Abbreviations

AM

Chapter 1

General Introduction

The arbuscular mycorrhiza symbiosis

Arbuscular mycorrhiza (AM) are a unique example of a ubiquitous symbiosis between soil borne fungi (Glomeromycota) and plant roots which leads to reciprocal benefits to both partners. The mutualistic symbiotic associations that AM fungi form with the roots of more than 80% of land plant families facilitate plant mineral nutrient uptake (e.g. P, N, trace elements,..), enhance tolerance to environmental stresses (e.g. salinity, heavy metals, drought) and reduce root diseases caused by pathogenic fungi or nematodes [START_REF] Smith | Mycorrhizal symbiosis (Third Edition)[END_REF]. In return, AM fungi obtain carbohydrates from their host plants.

The mycorrhizal symbiosis impacts on plant survival, ecosystem variability and productivity, and is consequently a major factor contributing to the maintenance of plant biodiversity and to ecosystem functioning [START_REF] Smith | Mycorrhizal symbiosis (Third Edition)[END_REF].

AM fungi form an independent phylum, the Glomeromycota, where they are presently subdivided into four orders (Glomerales, Archaeosporales, Paraglomerales and Diversisporales) [START_REF] Schüβler | A new fungal phylum, the Glomeromycota: phylogeny and evolution[END_REF]. Nowadays, new perspectives and new findings from gene-based studies are facilitating our understanding of the complex network of fungal-plant interections which regulate the AM symbiosis [START_REF] Lanfranco | Genetic and genomic glimpses of the elusive arbuscular mycorrhizal fungi[END_REF].

AM developmental stages

Because of their obligate biotrophy, AM fungi are incapable of completing their life cycle in the absence of a host root. Although they can persist in soil as spores [START_REF] Smith | Mycorrhizal symbiosis (Third Edition)[END_REF], asymbiotic spore germination and hyphal growth are important events which determine root colonization and the establishment of the mycorrhizal symbiosis. After spore germination, hyphal growth is very limited and the fungi must colonize root tissues of a host plant for their reproduction and long-term survival.

The different stages in the formation of the symbiosis between AM fungi and plant roots require a series of important morphological changes in each partner which can lead to structurally distinct types of AM (Paris vs Arum) depending on the plant and fungus involved. The Paris-type is characterized by the growth of hyphae directly from one root cell to the next whilst in the Arum-type, fungal spread along the root is by intercellular hyphae which grow in the spaces between plant cells [START_REF] Smith | Transley review No 96. Structural diversity in (vesicular)-arbuscular mycorrhizal symbioses[END_REF].

Early interactions between AM fungi and plants begin when spore germination and hyphal growth are stimulated by host root exudates [START_REF] Gianinazzi-Pearson | In vitro enhancement of spore germination and early hyphal growth of a vesicular-arbuscular mycorrhizal fungus by host root exudates and plant flavonoids[END_REF]) (Fig.

1.1). As hyphae from germinating soil-borne spores come into contact with the host root surface, they form a special type of appressorium called hyphopodium which is necessary for root penetration. Under a hyphopodium, plant cells produce a prepenetration apparatus (PPA) which guides the fungus through root cells towards the inner cortex. The plant cell nucleus moves ahead of the growing PPA and directs its orientation within the cell. After completion of the PPA, a fungal hypha extends from the hyphopodium, enters the PPA to cross the plant cell and, in the case of Arum-type AM, enters the intercellular space, where it grows laterally along the root axis between the root cells. The intercellular hyphae induce the development of PPA-like structures in the inner cortical cells, subsequently enter these cells and branch to form arbuscules (Fig. 1.2) [START_REF] Genre | Prepenetration apparatus assembly precedes and predicts the colonization patterns of arbuscular mycorrhizal fungi within the root cortex of both Medicago truncatula and Daucus carota[END_REF]. The arbuscule branches within the plant cell are surrounded by a plant-derived periarbuscular membrane (PAM) that is continuous with the plant peripheral plasma membrane and which excludes contact of the fungus with the plant cytoplasm. The resulting surface of contact between the plant protoplast and the fungal cell creates the symbiotic interface, a specialized apoplastic zone for nutrient exchange between the mycorrhizal symbionts [START_REF] Gianinazzi-Pearson | Plant cell responses to arbuscular mycorrhizal fungi: getting to the roots of the symbiosis[END_REF]. The apoplastic interface formed between the fungal plasma membrane and the plant-derived PAM is called the periarbuscular space (PAS) (Fig. 1.3) Because of the cell-wall synthesizing potential of both the fungal membrane and the PAM, the PAS comprises fungal and plant cell-wall material. Once the fungus is established within the host roots and arbuscules are formed, a network of extraradical hyphae develops out into the soil which serves for nutrient uptake and the formation of resting spores, thus completing the fungal life-cycle (Fig. 1.1).

Vesicles are also formed by the fungi, in roots or on soil mycelium, as storage organs.

Molecular mechanisms regulating the AM symbiosis

Interactions between plants and AM fungi begin with the recognition and the exchange of molecular signals which trigger the morphological changes necessary for symbiosis establishment [START_REF] Gianinazzi-Pearson | Plants and arbuscular mycorrhizal fungi: cues and communication in the early steps of symbiotic interactions[END_REF]. The resulting symbiosis is characterized by modifications in gene expression in both partners [START_REF] Balestrini | Fungal and plant gene expression in arbuscular mycorrhizal symbiosis[END_REF][START_REF] Massoumou | Medicago truncatula gene responses specific to arbuscular mycorrhiza interactions with different species and genera of Glomeromycota[END_REF].

Recognition and exchange of molecular signals for symbiosis establishment

Recent studies indicate that plants and AM fungi perceive each other prior to their physical interaction [START_REF] Gianinazzi-Pearson | Plants and arbuscular mycorrhizal fungi: cues and communication in the early steps of symbiotic interactions[END_REF][START_REF] Bonfante | Mechanisms underlying beneficial plant-fungus interactions in mycorrhizal symbiosis[END_REF][START_REF] Bonfante | Dating in the dark: how roots respond to fungal signals to establish arbuscular mycorrhizal symbiosis[END_REF]. There is much interest in the molecular identification of the plant and fungal signals involved. It was predicted that AM fungi can sense components from host root exudates in the rhizosphere and "branching factors" secreted from plant were found to be responsible for the induction of hyphal branching [START_REF] Buee | The presymbiotic growth of arbuscular mycorrhizal fungi is induced by a branching factor partially purified from plant root exudates[END_REF] and alterations in fungal mitochondrial activity, thus leading to the developmental switch from asymbiosis to presymbiosis [START_REF] Tamasloukht | Root factors induce mitochondrial-related gene expression and fungal respiration during the developmental switch from asymbiosis to presymbiosis in the arbuscular mycorrhizal fungus Gigaspora rosea[END_REF] . Flavonoids and strigolactones contained in host plant root exudates have been found to act as plant signals which enhance AM fungal activity, growth and root colonization [START_REF] Steinkellner | Flavonoids and strigolactones in root exudates as signals in symbiotic and pathogenic plant-fungus interactions[END_REF]. Meanwhile, the fungus produces diffusible signal molecules, termed 'Myc factors' in analogy to the Nod factors produced by symbiotic rhizobia, which affect plant gene expression, induce signal perception Ca 2+ spiking responses within host cells and stimulate lateral root formation [START_REF] Kosuta | A diffusible factor from arbuscular mycorrhizal fungi induces symbiosis-specific MtENOD11 expression in roots of Medicago truncatula[END_REF][START_REF] Weidmann | Fungal elicitation of signal transduction-related plant genes precedes mycorrhiza establishment and requires the dmi3 gene in Medicago truncatula[END_REF][START_REF] Navazio | Calcium opens the dialogue between plants and arbuscular mycorrhizal fungi[END_REF][START_REF] Chabaud | Arbuscular mycorrhizal hyphopodia and germinated spore exudates trigger Ca 2+ spiking in the legume and nonlegume root epidermis[END_REF]. Recently, an AM fungal lipochitooligosaccharide was identified as stimulating M. truncatula root growth and branching through a Ca 2+ -dependent symbiotic signalling pathway [START_REF] Maillet | Fungal lipochitooligosaccharide symbiotic signals in arbuscular mycorrhiza[END_REF].

Symbiosis-related transcriptional changes during AM formation

The plant partner

A large number of early and late mycorrhiza-induced genes have been identified in the plant partner [START_REF] Parniske | Arbuscular mycorrhiza: the mother of plant root endosymbioses[END_REF][START_REF] Oldroyd | Reprogramming plant cells for endosymbiosis[END_REF][START_REF] Bonfante | Dating in the dark: how roots respond to fungal signals to establish arbuscular mycorrhizal symbiosis[END_REF].

Molecular events associated with AM development and/or functioning include signal perception and transduction, plant defense responses, transmembrane nutrient transport and common properties between AM and the nodulation symbiosis [START_REF] Dumas-Gaudot | Proteomics as a way to identify extra-radicular fungal proteins from Glomus intraradices -RiT-DNA carrot root mycorrhizas[END_REF][START_REF] Lévy | A putative Ca 2+ and calmodulin-dependent protein kinase required for bacterial and fungal symbioses[END_REF][START_REF] Siciliano | Transcriptome analysis of arbuscular mycorrhizal roots during development of the prepenetration apparatus[END_REF]. Modifications in plant gene expression have been studied before and during appressorium development. For example, expression of the M. truncatula gene MtENOD11, coding for a putative cell wall repetitive proline-rich protein (RPRP) in roots, is induced by the diffusible factor from AM fungi [START_REF] Kosuta | A diffusible factor from arbuscular mycorrhizal fungi induces symbiosis-specific MtENOD11 expression in roots of Medicago truncatula[END_REF]. A plant Clp serine protease is activated in pea roots induced by Glomus mosseae during hyphopodium formation which may have multiple functions related to the cross-talk between plant and fungal partners and/or controlling levels of key regulatory proteins within a signal-transduction pathway during early interactions in arbuscular mycorrhiza [START_REF] Roussel | Signalling between arbuscular mycorrhizal fungi and plants: identification of a gene expressed during early interactions by differential RNA display analysis[END_REF]. Also, suppressive-subtractive cDNA libraries have identified a number of plant genes that are up-regulated during these early stages of fungal interactions with roots [START_REF] Weidmann | Fungal elicitation of signal transduction-related plant genes precedes mycorrhiza establishment and requires the dmi3 gene in Medicago truncatula[END_REF][START_REF] Siciliano | Transcriptome analysis of arbuscular mycorrhizal roots during development of the prepenetration apparatus[END_REF].

Initial steps of root colonization by AM fungi or rhizobia are regulated by a number of common plant genes through a so-called common Sym pathway [START_REF] Parniske | Arbuscular mycorrhiza: the mother of plant root endosymbioses[END_REF]. In M. truncatula, the common Sym genes DMI1, DMI2 and DMI3 have been particularly well studied [START_REF] Catoira | Four genes of Medicago truncatula controlling components of a Nod factor transduction pathway[END_REF][START_REF] Oldroyd | Peace talks and trade deals. Keys to long-term harmony in legume-microbe symbioses[END_REF][START_REF] Oldroyd | Reprogramming plant cells for endosymbiosis[END_REF]. In silico and microarray-based transcriptome profiling approaches have been carried out to uncover the transcriptome of developing root nodules and AM roots of Medicago truncatula. Hundreds of genes have been found to be activated in different stages of either symbiosis, of which almost 100 are co-induced during nodulation and AM development [START_REF] Küster | Identification and expression regulation of symbiotically activated legume genes[END_REF].

However, transcriptome studies have also indicated that the two symbioses involve independent recognition and signaling pathways, with early discrimination by plant roots between the different microbial symbionts [START_REF] Weidmann | Fungal elicitation of signal transduction-related plant genes precedes mycorrhiza establishment and requires the dmi3 gene in Medicago truncatula[END_REF][START_REF] Küster | Identification and expression regulation of symbiotically activated legume genes[END_REF].

Targeted and non-targeted approaches to search for differentially expressed genes have revealed the complexity of transcriptional changes also during late stages of the AM symbiosis, as well as the identification of plant genes exhibiting common responses to different AM fungi (see for example, [START_REF] Brechenmacher | Expression profiling of upregulated plant and fungal genes in early and late stages of Medicago truncatula-Glomus mosseae interactions[END_REF][START_REF] Küster | Identification and expression regulation of symbiotically activated legume genes[END_REF][START_REF] Massoumou | Medicago truncatula gene responses specific to arbuscular mycorrhiza interactions with different species and genera of Glomeromycota[END_REF]. Symbiosis-induced plant phosphate transporter genes have received particular attention. Several have been identified in different plant species, like potato, rice and M. truncatula, which are probably involved in the transfer of fungusdelivered phosphate into colonized plant cells [START_REF] Karandashov | Symbiotic phosphate transport in arbuscular mycorrhizas[END_REF][START_REF] Javot | Phosphate in the arbuscular mycorrhizal symbiosis: transport properties and regulatory roles[END_REF].

The fungal partner

Most of the effort put into identifying gene expression patterns in AM interactions has focused on plant genes. Research on AM fungi is hampered by their obligate biotrophic life style and the fact that they are so far not amenable to genetic manipulation, furthermore knowledge about fungal gene expression in the symbiosis has remained relatively limited [START_REF] Balestrini | Fungal and plant gene expression in arbuscular mycorrhizal symbiosis[END_REF][START_REF] Seddas | Symbiosis-related plant genes modulate molecular responses in an arbuscular mycorrhizal fungus during early root interactions[END_REF][START_REF] Bonfante | Mechanisms underlying beneficial plant-fungus interactions in mycorrhizal symbiosis[END_REF]Kutznetsova et al., 2010), although recently published genome-wide transcriptomic data open new opportunities [START_REF] Tisserant | The transcriptome of the arbuscular mycorrhizal fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate symbiont[END_REF][START_REF] Lanfranco | Genetic and genomic glimpses of the elusive arbuscular mycorrhizal fungi[END_REF]. [START_REF] Franken | RNA accumulation and genes expressed in spores of the arbuscular mycorrhizal fungus, Gigaspora rosea[END_REF] first reported the identification of expressed arbuscular mycorrhizal fungal genes in spores during asymbiosis stage. In recent years, progress has been made in studies of the fungal genes involved in mycorrhiza development and/or function using different approaches [START_REF] Gianinazzi-Pearson | Plants and arbuscular mycorrhizal fungi: cues and communication in the early steps of symbiotic interactions[END_REF][START_REF] Genre | Check-in procedures for plant cell entry by biotrophic microbes[END_REF]. For example, the construction of cDNA libraries and gene expression profiling during appressorium development by Glomus mosseae has identified some up-regulated genes related to Ca 2+dependent signaling, suggesting that calcium may play a role in the early stages of mycorrhiza formation [START_REF] Breuninger | Recognition events in AM symbiosis: analysis of fungal gene expression at the early appressorium stage[END_REF]. Also, the upregulation of a number of genes associated with increased cell activity has been detected in Gigaspora rosea during presymbiotic phase in the presence of plant root exudates [START_REF] Tamasloukht | Root exudate-stimulated RNA accumulation in the arbuscular mycorrhizal fungus Gigaspora rosea[END_REF].

Using differential RNA display, [START_REF] Tamasloukht | Root factors induce mitochondrial-related gene expression and fungal respiration during the developmental switch from asymbiosis to presymbiosis in the arbuscular mycorrhizal fungus Gigaspora rosea[END_REF] showed that extensive hyphal branching observed by Gigaspora rosea spores in response to the increased expression of several genes encoding mitochondrial proteins. Plant mutants have also been used to investigate the symbiosis-associated fungal molecular responses. Genotypes of M.

truncatula mutated for Ca 2+ -dependent signalisation cascades can down-regulate gene expression of G. intraradices at early stages of fungal development [START_REF] Seddas | Symbiosis-related plant genes modulate molecular responses in an arbuscular mycorrhizal fungus during early root interactions[END_REF]). In pea mutants where arbuscule formation is inhibited or fungal turnover modulated, most of the studied G. intraradices genes were down regulated when arbuscule formation was defective [START_REF] Kuznetsova | Symbiosisrelated pea genes modulate fungal and plant gene expression during the arbuscule stage of mycorrhiza with Glomus intraradices[END_REF]. Recent techniques like in situ RT-PCR and laser capture microdissection corroborate arbuscule-related fungal gene expression (Balestrini et al., 2007;[START_REF] Seddas | Spatial monitoring of gene activity in extraradical and intraradical developmental stages of arbuscular mycorrhizal fungi by direct fluorescent in situ RT-PCR[END_REF][START_REF] Kuznetsova | Symbiosisrelated pea genes modulate fungal and plant gene expression during the arbuscule stage of mycorrhiza with Glomus intraradices[END_REF], and heterologous complementation experiments are leading to the identification of gene function in systems using fungi other than yeast [START_REF] Tollot | An STE12 gene identified in the mycorrhizal fungus Glomus intraradices restores infectivity of a hemibiotrophic plant pathogen[END_REF][START_REF] Kloppholz | A secreted fungal effector of Glomus intraradices promotes symbiotic biotrophy[END_REF]. These approaches offer new perspectives for the detailed analysis of fungal developmental processes that occur during functional differentiation in symbiotic arbuscular mycorrhiza interactions.

Research on the AM fungal partner has focused particularly on genes associated with nutrient uptake, and a number of related fungal genes that are up-regulated during the symbiosis have been identified [START_REF] Govindarajulu | Nitrogen transfer in the arbuscular mycorrhizal symbiosis[END_REF][START_REF] Karandashov | Symbiotic phosphate transport in arbuscular mycorrhizas[END_REF][START_REF] López-Pedrosa | GintAMT1 encodes a functional high-affinity ammonium transporter that is expressed in the extraradical mycelium of Glomus intraradices[END_REF][START_REF] Balestrini | Laser microdissection reveals that transcripts for five plant and one fungal phosphate transporter genes are contemporaneously present in arbusculated cells[END_REF][START_REF] Cappellazzo | Characterization of an amino acid permease from the endomycorrhizal fungus Glomus mosseae[END_REF]. For example, an ammonium transporter GintAMT1 from G. intraradices is expressed in the extraradical mycelium where NH 4 + is taken up from the surrounding medium [START_REF] López-Pedrosa | GintAMT1 encodes a functional high-affinity ammonium transporter that is expressed in the extraradical mycelium of Glomus intraradices[END_REF]. The expression of gene GmosAAP1, coding an amino acid permease from G.

mosseae, was detected in the extraradical fungal structures, and transcript abundance increased upon exposure to organic nitrogen suggesting a role in the first steps of amino acid acquisition from the soil resources [START_REF] Cappellazzo | Characterization of an amino acid permease from the endomycorrhizal fungus Glomus mosseae[END_REF]. Three AM fungal phosphate transporter genes have been identified so far: GvPT, GiPT and GmosPT, from G.

versiforme, G. intraradices and G. mosseae, respectively [START_REF] Harrison | A phosphate transporter from the mycorrhizal fungus Glomus versiforme[END_REF][START_REF] Maldonado-Mendoza | A phosphate transporter gene from the extra-radical mycelium of an arbuscular mycorrhizal fungus Glomus intraradices is regulated in response to phosphate in the environment[END_REF][START_REF] Benedetto | Expression profiles of a phosphate transporter gene (GmosPT) from the endomycorrhizal fungus Glomus mosseae[END_REF]. The expression of the phosphate transporter genes from G. versiforme (GvPT) and G. intraradices (GiPT) has been detected predominantly in the extraradical mycelium, thus indicating a role in Pi acquisition from the soil (inport the Pi) [START_REF] Harrison | A phosphate transporter from the mycorrhizal fungus Glomus versiforme[END_REF][START_REF] Maldonado-Mendoza | A phosphate transporter gene from the extra-radical mycelium of an arbuscular mycorrhizal fungus Glomus intraradices is regulated in response to phosphate in the environment[END_REF].

