MAP3Ks upon diverse environmental cues and place those cues as potential activators of MAPK modules involving such MAP3Ks.

Table 2 .2: Testing the wounding-induced activation of MPK2 in map3k mutants led to variable results

 2

  wounding. I tested the ability of some of them to activate MPK2 when applied exogenously on plants. For simplicity, 10 day-old plantlets grown on liquid ½MS medium were used. The molecule was applied directly into the medium, to the roots (see Materials & Methods for

	more detail). The different molecules tested were: H 2 O 2 (hydrogen peroxide), ATP
	(Adenosine-Tri-Phosphate), OGs (Oligogalacturonides) and JA (Jasmonic Acid). MPK2
	activity was tested as previously after IP with α-MPK2 antibodies. No activation of MPK2
	was observed when Col-0 plants were treated with H 2 O 2 , ATP and OGs (figure 2.23A, B and

37: MAP3K19 is highly induced 48 hours after inoculation with B. cinerea MAP3Ks

  MPK2 was found activated in Col-0 plants 48 hours after inoculation with B. cinerea. This activation was lost in mkk3-1 plants. Interestingly, a decrease of activity can be noticed in Col-0 plants when plants are prewounded. This decrease was observed in at least two other independent experiments (not shown). It is likely due to the resistance of pre-wounded plants to the fungus. Indeed, prewounding induces a lower development of B. cinerea on Arabidopsis leaves which very likely leads to a lower activation of MPK2. Because MKK3-MPK2 module was activated by B. cinerea, we wondered which of our candidate MAP3Ks could act upstream the module in this context. As discussed previously, transcriptional regulation of MAP3Ks seem to be a key event in the activation of

			300
	Fold change (compared to non-	stressed)	0 50 100 150 200 250	B. cinerea Wounding + B. cinerea Wounding + B. cinerea B. cinerea Wounded Wounding
	Figure 2.
				. Based on
	this data I decided to test the activity of MPK2 in leaves submitted to wounding prior to the
	fungal inoculation. MPK2 activity was monitored as described above, 24 and 48 hours after

Botrytis or PDA inoculation of plants which had been pre-wounded with a needle or not. No MPK2 activation was observed in the absence of B. cinerea (with or without wounding) in expression levels were measured in Col-0 plants inoculated with B. cinerea spores, with or without a pre-wounding. Levels were measured by RT-qPCR using specific primers. Actin2 (AT3G18780) was used as a housekeeping gene. The experiment was performed only once (n=1).

both Col-0 and mkk3-1 plants (figure 2.36B).

MKK3-dependent modules. Col-0 plants were inoculated with B. cinerea spores or PDA alone with or without pre-wounding. Candidate MAP3Ks expression levels were measured 48 hours post-inoculation. Results are displayed as a ratio between gene expression levels in plants inoculated with B. cinerea (with or without pre-wounding) and those inoculated with PDA only. We found that MAP3Ks were not induced by wounding alone at this late timepoint (figure 2.37). After inoculation with B.cinerea, we observed a 250-fold induction of MAP3K19. Interestingly this induction was totally abolished when plants were wounded prior to the inoculation. This absence of MAP3K induction when plants were pre-wounded is consistent with the decrease of MPK2 activation and suggests a role for MAP3K19 as the potential MAP3K acting upstream MKK3-MPK2 in the response to B. cinerea. However this experiment was performed only once and would need to be repeated.

  and then test if our MAPK module of interest could play a role in this phenomenon. Therefore, I compared Col-0 and mkk3-1 plants in their ability to prime defenses against B. cinerea after wounding. I chose to wound leaves 1 hour prior to inoculation with fungal spores and favored the use of a needle. Leaves were punctured once on both sides of the midvein and were inoculated, on the wounding site, with B. cinerea spores or PDA as a mock control. The level of resistance of different plants was compared

based on the size of outgrowing lesions. These assays were carried out several times (n>3), mostly blindly with different controls. Overall, they gave highly heterogeneous results that I chose not to display extensively here (table 2.3). One example of Botrytis-caused lesions on

  . As stated above, orthologs of MPK3 and MPK6 in tobacco (WIPK and SIPK) are involved in wounding-induced JA production and I showed that JA is an important activator of MKK3-dependent modules. Taken together, these data indicate that the activation of MKK3-C-group MAPKs could depend on the MKK4/5-MPK3/6 module. To test this hypothesis, the use of the mkk4 mkk5 double mutant will be very useful. It would also be necessary to measure wounding-triggered JA levels in mkk4 mkk5 mutants to check whether hormonal regulations are conserved among species. Finally, in

	Arabidopsis, MPK3 and MPK6 were shown to regulate wounding induced ET production by
	regulating the expression of ACS genes involved in its biosynthesis. Therefore, if the
	dependency of MKK3-C-group MAPKs activation towards MPK3/6 is confirmed, it would be
	interesting to measure wounding-induced ET levels in plants impaired in MKK3. Our
	hypothetic model based on such interdependency is illustrated in figure 3.1.