However, transcripts of GvPT were also weakly detected in M. truncatula roots colonized by G. versiforme (Harrison and van Buuren,1995), and the phosphate transporter gene from G. mosseae (GmosPT) showed activity in arbuscules [START_REF] Benedetto | Expression profiles of a phosphate transporter gene (GmosPT) from the endomycorrhizal fungus Glomus mosseae[END_REF]. It was suggested that the presence of phosphate transporter transcripts in intraradical mycelium may indicate that, inside the root, the AM fungus somehow controls the amount of phosphate delivered to the plant by re-absorbing Pi released from neighboring plant cells [START_REF] Benedetto | Expression profiles of a phosphate transporter gene (GmosPT) from the endomycorrhizal fungus Glomus mosseae[END_REF][START_REF] Balestrini | Laser microdissection reveals that transcripts for five plant and one fungal phosphate transporter genes are contemporaneously present in arbusculated cells[END_REF][START_REF] Tisserant | The transcriptome of the arbuscular mycorrhizal fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate symbiont[END_REF].

Calcium-regulated signaling events in cells

Many cell processes depend on internal signals composed of small molecules which bind and activate protein kinases, ion channels, and other proteins, to initiate or continue signaling cascades [START_REF] Borges-Walmsley | cAMP signalling in pathogenic fungi: control of dimorphic switching and pathogenicity[END_REF][START_REF] Mansilla | Signalling pathways controlling fatty acid desaturation[END_REF][START_REF] Dahan | Nuclear protein kinases: still enigmatic components in plant cell signalling[END_REF]. These intracellular messengers include, amongst others, cyclic nucleotides (cAMP), inositol trisphosphate (IP 3 ), diacylglycerol (DAG) and calcium ions (Ca 2+ ) [START_REF] Wakelam | Diacylglycerol-when is it an intracellular messenger?[END_REF][START_REF] Borges-Walmsley | cAMP signalling in pathogenic fungi: control of dimorphic switching and pathogenicity[END_REF][START_REF] Nash | Intracellular signalling: receptor-specific messenger oscillations[END_REF].

Ca 2+ is an important signaling molecule in cell responses to various environmental stimuli, including abiotic stresses (e.g. salt, cold, drought, ozone, metals) as well as biotic elicitors from bacteria and fungi. The signature of Ca 2+ responses is cell type and organ specific [START_REF] Boudsocq | Stress signaling II: calcium sensing and signaling[END_REF]. Signaling occurs when the cell is stimulated to release calcium ions (Ca 2+ ) from intracellular stores, and/or when calcium enters the cell through plasma membrane ion channels [START_REF] Clapham | Calcium signaling[END_REF]. Stimuli from the external medium or from subcellular compartments elevate cytosolic free Ca 2+ concentrations in cells, often as repetitive oscillations or spiking [START_REF] Mcainsh | Shaping the calcium signature[END_REF]. Elevation of cytosolic Ca 2+ levels functions as a major secondary messenger in cell physiology and signaling where Ca 2+ channels and transporters, Ca 2+ sensors, and signal transducers are conserved essential components of the cellular Ca 2+ signaling machinery, named the "calciome" [START_REF] Ton | Functional expression of heterologous proteins in yeast: insights into Ca 2+ signaling and Ca 2+ -transporting ATPases[END_REF]. This can underlie Ca 2+ homeostasis in cells by regulating movement of ions between the extracellular environment, cells and subcellular compartments and so provide mechanisms for generating Ca 2+ signals [START_REF] Dodd | The language of calcium signaling[END_REF].

Ca 2+ entering cells can bind to and activate the universal Ca 2+ sensor protein calmodulin (CaM). Many downstream transcriptional and translational events are controlled by such Ca 2+ -mediated activation of CaM. A primary downstream interpreter of Ca 2+ signals is phosphorylation which is regulated by Ca 2+ -dependent kinases and phosphatases following the activation of CaM. As a result, transcription factors are activated through reversible phosphorylation by these proteins, leading to downstream gene expression [START_REF] Dodd | The language of calcium signaling[END_REF].

In yeast, Ca 2+ entering the cytosol via the plasma membrane channel complex Cch1/Mid1 binds to calmodulin and activates the CaM kinases CMK1 and CMK2 and the phosphatase calcineurin. Dephosphorylation by calcineurin activates the transcriptional regulator Crz1 (Calcineurin-Responsive Zinc Finger) /Tcn1, leading to nuclear translocation and transcription of target genes involved in cell physiological processes.

Two Ca 2+ -ATPases, located in the vacuolar (PMC1) and the Golgi (PMR1) membranes, and a vacuolar calcium ion transporter (vacuolar Ca 2+ /H + exchanger) (VCX1) are responsible for depleting the cytosolic Ca 2+ to maintain low levels compatible for growth.

Activation of calcineurin leads to the transcriptional induction of the Pmc1 and Pmr1

genes via Crz1 but inhibits the function of VCX1. Consequently, PMC1 and VCX1 independently transport cytosolic Ca 2+ into vacuoles.

Ca 2+ flux in organelles

Calcium elevation is not only induced in the cytosol but also in other cellular compartments, including the nucleus and mitochondria. Calcium influxes into the cytosol or organelles via ion channels in the plasma membrane and endomembranes, and Ca 2+ effluxes via transporters which remove Ca 2+ from the cytosol or organelles. This allows replenishment of internal and external stores in the cell, and a return to basal Ca 2+ levels, which may contribute to shaping specific and distinct Ca 2+ signatures in the different cell compartments [START_REF] Mcainsh | Shaping the calcium signature[END_REF]. Endoplasmic reticulum, vacuoles and mitochondria have important functions in Ca 2+ homeostasis where Ca 2+ pumps and/or exchangers are regulated to maintain cytoplasmic Ca 2+ balance [START_REF] Ton | Functional expression of heterologous proteins in yeast: insights into Ca 2+ signaling and Ca 2+ -transporting ATPases[END_REF].

The large vacuole in fungi and plants serves as a major store for Ca 2+ , for the purposes of both detoxification and signaling [START_REF] Ton | Functional expression of heterologous proteins in yeast: insights into Ca 2+ signaling and Ca 2+ -transporting ATPases[END_REF]. A remarkable feature of yeast vacuoles is their ability to sequester large amounts of Ca 2+ and release it in response to particular stimuli. The vacuole probably receives a small amount of Ca 2+ through the fusion of vesicles derived from the Golgi complex, which acquires Ca 2+ directly from secretory pathway Ca 2+ -ATPases (SPCAs) or indirectly from the environment through endocytosis, but the large majority of vacuolar Ca 2+ comes directly from the cytoplasm through the action of Ca 2+ pumps and Ca 2+ /H + exchangers that are specifically localized in the vacuole membrane [START_REF] Cunningham | Acidic calcium stores of Saccharomyces cerevisiae[END_REF].

Most of the Ca 2+ in the fungal and plant vacuoles is immobilized in the form of complexes with inorganic polyphosphate [START_REF] Cunningham | Acidic calcium stores of Saccharomyces cerevisiae[END_REF]. AM fungi are characterized by an extensive vacuolar system which is considered central for sequestering and transfering phosphate that accumulates within the hyphae after uptake from soil [START_REF] Smith | Physiological Interactions between Symbionts in Vesicular-Arbuscular Mycorrhizal Plants[END_REF]. Ca 2+ has been co-localized with polyphosphate in the vacuoles of the AM fungal cells, suggesting a role of Ca 2+ in the phosphate metabolism in mycorrhizal tissues [START_REF] Strullu | Ultrastructure and electronprobe microanalisis of the metachromatic vacuolar granules occurring in taxus mycorrhizas[END_REF]. In this context, it has been proposed that the vacuolar H + /Ca 2+ transporters CAX1 and CAX3 in Arabidopsis may be involved in Pi homeostasis because a cax1/cax3 double mutant is affected in Ca 2+ homeostasis and shows 66% more shoot Pi than the wild type [START_REF] Cheng | Functional association of Arabidopsis CAX1 and CAX3 is required for normal growth and ion homeostasis[END_REF].

To maintain a low cytosolic Ca 2+ concentration, cells also actively pump the ion from the cytosol into the endoplasmic reticulum (ER) or Golgi and also mitochondria, as well as into the extracellular space. During Ca 2+ -signalling in animal cells, the sarco (endo) plasmic reticulum Ca 2+ -ATPase (SERCA pump) regulates Ca 2+ levels by effluxing the ion into the endoplasmic reticulum lumen which also serves as an intracellular Ca 2+ store [START_REF] Berridge | Calcium signalling: dynamics, homeostasis and remodelling[END_REF]. In Arabidopsis, the ER-type Ca 2+ -ATPases (ECA) play an important role in regulating cytosolic cation levels. ECA1 and ECA3 also have an important role in tolerance to Mn 2+ stress. ECA1 was found to be localized at the ER, while ECA3 was found associated with Golgi and post-Golgi vesicles [START_REF] Dodd | The language of calcium signaling[END_REF]. receptor), which is a Ca 2+ channel, and thus releases Ca 2+ from the ER. After such store depletion, a Ca 2+ entry mechanism, "Store-Operated Channels" (SOCs) is activated. The inflowing calcium current that results after ER calcium have been released is referred to as Ca 2+ -release-activated Ca 2+ current (CRAC). The four-transmembrane domain plasmamembrane protein, Orai1 and the EF-hand protein STIM 1 are required for CRAC activity, that is, Orail and STIM1 are possible mediators of CRAC [START_REF] Clapham | Calcium signaling[END_REF].

Mitochondria also regulate internal Ca 2+ levels which are important for signaling and for driving mitochondrial function in cells. The main mechanism depends on the rapid sequestration of the ion from the cytoplasm via Ca 2+ -binding mitochondrial carriers [START_REF] Satrústegui | Mitochondrial transporters as novel targets for intracellular calcium signaling[END_REF]. Mitochondria not only control their own intra-organelle Ca 2+ concentration but they also influence the entire cellular network of cellular Ca 2+ signaling, including the endoplasmic reticulum, the plasma membrane, and the nucleus. Ca 2+ channeling between mitochondria and the endoplasmic reticulum collaborate in the regulation of Ca 2+ influx and extrusion through the plasma membrane Ca 2+ ATPases (PMCA). In addition, mitochondrial and nucleus communicate through a Ca 2+ signal [START_REF] Szabadkai | Mitochondria: The hub of cellular Ca 2+ signaling[END_REF].

There is some evidence that the nuclei of plant cells may generate Ca 2+ signals independently of the cytosol [START_REF] Mazars | Calcium homeostasis in plant cell nuclei[END_REF]. Studies of signaling events involved in the nodulation process in legumes have clearly shown that Nod factor-induced changes in intracellular Ca 2+ are frequently observed as Ca 2+ oscillations in the nuclear region [START_REF] Oldroyd | Nuclear calcium changes at the core of symbiosis signalling[END_REF]. It has been hypothesized that Ca [START_REF] Corcoran | Defining Ca 2+ /calmodulin-dependent protein kinase cascades in transcriptional regulation[END_REF]. CaM regulates two CaMKK-encoding genes which in turn activate CaMKI and CaMKIV. In mammalian cells, CaMKI is cytoplasmic, CaMKII is cytoplasmic or nuclear whilst CaMKIV is predominantly in the nucleus [START_REF] Kahl | Regulation of cell cycle progression by calcium/calmodulin-dependent pathways[END_REF]. The CaMK cascade is thus well positioned to affect cytoplasmic events, but it is more difficult to explain its effects on transcription. Whether CaMKI enters the nucleus or affects transcription through a cytoplasmic intermediate has not been well

studied, but it can stimulate transcription of reporters in transient transfection assays, and the molecular mechanisms that regulate the translocation of particular proteins into the nucleus are unknown [START_REF] Kahl | Regulation of cell cycle progression by calcium/calmodulin-dependent pathways[END_REF][START_REF] Dahan | Nuclear protein kinases: still enigmatic components in plant cell signalling[END_REF].

Ca 2+ -related gene expression related to AM establishment

Calcium is a key secondary messenger in cell functional processes and changes in Ca 2+ levels represent intracellular signaling events in biological systems, including mutualistic and pathogenic plant-microbe interactions [START_REF] Vadassery | Calcium signaling in pathogenic and beneficial plant microbe interactions: what can we learn from the interaction between Piriformospora indica and Arabidopsis thaliana[END_REF].

Convincing evidence for the implication of plant genes regulating Ca 2+ signaling in recognition responses of roots to symbiotic AM fungi has come from studies of various host plants and their symbiosis-defective genotypes mutated for Ca 2+ -dependent signalisation cascades [START_REF] Weidmann | Fungal elicitation of signal transduction-related plant genes precedes mycorrhiza establishment and requires the dmi3 gene in Medicago truncatula[END_REF][START_REF] Sanchez | Pseudomonas fluorescens and Glomus mosseae trigger DMI3-dependent activation of genes related to a signal transduction pathway in roots of Medicago truncatula[END_REF][START_REF] Oldroyd | Nuclear calcium changes at the core of symbiosis signalling[END_REF][START_REF] Gianinazzi-Pearson | Plants and arbuscular mycorrhizal fungi: cues and communication in the early steps of symbiotic interactions[END_REF]. In M. truncatula, for example, three nodulation- signal [START_REF] Ané | Medicago truncatula DMI1 required for bacterial and fungal symbioses in legumes[END_REF][START_REF] Lévy | A putative Ca 2+ and calmodulin-dependent protein kinase required for bacterial and fungal symbioses[END_REF][START_REF] Mitra | A Ca 2+ /calmodulin-dependent protein kinase required for symbiotic nodule development: Gene identification by transcript-based cloning[END_REF][START_REF] Oldroyd | Nuclear calcium changes at the core of symbiosis signalling[END_REF].

In fact, some putative signal transduction-related M. truncatula genes show activation during early stages of AM fungal interactions with wild-type M. truncatula, but none respond in the mycorrhiza-resistant dmi3 mutant [START_REF] Weidmann | Fungal elicitation of signal transduction-related plant genes precedes mycorrhiza establishment and requires the dmi3 gene in Medicago truncatula[END_REF][START_REF] Sanchez | Pseudomonas fluorescens and Glomus mosseae trigger DMI3-dependent activation of genes related to a signal transduction pathway in roots of Medicago truncatula[END_REF]. Such non-symbiotic plant mutants can also down-regulate the expression of G.

intraradices genes prior to and during appressorium formation by the AM fungus [START_REF] Seddas | Symbiosis-related plant genes modulate molecular responses in an arbuscular mycorrhizal fungus during early root interactions[END_REF]. However, nothing is known about the regulation or role of Ca 2+ in the fungal cells during the development and functioning of the AM symbiosis, apart from the reported up-regulation of one Gigaspora rosea P-type IIA ATPase by root exudates [START_REF] Tamasloukht | Root exudate-stimulated RNA accumulation in the arbuscular mycorrhizal fungus Gigaspora rosea[END_REF], and the reported activation of some G. mosseae genes with a potential role in Ca 2+ -dependent signaling, in synchrony with appressorium formation on parsley roots [START_REF] Breuninger | Recognition events in AM symbiosis: analysis of fungal gene expression at the early appressorium stage[END_REF].

Ca 2+ involvement in plant-biotrophic pathogen interactions

Calcium signaling has been implicated in the induction of appressoria, development and pathogenesis of leaf pathogenic fungi [START_REF] Ebbole | Magnaporthe as a model for understanding host-pathogen interactions[END_REF][START_REF] Rho | Phospholipase C-mediated calcium signalling is required for fungal development and pathogenicity in Magnaporthe oryzae[END_REF]. Several Ca 2+related proteins have been identified in biotrophic pathogens. For example, a P-type ATPase (PDE1) has a role in the development of penetration hyphae and pathogenicity in the rice blast fungus Magnaporthe grisea [START_REF] Balhadère | PDE1 encodes a P-Type ATPase involved in appressorium-mediated plant infection by the rice blast fungus Magnaporthe grisea[END_REF]. Also, a Ca 2+ /calmodulin-dependent signaling system is involved in appressorium formation by M.

grisea and calmodulin gene expression is induced concomitantly with appressorium formation [START_REF] Lee | Calcium/calmodulin-dependent signaling for appressorium formation in the plant pathogenic fungus Magnaporthe grisea[END_REF][START_REF] Liu | Early expression of the calmodulin gene, which precedes appressorium formation in Magnaporthe grisea, is inhibited by selfinhibitors and requires surface attachment[END_REF]. In addition, an endoplasmic sarcoplasmic/reticulum Ca 2+ -ATPase (Eca1) has been identified in the fungal pathogen

Ustilago maydis which restores growth of a yeast mutant defective for calcium homeostasis [START_REF] Adamíková | Calcium signaling is involved in dynein-dependent microtubule organization[END_REF]. These studies point to an importance of Ca 2+dependent signaling processes in fungal events leading to interactions with plant tissues.

It has been hypothesized that pathogenic and symbiotic fungi may share some common regulatory mechanisms for the invasion of plant tissues [START_REF] Genre | Check-in procedures for plant cell entry by biotrophic microbes[END_REF], and evidence to support this hypothesis comes from recent investigations. It has been shown that transformation with the G. intraradices transcription factor gene GintSTE12 restores pathogenicity of a clste12Δ mutant of the hemi-biotrophic pathogen

Colletotrichum lindemuthianum, providing evidence for the possible existence of common genetic elements regulating the penetration of plant tissues by the two fungi [START_REF] Tollot | An STE12 gene identified in the mycorrhizal fungus Glomus intraradices restores infectivity of a hemibiotrophic plant pathogen[END_REF]. Also, the expression of another G. intraradices gene, SP7, which interacts with a plant pathogenesis-related transcription factor, in the rice blast fungus Magnaporthe oryzae attenuates root decay symptoms caused by the biotrophic pathogen [START_REF] Kloppholz | A secreted fungal effector of Glomus intraradices promotes symbiotic biotrophy[END_REF].

Furthermore, a gene (Erl1) encoding an Era (Escherichia coli Ras)-like GTPase which might be involved in the control of in planta growth of M. oryzae is orthologous to the mature amino terminal part of the Gin1 protein from G. intraradices [START_REF] Heupel | Erl1, a novel era-like GTPase from Magnaporthe oryzae, is required for full root virulence and is conserved in the mutualistic symbiont Glomus intraradices[END_REF].

Thesis objectives

The aim of my thesis work was to provide a first insight into whether Ca 2+ may also be an important intracellular component in fungal events leading to successful mycorrhizal interactions with plant tissues. In this context, Ca 2+ -related genes were identified in isolates of the AM fungi G. mosseae and G. intraradices, expression was analysed at different stages of interactions with host roots and full-length cDNA sequences were obtained for candidate genes. Two G. mosseae genes were targeted based on sequences previously identified in an appressorium stage cDNA library [START_REF] Breuninger | Recognition events in AM symbiosis: analysis of fungal gene expression at the early appressorium stage[END_REF], and seven G. intraradices genes were selected from widescale transcriptome sequencing data covering different stages of fungal development [START_REF] Tisserant | The transcriptome of the arbuscular mycorrhizal fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate symbiont[END_REF] For experiments involving the early interactions of the symbiosis, 2000 spores of G.

mosseae were germinated in water at 25 °C in the dark for 7 days and seeds of Astragalus sinicus L. (a legume used as rice field green manure in China) were surface sterilized (5 min in 75 % alcohol, 10 min in 3 % sodium hypochlorite), and germinated at 28 °C in the dark on 0.7% sterile water-agar plates for 3 days. Seedlings were then transferred to the sterile water containing the germinated spores in order to stimulate them by crude plant root exudates for 7 days (Fig. 2.1), then spores were frozen for RNA extraction. Mycorrhizal root material was provided by the laboratory in Wuhan. A. sinicus seeds were surface sterilised for 5min in 5% NaClO, then rinsed in sterile demineralised water.

They were germinated for 48 h on 0.7% sterile agar at 28 °C in the dark then transplanted into 400 ml plastic pots containing a mix (1:1 v/v) of soil (loam, pH 6.59, P: 11.83 mg/kg) and sand with 10% soil-based inoculum (spores, roots and hyphae) of G. mosseae (BEG225, China) produced on Sorghum. Plants were grown for 40 days under controlled conditions (100-200Wm-2, 24 and 18 °C, 16 and 8 h, day and night, respectively) and watered each day with demineralised water and once a week with Hoagland nutrient solution [START_REF] Hoagland | The water-culture method for growing plants without soil[END_REF]) (Table 2.1).

Medicago truncatula + Glomus intraradices BEG141

Wild-type M. truncatula (line J5) and the Myc -dmi3/Mtsym13 mutant TRV25

(provided by G. Duc, INRA-Dijon, France) genotypes were used. The M. truncatula Myc-dmi3/Mtsym13 mutant is mutated for the gene DMI3/MtSYM13 encoding a calcium calmodulin-dependent protein kinase [START_REF] Lévy | A putative Ca 2+ and calmodulin-dependent protein kinase required for bacterial and fungal symbioses[END_REF]. It presents a mycorrhizadefective phenotype allowing only appressoria formation development of AM colonization [START_REF] Morandi | Characterisation of new symbiotic Medicago truncatula (Gaertn.) mutants, and phenotypic or genotypic complementary information on previously described mutants[END_REF] Glomus intraradices BEG141 spores were extracted by laceration of colonized leek roots (6-month-old leek pot culture) in sterile water using a blender. Two biological repetitions of spores (5,000) were activated for 24h at 25 °C in water and 2 % CO 2 (IG150, Jouan incubator). Root exudates of M. truncatula were collected according to [START_REF] Seddas | Symbiosis-related plant genes modulate molecular responses in an arbuscular mycorrhizal fungus during early root interactions[END_REF]. Briefly, five germinated seedlings of M. truncatula per each well were incubated in 3 ml of sterile water in a six-well plate (Falcon Multiwell, BD Sciences, San Jose, CA, U.S.A.) for 7 days. After elimination of all plant residues, this crude plant root exudates were directly used with spores. Two biological repetitions of spores (5,000) were germinated in wild-type J5 or TRV25 mutant root exudates separately for 6 days and two biological repetitions of 5,000 spores were germinated in water for 6 days as control.

For a time-course study of gene expression during mycorrhiza development, surface sterilized seeds (6 min in 98 % sulphuric acid, 10 min in 3 % sodium hypochlorite) of M.

truncatula line J5 and the Myc -TRV25 mutant were germinated for 3 days on 0.7 % Bactoagar (Difco Laboratories, Detroit) at 25 °C in the dark and then transplanted into 50ml or 75-ml pots containing a mixture (2:1, vol / vol) of Terragreen (OilDri-US special, Mettman, Germany) and a neutral clay soil-based inoculum (spores, roots, and hyphae) of G. intraradices BEG141 produced on leek. Inoculum was checked for the absence of rhizobia by growing with the hypernodulating M. truncatula genotype TR122 for 3 weeks.

Plants were grown under constant conditions: 355 μE/m2/s, 16h-24 °C and 8h-21 °C, day and night, respectively, 70 % humidity. They were fertilized with 3 ml (50-ml pot) or 5 ml/pot (75-ml pot) of a modified Long Ashton solution [START_REF] Hewitt | Sand and water culture methods used in the study of plant nutrition (2nd revised edn)[END_REF]

Estimation of mycorrhizal root colonization

To determine the number of appressoria and total root colonization for M. truncatula, fresh roots were washed under running tap water, cleared for 48 h in 10 % KOH at room temperature and stained overnight with 0.05 % trypan blue in glycerol (4 and 6 dai roots)

or 30 min in 10 % KOH at 90 °C and 15 min in 0.05 % trypan blue at 90 °C (17, 21 dai roots). For P. sativum roots were cleared 1 hour at 90°C in 10 % KOH and stained 15 min in 0.05 % trypan blue at 90 °C. Root systems were cut into 1 cm pieces and 30 root fragments/plant were observed microscopically according to [START_REF] Trouvelot | Mesure du taux de mycorrhization VA d'un systeme radiculaire. Recherche des methodes d'estimation ayant une signification fonctionnelle[END_REF] (Fig.

2.2). Mycorrhiza parameters were calculated using the MYCOCALC program (http://www.dijon.inra.fr/mychintec/Mycocalc-prg/download.html). The parameters used were F % (frequency of mycorrhizal colonization in the root system), M % (intensity of mycorrhizal colonization in the root cortex), m % (intensity of mycorrhizal colonization in mycorrhizal root fragments), A % (arbuscule abundance in the root system), a % (arbuscule abundance in mycorrhizal root fragments). Also the development of vesicles in roots was estimated as V % (vesicle abundance in the root system), v % (vesicle abundance in mycorrhizal root fragments), using the same scoring as for arbuscules

Laser Capture Microdissection (LCM) of arbusculecontaining cortical root cells

Tissue preparation

Roots of M. truncatula J5 colonized by G. intraradices BEG141, harvested between 17 and 21 dai, were cut into 0.5-cm pieces and fixed in Tissue-Tek® O.C.T.™ Compound at -24 °C. Sectioning was performed at -24 °C using a CRYOCUT 1800 microtome (Leica, Germany) and RNAse-free tools. 35 μm thick sections were placed on RNA-free slides (Probe On Plus Microscope Slides, Fisher Scientific, France). To remove the medium, slides were incubated on ice as follows: 3 min in 70 % ethanol, then 40 min in DEPCtreated water and rinsed twice in 100 % ethanol for 2 min. After the last ethanol step, slides were kept on a warmer at 37 °C for 25 min. After that, the root tissues were used directly for microdissection or stored at -80 °C then placed in a desiccator before microdissection.

Dissection of arbuscule-containing cells

Dissection was performed using an Arcturus™ XT Microdissection instrument (Life Sciences ∕ Roche Applied Biosystems, France). Sections on the slides were visualized under a computer monitor through a video camera, and selected cells were marked using graphical software, isolated using an infrared (IR) laser and captured automatically into CapSure HS laser capture microdissection caps (Arcturus Picopure isolation kit (Life Sciences ∕ Roche Applied Biosystems, France). To prevent RNA degradation, dissection in one cap was performed within 30 min and then transferred into RNA extraction buffer provided in the Picopure RNA Isolation Kit.

RNA extraction from laser-dissected cells

Harvested laser-dissected cells were incubated in the RNA extraction buffer for 45 min at 42 °C, centrifuged at 800 × g for 2 min, and stored at -80 °C until use. RNA was extracted using the Picopure RNA Isolation Kit according to the manufacturer's protocol.

The quantity and quality of RNA were checked with an Agilent Bioanalyzer 2100 (http://www.home.agilent.com/).

One-step RT-PCR

After RNA extraction, the RT-PCR experiments were performed using a One-Step RT-PCR kit (Qiagen, Valencia, CA, U.S.A.). Reactions were carried out in a final volume of 50 μl containing 10 μl of 5× buffer, 2 μl of 10mM dNTPs, 3 μl of each primer (10 μM), 2 μl of One-Step RT-PCR enzyme mix, 8 units of RNAse inhibitor, and 3 ng of total RNA from arbusculated cells and noncolonized cortical cells from mycorrhizal roots. The samples were incubated for 30 min at 58 °C, followed by 15-min incubation at 95 °C.

Amplification reactions (the primers and conditions are indicated in Table 2.5 and2.6) were run for 35 to 40 cycles. The PCR products were separated by agarose gel electrophoresis in a Trisacetate-EDTA 0.5× buffer, stained with ethyl bromide, and visualized using a VersaDoc Imaging System (Bio-Rad Laboratories, CA, U.S.A.).

Experiments were repeated using arbusculated cells from two different biological repetitions (C1 and C2) and representative results are shown.

Nucleic acid preparation from spores and roots

RNA extraction

Total RNA was extracted from G. mosseae quiescent spores and spores germinated in water or stimulated by plant root exudates (2000 per treatment) using a E.Z.N.A. fungal RNA kit (Omega Bio-Tek Inc, USA) (E.Z.N.A., EaZy Nucleic Acid), and DNA removed by RNase-Free DNase (Omega Bio-Tek Inc, USA) according to the manufacturer's protocol. RNA quantity was estimated using an Eppendorf biophotometer (Eppendorf, Germany).

Total RNA was extracted from G. intraradices DAOM197198 spores, G. intraradices BEG141 spores and mycorrhizal (G. intraradices BEG141) M. truncatula roots using a SV Total RNA Isolation System (Promega, Madison, WI, USA), according to the manufacturer's protocol. The DNase-containing columns of the Promega kit for total RNA isolation (SV Total RNA Isolation System) were used to remove any DNA contamination.

Total RNA was isolated from mycorrhizal (G. intraradices DAOM197198) M. truncatula roots by A. Aloui using the phenol-chloroform method according to Franken and Gnadinger (1994). In order to test the specificity of the fungal primers, cDNA from non-mycorrhizal M. truncatula root was used as control; total RNA was extracted from non-mycorrhizal M. truncatula roots using the Qiagen RNeasy Plant Mini Kit (74904) following the manufacturer's instructions (Qiagen, Hilden, Germany). RNA integrity, quantity and quality were controlled by 1.2 % denaturing agarose gel electrophoresis and photometric analysis (Eppendorp biophotometer).

cDNA synthesis

Genomic DNA extraction from fungal spores

G. mosseae genomic DNA was extracted from 2000 spores using the protocol described by Zézé et al. (1994). Briefly, spores were gently crushed in TE buffer containing 2 % mercaptoethanol, the suspension was centrifuged and nuclei were resuspended in lysis buffer. After centrifugation, the supernatant was collected and proteins were precipitated with potassium acetate. Nucleic acids contained in the supernatant were treated with DNAse-free RNAse A, and then purified with phenol-chloroform. The purified DNA was precipitated in ethanol at -70 °C, centrifuged, washed with 70 % ethanol, air dry and resuspended in TE buffer or sterile water.

Plasmid DNA preparation

Amplified fragments of cDNA (see below) were cloned into a plasmid vector using the TOPO TA Cloning kit (Invitrogen Corporation, Carlsbad, CA, USA). The plasmids were recuperated by white-blue selection and PCR. Recombinants (white colonies) were multiplied in liquid LB culture containing ampicillin (50 μg / ml), and plasmid DNA was isolated using the NucleoSpin®Plasmid kit (Macherey-Nagel, Düren, Germany) following the manufacturer's instructions. The yield and purity of plasmid DNA were estimated using an Eppendorp BioPhotometer. Cloned PCR products were sent for sequencing to MWG-Biotech (Ebersberg, Germany).

Fungal gene selection and primer design

Gene selection

ESTs of two calcium-related G. mosseae genes (Gm2 and Gm152) were selected from amongst those reported to be up-regulated with appressorium formation; these putatively code for a MAP3k-like protein kinase and a P-type ATPase respectively [START_REF] Breuninger | Recognition events in AM symbiosis: analysis of fungal gene expression at the early appressorium stage[END_REF]. The G. mosseae gene coding for the H + -ATPase PMA1, whose expression was previously shown not to vary during appressorium formation, was used for comparison as described by [START_REF] Breuninger | Recognition events in AM symbiosis: analysis of fungal gene expression at the early appressorium stage[END_REF]. EST sequences and similarity to genes in other eukaryotes are given in Table 2.3 Seven fungal ESTs encoding Ca 2+ -related proteins were selected from the transcriptome sequencing programme of G. intraradices DAOM197198 (syn. R.

irregularis) according to the microarray expression analysis results, on the basis of their putative function and expression profile in microarray analyses. Most of the selected genes were up-regulated in mycorrhizal roots as compared to the spores at least 2.5 fold with a p value <0.05 [START_REF] Tisserant | The transcriptome of the arbuscular mycorrhizal fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate symbiont[END_REF]. Homology searches were carried out using NCBI and Swissprot databases by blastx. Six ESTs putatively encoded proteins involved in membrane transport (two vacuolar Ca 2+ ion transporters, three Ca 2+ ATPases, one mitochondrial Ca 2+ -binding carrier) and one Ca 2+ /calmodulin-dependent protein kinase (CCaMK). Six of the corresponding genes encoded proteins involved in membrane transport: plasma membrane/tonoplast, endoplasmic reticulum, golgi reticulum, mitochondria, and one is a nuclear protein (Table 2.4). Two symbiosis-related G.

intraradices genes, stearoyl-CoA desaturase (DESAT) and a phosphate transporter (GiPT), were used as controls to monitor fungal activity; G. intraradices large-subunit (LSU)

rRNA was used to confirm the presence of fungal RNA for transcript analyses, and α subunit of translation elongation factor 1 (TEF) as the fungal reference house-keeping gene [START_REF] Maldonado-Mendoza | A phosphate transporter gene from the extra-radical mycelium of an arbuscular mycorrhizal fungus Glomus intraradices is regulated in response to phosphate in the environment[END_REF][START_REF] Seddas | Symbiosis-related plant genes modulate molecular responses in an arbuscular mycorrhizal fungus during early root interactions[END_REF]. EST sequences and similarity to genes in other eukaryotes are given in Table 2.4. 2.3).

Primers were also manually designed for the selected ESTs of G. intraradices Gi-TEFrev GCACAATCGGCCTGA GAAGTAC 1) Amplicon size

DAOM197198 (Glomus_c17763, Glomus_lrc1694, Glomus_c3514, Glomus_c2781, Glomus_c23149, Glomus_995) (

Polymerase chain reaction (PCR)

Reverse-transcription (RT-PCR)

Transcripts of each selected gene were amplified by RT-PCR. Reactions were carried out in 20 µl PCR mix containing 0.2 U of Taq polymerase and 2 µl 10X buffer with 1.5 mM MgCl 2 (Invitrogen, USA), 125 µM dNTPs, 0.5 µM of each gene-specific primer pair (Table 2.3, 2.5 and 2.6), and 1:10 diluted cDNA from spores or roots. Reactions were conducted in a T3000 thermocycler (Biometra, Germany). For the G. mosseae genes, the amplification programme was 60 °C with 33 amplification cycles, and for G. intraradices genes the following program was used: 94 °C for 3 min, 30 cycles (94 °C for 1 min, 58 or 60 °C for 1 min, 72 °C for 1 min), 10 min at 72 °C. PCR products were separated by 1.2 % agarose gel electrophoresis for 25 min at 100 volts, gels were stained 10 min in ethidium bromide and documented under UV light using GelDoc EQ apparatus (BioRad, USA).

Relative quantitative real-time PCR

Targeted genes of G. intraradices DAOM197198

Relative real-time PCR reactions were carried out to quantify gene transcripts using an ABI PRISM 7900 real-time cycler (Applied BioSystems, Foster City, CA, USA), and the ABsolute SYBR green ROX MIX (ABsoluteTM QPCR® SYBR Green ROX Mix 2x; Thermo Scientific, UK) as fluorescent dye. The gene specific primers are described above (see tables 2.5, 2.6). Each reaction (15 µl) contained 2 µl of 1:20 diluted cDNA from spores and root-extracted RNA, 7.5 µl SYB green mix (from Absolute QPCR SYBR Green kit), 1 pM of each primer and sterile water. The amplification program was performed as follows: 95 °C for 15 min, 40 cycles (95 °C for 15 s, 58 °C for 30 s, 72 °C for 20 s, 75 °C for 15 s). A melting curve (95 °C for 15 s, 55 °C for 15 s, 95 °C for 15 s) was recorded at the end of every run to verify the specificity of amplification. Primer efficiency (E) was calculated using the formula: E=10 (-1/slope) -1 (Invitrogen guide for important parameters of quantitative PCR analysis). cDNA amplifications were quantified using 5 cDNA dilutions to produce a linear slope. Three biological repetitions, each with two technical repetitions, were analyzed for each treatment. The threshold was set at 0.2 and Ct (threshold cycle) values were automatically calculated by the SDS 2.3 program (Applied Biosystems, Foster City, USA). To compare to microarray data, the relative expression ratio between mycorrhizal M. truncatula roots and water-activated spores was calculated according to the formula of [START_REF] Pfaffl | A new mathematical model for relative quantification in real-time RT-PCR[END_REF] using the reference gene GiTEF: PCR System Thermal Cycling Block (Applied Biosystems, USA) using the ABsolute SYBR green ROX MIX (ABgene, Epsom, UK), and the gene specific primers described above (see tables 2.5, 2.6). Assays were performed on cDNA from two biological repetitions of differently treated spores and three biological repetitions of mycorrhizal M. truncatula roots for each time point. For each sample, PCR reactions were carried out in technical duplicates using 2 μl of 1:20 diluted cDNA from spores and 1:4 of diluted cDNA from mycorrhizal root as template. PCR was performed as follows: 95 °C for 15 min, 40 cycles (95 °C for 15 s, 58 °C for 30 s, 72 °C for 30 s, 75 °C for 15 s) and for the melting curve: 95 °C for 15 s, 72 °C for 1 min, 95 °C for 15 s. Amplification efficiency of each primer pair was determined using a serial dilution of cDNA. The generated data were analysed by StepOne software v2.1 (Applied Biosystems, USA) using a threshold set at 0.2.

ratio= (E target ) ΔCt target (control-sample) (E ref ) ΔCt ref (control-sample)
The GiTEF gene served as reference, and relative gene expression was calculated using the formula R=2 ΔCT(reference-target) .

Ct target : the threshold cycle of target gene transcript Ct reference : the threshold cycle of reference gene transcript

Absolute quantitative real-time PCR

Transcript abundance of genes corresponding to BEG141_c2781, BEG141_c10704, BEG141_lrc1694 and BEG141_lrc995 was quantified by absolute Q-PCR, using the Step One Plus Real-Time PCR System Thermal Cycling Block (Applied Biosystems, USA) and the ABsolute SYBR green ROX MIX (ABgene, Epsom, UK). The expression of each gene was assayed in two technical repetitions and analyzed by the StepOne software v2.1 (Applied Biosystems, USA). To calculate the absolute number of transcripts present in the original samples, plasmid DNA containing each amplicon was quantified by UV absorbance spectroscopy and linearized by digestion with the restriction enzyme BamH1. A standard curve of each primer pair was determined using a serial dilution of linearized plasmid DNA at dilutions of 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , and 10 7 copies for each assay. To verify the specificity of each amplification, a melting-curve analysis was included at the end of each PCR run. Relative number of transcripts for each gene was calculated as a ratio to the amount of the reference gene TEF transcripts.

In situ RT-PCR

In situ RT-PCR was performed according to [START_REF] Seddas | Spatial monitoring of gene activity in extraradical and intraradical developmental stages of arbuscular mycorrhizal fungi by direct fluorescent in situ RT-PCR[END_REF] on spores from pot cultures or roots prepared as follows. Surface sterilized seeds of M. truncatula line J5 were germinated as described above, and half the seedlings were transplanted into 75-ml pots containing the Terragreen/soil-based G. intraradices BEG141 inoculum mix for inoculated plants. The other half of the seedlings were grown as controls with autoclaved inoculum and 1ml bacterial filtrate for each pot (15ml inoculum in 15ml water filtered with Whatman 2v filter paper). Plants, grown under constant conditions as described above, were harvested from 17 to 21 dai, root systems were washed in ice-cold water and fresh roots were immediately fixed for in situ RT-PCR.

Spores or roots were fixed in 67 % (v / v) ethanol and 23 % (v / v) acetic acid containing 10 % (v / v) DMSO. After 1 h incubation at 4 °C under vacuum, samples were placed in 200 µl fresh fixative solution during 16 h at 4 °C. Samples of fixed materials were washed twice in 67 % (v / v) ethanol and 23 % (v / v) acetic acid then once in DEPCtreated water. Cell walls were permeabilised by digestion with chitinase from Streptomyces griseus (Sigma) and pectinase from Aspergillus niger (Sigma). After washing in the same buffer, samples were digested with proteinase K. Genomic DNA was digested with Hae III (Promega) and Hpa II (Promega) in presence of RNAsin (Promega), then eliminated with DNAse. RNA controls were carried out with RNase before performing the DNAse treatment.