  Chardin and Sebastian Schenk on MPK7 activation in response to nitrogen (which has been performed at the same time in the laboratory, in the frame of a collaboration with Anne Krapp not published) enlarged this working model to other stimuli, suggesting that the ABA-

	dependent regulation of MAP3K18 is not anecdotic, but rather a shared mechanism for the
	activation of MAPK modules. The starting point of the study of MKK3-dependent modules in
	nitrogen signaling was that MAP3K13 and MAP3K14 appeared strongly and rapidly regulated
	by NO 3	-(nitrate) resupply of previously starved plantlets (Marchive et al., 2013). This
	regulation is under the control of NIN-like protein 7 (NLP7), a major transcription factor
	involved in nitrate signaling in roots which relocates from cytosol to nucleus upon NO 3	-and
	is able to bind promoters of many NO 3	--responsive genes including MAP3K13, 14, 17, 18 and
	19. All those MAP3Ks are members of the sub-clade III MAP3Ks. Therefore according to our
	model, MKK3 and C-group MAPKs should be downstream of those MAP3Ks in a nitrate
	context. Camille Chardin and Sebastian Schenk assayed the activity of MPK7 in Col-0, mkk3-
	1 and nlp7 plants after nitrate resupply (figure 3.2). MPK7 was activated by nitrate, less by
	nitrite (NO 2	-

  . And as a matter of fact, 6 MAP3Ks of sub-clade III are able to interact with MKK3 in Y2H and the whole clade activates MKK3-MPK2 in Arabidopsis mesophyll protoplasts. It would be wise to follow up with the CRISPR-Cas9 technology, that we managed to use to build some map3k single and double mutants, in order to build multiple mutants and thus identify exactly the MAP3Ks acting upstream our module in the context of various stresses.Throughout this work, mkk3-1 was used under the assumption that plants impaired in MKK3 are also indirectly impaired in MPK1, 2, 7 and 14. Those 4 MAPKs being closely related, it is likely that they also share functional similarities. And indeed, we assessed several times MPK1, MPK2 and MPK7 activation in the same samples in response to nitrate or wounding and found that they behave very similarly. Once the role of mkk3-1 is assessed it would be interesting to confirm it using a quadruple mpk1/2/7/14 mutant which is supposed to have an identical phenotype. Two simple hypotheses may explain the duplication of C-group MAPKs. A first possibility is that they have the same function but are not expressed in the same cell types or at the same moment, complementing the lack of each other. The study of promoter::GUS lines will help to address this question. The other possibility is that they do not share same substrates. Identifying them will clearly help to validate this possibility.

	candidate MAP3Ks upon wounding as well as initiate biochemical approaches to identify
	PTMs on MAP3Ks.
	c. A m ysterious and conserved NTF2 -like domain in the C -terminal end
	of MKK3
	MKK3 is the only member of group B and is characterized by the presence of a
	NTF2-like domain at its C-terminal end. This domain, which is conserved in all plant
	MKK3s, was not characterized so far. It could have a role in the nuclear addressing of MKK3
	upon a given stress. Once the cellular localization of MKK3 upon stresses is assessed, it could
	be interesting to compare it to plants expressing a modified MKK3 lacking its NTF2 domain.
	b. Are MAP3Ks also regulated by phosphorylation?
	Although sub-clade III MAP3Ks are transcriptionally induced by numerous stresses,
	a post-translational regulation is not to exclude. MAP3K18 de novo synthesis is required for
	the activation of MKK3-MPK7 by ABA with a rather slow kinetics (4 hours post stress).
	However, other studies have shown a more rapid activation of C-group MAPKs by ABA
	(from 15 minutes post-stress) (Ortiz-Masia et al.; 2007; Umezawa et al., 2013) which
	suggests that a post-translational activation of MAP3Ks is possible. Using Arabidopsis lines
	constitutively expressing tagged versions of MAP3K18, another study showed the increase of
	MAP3K18 activity 15 minutes after ABA treatment (Matsuoka et al., 2015). Such differences
	could be explained by different experimental conditions between laboratories. In any case, it
	would be interesting to develop genetic tools to be able to test directly the kinase activity of

Table 4 . 1 :

 41 Mutant lines mentioned in this manuscriptConidial stock suspension of Botrytis cinerea is taken from the -80°C stock and let to thaw at room temperature. The conidial suspension is then spread on Petri dishes containing homemade PDA medium (20 % potato infusion, 2% sucrose and 2% agar) and is let to develop at room temperature. Botrytis cinerea cultures are freshly prepared prior each experiment.