The large ribosomal subunit gene (LSU rRNA) of G. intraradices BEG141 was used to standardize the in situ RT-PCR methodology. Genes putatively encoding two P-type Ca 2+ ATPases (BEG141_c2781, BEG141_c10704) and one tonoplast Ca/Mn transporter (2) (BEG141_lrc1694), as well as the control genes DESAT, TEF and GiPT, were chosen to localize gene activities in spores and mycorrhizal roots. Gene-specific primers (Table 2.5 and 2.6) were labeled with Texas Red (MWG-Biotech). Controls were provided by omission of primers in the reverse transcription reaction.

Following amplification, the PCR-mix was removed and samples were post-fixed in 100 % ethanol during 10 min at room temperature then quickly wash in 70 % ethanol.

Each sample was deposited on a microscope slide in anti-fading medium (DAKO) and stored at 4 °C in the dark until observation. Fluorescence was observed using a confocal microscope (LEICA TCS SP2 AOBS; Leica Microsystems, Germany). Excitation was carried out with a 594 nm laser used at 27 % of its maximum power with a photomultiplicator at 687 V. The resulting signal was collected between 606 and 640 nm.

Autofluorescence of plant and fungal tissues was avoided under these conditions of excitation and signal collection. A 40× oil objective was used to seize images. Each fluorescent image corresponded to the maximum projection of optical sections from a z series, using Leica Confocal software. The resultant depth (z) of each projection was about 300 nm. The optical section number of each projection was between 20 and 40. A Nomarski image was taken in parallel each time.

The same treatment was also done on the 40 day old G. mosseae-inoculated A. sinicus roots (see chapter 2.1.1) to locate gene expression of Gm152. The Gm152 gene-specific primers (Table 2.3) were labeled with TET (Tetrachlorofluorescein, a fluorescent dye labeling) (Sangon Shanghai, China). Fluorescence was observed using a confocal microscope (Zeiss LSM510 META, Germany); a 488 nm laser was used for excitation and the resulting signal was collected at 514 nm for the mycorrhizal root tissues.

Rapid Amplification of cDNA Ends (RACE)

Since the 5′end cDNA sequence of Gm152 was available in the G. mosseas SSH library database, only the 3′end cDNA sequence was obtained using rapid amplification of cDNA ends (RACE) according to [START_REF] Scotto-Lavino | 3′ end cDNA amplification using classic RACE[END_REF]. Briefly, to generate the 3' end, mRNA was reverse transcribed using a primer QI-QO that terminates in two mixed bases (GATC / GAC) followed by 17 Ts and a unique primer sequence. The end was amplified using the primer QO that contains part of this sequence and that binds to these cDNAs at their 3'ends, and a primer that matches the gene of interest, 152GSP1. A second amplification series was then performed using internal primers QI and 152GSP2 to suppress the amplification of non-specific products. Primers were listed in Table 2.7. A LA Taq DNA Polymerase (TaKaRa, Japan) was used for this LA (Long and Accurate) PCR amplification from G. mosseas cDNAs according to the manufacturer's instructions.

PCR products were visualized by electrophoresis on 1.0% agarose gels stained with ethidium bromide. Bands were excised and purified using the TIANgel Midi Purification Kit (Tiangen Biotech, Beijing, China). PCR products were sequenced (AuGCT Biotechnology, Beijing, China) and homology searches were carried out using NCBI databases by BLAST search. Multiple sequence alignments of translated gene sequences were carried out with the program CLUSTALW (http://www.ebi.ac.uk/clustalW/). The complete cDNA sequence of three selected G. intraradices BEG141 ESTs was obtained after 3′ and 5′ rapid amplification of cDNA ends (3′, 5′ RACE) using the Generacer kit (Invitrogen Corporation, Carlsbad, CA, USA) according to the manufacturer's instructions. The gene-specific primers for 3' and 5' RACE, used in conjunction with GeneRacer primers for PCR amplification, are listed in

PCR amplification on genomic DNA

The full genomic sequence of Gm152 was obtained by PCR amplification of G. mosseae genomic DNA using Inverse PCR (Molecular Cloning: A Laboratory Manual) and TAIL-PCR [START_REF] Liu | High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences[END_REF]. Based on a known (429bp) cDNA sequence, specific primers were designed for Gm152 and a series of PCR reactions were carried out using primers listed in Table 2.9 and Table 2.10.

Inverse PCR

Inverse PCR, which is a method used to amplify DNA with only one known sequence, involves restriction digestion and ligation resulting in a looped fragment that can be primed for PCR from a single section of the known sequence.

Genomic DNA was digested into fragments using the restriction enzyme EcoR1 (Promega, USA) according to the manufacturer's instructions. After, self-ligation with T4

DNA ligase (Promega, USA) was induced to give a circular DNA product. PCR was carried out with the primer pair 152f-2/152AR and 152f-3/152AR (Table 2.9), which are complementary to sections of the known internal sequence. PCR was done in a 20 µl volume containing 5 µg circularized DNA, 0.2 U LA Taq (TaKaRa, JAPAN), 2µl 10×PCR buffer (Mg 2+ ), 2.5 mM dNTP and 10 μM primers. The amplification program was performed as follows: 94°C for 3 min, 30 cycles (94°C for 1 min, 58 or 60°C for 1 min, 72°C for 3 min), 72°C for 5 min. PCR products were sequenced (AuGCT Biotechnology, Beijing, China) and homology searches were carried out using NCBI databases by BLAST search. Finally, the sequence was verified by comparing with the sequence available in the data base.

TAIL-PCR

The Thermal Asymmetric Interlaced PCR (TAIL-PCR) method is a technique for genomic walking which does not require any restriction or ligation steps as compared to inverse PCR. It utilizes nested known long sequence-specific primers with high melting temperature in combination with short (15-16 nucleotides) degenerate primers of arbitrary sequences (AD primer). A thermal cycling program composed of low-stringency and highstringency cycles, allows only sequence-specific fragments to be exponentially amplified.

AD primers with higher degrees of degeneracy in the low-stringency PCR cycle may have more chances to bind to the target sequences. In the high-stringency PCR cycles with high annealing temperatures, only the specific primer with the higher melting temperature can efficiently anneal to target molecules [START_REF] Liu | High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences[END_REF]. Primers used for TAIL-PCR of Gm152 are given in Table 2.9 and the PCR conditions are listed in Table 2.10.

The protocol for TAIL-PCR was as follows:

-Primary reaction Each primary reaction mixture (20 µl) consisted of 2 µl 10×PCR buffer, 200 µM each of dNTPs, 0.2 µM specific primer (152f1 or F1), 1.2 µM AD primer, 1.25 unit of Taq DNA polymerase, and 1-10 ng of DNA.

-Secondary reaction

The secondary PCR mixtures (20µl) contained 2 µl 10×PCR buffer, 200 µM each of dNTPs1, 0.2 µM internal specific primer (152f2 or F2),1.2 µM the same AD primer, 1.25 units of Taq DNA polymerase and 1 µl of the 40 time diluted PCR product from the primary reaction.

-Tertiary reaction

The tertiary PCR mixtures (20 µl) contained 2 µl 10×PCR buffer, 200µM each of dNTPs, 0.5 µM the innermost specific primer (152f3 or F3), 3µM the same AD primer, 1.25 units of Taq DNA polymerase and 1 µl of the 10 time diluted PCR product from the secondary reaction.

The secondary and tertiary PCR products were visualized by electrophoresis on 1.0% agarose gels stained with ethidium bromide. The specificity of the products was confirmed by the expected differences of size between the secondary and tertiary products. MATa, ura3-52, trp1-Δ2, leu2-3_112, his3-11, ade2-1, can1-100 Open Biosystems (Thermo Scientific) YGL167C MATa, ura3-52, trp1-Δ2, leu2-3_112, his3-11, ade2-1, can1-100, pmr1Δ

Open Biosystems (Thermo Scientific) YDL128W MATa, ura3-52, trp1-Δ2, leu2-3_112, his3-11, ade2-1, can1-100, vcx1Δ

Open Biosystems (Thermo Scientific) YFR014C MATa, ura3-52, trp1-Δ2, leu2-3_112, his3-11, ade2-1, can1-100, cmk1Δ

Open Biosystems (Thermo Scientific) K667

MATa, ura3-1, trp1-1, leu2-3, his3-11, ade2-1, can1-100,cnbΔ:: LEU2, pmc1Δ::TRP1, vcx1Δ

Cagnac et al 2010

Plasmid construction and yeast transformation

The yeast complementation vectors were constructed with Gateway technology with Clonase™ II (Invitrogen Corporation, Carlsbad, CA, USA) according to the manufacturer's instructions. Destination vector pDRf1-GW used for gateway was from [START_REF] Loqué | A cytosolic trans-activation domain essential for ammonium uptake[END_REF]. The complete gene coding sequences were amplified using proofreading DNA polymerase (Platinum® Taq DNA polymerase High Fidelity; Invitrogen Corporation, Carlsbad, CA, USA). The primers used are listed in Table 2.13. All the plasmids were multiplied in E. coli grown at 37 °C in LB broth or on agar supplemented with 50 μg / ml of kanamycin or ampicillin according to the manufacturer's instructions. Yeast transformations were performed using a protocol adapted from [START_REF] Gietz | Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure[END_REF]. Mutant strains received either the pDRf1-GW -gene vector or the pDRf1-GW empty vector, both containing a URA3 auxotrophic marker.

Growth media and tests

Transformants were selected on a SD media (0.67 % yeast nitrogen base, 2 % glucose)

with the appropriate amino acids and lacking uracil. The medium for growth tests were YPD medium (1 % yeast extract, 2 % peptone, 2 % glucose) or SD medium with / without uracil. The medium adding a series of concentration of CaCl 2 were used for knockout strain phenotypic selection according to [START_REF] Szigeti | Calcium and magnesium competitively influence the growth of a PMR1 deficient Saccharomyces cerevisiae strain[END_REF] and [START_REF] Kmetzsch | The vacuolar Ca 2+ exchanger Vcx1 is involved in calcineurin-dependent Ca 2+ tolerance and virulence in Cryptococcus neoformans[END_REF].

Cells were grown at 30 °C and 37 °C for 2 d.

Statistical analysis

All data were statistically compared between treatments using one-way ANOVA using the Systat version 10 program (Systat Software 2000). Significant differences between means were established by the Tukey's pairwise comparison test at P≤0.05.

Where ANOVA gave P values just above 0.05 between wild type and mutant root data, gene expression was also compared separately for different time points using the Student's t-test at P≤0.05.

CHAPTER 3

Studies of G. mosseae genes expressed before root contact with A. sinicum

Evidence for the implication of calcium in early recognition responses of roots to the symbiotic fungi has been obtained from gene expression studies, but little is known for the molecular mechanism on fungal partner. [START_REF] Breuninger | Recognition events in AM symbiosis: analysis of fungal gene expression at the early appressorium stage[END_REF] reported that gene expression in G. mosseae during appressorium development was up-regulated. In their work, the expression of genes (named Gm2 and Gm152), putatively encoding a MAP3klike protein kinase (clone 2) and a P-type ATPase (clone 152), were up-regulated with appressorium formation on parsley roots and could potentially be involved in cellular Ca 2+ homeostasis and signaling. Consequently, we targeted these two genes for further study.

This work (the 1 st year of my PhD research) was carried out at Huazhong Agriculture University, Wuhan, China. A. sinicus was used as host plant since much work about AM fungi -A. sinicus interactions has been carried out in the laboratory in Wuhan.

Fungal gene expression monitored by reversetranscription (RT)-PCR

Transcript analyses of the two selected G. mosseae genes and PMA1 by RT-PCR indicated significant activation of Gm2 and slight up-regulation of Gm152 in germinated spores stimulated by crude A. sinicus root exudates as compared to spores germinated in water (Fig. 3.1). PMA1 expression did not appear to be affected by root exudates; this gene is highly expressed during asymbiotic growth but is down-regulated during the symbiotic phase of fungal growth in roots [START_REF] Requena | Symbiotic status, phosphate, and sucrose regulate the expression of two plasma membrane H + -ATPase genes from the mycorrhizal fungus Glomus mosseae[END_REF].

Localization of G. mosseae Gm152 gene activity in mycorrhizal roots of A. sinicus

Results from in situ RT-PCR of Gm152 transcripts are given as Nomarski images, confocal fluorescence images, and a superposition of fluorescence and Nomarski images (Figs. 3.5,3.6). Samples with RNAse digestion served as negative controls (Fig. 3.5).

Autofluorescence of plant and fungal tissues was eliminated by analysing autofluorescence under different wavelength regions; where wavelength regions gave strong fluorescence in the negative control samples, these regions were avoided and only regions where autofluorescence in the negative controls was weaker were used to observe samples. When green fluorescent-labeled amplified cDNA was observed in situ, only a weak signal was detected in extraradical mycelium, whereas no signal was detected in the any cells of mycorrhizal A. sinicus root tissues (Fig. 3.6). Gm152 appears to be expressed in extraradical mycelium but it was not possible to conclude to the localization of Gm152 expression in intraradical fungal structures as the in situ RT-PCR gave a very weak signal or important background.

Discussion and conclusions

In a first step to investigate the role of Ca 2+ in the molecular responses of the fungal partner during processes leading to the establishment of the mycorrhizal symbiosis, we selected two G. mosseae genes, Gm2 and Gm152, which had previously been reported to be up-regulated at the early stage of appressoria formation on parsley roots [START_REF] Breuninger | Recognition events in AM symbiosis: analysis of fungal gene expression at the early appressorium stage[END_REF]. The putative function and expression profiles of these two genes suggested that they may be involved in Ca 2+ -regulated processes related to mycorrhiza interactions. Following the expression pattern observed by RT-PCR for Gm2 and Gm152 in root exudates of A. sinicus, both fungal genes appear to be activated also prior to contact with the host plant.

The full-length sequence obtained for Gm152 confirmed a probable function of Ptype ATPase for this G. mosseae gene. ATPases carry out many energy-driven fundamental processes in biology, and P-type ATPases are members of a large family of biological energy transducers that utilize the chemical energy of ATP hydrolysis to generate transmembrane electrochemical ion gradients. The Gm152 protein contains two functional regions, E1-E2_ATPase and a hydrolase, and it shows high similarity to the endoplasmic reticulum Ca 2+ -transporting P-type ATPase from the ectomycorrhizal fungus L. bicolor.

Consequently, this gene product might be involved in pumping Ca 2+ across the membrane to produce ion fluxes for Ca 2+ -dependent signaling processes. Furthermore, a Gigaspoara rosea gene, Gros(ASP)1, putatively encoding a Type IIB calcium ATPase was upregulated during the developmental switch from asymbiosis to presymbiosis [START_REF] Tamasloukht | Root exudate-stimulated RNA accumulation in the arbuscular mycorrhizal fungus Gigaspora rosea[END_REF]. In this case, Gm152 could be one component of Ca 2+ -related proteins that play a role in the adaptation of the cell to variations in Ca 2+ concentrations (homeostasis) during symbiotic interactions.

However, further investigations of G. mosseae Ca 2+ -related genes that could be involved in cellular Ca 2+ homeostasis and signaling during successful mycorrhizal interactions are hampered by the lack of sequence information for this fungus. It was therefore decided to continue this work using another AM fungus, G. intraradices, for which a large amount of genome and transcriptome data has recently become available [START_REF] Martin | The long hard road to a completed Glomus intraradices genome[END_REF][START_REF] Tisserant | The transcriptome of the arbuscular mycorrhizal fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate symbiont[END_REF]. A preliminary search showed the presence of a gene (Glomus_c14022) homologous (84% similarities) to Gm152 in the G. intraradices genome. However, the microarray analysis showed that gene Glomus c14022 was down-

Medicago truncatula wild-type J5 and the mutant TRV25

Fresh shoot and root weights and fungal development were determined at each time point after inoculation with G. intraradices BEG141. Plant growth (Table 4.1) and appressoria formation by the AM fungus (Table 4.2) did not differ significantly (t-test) between wild-type M. truncatula J5 and the symbiosis-defective mutant TRV25 at 4 and 6 days after inoculation (dai). At later stages of interactions with (17, 21 dai), hyphae had penetrated the roots of wild-type M. truncatula, forming arbuscules within cortical cells and vesicles (Fig. 4.1), whilst root penetration beyond the epidermis did not occur on the mutant roots. Timecourse results of mycorrhiza colonization showed that G. intraradices developed well in wild-type M. truncatula roots (Table 4.3). The AM fungus had penetrated wild-type J5 roots forming rare arbuscules (A % = 0.09) at 6 dai, and by 21 dai, most of the root system of wild-type M. truncatula was colonized (M % = 80.4) and arbuscules were abundant (A% = 67.5) (Fig. 4.2).

Wild type Pisum sativum L. cv Finale and the mutant

Pssym36

No significant difference (t-test) in the weight of roots or shoots was observed between the two pea genotypes (Tables 4.4), indicating that the mutation in the Pssym36 gene did not affect biomass accumulation at 21dai as compared with wild type cv Finale plants. G. intraradices developed less in Pssym36 mutant roots than in wild type cv Finale according to the mycorrhization parameters (Tables 4.5). The AM fungus penetrated mutant Pssym36 roots but rarely formed vesicles, and only aborted arbuscules with stumpy branches were observed within root cortical cells at 21 dai as compared to wild type cv Finale (Fig. 4.3).

Genotypes F % M % A % V % m % a % v % Finale

71.1 ± 2.2 35.2 ± 2.3 21.9 ± 2.7 15.7 ± 3.1 49.8 ± 4.6 61.7 ± 3.6 43.9 ± 6.1 Pssym36 56.7 ± 8.4 13.0 ± 6.7 1.43 ± 0.9 0.20 ± 0.2 20.7 ± 7.6 9.87 ± 1.3 0.81 ± 0.7 Data presented are mean values with standard errors from three biological repetitions.

Discussion and conclusions

Microscope observations of G. intraradices BEG141 development confirmed that the M. truncatula and P. sativum mutants have modified AM phenotypes as compared to the wild type plants. AM colonization of roots in the M. truncatula mutant TRV25 (mutated for the DMI3/MtSYM13 gene) was blocked after the appressorium stage and characterized by the absence of intraradical mycelium, as already described [START_REF] Catoira | Four genes of Medicago truncatula controlling components of a Nod factor transduction pathway[END_REF][START_REF] Morandi | Characterisation of new symbiotic Medicago truncatula (Gaertn.) mutants, and phenotypic or genotypic complementary information on previously described mutants[END_REF]. Mutation in the PsSym36 gene of P. sativum influenced arbuscule formation and limited it to aborted branches after the penetration of root cortical cells. The production of vesicles was also blocked which may result from insufficient carbon transfer from the plant to the AM fungus in the absence of functional arbuscules [START_REF] Kuznetsova | Symbiosisrelated pea genes modulate fungal and plant gene expression during the arbuscule stage of mycorrhiza with Glomus intraradices[END_REF]. However, the mutations in the DMI3/MtSYM13 and PsSym36 genes did not affect plant development (shoot and root biomass accumulation) at 4, 6, 17 and 21 dai respectively.

The biological systems developed for M. truncatula and P. sativum were used in the studies that follow to investigate the role of G. intraradices genes related to calcium homeostasis and/or signalling in arbuscular mycorrhiza interactions.

R. irregularis)

Seven ESTs encoding Ca 2+ -related proteins were selected from the transcriptome sequencing programme of G. intraradices DAOM197198 (syn. R. irregularis) [START_REF] Tisserant | The transcriptome of the arbuscular mycorrhizal fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate symbiont[END_REF]. Six of the corresponding genes putatively encode proteins involved in membrane transport (two vacuolar Ca 2+ ion transporters, three Ca 2+ ATPases, one mitochondrial Ca 2+ -binding carrier) and one Ca 2+ /calmodulin-dependent protein kinase (CCaMK) (Table 5.1). More detailed homology searches were carried out by Blast p (Tables 5.2 and 5.3) for the three genes putatively encoding P-type ATPases, that is, the ESTs Glomus_c3514, Primers were manually designed from the ESTs Glomus_c17763, Glomus_lrc1694, Glomus_c3514, Glomus_c2781, Glomus_c10704, Glomus_c23149 and Glomus_995 (Table 5.4). PCR performed on cDNA from mycorrhizal roots or spores of G. intraradices DAOM197198 and sequencing of PCR products confirmed their specificity (data not shown).

Fungal gene expression monitored by reversetranscription (RT)-PCR

Gene expression in G. intraradices DAOM197198 spores and mycorrhizal roots of M. truncatula

Transcript analyses by RT-PCR showed that most of the G. intraradices DAOM197198 genes were expressed in spores activated by incubation in water and that the expression of five of the genes was clearly enhanced in mycorrhizal roots (Fig. 5.1).

The latter putatively encode the endoplasmic reticulum P-type ATPase, Golgi reticulum Ptype ATPase, two vacuolar Ca/Mn transporters and the nuclear CCaMK. For most of the genes, their expression profile observed by RT-PCR corresponded to results from hybridisation experiments (asterisks), using the Nimblegen microarray [START_REF] Tisserant | The transcriptome of the arbuscular mycorrhizal fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate symbiont[END_REF]. In order to use G. intraradices BEG141, which is a highly effective isolate, as a model AM fungus for the molecular work, the primers designed for the ESTs of G.

intraradices DAOM197198 were also used to amplify G. intraradices BEG141 cDNA.

Corresponding transcripts were monitored in mycorrhizal M. truncatula roots 17 dai with G. intraradices BEG141 cDNA, compared to cDNA from non-mycorrhizal M. truncatula roots as control, by gel RT-PCR using specific primers (see Tables 5.4) for each gene at After sequencing, the amplification products from G. intraradices BEG141 genes were compared to those from the isolate DAOM 197198, used to design the primers. High similarities between the nucleotide sequences indicated that the same genes were detected in the two fungal isolates (Table 5.5).

Transcript accumulation of the seven G. intraradices BEG141 ESTs was then monitored in quiescent spores, water-activated spores (24h) and mycorrhizal roots (17 dai) of the AM fungal isolate. G. intraradices large-subunit (LSU) rRNA was included in the analyses to ensure equivalent levels of fungal RNA at the different developmental stages.

Discussion and conclusions

In order to identify calcium-dependent processes that may be important in fungal events leading to mycorrhizal interactions with plant tissues, we selected seven fungal Ca 2+ -related proteins from the AM fungus G. intraradices DAOM 197198 according to their expression profiles in microarray analyses [START_REF] Tisserant | The transcriptome of the arbuscular mycorrhizal fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate symbiont[END_REF]. Two genes encode SERCA ATPases and one is related to PMCA ATPase. SERCA, or sarco/endoplasmic reticulum Ca 2+ -ATPases transfer Ca 2+ from the cytosol of the cell into the endoplasmic reticulum lumen [START_REF] Berridge | Calcium signalling: dynamics, homeostasis and remodelling[END_REF], whilst the plasma membrane Ca 2+ ATPase (PMCA) is a transport protein in the plasma membrane of cells that removes calcium from the cell, that is, transports Ca 2+ into the extracellular space [START_REF] Mcainsh | Shaping the calcium signature[END_REF]. Thus these proteins regulate the amount of Ca 2+ within cells to maintain low concentrations of Ca 2+ for proper cell signaling. Attempt to also design primers for a Gm152 (in chapter 3, which encoding an endoplasmic reticulum Ca 2+ -transporting P-type ATPase) homologue (Glomus _c14022) in G. intraradices have been unsuccessful. (asterisks) (Table 6.1). Taken together with the RT-PCR results, slight discrepancies in gene expression may be expected compared to the microarray analysis since microarray hybridizations may provide data for the overall expression of multigene families, with members having different expression levels, while the primers used in Q-PCR are more discriminatory.

Moreover, the M. truncatula mycorrhizal roots used for microarray and Q-PCR analyses were produced in different laboratories under different conditions.

Gene expression in G. intraradices BEG141 spores

At the asymbiosis stage (spores in absence of root exudates) and pre-symbiotic stage (spores in presence of root exudates), the relative expression of all the genes remained low as compared to the reference TEF gene, except for DESAT in quiescent spores. Most of the fungal genes showed no significant variation in gene expression between treaments and none of the genes were activated by root exudates, as compared to quiescent spores (Fig. 6.1). Expression of two Ca 2+ -related genes decreased with spore germination. The gene encoding a plasma membrane/vacuolar Ca 2+ -transporting ATPase (BEG141_c3514) was down-regulated during spore germination and no transcripts were detected after 6 days in water or root exudates, whilst the mitochondrial membrane Ca 2+ -dependent carrier (BEG141_c23149) gene was inhibited by root exudates from wild-type M. truncatula.

Only one gene was activated after incubation of spores 24h in water, the vacuolar Ca/Mn transporter 1 (BEG141_c17763), but expression decreased with spore germination after 6 days in water or root exudates (Fig. 6.1).

Gene expression in G. intraradices BEG141 associated with wild-type and mutant M. truncatula roots

The expression profiles of all the studied genes varied between early (4, 6 dai) and late (17, 21 dai) stages of development of G. intraradices with wild-type M. truncatula roots (Fig. 6.2). Expression of the symbiosis-related marker genes DESAT and GiPT relative to the reference TEF gene increased to high levels as G. intraradices developed arbuscules in roots of wild-type M. truncatula, indicating the formation of an active symbiosis. Very little expression of these genes was associated with appressoria formation on roots of either the wild-type or mutant genotypes.

Five of the seven selected Ca 2+ -related G. intraradices genes had very low levels of expression relative to the reference TEF gene during fungal development with M. truncatula roots. Expression was highest for the two P-type Ca 2+ ATPases (BEG141_c2781 and BEG141_c10704) and lowest for the plasma membrane/vacuolar Ca 2+ -transporting ATPase (BEG141_c3514). Most of the Ca 2+ related fungal genes were more weakly or not expressed when G. intraradices came into contact with roots of the dmi3/Mtsym13 mutant, as compared to wild-type roots (Fig. 6.2). Exceptions were the vacuolar Ca/Mn transporter 1 (BEG141_c17763) and the nuclear CCaMK (BEG141_lrc995) genes.

Expression of four genes followed fungal development from appressoria formation (4, 6 dai) on both M. truncatula genotypes, and to root colonization (17, 21 dai) of the wildtype genotype; these encode the endoplasmic reticulum and Golgi reticulum P-type Ca 2+

ATPases (BEG141_c2781 and BEG141_c10704), the vacuolar Ca/Mn transporter 1 (BEG141_c17763) and the nuclear CCaMK (BEG141_lrc995). For the first three genes, expression tended to decrease but not significantly from 17 to 21 dai. In contrast, expression of three fungal genes was only detected during arbuscule development after root colonization (17, 21 dai) and none of them were active during appressorium formation on wild-type or mutant roots; these encode the mitochondria Ca 2+ -dependent carrier (BEG141_c23149), the plasma membrane/vacuolar Ca 2+ -transporting ATPase and the vacuolar Ca/Mn transporter 2 (BEG141_lrc1694). Expression of the mitochondria Ca 2+dependent carrier and the plasma membrane/vacuolar Ca 2+ -transporting ATPase increased significantly from 17 to 21 dai whilst that of the vacuolar Ca/Mn transporter 2 remained constant (Fig. 6.2). Relative expression

Absolute quantitative Real-time RT-PCR

Expression of the G. intraradices genes was validated by measuring transcript abundance relative to that of the TEF gene using absolute Q-PCR. For this, the two symbiosis-related marker genes DESAT and GiPT, and four Ca 2+ -related genes showing different expression profiles during colonization of M. truncatula roots (endoplasmic reticulum P-type ATPase, Golgi reticulum P-type ATPase, vacuolar Ca/Mn transporter 2, nuclear CCaMK), were selected. Transcripts of all of the selected genes were detected in spores and mycorrhizal roots. Expression profiles at the asybiosis and pre-symbiotic stage of all the genes were similar to those from the relative Q-PCR analysis, with no significant variation in relative transcript abundance during spore germination in the different treatments (Fig. 6.3).

Increasing expression of the DESAT and GiPT genes with mycorrhiza formation in wild-type M. truncatula roots was confirmed by absolute Q-PCR (Fig. 6.4). Likewise, the golgi reticulum P-type ATPase and vacuolar Ca/Mn transporter 2 genes were weakly or not expressed in contact with roots of either M. truncatula genotype then up-regulated in mycorrhizal roots, whilst the vacuolar Ca/Mn transporter 1 (BEG141_c17763) and the nuclear CCaMK (BEG141_lrc995) genes were active throughout fungal development.

Discussion and conclusions

The relative and absolute Q-PCR analyses showed that the G. intraradices genes encoding Ca 2+ -related proteins vary in their expression profiles between pre-symbiotic and symbiotic stages, suggesting different functions in fungal development. None of the genes were significantly activated by root exudates and maximum expression for them all was clearly linked to the functional symbiosis in wild-type M. truncatula roots, which was desaturase catalyses the biosynthesis of mono-unsaturated fatty acids (UFAs) from saturated fatty acids, and UFA homeostasis in many organisms is achieved by feedback regulation of fatty acid desaturase gene transcription [START_REF] Mansilla | Signalling pathways controlling fatty acid desaturation[END_REF]. The DESAT gene also showed high activity in quiescent spores. As lipids are the major form of carbon storage in spores of AM fungi, DESAT may be involved in regulation of fatty acid at this stage as well as in lipid synthesis from host derived carbon in intraradical structures.

Calcium signaling involves calcium spiking which stimulates several Ca 2+ -binding proteins and elicits downstream signaling pathways. The calcium signal is terminated when cytosolic-free Ca 2+ concentrations are reduced to basal levels by moving calcium between cell components (McAinsh and Pittman, 2009). Vacuoles, endoplasmic reticulum, and mitochondria have important functions in Ca 2+ homeostasis. Vacuolar calcium ion transporters have a role in promoting intracellular calcium ion sequestration via the exchange of calcium ions for hydrogen ions across the vacuolar membrane; they can also be involved in manganese ion homeostasis via its uptake into the vacuole and belong to the sodium/potassium/calcium exchanger family [START_REF] Geisler | The ACA4 gene of Arabidopsis encodes a vacuolar membrane calcium pump that improves salt tolerance in yeast[END_REF]. The two vacuolar calcium ion transporters 1 and 2 of G. intraradices showed significant differences in gene expression during spore germination and fungal development. Expression of the calcium ion transporter 1 gene was significantly increased with spore activation in water but show little response with spore germination to plant root exudates. Nevertheless, expression was detected with appressorium development on wild-type or myc -mutant M. truncatula roots, and it increased when arbuscules formed in roots. In comparison, expression of the calcium ion transporter 2, which also showed no variation with spore germination, was only detected with arbuscule formation. Accordingly, these two genes could be putatively involved in different symbiotic processes. G. intraradices vacuolar calcium ion transporter 1 showed 49% identities and calcium ion transporter 2 showed 44% identities (blast p) to the membrane protein CrCAX1_CAR92574, a cation antiporter of the CAX (cation exchanger) family in Chlamydomonas reinhardtii [START_REF] Pittman | A cationregulated and proton gradient-dependent cation transporter from Chlamydomonas reinhardtii has a role in calcium and sodium homeostasis[END_REF]. In Arabidopsis, the calcium-proton exchangers CAX1 and CAX3 appear to play a prominent role in Ca 2+

homeostasis. Mutants for cax1, cax3 or cax1/cax3 have significantly severe phenotypes including leaf tip necrosis and ionic content. Also CAX1 and CAX3 may be involved in Pi homeostasis [START_REF] Cheng | Functional association of Arabidopsis CAX1 and CAX3 is required for normal growth and ion homeostasis[END_REF], which could be relevant to the AM symbiosis since high concentrations of Ca 2+ and polyphosphate have been co-localized in vacuoles of arbuscules suggesting a role of Ca 2+ in phosphate metabolism in mycorrhizal tissues [START_REF] Strullu | Ultrastructure and electronprobe microanalisis of the metachromatic vacuolar granules occurring in taxus mycorrhizas[END_REF].

ATPases carry out many energy-driven fundamental processes in biology. The P-type II ATPase gene family codes for proteins with an important role as ion pumps in adaptation of the cell to variations in external K + , Ca 2+ and Na 2+ concentrations. During Ca 2+signalling in animal cells, for example, the SERCA pump regulates Ca 2+ levels by effluxing the ion into the endoplasmic reticulum which serves as an intracellular Ca 2+ store [START_REF] Berridge | Calcium signalling: dynamics, homeostasis and remodelling[END_REF]. From the present gene expression analysis, the two P-type ATPases (endoplasmic reticulum P-type ATPase and Golgi reticulum P-type ATPase) of G.

intraradices showed low activity in the pre-symbiotic stage as compared to their expression levels during fungal development within roots of the wild-type M. truncatula genotype. The expression profiles of these two genes suggest that they may regulate the intracellular Ca 2+ homeostasis when G. intraradices comes into contact with plant cell, and thus be involved in a Ca 2+ -based signaling process. In this context, a P-type ATPase has been reported to have a role in penetration hypha development and pathogenicity in the rice blast fungus Magnaporthe grisea [START_REF] Balhadère | PDE1 encodes a P-Type ATPase involved in appressorium-mediated plant infection by the rice blast fungus Magnaporthe grisea[END_REF]. Also, an endoplasmic sarcoplasmic/reticulum Ca 2+ -ATPase (Eca1) has been identified in the plant pathogenic fungus Ustilago maydis which restores growth of a yeast mutant defective in calcium homeostasis [START_REF] Adamíková | Calcium signaling is involved in dynein-dependent microtubule organization[END_REF].

In contrast, the vacuolar Ca 2+ -transporting ATPase was inhibited with spore germination and specifically activated at the arbuscule stage during intraradical development. In this case, the Ca 2+ -transporting ATPase could be putatively linked to cation mediation in the functional symbiosis.

The G. intraradices gene encoding the mitochondrial Ca 2+ -dependent carrier protein showed decreased expression with spore germination and activation in intraradical mycelium at the late stage of arbuscule formation. Consequently this gene may be linked to symbiosis functioning. Two human mitochondrial carrier genes with calcium-binding domains, ARALAR1 and ARALAR2, have been proposed to be involved in calciumregulated metabolite transport in mitochondria and/or participate in calcium-modulated mitochondrial functions [START_REF] Del Arco | Characterization of a second member of the subfamily of calcium-binding mitochondrial carriers expressed in human non-excitable tissues[END_REF]. Mitochondria not only control their own intra-organelle Ca 2+ concentration but also influence the entire cellular network of cellular Ca 2+ signaling, including the endoplasmic reticulum, the plasma membrane, and the nucleus. Ca 2+ channeling between the endoplasmic reticulum and mitochondria collaborate in the regulation of Ca 2+ influx and extrusion through the plasma membrane Ca 2+ ATPases

Laser cryo-microdissection

Samples of mycorrhizal roots of M. truncatula were visualized on a computer monitor (Fig. 7.2 A, B); the targeted cells were marked using graphical software and cut out using an infrared (IR) laser (Fig. 7.2 C, D). Dissected cells were captured automatically into the CapSure HS LCM Caps (Fig. 7.2 E, F). Between 1,000 and 2,000 cells of each cell population were collected and processed for RNA extraction. RNA from the same cell population was combined to give four samples (C1, C2, C3 and V1) (Table 7.1).The quantification and quality of RNA were checked by Agilent Bioanalyzer 2100 (Table 7.1). Experiments were repeated two times using arbusculated cells from different biological experiments (C1 and C2) and representative results are shown.

Discussion and conclusions

The three approaches used to localize transcripts of the different G. intraradices genes gave complementary information concerning gene expression topography. The technique of in situ RT-PCR showed DESAT, LSU (as reported by [START_REF] Seddas | Symbiosis-related plant genes modulate molecular responses in an arbuscular mycorrhizal fungus during early root interactions[END_REF] and TEF expression in spores, but only LSU activity was detected in mycelium in roots and no transcripts of the Ca 2+ -related genes could be located in any fungal structures. The reason for these results may be two-fold: technical, or gene expression may be too low to detect under the present conditions. In situ RT-PCR also gave a very weak signal and important background for the fungal gene Gm152 (see fig. 3.5 in Chapter 3).

Using microdissection and RT-PCR to target G. intraradices transcripts in tissues of mycorrhizal M. truncatula roots, on the contrary, clearly showed that activity of all four marker genes (DESAT, GiPT, TEF, LSU) is associated both with arbuscules and with intercellular hyphae in non-arbuscular, vesicle-free root tissues. As reported by [START_REF] Seddas | Spatial monitoring of gene activity in extraradical and intraradical developmental stages of arbuscular mycorrhizal fungi by direct fluorescent in situ RT-PCR[END_REF], DESAT transcripts were detected not only in arbuscules but also in vesicles. In contrast, expression of phosphate transporter genes from G. versiforme (GvPT) and G. intraradices (GiPT) has been detected predominantly in the extraradical mycelium, the initial site of phosphate aquisition from the soil [START_REF] Harrison | A phosphate transporter from the mycorrhizal fungus Glomus versiforme[END_REF][START_REF] Maldonado-Mendoza | A phosphate transporter gene from the extra-radical mycelium of an arbuscular mycorrhizal fungus Glomus intraradices is regulated in response to phosphate in the environment[END_REF]. However, as for G. intraradices BEG141, the phosphate transporter gene from G. mosseae (GmosPT) also shows activity in arbuscules where it has been suggested that the AM fungus may somehow control the amount of phosphate delivered to the plant by re-absorbing Pi released from neighbouring plant cells [START_REF] Benedetto | Expression profiles of a phosphate transporter gene (GmosPT) from the endomycorrhizal fungus Glomus mosseae[END_REF][START_REF] Balestrini | Laser microdissection reveals that transcripts for five plant and one fungal phosphate transporter genes are contemporaneously present in arbusculated cells[END_REF][START_REF] Tisserant | The transcriptome of the arbuscular mycorrhizal fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate symbiont[END_REF]. These observations were supported by monitoring of gene expression in G. intraradices-colonized roots of P.

sativum which showed that, as compared to wild type cv Finale, the expression of GiPT was present but significantly down-regulated in the Pssym36 mutant where AM fungi penetrate and colonize roots forming intercellular hyphae, but complete arbuscule formation is prevented. The incomplete arbuscule development results in the absence of the main functional symbiotic interface between the fungal and plant cells thus reducing nutrient exchange. This could explain the repression not only of GiPT but also of DESAT in the Pssym36 mutant which confirms the observations by Kutznetsova et al. (2010) who concluded that this could result from reduced biosynthesis of fatty acids by the fungus due to limited uptake of plant carbon in the absence of a functional arbuscule interface. Since transcripts of these two marker genes can also be detected by microdissection and RT-PCR in non-arbusculated tissues, that is in intercellular hyphae, it is suggested that carbon is also transferred to intercellular hyphae from cortical cells and that Pi can be re-absorbed from plant cells via the intercellular space.

For the selected Ca 2+ -related genes of G. intraradices, most of the transcripts could be detected in arbuscules and/or intercellular hyphae by laser microdissection and RT-PCR.

B.

The plates were incubated for 2 days at 30°C, 35°C or 37°C and photographed. The results are presented in Figs.8.4 to 8.8. For the knockout strain cmk1Δ (YFR014C), no obvious calcium-related phenotypes were mentioned in the previous studies.