	Allele name	Ecotype	Type of mutation	Provided by	Reference
	aos	Col-0	T-DNA		Park et al., 2002
	coi1-16*	Col-0	Substitution		Ellis & Turner, 2002
	coi1-34*	Col-0	Substitution	E.E. Farmer	Acosta et al., 2013
	dorn1-1	Col-0	Substitution	G. Stacey	Choi et al., 2014
	HAB	Col-0	Substitution		Robert et al., 2006
	map3k13-1	Col-0	T-DNA (GABI 277E09)	A. Krapp
	map3k14-1	Col-0	T-DNA (GABI 653B01)	A. Krapp
	map3k15 map3k16	Col-0	T-DNA (SALK 084817/SALK 003255)
	map3k17 map3k18	Col-0	T-DNA (SALK137069/GABI244G02)		Danquah et al., 2015
	map3k19	Col-0	T-DNA (WiscDsLoxHs225_01B)
	mkk3-1	Col-0	T-DNA (SALK 051970)		Dóczi et al., 2007
	mkk3-2	Col-0	T-DNA (SALK 208528)
	mpk1 mpk2	Col-0	T-DNA (SALK 063847/SALK019507)	M.J. Marcote	Ortiz et al., 2007
	mpk3-1	Col-0	T-DNA (SALK 151594)	S. Ferrari	Galletti et al., 2011
	mpk6-2	Col-0	T-DNA (SALK 073907)	S. Ferrari	Galletti et al., 2011
	opr3	WS-0	T-DNA (intron)		Chehab et al., 2011
	pepr1 pepr2	Col-0	T-DNA (SALK 059281/SALK 098161)		Krol et al., 2010
	rbohd rbohf	Col-0	T-DNA		Torres et al., 2002
	wak2-1	Col-0	T-DNA (SAIL 286E03)	M. Fagard	Kohorn et al., 2006
	wak2-2	WS-0	insertion (FLAG 031E03)	M. Fagard
	* coi1-16 plants contain a second mutation in the PEN2 gene (Ellis & Turner, 2002) and
	are male fertile under 20°C. coi1-34 plants are partially male sterile.

  For the study of systemic responses, Arabidopsis plants were grown on soil in Percival chambers. Leaves were wounded following the online Nature protocol "Leaf numbering for experiments on long distance signaling in Arabidopsis" from Farmer lab(Mousavi et al., This protocol was developed by Jane Sheen's laboratory(Niu & Sheen, 2007).

	d. Leaf numbering g. Production of Arabidopsis mesophyll protoplasts
	Table 4.3: Preparation of the enzymatic solution
			Final [C]	For 10 mL
	2013).		Cellulase R10	1,50%	0,15 g
			Macerozyme R10	0,40%	0,04 g
	Mannitol 800 mM e. Trichome counting KCl 2 M	400 mM 20 mM	5 mL 100 µL
	Plants were grown on soil in Percival chambers. The protocol was adapted from Poudel et al., MES 200 mM pH 5,7 20 mM 1 mL
	2016. Cotyledons and two first leaves of 2 week-old Arabidopsis plantlets were wounded with H 2 O 3,7 mL
	Heat at 55°C for 10 min intervals over the course of 12 days. At day12, trichomes were observed on leaf n°5 under a and cool down at RT°
	stereomicroscope. Pictures were taken and trichomes were counted manually with ImageJ CaCl 2 1M 10 mM 100 µL
	software.		BSA 10 %	0,10%	100 µL
	Day time
	Corresponding time point	4h	2h	1h	30'	15' Collection
	Table 4.2: Exogenous treatments applied on Arabidopsis plantlets
	Applied molecule		Reference	Stock concentration	Diluted in Stored at	Final concentration
	JA	Sigma Aldrich J2500	50 mM		Ethanol	-20 °C	50 µM
	peptide QRLSTGSRINSAKDDAAGLQIA
	flg22	was synthesized by GeneCust	1 mM		H 2 O	-80°C	1 µM
		to a purity > 85%
	ATP			10 mM		H 2 O	-20 °C	100 µM
	OGs	provided by Pr. Felice Cervone	5 mg/mL		H 2 O	-20 °C	50 µg/mL
	ABA	Sigma Aldrich A1049	50 mM		Ethanol	-20 °C	50 µM
	H 2 O 2	Sigma Aldrich 95294	≈10 M		H 2 O	4 °C	1 mM