Complementation of vcx1 mutants

In order to confirm the function of the vacuolar Vcx1 gene from Glomus intraradices (GiVCX1/BEG141_lrc1694), complementation of a VCX1 deficient yeast strain (YDL128W) was performed. However, the vcx1 mutant did not display Ca 2+ sensitivity (Figs. 8.4 and 8.5) nor calcineurin-dependent Ca 2+ sensitivity using the calcineurin inhibitor cyclosporine (Fig. 8.6). [START_REF] Kmetzsch | The vacuolar Ca 2+ exchanger Vcx1 is involved in calcineurin-dependent Ca 2+ tolerance and virulence in Cryptococcus neoformans[END_REF] described the effect of calcineurin on a mutant of Cryptococcus neoformans vcx1Δ which was strongly inhibited in its growth with cyclosporine at 35°C. It was expected to replicate this phenotype in the yeast mutant for the gene vcx1 (YDL128W) and thus test complementation, but the phenotype described by [START_REF] Kmetzsch | The vacuolar Ca 2+ exchanger Vcx1 is involved in calcineurin-dependent Ca 2+ tolerance and virulence in Cryptococcus neoformans[END_REF] showing lack of growth in the presence of calcium could not be reproduced. In S. cerevisiae calcineurin negatively regulates the activity of Vcx1, but increases positively the transcription of two Ca 2+ ATPases of the vacuole (PMC1) and the Golgi (PMR1) via the factor Crz1 transcription. So the use of calcineurin inhibitors like cyclosporine A or FK506, or of the mutant cnb1 is not an absolute way to repress the Ca 2+ regulation. What emerges from the experiments by [START_REF] Cunningham | Calcineurin inhibits VCX1-dependent H + /Ca 2+ exchange and induces Ca 2+ ATPases in Saccharomyces cerevisiae[END_REF] or [START_REF] Cagnac | Vacuolar cation/H + antiporters of Saccharomyces cerevisiae[END_REF] is that there are one or more regulatory elements by which calcineurin plays a role in resistance to Ca 2+ , most likely counterparts of PMC1 and PMR1.

For this reason, the yeast strain K667 which is triply mutated for the genes pmc1, cnb1 and vcx1 and cannot grow in concentrations of Ca 2+ higher than 20 to 30 mM (according to [START_REF] Cagnac | Vacuolar cation/H + antiporters of Saccharomyces cerevisiae[END_REF]) was used. Because the yeast strain K677 lacks a functional calcineurin (CNB1), the endogenous vacuolar Ca 2+ -ATPase (PMC1) and the vacuolar antiporter H + /Ca 2+ (VCX1), it is unable to uptake calcium into the vacuole and therefore to grow in the presence of high calcium concentrations [START_REF] Cunningham | Calcineurin inhibits VCX1-dependent H + /Ca 2+ exchange and induces Ca 2+ ATPases in Saccharomyces cerevisiae[END_REF]. This phenotype can be reverted when complemented by a functional homolog of the VCX1 gene [START_REF] Hirschi | CAX1, an H + /Ca 2+ antiporter from Arabidopsis[END_REF][START_REF] Cheng | Functional association of Arabidopsis CAX1 and CAX3 is required for normal growth and ion homeostasis[END_REF][START_REF] Cagnac | Vacuolar cation/H + antiporters of Saccharomyces cerevisiae[END_REF]. A successful complementation was not achieved; the K667 yeast strain transformed with the full length encoding sequence of VCX1from G. intraradices (GiVCX1/BEG141_lrc1694), was unable to grow on high calcium levels, like the untransformed K667 strain. Several hypotheses could be formulated to explain this result. VCX1 of Saccharomyces cerevisiae and G. intraradices have predicted phosphorylation sites, and according to mass spectrometry phosphopeptide sequencing, serine 13 of yeast VCX1 is phosphorylated [START_REF] Gruhler | Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway[END_REF]. The function and the conditions of this phosphorylation event are unknown [START_REF] Gruhler | Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway[END_REF]. As the amino acids surrounding this serine (S13) differ between the two organisms, it is possible that the equivalent residue is not phosphorylated in the case of G. intraradices, and that this could hamper the activity of GiVCX1/BEG141_lrc1694, thereby not allowing a complete complementation. Another cause of lack of complementation could arise from differences in codon usage between G. intraradices and S. cerevisiae.

Complementation of a pmr1 mutant

A PMR1 deficient yeast strain (YGL167C) showing Ca 2+ sensitivity [START_REF] Szigeti | Calcium and magnesium competitively influence the growth of a PMR1 deficient Saccharomyces cerevisiae strain[END_REF] was acquired for complementation with GiPMR1/BEG141_c2781. However, the phenotype of the pmr1 mutant from Open Biosystems (Thermo Scientific) was not correct as it was not sensitive to high levels of environmental Ca 2+ (Fig. 8.7). Also, the YGL167C yeast strain transformed with PMR1/BEG141_c2781 of G. intraradices and the empty vector showed no differences in growth, although transformation was successful because the YGL167C yeast strain complemented with GiPMR1/BEG141_c2781was able to grow on SD agar without uracil (Fig. 8.8), but it did not show a Ca 2+ sensitive phenotype in high concentrations of Ca 2+ (as reported for yeast PMR1 by [START_REF] Szigeti | Calcium and magnesium competitively influence the growth of a PMR1 deficient Saccharomyces cerevisiae strain[END_REF]. Consequently, it was not possible to conclude to the function of the GiPMR1 encoding gene.

Discussion and conclusions

Complete coding sequences for three genes from G. intraradices BEG141 have been identified which are putatively important in Ca 2+ homeostasis and/or Ca 2+ -based signaling: a tonoplast calcium ion transporter 2 (GiVCX1/BEG141_lrc1694), an endoplasmic reticulum calcium ATPase (GiPMR1/BEG141_c2781), and a nuclear CCaMK (GiCMK1/BEG141_ lrc995). Unfortunately, the yeast complementation assays in this study were inconclusive because the vcx1 mutant YDL128W and the pmr1 mutant YGL167C did not show an expected Ca 2+ sensitive phenotype, and the triple K667 mutant of yeast, which cannot grow in the presence of Ca 2+ , was not rescued after transformation with the BEG141_lrc1694 attB-pDONR plasmid. However, the phylogenetic trees clearly showed that full length BEG141_lrc1694 has a high homology with vcx1 and BEG141_c2781 with pmr1, and that the G. intraradices genes have a domain structure clste12Δ mutant even though the level of similarity was not high (45%) [START_REF] Tollot | An STE12 gene identified in the mycorrhizal fungus Glomus intraradices restores infectivity of a hemibiotrophic plant pathogen[END_REF]. This illustrates the possibility of investigating gene function of AM fungi where corresponding mutants exist in fungi other than yeast, which is not at present the case for VCX1 or PMR1.

The role of calcineurin in the regulation of Pmc1, Pmr1 and Vcx1, and vice-versa, in organisms like yeast is well-known. Ca 2+ signaling involves Ca 2+ spiking which is induced by external environmental stimuli. In response to these changes, Ca 2+ binds to and activates the universal Ca 2+ sensor protein calmodulin (CaM) which in turn may activate various proteins, including the phosphatase calcineurin. Activation of calcineurin leads to transcriptional induction of the vacuolar Pmc1 and the Golgi Pmr1 genes, both Ca 2+ pumps responsible for depleting the cytosolic Ca 2+ to maintain the low levels for growing [START_REF] Ton | Functional expression of heterologous proteins in yeast: insights into Ca 2+ signaling and Ca 2+ -transporting ATPases[END_REF], whilst the vacuolar Vcx1 Ca 2+ /H + exchanger is inhibited by activation of calcineurin. Thus, Pmc1 and Vcx1 independently transport cytosolic Ca 2+ into vacuoles, the major Ca 2+ cell store [START_REF] Cunningham | Acidic calcium stores of Saccharomyces cerevisiae[END_REF]. In fine, removal from the cytoplasm of Ca 2+ necessary for calcineurin activation by calmodulin inhibits calcineurin activity and downstream Ca 2+ -dependent transcriptional and translational events.

Ca 2+ is a key secondary messenger in cell physiology and signalling. The Ca 2+ channels, pumps and transporters, Ca 2+ sensors, and signal transducers are conserved essential components of the cellular Ca 2+ signaling machinery which can underlie Ca 2+ homeostasis in cells by regulating movement of ions between the extracellular environment, cells and subcellular compartments, and so provide mechanisms for generating Ca 2+ signals [START_REF] Dodd | The language of calcium signaling[END_REF]. In this study, AM fungal genes coding Ca 2+related proteins were targeted in order to investigate the regulation or role of Ca 2+ in the molecular responses of the fungal partner during processes leading to the establishment and functioning of the mycorrhizal symbiosis.

Two genes investigated from G. mosseae (putatively encoding a MAP3k-like protein kinase and a P-type ATPase) were up-regulated in the presence of host root exudates from where it could be putatively linked to cation mediation.

Ca 2+ /H + antiporters have a role in promoting intracellular calcium ion sequestration into organelles via the exchange of calcium ions for hydrogen ions across the membrane.

The significant differences in the spatio-temporal expression of the G. intraradices genes encoding the two vacuolar calcium ion transporters 1 and 2 suggest independent functions in the symbiotic process during mycorrhiza interactions. Neither of the genes responded to root exudates but both were expressed in arbuscules. However, the genes showed different expression profiles in that only the former was also active at the appressoria stage which may indicate different implications according to the stage of symbiosis development.

Expression of the Vcx1-like vacuolar calcium ion transporter 1 gene was detected with appressorium development on both wild-type and mutant M. truncatula roots, and it increased when arbuscules formed in roots. In comparison, expression of the calcium ion transporter 2 was only detected with arbuscule formation which corresponded to its transcript location. Vacuoles represent a major organelle for Ca 2+ storage in highly vacuolated cells, like those of fungi and plants, where the ion is sequestered in the form of complexes with polyphosphate [START_REF] Cunningham | Acidic calcium stores of Saccharomyces cerevisiae[END_REF]Pittman, 2011). AM fungi are in fact characterized by an extensive vacuolar system which is considered central for the accumulation and transfer of phosphate within the hyphae [START_REF] Smith | Physiological Interactions between Symbionts in Vesicular-Arbuscular Mycorrhizal Plants[END_REF]. High concentrations of Ca 2+ and polyphosphate have been co-localized in vacuoles of arbuscules suggesting a role of Ca 2+ in phosphate sequestration in fungal tissues [START_REF] Strullu | Ultrastructure and electronprobe microanalisis of the metachromatic vacuolar granules occurring in taxus mycorrhizas[END_REF]. In G. intraradices, the action of the two vacuolar Ca 2+ ion transporters 1 and 2, together with the PMC1-like plasma membrane/vacuolar Ca 2+ -transporting ATPase, may contribute to this phenomenon.

Mitochondrial carriers transfer molecules across the mitochondrial membranes involved in energy transfer. The G. intraradices gene coding for the mitochondrial Ca 2+dependent carrier protein was inhibited by wild type M. truncatula root exudates and weakly activated in intraradical mycelium at the late stage of arbuscule formation, which may reflect an eventual role in symbiosis functioning. Mitochondria couple cellular metabolic state with Ca 2+ transport processes, that is they control not only their own intraorganelle Ca 2+ level but also more generally impact on cellular concentrations and therefore Ca 2+ related signaling events. For example, Ca 2+ channeling between mitochondria and the endoplasmic reticulum collaborate in the regulation of cytosolic Ca 2+ that results from influx and extrusion through plasma membrane Ca 2+ ATPases [START_REF] Szabadkai | Mitochondria: The hub of cellular Ca 2+ signaling[END_REF]. Indeed, the cooperation of these Ca 2+ related proteins located in different organelles forms a complex calcium signaling network and maintains cytosolic calcium at concentrations compatible with growth.

CCaMK proteins are involved in signalling pathways leading to gene regulation. Several studies on plant pathogenic fungi have indicated the involvement of Ca 2+related processes in interactions with hosts. For example, it has been reported that a P-type ATPase (PDE1) of the rice blast fungus Magnaporthe grisea is necessary for penetration of host tissues, hypha development and pathogenicity (Balhadere and Talbot, 2001). Also, an endoplasmic sarcoplasmic/reticulum Ca 2+ -ATPase (Eca1) identified in the plant pathogen Ustilago maydis restores growth of a yeast mutant defective in calcium homeostasis [START_REF] Adamíková | Calcium signaling is involved in dynein-dependent microtubule organization[END_REF]. In the fungal pathogen Magnaporthe grisea, a calcium/calmodulin-dependent signaling system is involved in appressorium formation.

CCaMK activity is modulated by calmodulin isoforms and in M. grisea calmodulin gene expression is induced concomitantly with appressorium formation [START_REF] Lee | Calcium/calmodulin-dependent signaling for appressorium formation in the plant pathogenic fungus Magnaporthe grisea[END_REF][START_REF] Liu | Early expression of the calmodulin gene, which precedes appressorium formation in Magnaporthe grisea, is inhibited by selfinhibitors and requires surface attachment[END_REF].

Calcium is well known as an important signaling molecule in plant responses to various environmental stimuli. Abiotic stress signals, such as salinity, osmotic stress, drought, oxidative stress or low temperature, and biotic signals, for example from pathogens or symbionts, serve as elicitors for the plant cells. These different signals induce

Ca 2+ elevation in the cytosol and other cellular compartments independently, including the nucleus and mitochondria. Calcium efflux from the cytosol into organelles allows replenishment of internal stores, and a return to resting calcium levels, which may contribute to shaping the specific and distinct calcium signatures [START_REF] Mahajan | Cold, salinity and drought stresses: An overview[END_REF][START_REF] Boudsocq | Stress signaling II: calcium sensing and signaling[END_REF]. Calcium influxes the cytosol via Ca 2+ -permeable channels and effluxes out of the cytosol through energy-dependent Ca 2+ -ATPases and transporters. In Arabidopsis, Ca 2+ pumps, including the endoplasmic reticulum (ER)-type Ca 2+ -ATPases (ECA or type IIA) and the auto-inhibited Ca 2+ -ATPases (ACA or type IIB) are proposed to be involved in calcium signaling in response to abiotic stresses. For instance, AtECA1 and AtECA3 have an important role in tolerance to Mn 2+ stress; ECA1 expression restores growth of yeast pmr1 mutants on Mn 2+ -containing medium. Also, the vacuolar Ca 2+ /H + antiporter CAX1, which is induced by cold, has been shown to negatively regulate the cold-acclimation response in Arabidopsis by repressing the expression of CBF/DREB1 genes and their downstream targets. Moreover, a nuclear CCaMK from pea roots (PsCamPK) is up-regulated in roots in response to low temperature and increased salinity [START_REF] Pandey | A Ca 2+ /CaM-dependent kinase from pea is stress regulated and in vitro phosphorylates a protein that binds to AtCaM5 promoter[END_REF]. In legume plants, CCaMKs play a critical role in signaling and gene regulation in interactions with root symbionts where a plant CCaMK is a prime candidate for the response to calcium signatures that are induced by AM fungi or Nod factors [START_REF] Lévy | A putative Ca 2+ and calmodulin-dependent protein kinase required for bacterial and fungal symbioses[END_REF][START_REF] Mitra | A Ca 2+ /calmodulin-dependent protein kinase required for symbiotic nodule development: Gene identification by transcript-based cloning[END_REF].

A hypothesis can be formulated for Ca 2+ homeostasis and signaling in G. intraradices based on the data from the present thesis research together with what is known from studies on yeast. In yeast (Fig. 9.1), Ca 2+ enters the cytosol via the plasma membrane channel complex Cch1/Mid1. In response to changes in Ca 2+ levels, Ca 2+ binds to and activates the universal Ca 2+ sensor protein calmodulin which in turn activates the serine/threonine phosphatase calcineurin and multifunctional Ca 2+ /CaM-dependent protein kinases (CaMKs). Dephosphorylation by activated calcineurin induces the transcriptional regulator Crz1/Tcn1, leading to nuclear translocation and transcriptional activation of target genes including the vacuolar Pmc1 and the Golgi Pmr1 genes, both encoding Ca 2+

pumps ATPases in the tonoplast and responsible for depleting the cytosolic Ca 2+ to maintain low levels that are compatible for yeast growth [START_REF] Ton | Functional expression of heterologous proteins in yeast: insights into Ca 2+ signaling and Ca 2+ -transporting ATPases[END_REF]. The vacuolar Ca 2+ /H + exchanger Vcx1 gene is inhibited by activation of calcineurin so that the Pmc1 and Vcx1 proteins transport cytosolic Ca 2+ independently into vacuoles, the major Ca 2+ cell store in the fungal cells [START_REF] Cunningham | Acidic calcium stores of Saccharomyces cerevisiae[END_REF]. In addition, Vcx1 inhibits calcineurin by removing Ca 2+ (necessary for calcineurin activation by calmodulin) from the cytoplasm, so that Vcx1 and calcineurin form a double-negative feedback interaction:

the Vcx1-off/calcineurin-on state leads to activation of the Pmc1 and Pmr1 genes to reduce cytosolic Ca 2+ levels, switching on a Vcx1-on/calcineurin-off state. This dynamic process of homeostasis will be turned on and off during Ca 2+ spiking. However, yeast complementation assays to obtain valid experimental evidence for G.

intraradices gene function were unfortunately unsuccessful in the present study. In order to gain insight into the role of the different genes in the mycorrhizal symbiosis, an alternative approach for functional analysis should be developed. Heterologous complementation using fungi other than yeast has recently been reported to investigate gene function of AM fungi. For example, insertion of the G. intraradices transcription factor gene GintSTE12 into a clste12Δ mutant of Colletotrichum lindemuthianum restores pathogenicity in the fungal pathogen, providing evidence for the possible existence of common genetic elements regulating penetration of plant tissues by symbiotic and pathogenic fungi [START_REF] Tollot | An STE12 gene identified in the mycorrhizal fungus Glomus intraradices restores infectivity of a hemibiotrophic plant pathogen[END_REF]. Likewise, expression of the G. intraradices gene SP7, which interacts with a plant pathogenesis-related transcription factor, in the rice blast fungus Magnaporthe oryzae attenuates root decay symptoms caused by the biotrophic pathogen [START_REF] Kloppholz | A secreted fungal effector of Glomus intraradices promotes symbiotic biotrophy[END_REF].

Such an approach, together with the use of microdissection to localize the gene expression, offers new perspectives for the detailed analysis of fungal developmental processes that occur during functional differentiation in symbiotic arbuscular mycorrhiza interactions.

In conclusion, the present study of Ca 2+ -related genes in G. mosseae and G. intraradices provides the first insight into the network of Ca 2+ pumps and transporters which may be involved in regulating cytosolic Ca 2+ in an AM fungus, and consequently in processes of homeostasis regulating changes in Ca 2+ concentration that drive downstream CCaMK-dependent signaling events that are essential to the establishment and/or functioning of the mycorrhizal symbiosis.

Zézé, A., Dulieu, H., and Gianinazzi-Pearson, V. (1994). DNA cloning and screening of a partial genomic library from an arbuscular mycorrhizal fungus, Scutellospora castanea. Mycorrhiza 4, 251-254.

Fig

  Fig. 1.1 Different stages of root colonization by an arbuscular mycorrhizal fungus (taken from Balestrini and Lanfranco, 2006).

Fig. 1 . 2 .

 12 Fig. 1.2. Schema summarizing the Arum-type AM colonization pattern and associated cellular dynamics in M. truncatula (taken from Genre et al., 2008). (1) The adhesion of a fungal hyphopodium to the root surface triggers repositioning of the host nucleus and the assembly of a PPA in the contacted epidermal cell; (2) PPA formation in the underlying outer cortical cell; (3) Hypha crossing the outer root layers; (4) Nuclear reposition in inner cortical cells upon hyphal contact; (5) Formation of PPAs in the inner cortical cells; (6) Inner cortical cell penetration; (7) Arbuscule branch formation; (8) Completed arbuscule development.

Fig. 1 . 3 .

 13 Fig. 1.3. Structure of the intracellular arbuscule (taken from Parniske, 2008). The periarbuscular memebrane (PAM) is continuous with the plant plasma membrane and excludes the fungus from the plant cytoplasm. The periarbuscular space (PAS) is the apoplastic interface between the fungal cell wall and the plant-derived PAM.

  Ca 2+ is constantly seeping out of the ER into the cytoplasm and SERCAs pump it back into the ER. Specific signals can increase the cytoplasmic Ca 2+ level by opening channels in the ERor the plasma membrane. The most common signaling pathway that increases cytoplasmic calcium concentration is the phospholipase C (PLC) pathway. Many cell surface receptors, including G protein-coupled receptors and receptor tyrosine kinases, activate the phospholipase C enzyme. PLC hydrolyses the membrane phospholipid PIP 2 to form two second messengers, IP3 and diacyglycerol (DAG). DAG activates the protein kinase C enzyme, while IP 3 diffuses to the endoplasmic reticulum, binds to its receptor (IP 3

Fig. 2 . 1 .

 21 Fig. 2.1. Stimulation of germinated spores by root exudates of A. sinicus

Fig

  Fig. 2.2. Estimation of mycorrhiza development by scoring (Trouvelot et al. 1986). For each root fragment, mycorrhizal colonization is scored from 0 to 5 and arbuscule abundance is classed in 4 categories (A0, A1, A2 and A3).

  RNA from G. mosseae quiescent spores (0.030 µg/µl), from spores germinated in water (0.028 μg/μl) and from spores stimulated by crude plant root exudates (0.083 μg/μl) (Eppendorf biophotometer) were used to synthesize cDNA according to the protocol of the Superscript III Reverse Transcriptase kit (Invitrogen, USA). 0.2 μg DNA-free RNA from spores were reverse transcribed with 1 μg oligo(dT)18 (Promega, USA) and 200 U M-MLV Reverse transcriptase RNase H Minus (Promega) according to the Promega protocol. cDNA was amplified via the protocol as follows: 42 °C for 1 h and 70 °C for 2 min and stored at -20 °C until use. RNA from two batches of spores and three biological repetitions of mycorrhizal M. truncatula roots and non-mycorrhizal M. truncatula were used to synthesize cDNA with Superscript III Reverse Transcriptase according to the Invitrogen protocol. 0.15 μg or 1 μg DNA-free RNA from spores and mycorrhizal roots were separately reverse transcribed into first-strand cDNA. The reaction mix containing RNA, 1 μg oligo(dT) 15 (Promega) and 0.5 mM dNTPs (Final concentration) was preheated at 70 °C for 5 min and cooled on ice for 3 min. 40 U RNasine RNA inhibitor (Promega), 300 U M-MLV Reverse transcriptase RNase H Minus (Promega) and reaction buffer were added to obtain a final mix volume of 25 μl and amplified using the following programme: 25 °C for 15 min, 42 °C for 1 h and 96 °C for 2 min. cDNA was stored at -20 °C until use.

  E target : efficiency of target gene transcript E ref : efficiency of reference gene transcript Control: activated spores Sample: mycorrhizal roots Targeted genes of G. intraradices BEG141 Gene expression was monitored by relative Q-PCR on the Step One Plus Real-Time

Fig. 3 . 1

 31 Fig. 3.1 Transcript levels of G. mosseae genes in germinated spores treated with A. sinicus root exudates (A) or not (B) (RT-PCR 33cycles, 60 °C)

Fig. 3 . 5

 35 Fig. 3.5 Negative control for direct fluorescent in situ RT-PCR in A. sinicus roots after RNAse digestion to eliminate RNA.

Fig. 3 . 6

 36 Fig. 3.6 Detection of Gm152 gene activity by direct fluorescent in situ RT-PCR in G. mosseae hyphae associated with A. sinicus roots after DNAse digestion to eliminate genomic DNA.

Fig. 4

 4 Fig. 4.1. Light micrographs of G. intraradices BEG141 development in wild-type M. truncatula J5 roots. Bars= 50 μm

  Fig 4.2. Glomus intraradices BEG141 development in M. truncatula wild-type (J5) roots (M %: intensity of the mycorrhizal colonisation; A %: arbuscule abundance). Bars = standard errors

Table 4. 5 .

 5 Mycorrhization parameters of the wild type Finale and mutant Pssym36 at 21 dai with G. intraradices BEG141 (Ffrequency of the AM fungus in the root system; M -intensity of mycorrhizal colonization in the root system; mintensity of mycorrhizal colonization in mycorrhizal root fragments; A -arbuscule abundance in the root system; a -arbuscule abundance in mycorrhizal root fragments; V -vesicle abundance in the root system; v -vesicle abundance in mycorrhizal root fragments).

Fig. 4

 4 Fig. 4.3. Light micrographs of G. intraradices BEG141 development in roots of wild type and Pssym36 mutant P. sativum plants. Bars=50 μm

  Glomus_c2781 and Glomus_c10704. According to comparisons with data from Corradi and Sanders (2006), Glomus_c2781 and Glomus_c10704 are closely related to P-type II A ATPases (SERCA ATPase) whilst Glomus_c3514 is closely related to a P-type II B ATPase (PMCA ATPase).

Fig. 5 . 1 .

 51 Fig. 5.1. Expression analyses by RT-PCR of G. intraradices DAOM197198 genes: amplification signals from cDNA of activated (act.) spores and mycorrhizal (AM) roots. Gene expression profiles from microarray hybridisation experiments are given for comparison.

  35 amplification cycles. DESAT and GiPT were included as symbiosis marker genes. Transcripts of all the genes were detected in mycorrhizal M. truncatula roots and none was detected in non-mycorrhizal M. truncatula roots (Fig.5.2), which demonstrated the specificity of the fungal primers also for G. intraradices BEG141.

Fig. 5

 5 Fig. 5.2. Transcripts of Glomus intraradices BEG141 genes detected in mycorrhizal M. truncatula roots (M) and not in non-mycorrhizal roots (NM). Marker: 1 KB plus DNA ladder; 1. DESAT; 2. GiPT; 3. BEG141_c2781; 4. BEG141_c10704; 5. BEG141_c23149; 6. BEG141_c3514; 7 BEG141_c17763; 8. BEG141_lrc1694; 9. BEG141_lrc995.

Fig

  Fig. 5.3. RT-PCR of transcripts of Glomus intraradices BEG141 genes in quiescent spores, activated spores and mycorrhizal roots (17 dai). Gene expression analyses from microarray hybridisation of the DAOM 197198 isolate are given for comparison.

(

  Most of the selected genes were up-regulated in mycorrhizal M. truncatula roots as compared to spores, indicating an eventual role in the establishment or functioning of the mycorrhizal symbiosis. Six of these genes encoded membrane proteins which are potentially involved in calcium homeostasis necessary for intracellular signaling, via the regulation of Ca 2+ transport between different cell compartments. Gene specific primers were designed and tested on cDNA from G. intraradices DAOM197198 and G. intraradices BEG141. The high similarities between the nucleotide sequences indicated the presence of the same genes in these two fungal isolates. Detection of transcripts by RT-PCR confirmed expression of all the targeted genes in the spores and intraradical mycelium of the two fungal isolates, but to different extents. In order to gain a more precise insight into the eventual role of the different Ca 2+ -related G. intraradices genes in mycorrhizal interactions, further research was carried out by monitoring gene expression during fungal development (Chapter 6), localizing transcripts in different fungal structures (Chapter 7) and functional analysis using yeast complementation systems Fungal transcripts were quantified in mycorrhizal roots of wild-type M. truncatula as compared to water-activated spores by relative quantitative real-time RT-PCR (Q-PCR), using the constitutively expressed α subunit of the fungal translation elongation factor 1 (TEF) as the reference gene. With the exception of the vacuolar Ca/Mn transporter 2, the expression pattern observed by Q-PCR confirmed the results from the NimbleGen analyses

Fig. 6 . 1 .Stearoyl

 61 Fig. 6.1. Expression of selected G. intraradices BEG141 genes (relative to TEF) profiled by Q-PCR. Experiments were carried out on RNA extracted from spores. Data represent means of values from two independent biological experiments. Bars indicate standard errors of means and different letters indicate significant differences between treatments (P ≥ 0.05) using one-way analysis of variance (Tukey's pairwise comparisons test); n.d. = not detected

Fig. 6 . 3 .

 63 Fig. 6.3. Transcript accumulation, relative to TEF, of G. intraradices BEG141 genes quantified by absolute Q-PCR. Experiments were carried out on RNAs extracted from spores. Data are represent means of values from two independent experiments. Bars indicate standard errors of means and different letters indicate significant differences between values (P ≥ 0.05) detected using one-way analysis of variance (Tukey's pairwise comparisons test).

  high fungal DESAT and GiPT gene activity at 17-21 dai. Stearoyl-CoA

Fig. 7 . 2 .

 72 Fig. 7.2. Laser cryo-microdissection of M. truncatula root cortical cells containing arbuscules of G.intraradices BEG141. A and B (autofluorescence), before dissection; C and D (autofluorescence), tissue remaining after dissection; E and F (autofluorescence), collected arbuscule-containing cells. Bars = 50 μm

  Fig. 7.3. An LSU rRNA probe was used to confirm the presence of fungal tissue in the samples and the G. intraradices genes TEF, DESAT and GiPT were used as controls to monitor fungal activity. As expected, cells with arbuscules contained transcripts of these four marker genes; in addition, they were detected in non-arbusculated, vesicle-free tissues (Fig. 7.3A), indicating the presence of RNA from intercellular hyphae. The DESAT gene was also expressed in vesicles (Fig. 7.3C).

Fig. 7

 7 Fig. 7.3. (A) Transcripts of G. intraradices BEG141 genes in mycorrhizal roots (AM roots), arbuscule-containing cortical root cells (Arbuscules) and non-arbuscule tissues. PCR was run for 35 to 40 cycles for microdissected cells and 30 cycles for AM roots (*except for Mm Ca 2+ carrier where 40 PCR cycles were necessary for detection). (B) Increased amounts of PCR products (40 cycles) from transcripts of arbuscules and non-arbuscule tissues loaded onto gels. Vac Ca 2+ transporter 1 = vacuolar Ca 2+ ion transporter 1; Vac Ca 2+ transporter 2 = vacuolar Ca 2+ ion transporter 2; PM/Vac Ca 2+ ATPase = plasma membrane/vacuolar Ca 2+ transporting ATPase; ER Ca 2+ ATPase = endoplasmic reticulum Ca 2+ ATPase; GR Ca 2+ ATPase = Golgi reticulum Ptype Ca 2+ ATPase (BEG141_c10704); Mm Ca 2+ carrier = mitochondria membrane Ca 2+ -binding carrier; CCaMK = Ca 2+ /calmodulin protein kinase.

Fig. 7 . 4 .

 74 Fig. 7.4. Expression of G. intraradices BEG141 genes (relative to TEF) profiled by Q-PCR. Experiments were carried out on RNAs extracted from roots of P. sativum genotypes Finale and Pssym36. Data represent means of values from three independent biological experiments. Bars indicate standard errors of means and different letters indicate significant differences between values (P ≥ 0.05) using one-way analysis of variance (Tukey's pairwise comparisons test); n.d. = not detected.

Fig. 8

 8 Fig. 8.2. A. Phylogenetic analysis of G. intraradices BEG141 endoplasmic reticulum calcium ATPase in relation to ortholog proteins from other eukaryotic organisms: C. cinereus (XP_001833748), C. neoformans (XP_572412), A. fumigatus (EAL88529.1), M.oryzae (XP362105), U. maydis (CAE11789.1), A. thaliana (AAF75073.1), M. truncatula (AAL35972), S. cerevisiae (CAA96880), and S. pombe (NP_595098). The bar marker indicates the difference in percent. G. intraradices used in this study is indicated with a red point. B. Domain structure of endoplasmic reticulum calcium ATPase ortholog proteins from different eukaryotic organisms. White boxes correspond to transmembrane domains, green boxes to the N terminus of the cation ATPase pfam PF00690.21, rose boxes to E1-E2_ATPase pfam PF00122.15, blue boxes to the hydrolase domain PFAM PF00702.21 and yellow boxes to the C terminus of the cation ATPase PFAM PF00689. The lengths (in amino acids) of each of the proteins are indicated on the right.. Gi_Beg141_2781: G. intraradices BEG141; Cc_XP_001833748: C. cinereus; Cnof_XP_572412: C. neoformans; Af_EAL88529.1: A. fumigatus; Mo_XP362105: M.oryzae; Um_CAE11789.1: U. maydis; Sc_Pmr1m: S. cerevisiae (CAA96880); Sp_Pmr1: S. pombe (NP_595098).

Fig. 8

 8 Fig. 8.4.The vcx1 yeast mutant did not display Ca 2+ sensitivity. Ten-fold serial dilutions of wildtype W303, vcx1 mutant YDL128W (vcx1Δ), vcx1::VCX1-complemented (vcx1Δ::1694) and vcx1::empty vector-complemented (vcx1Δ:: pDRf1-GW) cells were plated in YPD agar containing 50-400 mM CaCl 2 .The plates were incubated for 2 days at 30°C or 37°C, as indicated. As control, cells were grown in YPD agar only.

  in other eukaryotes. The lack of complementation could be due to differences in the codon usage between yeast and G. intraradices. The G. intraradice vcx1and pmr1 show 48 % and 33 % identity to yeast vcx1 and pmr1, respectively. In another study, heterologous complementation with the G. intraradices transcription factor gene GintSTE12 successfully restored pathogenicity of a Colletotrichum lindemuthianum

A

  . sinicum and, based on in situ RT-PCR, the latter appeared to be active in extraradicular hyphae. More detailed studies on seven G. intraradices identified Ca 2+ -related genes, which putatively encode six membrane proteins and one nuclear kinase. The full-length coding sequences obtained for three of them confirmed a putative function already described in other fungi (PMR1-like endoplasmic reticulum calcium ATPase, VCX1-like vacuolar Ca/Mn transporter, CCaMK). None of the genes from G. intraradices responded positively to root exudates from the host plant M. truncatula but expression profiles during symbiotic interactions suggest different functions in fungal development or function. Four of the fungal genes are active during appressoria development and up-regulated during root colonization. They are putatively linked to Ca 2+ homeostasis or perception during AM symbiosis development and code for the PMR1-like endoplasmic reticulum Ca 2+ ATPase, a Golgi reticulum P-type ATPase, the VCX1-like vacuolar Ca/Mn transporter 1 and a nuclear CCaMK. The expression of these four genes during appressoria development also on roots of the mycorrhiza-resistant M. truncatula mutant Mtdmi3, as well as in G. intraradices-colonized roots of the mycorrhiza-defective pea mutant Pssym36, suggests a possible function in the general development of the fungus. Expression of the remaining three genes putatively linked to Ca 2+ homeostasis is directly related to the development of intraradical structures in mycorrhizal roots. These encode a mitochondria membrane Ca 2+ -dependent carrier, a vacuolar Ca/Mn transporter (2) and a PMC1-like plasma membrane/vacuolar Ca 2+transporting ATPase. Their expression profile suggests a role in the functional symbiosis since transcripts are only detected at the arbuscule stage of G. intraradices in roots of wild type M. truncatula, and none is activated in interactions with the mycorrhiza-resistant M. truncatula mutant Mtdmi3 or during intercellular hypha development in roots of the mycorrhiza-defective pea mutant Pssym36 (except vacuolar Ca/Mn transporter 2). Targeted localization of transcripts by microdissection and RT-PCR indicated that the mitochondria membrane Ca 2+ -dependent carrier and vacuolar Ca/Mn transporter genes are in fact only active in arbuscules formed in wild type M. truncatula roots, whilst those encoding PMR1like endoplasmic reticulum Ca 2+ ATPase and nuclear CCaMK are expressed both in arbuscules and intercellular hyphae, and the plasma membrane/ tonoplast Ca 2+ -transporting ATPase gene only in the latter. Endoplasmic reticulum, vacuoles and mitochondria have important functions in Ca 2+homeostasis which is regulated through membrane-bound proteins. The P-type II ATPase gene family codes for proteins with an important role as ion pumps in adaptation of the cell to variations in external K + , Ca 2+ and Na 2+ concentrations. During Ca 2+ -signalling in animal cells, a P-type II ATPase (SERCA pump) regulates Ca 2+ levels by effluxing the ion into the endoplasmic reticulum which serves as an intracellular Ca 2+ store[START_REF] Berridge | Calcium signalling: dynamics, homeostasis and remodelling[END_REF]. The Pmr1-like endoplasmic reticulum and the Golgi reticulum P-type ATPase genes of G. intraradices showed low activity in the pre-symbiotic stage as compared to their expression levels during fungal development within roots of the wild-type M. truncatula genotype. It suggests that they may regulate the intracellular Ca 2+ homeostasis when G. intraradices comes into contact with plant cells, and thus be involved in the regulation of a Ca 2+ -based signaling process. In contrast, the Pmc1-like plasma membrane/vacuolar Ca 2+ -transporting ATPase gene was inhibited with spore germination and specifically activated in the full symbiosis during intercellular hypha development,

  Expression of the G. intraradices nuclear CCaMK gene was unaffected by root exudates during presymbiotic fungal development but the gene was clearly up-regulated during root colonization in the symbiotic stage. Interestingly, this gene followed very similar patterns of expression and transcript localization from appressoria to root colonization as the Pmr1like endoplasmic reticulum Ca 2+ -ATPase gene, suggesting a possible role of the G. intraradices nuclear CCaMK in Ca 2+ perception following fungal responses to plant signals.

Fig

  Fig. 9.2. The supposed sub-cellular localization of the different Ca 2+ -related genes of the AM fungus G. intraradices BEG141: 1. Vacuolar calcium ion transporter 1; 2. Vacuolar calcium ion transporter 2; 3. Vacuolar calcium transporting ATPase; 4. Endoplasmic reticulum calcium ATPase; 5. Golgi reticulum calcium transporting ATPase; 6. Mitochondria membrane calciumbinding carrier; 7. Nuclear calcium/calmodulin-dependent protein kinase (CCaMK).

  2+

Chapter 2 Material and Methods 2.1. Biological materials and growth conditions 2.1.1. Astragalus sinicus L. + Glomus mosseae (BEG225)

	. Based on the
	results obtained, a mechanism for Ca 2+ homeostasis in AM fungal cells is proposed and the
	possible role of Ca 2+ -related proteins in intracellular signaling during symbiotic
	mycorrhizal interactions is discussed. Spores of Glomus mosseae Nicol. & Gerd. (isolate BEG225), from the Bank of
	Glomales in China (BGC), were collected from pot cultures of mycorrhizal Sorghum by
	wet sieving. For pre-symbiotic stages, spores were washed several times with sterile
	distilled water and germinated in 3 ml of water in multiwell dishes (Fig. 2.1) at 25°C in the
	dark. After 7 days, the germinated spores were harvested and immediately frozen in liquid
	nitrogen for RNA extraction.

Table 2 .1. Modified Hoagland nutrient solution with low phosphorus

 2

	Component	Concentration (mol / L)
	KNO 3 Ca(NO3) 2 NH 4 NO 3 MgSO4 MnCl 2 CuSO4 × 5H 2 O 3 × 10 -7 2 × 10 -3 5 × 10 -4 1 × 10 -3 5 × 10 -4 9 × 10 -6 ZnSO 4 × 7H 2 O 8 × 10 -6 H 3 BO 3 4.6 × 10 -5 CaCl 2 2.5 × 10 -4 (NH4) 2 MoO 4 3 × 10 -7 FeNa-EDTA 2 × 10 -5 KH 2 PO4 6.5 × 10 -5
	2.1

.2. Medicago truncatula +Glomus intraradices DAOM197198 (syn. Rhizophagus irregularis Blaszk., Wubet, Renker & Buscot)

	Mycorrhizal root material was kindly provided by A. Aloui (INRA, Dijon). Medicago
	truncatula Gaertn. cv. Jemalong wild-type (line J5) was inoculated with the Glomus
	intraradices isolate DAOM197198 (syn. Rhizophagus irregularis; Krüger et al., 2012) and
	plants were grown for 3 weeks according to Aloui et al. (2009). Roots were harvested,
	washed in ice-cold osmosed water and stored in liquid nitrogen until RNA extraction.
	Spores (10,000) of G. intraradices DAOM197198, provided by P. Seddas (INRA,
	Dijon), were activated for 24h at 25 °C in sterile water and 2 % CO2 (IG150, Jouan
	incubator) and stored in liquid nitrogen until RNA extraction.