f. Exogenous treatment of Arabidopsis plantlets

Seeds were sterilized in a solution of EtOH 76°C + 5% Triton for 10 minutes and a second time in EtOH 76 °C only. 10 seeds were sown on each small Petri dish containing 8mL of the previously prepared ½ MS medium. All exogenous treatments were applied directly in the ½ MS medium, to the roots of 10 day-old Arabidopsis plantlets.

Table 4

 4 Protoplasts are counted on a hematometer and resuspended in MMg buffer for a final concentration of 2.10 5 protoplasts/mL. Bottoms of small Petri dishes are covered with 1mL CS 5% (calf serum). This coating prevents the protoplasts from stick to plastic walls. 800 µl of buffer WI is added to each Petri dish. For each transformation: 200 µL of protoplasts (2.10 5 /mL), 20 µL of DNA (2µg/µL), and 200 µL of PEG solution are used. The whole is left to incubate 5 minutes at room temperature. Botrytis cinerea inoculation Conidia were resuspended in liquid potato/dextrose (PD) medium and filtered through Miracloth. Using a microscope, spore density was adjusted to 5.10 5 conidiospores/mL. Spodoptera littoralis feeding assays were all performed by our collaborators in the Max Planck Institute of Jena (Germany). For more details see[START_REF] Vadassery | CML42-mediated calcium signaling coordinates responses to Spodoptera herbivory and abiotic stresses in Arabidopsis[END_REF]

	.4: Composition of W5 and WI solutions
	W5	Final [C]	For 500 mL	WI	Final [C]	For 500 mL
	NaCl 5 M	154 mM	15,4 mL	Mannitol 800 mM	500 mM	312,5 mL
	CaCl 2 1 M	125 mM	62,5 mL	MES 200 mM pH 5,7	4 mM	10 mL
	KCl 2 M	5 mM	1,25 mL	KCl 2 M	20 mM	5 mL
	MES 200 mM pH 5,7	2 mM	5 mL	H 2 O	qs	172,5 mL
	H 2 O	qs	415,85 mL

 Protoplasts transformation:

h.

Table 4 .8: Composition of the RT mix

 4 Vector and insert were ligated and positive clones selected following the

	previous protocol.
	d. RNA extraction from plants
	NucleoSpin® RNA Plant (Macherey Nagel) and RNeasy Plant Mini Kit (Qiagen) kits were
	used to extract RNA from plants. Extractions were realized according to instructions included
	in the kits. DNAse treatment was included in the extraction protocol. RNA was quantified
	using a Nanodrop (ThermoScientific).
	e. RT-qPCR
	 Reverse-transcription (RT): 1 µg of RNA was used for each reaction, diluted in a total
	of 14 µl water. RNAs were heated 10 minutes at 70°C for denaturation. 16 µl of the RT
	mix was then added to RNAs and the whole was incubated for 1 hour at 42°C followed
	by 10 minutes at 75°C.

The latter was amplified with appropriate primers (see table 4.7), cleaned and digested as described above and pGREEN0229-MAP3K14locus-YPF-Tnos was linearized by corresponding enzymes in presence of CIP.

Table 4 . 9 :

 49 Genes used for qPCR experiments and corresponding primer couples

	3. Biochemistry methods
	a. Protein extraction
	 for subsequent immuno-precipitation: frozen plants were ground directly using Tissue-
	LyzerII (Qiagen) in 2 mL Eppendorf tubes containing iron beads. 1 mL extraction buffer
	was used for each sample. Tubes were vortexed and centrifuged (10 minutes, 14000 rpm,
	4°C) to recover soluble proteins. When extraction was done from protoplasts, 200 µL of
	extraction buffer was added to each sample.
	 for subsequent western blot: proteins were extracted with Laemmli buffer and heated 5
	minutes at 95°C.
	b. Protein quantification and normalization
	10 µL of each protein extract (previously diluted 10 times) was mixed with 250 µL Bradford
	Laemmli and incubated at 95°C for 5 minutes. This quantification step was not carried out for
	protoplast extracts.
	c. Immuno-precipitation (IP)
	For each protein extract a mix was prepared containing 30 µL of extraction buffer, 10 µL of
	Protein G sepharose beads (GE Healthcare) and 3 µL of the appropriate antibody. Beads were
	previously washed 4 times with 1 mL extraction buffer. Next, samples were incubated on a
	rotating wheel at 4°C for 2 hours. Beads were then washed twice with extraction buffer and
	once with kinase buffer that are used at the subsequent step. Washing consists in adding 1 mL
	of buffer per sample and centrifuging a few seconds at 4000 rpm. After the last washing step,
	all liquid is removed and beads are kept on ice. When IP was performed from protoplast
	extracts, the protocol was the same except only 1 µL of antibody was used.

reagent (Coomassie Protein Assay Reagent, Pierce) and dropped on a 96-wells Elisa plate.

Each sample was run in triplicate and a BSA range was used for standard protein concentrations (0.5 to 2.5 µg / 10 µL). Absorbance at 595 nm was measured using a TECAN reader and all concentrations were normalized to the less concentrated sample by dilution in the extraction buffer. For all further experiments, > 300µg proteins were used. Samples meant for immuno-precipitation were kept on ice and those meant for SDS-PAGE were mixed with e. Western blot Samples are run by SDS PAGE at constant 30 mA / gel for around 1 hour. Migrated proteins are transferred on ethanol-activated PVDF membranes (GE Healthcare) under constant 100V

Table 4 .11:

 4 Dilutions of antibodies used for western blots

	Primary antibody Dilution	In
	α-pTpY	1/5000	5% BSA
	α-YFP	1/10000	5% milk
	α-Myc	1/10000	5% milk
	α-HA	1/5000	5% milk
	α-MPK3	1/3000	5% milk
	α-MPK6	1/3000	5% milk

After revelation, membranes can be stained by incubation in Coomassie blue to visualize total protein loading.

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December 2016 | Volume 7 | Article 1941

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FUNDING

This work has benefited from a grant (LabEx Saclay Plant Sciences-SPS, reference ANR-10-LABX-0040-SPS), managed by the French National Research Agency under an Investments for the Future program (reference no. ANR-11-IDEX-0003-02).

LIST OF FIGURES & TABLES

 Data exploitation: amplification curves allow the identification of a Cp ("Crossing point") for each sample. The Cp corresponds to the number of cycles necessary to dissociate the fluorescence level from a threshold value and is directly correlated to the gene expression level. The latter is calculated through the following equation and the average of the three replicates is used to be represented in graphics.

2 Cp(housekeeping gene) -Cp(target gene)

f. Microarray 4 week-old Col-0 and mkk3-1 Arabidopsis plants were wounded with a forceps. Three leaves / plant were wounded, three times each covering ~ 90% of the leaf surface. For each genotype 7 plants were wounded and leaves were harvested 2 hours later. Non-wounded plants were used as control. Three independent experiments were performed. Total RNAs were extracted and micro array analysis was carried out using the CATMAv7 (Two-Color Microarray-Based Gene Expression Analysis) array (AGILENT technology). The experiment was performed by the Transcriptomic platform in our institute (IPS2, Orsay).

Samples were compared by pairs as follows:

Col-0 non-wounded mkk3-1 non-wounded Col-0 2h post-wounding mkk3-1 2h post-wounding and C-type MAPKs define functional modules activated by many stresses, one of the main mechanisms of activation being the transcriptional regulation of the upstream MAP3Ks (Figure 2C). This obviously does not exclude the possibility that these modules are also directly activated through phosphorylation of these MAP3Ks in response to various stresses.

CONCLUSION

Recent evidence suggests that MKK3 modules integrate many signals and represent a new landscape of phosphorylation cascades which deserve further attention as they were recently suggested to be important crop traits in various crops. In the future, besides a functional characterization of these modules, a major challenge will be the identification of the downstream responses controlled by the modules. Similar to the iconic stressactivated MAPKs which are also involved in development, we will have to clarify how MAPKs choose their proper targets when activated by different input signals. A seducing hypothesis is that the MKK3 modules regulate a set of responses which are shared by several signals. Transcriptome experiments using mutants of the different MAP3Ks or the MKK3 hub kinase in response to specific stresses will help to clarify this point and will be of particular interest in the context of the integration of environmental cues.

AUTHOR CONTRIBUTIONS

All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.

ACKNOWLEDGMENT

The authors thank all the members of the Stress Signaling group (IPS2) for constructive discussions.

Conflict of Interest Statement:

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Colcombet, Sözen and Hirt. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Caractérisation d'un module MAPK impliqué dans la réponse à la blessure chez

Arabidopsis thaliana