Table 2 .2. Long Ashton solution without phosphorus Component Concentration (mg/l)

 2

	KNO 3	808
	Ca(NO3) 2 × 4H 2 O	1888
	MgSO4 × 7H 2 O	368
	MnSO4 × H 2 O	2.23
	CuSO4 × 5H 2 O	0.25
	ZnSO 4 × 7H 2 O	0.29
	H 3 BO 3	3.10
	NaCl	5.90
	H 24 Mo 7 N 5 O 24 × 4H 2 O 0.088
	FeNa-EDTA	22
	2.1

.4. Pisum sativum L. + Glomus intraradices BEG141

	Wild-type pea (P. sativum L.) cv. Finale and the symbiotic mutant Pssym36
	(previously denoted RisNod24; provided by A. Borisov, ARRIAM, St. Petersburg, Russia)
	were used. The mutant Pssym36 is defective for arbuscule formation (Gianinazzi-Pearson,
	1996).
	Seeds were surface-disinfected 10-min in a sodium hypochlorite solution (3 %) at
	room temperature, washed with distilled water and germinated in Petri dishes (12-cm
	diameter) on humid filter paper at 24 °C in the dark for 5 days. Seedlings were planted into
	pots containing 200 ml of a 1:1 mixture of quartz sand and mycorrhizal soil-based
	inoculum (spores, roots, and hyphae) of G. intraradices BEG141. Plants were grown under
	control conditions (326 μ mol m -2 s -1 , 24/22 °C, 16 h light, 70 % relative humidity),
	fertilized with 10 ml / pot of a modified Long Ashton solution without phosphorus,
	enriched in nitrogen (Table 2.2) three times a week, or 10 ml of distilled water on other
	days. Plants were harvested at 21 days after inoculation (dai). Root systems were washed

in cold water, root and shoot fresh weights were recorded, and roots were stored in liquid nitrogen for RNA extraction or directly stained to determine the AM fungal development on randomly sampled root pieces (see below).

Table 2 .3. Putative function and primer design of selected G. mosseae genes Selected ESTs Primer Sequence 5'-3'

 2 .

	Putative function Organism	E-value

Table 2 .4. Putative function of studied G. intraradices genes

 2

	ESTs	Putative function	Organism	E-value
	Glomus_c17763 Vacuolar Ca/Mn transporter (1)		Saccharomyces	7e-20
					cerevisiae
	Glomus_lrc1694 Vacuolar Ca/Mn transporter (2)		Saccharomyces	3e-53
	Glomus_c3514	Vacuolar Ca 2+ -transporting ATPase II	cerevisiae Schizosaccharomyces	9e-21
		Plasma membrane Ca 2+ -transporting ATPase II	pombe Oreochromis	2e-12
					mossambicus
					(Mozambique tilapia)
	Glomus_c2781	Sarcoplasmic/endoplasmic reticulum calcium	Rana esculenta	2e-40
	ATPase II Glomus_c10704 Golgi reticulum P-type Ca 2+ ATPase II	(edible frog) Pongo abelii	1e-10
	Glomus_c23149 Mitochondria	membrane	Ca 2+ -dependent	(Sumatran orangutan) Homo sapiens	5e-92
		carrier			(human)
	Glomus_lrc995	Nuclear CCaMK			Mus musculus	4e-69
					(house mouse)

2.5.2. Primer design

Primers were manually designed for Gm2 and Gm152 sequences and checked by the programme DNAMAN (Lynnon Biosoft, USA). They were tested by PCR on cDNA synthesized from G. mosseae spores by gel RT-PCR at 60 °C and with 33 amplification cycles. PCR products were sequenced (AuGCT biotechnology, Beijing, China) and EST sequence homology verified by blastx (Table

Table 2

 2

.5) and checked by the programme Amplify 3X (http://engels.genetics.wisc.edu/amplify/). They were tested on cDNA synthesized from equal amounts of total RNA from mycorrhizal (G. intraradices DAOM197198 or BEG141) M. truncatula roots and from spores by gel RT-PCR at 58 °C and with 30 amplification cycles. PCR products were sequenced (MWG-Biotech, Ebersberg, Germany) and EST sequence homology verified by blastx (Table

2

.4). Previously published primers from G. intraradices BEG141 and other fungi described as G. intraradices were used for the control genes LSU, TEF,

DESAT and GiPT (Maldonado-Mendoza et al., 2001;[START_REF] Farmer | Molecular monitoring of fieldinoculated AMF to evaluate persistence in sweet potato crops in China[END_REF][START_REF] Seddas | Symbiosis-related plant genes modulate molecular responses in an arbuscular mycorrhizal fungus during early root interactions[END_REF]

(Table 2.6

).

Table 2 .5. Primer design for G. intraradices genes

 2

	Gene	Primer	Sequence 5'-3'	Size 1
		name		(bp)
	Vacuolar calcium ion transporter 1 C17763_F2 GATGGTATAACGAAAAGCGGAC	331
		C17763_R2 AATAGCGGTAACAAACTCAGCA
	Vacuolar calcium ion transporter 2 1694_F2	CGTCTTCTCATATCATTCTAACTAAAG 114
		1694_R3	TTGGTTTGAAGGTGCTATGTTGC
	Plasma membrane /vacuolar	C3514_F	TGGAGGATTAGATGGATTGGT	247
	calcium transporting ATPase II	C3514_R2	GAATAGACTTTGTTTTCTTTACTGG
	Endoplasmic reticulum calcium	C2781_F	CGACGAATACTCTGCTCACG	249
	ATPase II	C2781_R3	TCAATGCCACTAATTCTCTTAGC
	Golgi reticulum calcium	10704_F3	GGAGCTGAAATTGTCCTGATGC	198
	transporting ATPase II	10704_R3	CGTTGCATATTTACCAAGCACAGG
	Mitochondria membrane calcium-	23149F5	TAAGAGATGTACCATTTTCAGCCA	249
	binding carrier	23149R5	TCCGCTAATATCCTTCTTCCTGC
	Nuclear calcium/calmodulin-	lrc995_F4	ATTATGTGGATACACTCCATTCTG	195
	dependent protein kinase	lrc995_R2	AACCAAGGGTGTTTCAATGCTTC
	(CCaMK)
	1) Amplicon size

Table 2 .6. Primer design for control G. intraradices genes

 2

	Gene	Primer	Sequence 5'-3'	Size 1	Target	Reference
		name		(bp)	organism
	Stearoyl-CoA-	DESAT for TCGTGTTCCTGAAAA	269	G. intraradices	Seddas et al. (2009)
	desaturase		TGAAG		BEG141
	(DESAT)	DESAT rev GCTTTAGTGGAGTCT
			TTACC
	Phosphate	GiPT1	CTCCTGCAAATATCG	243	G. intraradices	Maldonado-Mendoza
	transporter		ACTTGGGT		(AF359112)	et al. (2001)
	(GiPT)	GiPT2	GATAATCTCCTCCAAT
			ACCGACA
	Large subunit	FLR3	TTGAAAGGGAAACG	300	Glomeromycota Gollotte et al. (2004)
	rRNA (LSU)		ATTGAAGT
		8.24	CGATCAGAGACCAG		G. intraradices	Farmer et al. (2007)
			ACAGGT
	Translation	Gi-TEFfor	AGCCGAACGTGAAC	144	G. intraradices	Seddas et al. (2009)
	elongation		GTGG		BEG141
	factor (TEF)

Table 2 .7. 3′ RACE primers for Gm152 Primer name

 2

		Sequence 5'-3'	Temp 1
			(°C)
	QI-QO	CCAGTGAGCAGAGTGACGAGGAC	68
		TCGAGCTCAAGCTTTTTTTTTTTTT
		TTTT
	QO	CCAGTGAGCAGAGTGACG	60
	QI	GAGGACTCGAGCTCAAGC	60
	152GSP1 GCGGGGACAGGTTTATAAATTTC	60
	152GSP2 TGTTTTGGCATGGATACGTATGG	60
	1) Annealing temperature.

Table 2 .8. 3′ and 5′ RACE primers for the selected ESTs of Glomus intraradices BEG141 ESTs Primer name

 2 Table 2.8. These primers were used for PCR amplification from G. intraradices BEG141 cDNAs with a proofreading DNA polymerase (Platinum® Taq DNA polymerase High Fidelity; Invitrogen Corporation, Carlsbad, CA, USA). For the RACE amplification results, homology searches were carried out using NCBI databases by BLAST search (http://blast.ncbi.nlm.nih.gov/). Multiple sequence alignments of translated gene sequences were carried out with the program CLUSTALW2 (http://www.ebi.ac.uk/clustalW/). The amino acid sequences of orthologs from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Coprinopsis cinerea, Cryptococcus neoformans, Aspergillus fumigatus, Magnaporthe grisea, Ustilago maydis, Arabidopsis thaliana and Medicago truncatula were aligned and the program Mega4 was used for phylogenetic analysis.

			Sequence 5'-3'		Temp 1
					(°C)
	BEG141_ lrc1694	1694_R3	TTGGTTTGAAGGTGCTATGTTGC	3′ end	58
		1694-F6	TAACAGCAAAATTAAAAGCAGC	RACE 5′ end	58
	BEG141_c2781	R-2781-F5	AATTCGTGAGCAGAGTATTCGTCG	RACE 3′ end	65
		R2781R5	RACE CTCATTTTCGCTAAGAGAATTAGTGGC 5′ end	65
		R2781-R22 AGCTGGAGCATCATTAACACC	RACE 5′ end	60
		R2781-R32 CAGGAATTTTATCACCCACAGC	RACE 5′ end	58
	BEG141_ lrc995	lrc995_F6	ATTGATAAAATGTTAGCTTATGATGCTG	RACE 3′ end	62
		lrc995_R3	CTT GAGCATAATCACTAATATTTGACC	RACE 5′ end	60
				RACE
	1) Annealing temperature.

Table 2 .9. Primers designed for Gm152 PCR amplification Primer name Sequence 5'-3' Temp 1 (°C)

 2

	Inverse PCR
	152f-2	ACTGCTTGTCATGTGGCTCG	60
	152f-3	ATG G G TATATG C TCG CG TG TC	60
	152AR	GGAAACAGCATCGTCCTTCAG	60
	TAIL-PCR
	152f1	TGGTTCGGGCTTTGATGGGTTG	60
	152f2	TTGAGGTGTCATGATATCTTGCGG	60
	152f3	GTTTTGGCATGGATACGTATGG	58
	F1	CTGCCAGTAAGGATCAAGTTTGC	60
	F2	CGCAAATATTATACGTCAAGGTCG	60
	F3	GCAAACGACGACCACAAACC	60
	AD1	NTCGA(G/C)T(A/T)T(G/C)G(A/T)GTT 45

Table 2 .11. Comparison with the amino acid sequence databases of Saccharomyces cerevisiae (S288C)

 2 The triple mutant K667[START_REF] Cagnac | Vacuolar cation/H + antiporters of Saccharomyces cerevisiae[END_REF], was generously provided by O.Cagnac (Estación Experimental del Zaidin, CSIC, Grenada, Spain). All yeast strains used in this study are listed in Table2.12.

	2.8. Yeast complementation
	2.8.1. Yeast strains
	The homology searches for three selected genes were carried out by BLASTX in the
	Saccharomyces Genome Database (SGD; Stanford University, Stanford, CA), available on
	line (http://www.yeastgenome.org) (Table 2.11). Three yeast strains YGL167C,
	YDL128W and YFR014C, respectively knocked out for PMR1, VCX1 and CMK1 and the
	parental wild type strain MATa W303 were obtained from Open Biosystems (Thermo
	Scientific). Genes	Putative protein	Location	E-value
	BEG141_lrc1694 YDL128W (VCX1) Vacuolar membrane 6.2e-79
	BEG141_c2781	YGL167C (PMR1) Golgi reticulum	3.1e-120
	BEG141_lrc995 YFR014C (CMK1) Nucleus	1.9e-68

Table 2 .12. Strains used in this study Strain Genotype Source W303

 2

Table 2 .13. Primers for the complete gene coding sequences with the attB adaptor

 2

	Genes	Primer	sequences
		name
	BEG141_lrc1694 1694_attB_F GGGGACAAGTTTGTACAAAAAAG
			CAGGCTTCATGGCAGAGGGACAACCACTGGTCTTAAAC
		1694_attB_R GGGGACCACTTTGTACAAGAAAG
			CTGGGTCTTACAATTTATCGGGATATAAAAAGAAAGC
	BEG141_c2781	2781_attB_F GGGGACAAGTTTGTACAAAAAAG
			CAGGCTTCATGGATGACGCATTTACAAAAAGTCC
		2781_attB_R GGGGACCACTTTGTACAAGAAAG
			CTGGGTCCTAATCAACTTTAATTTTTGTTGGTGG
	BEG141_lrc995	995_attB_F	GGGGACAAGTTTGTACAAAAAAG
			CAGGCTTCATGCGTAAATATTATAGAAATACTCCG
		995_attB_R	GGGGACCACTTTGTACAAGAAAG
			CTGGGTCTTAAACCGGTATAACTTTGTTTTTCG

Table 4 .1. Development of the two Medicago truncatula genotypes at 4 dai and 6 dai M. truncatula genotype Fresh shoots (g)

 4 Values followed by the same letter are not significantly different (P≥0.05) (ANOVA, Tukey test).

	Fresh roots (g)

Table 4 .2. Number of appressoria formed by Glomus intraradices BEG141 on the two Medicago truncatula genotypes 4 and 6 days after inoculation M. truncatula genotype Appressoria per cm root

 4

		4 dai	6 dai
	J5	0.86 a	1.66 a
	TRV25	0.94 a	0.76 a
	Values followed by the same letter are not significantly different (P≥0.05) (ANOVA, Tukey test).

Table 4 .4. Development of the two P. sativum L. genotypes at 21 days after inoculation

 4 Values followed by the same letter are not significantly different (P≥0.05) (ANOVA, Tukey test).

	Genotypes	Fresh shoots (g)	Fresh roots (g)
	Finale	1.83 a	4.49 a
	Pssym36	2.01 a	4.22 a

Table 5 .1. Putative functions of selected G. intraradices DAOM197198 genes

 5

	ESTs	Putative function	Organism	E-value
	Glomus_c17763 Vacuolar Ca/Mn transporter (1)		Saccharomyces	7e-20
					cerevisiae
	Glomus_lrc1694 Vacuolar Ca/Mn transporter (2)		Saccharomyces	3e-53
	Glomus_c3514	Vacuolar Ca 2+ -transporting ATPase II	cerevisiae Schizosaccharomyces	9e-21
		Plasma membrane Ca 2+ -transporting ATPase II	pombe Oreochromis	2e-12
					mossambicus
					(Mozambique tilapia)
	Glomus_c2781	Sarcoplasmic/endoplasmic reticulum calcium	Rana esculenta	2e-40
	ATPase II Glomus_c10704 Golgi reticulum P-type Ca 2+ ATPase II	(edible frog) Pongo abelii	1e-10
	Glomus_c23149 Mitochondria	membrane	Ca 2+ -dependent	(Sumatran orangutan) Homo sapiens	5e-92
		carrier			(human)
	Glomus_lrc995	Nuclear CCaMK			Mus musculus	4e-69
					(house mouse)

Table 5 .3. Comparison with the amino acid sequence databases of Type IIB ATPase (PMCA ATPase) according to Corradi and Sanders (2006)

 5

	Type IIB (PMCA ATPase)		E-value (Blast p)
	Organism	Putative protein	Glomus_	Glomus_	Glomus_
			c 2781	c 10704	c 3514
	Fungi
	Blakeslea trispora	Partial bca1 gene for putative	4.8		0.15
		calcium ATPase, exon 1
	Phycomyces	Partial pca1 gene for putative	3.1	1.8
	blakesleeanus	calcium ATPase, exon 1
	Schizosaccharomyces	Vacuolar calcium transporting P-	0.021	2.00E-07 7.00E-32
	pombe	type ATPase, Pmc1
	Neurospora crassa	Putative calcium p-type ATPase	0.006	5.00E-07 9.00E-45
		NCA-3
	Gibberella zeae	Hypothetical protein FG09515.1		2.00E-06 1.00E-38
	Magnaporthe grisea	Hypothetical protein MG04890.4	2.00E-04	4.00E-07 6.00E-42
	Aspergillus nidulans	Hypothetical protein AN1189.2	0.021	2.00E-06 4.00E-43
	Glomus intraradices	Partial pmca1 gene for P-Type II B	2.2	0.12	4.8
		ATPase
		Partial pmca2 gene for P-Type II B	0.48	0.13
		ATPase
	Glomus diaphanum	Partial pmca gene for P-type II B	1.8	0.12
		ATPase
	Glomus proliferum	Partial pmca gene for P-type II B	2.5	0.12	1.8
		gene
	Cryptococcus neoformans Hypothetical protein CNBD2820	0.01	5.00E-08 3.00E-36
	Ustilago maydis	Signal peptide, repeats, gene	0.65
		anchored to telomere
	Metazoa
	Homo sapiens	Hypothetical protein	0.003	1.00E-08 2.00E-22
	Rana catesbiana	PMCA2av	0.003	5.00E-08 8.00E-14
	Viridiplantae
	Arabidopsis thaliana	Putative Ca2+-ATPase	1.00E-05	8.00E-08 2.00E-20
	Brassica oleracea	Calmodulin-stimulated calcium-	0.011	6.00E-08 3.00E-20
		ATPase
	Blank: no significant similarity found.

Table 5 .4. Primer design for G. intraradices DAOM197198 genes Gene Primer name

 5

	Sequence 5'-3'	Size 1
		(bp)

Table 6 .1. Relative quantification of G. intraradices DAOM197198 transcripts

 6

	ESTs	Q-PCR expression	µarray expression
		AM roots/act. spores	AM roots/act. spores
	Glomus_c17763	24.9 ±1.38	7.99	*
	Glomus_lrc1694	1.85 ± 0.21	0.79
	Glomus_c3514	0.88 ± 0.28	1.61	*
	Glomus_c2781	1.76 ± 0.14	2.66	*
	Glomus_c10704	2.24 ± 0.32	2.97	*
	Glomus_c23149	0.54 ±0.04	0.41	*
	Glomus_lrc995	2.07 ±0.18	1.88	*

Data presented from Q-PCR are mean values with standard errors from three biological repetitions.

Table 7 .1. Microdissected cell populations and RNA yield RNA samples Number of cells RNA yield (ng) RNA (ng/μl) RIN

 7

	Arbusculated cells Non-arbusculated tissues C3 ≈ 6,540 C1 ≈ 11,400 C2 ≈ 4,900 Vesicles V1 ≈ 28	50.658 192.384 127.782 10.504	1.6886 8.016 4.122 1.313	3.3 3.5 3.7 3.6
	RT-PCR was performed on the extracted RNA by a One-Step RT-PCR and
	compared to whole mycorrhizal root extracts. PCR products are presented in

  Expression of the mitochondria membrane calcium-binding carrier gene (BEG141_c23149) in arbusculated tissues of mycorrhizal M. truncatula roots and the lack of transcripts in intercellular hyphae or roots of the Pssym36 pea mutant clearly indicate that it is specifically active in functional arbuscules. The two vacuolar Ca/Mn transporter genes (BEG141_c17763, BEG141_lrc1694) show the same arbuscular location using laser microdissection but differ by their expression profiles in the Pssym36 mutant. From the microdissection results, these two genes are only expressed in arbuscules whilst both are active in the Pssym36 mutant. Since transcripts could not be detected in intercellular hyphae by laser microdissection, this may indicate a function not only in normal but also in aborted arbuscules or that different host plants may somehow stimulate different expression profiles. For example, up-regulation of the vacuolar calcium ion transporter 1

	cation-transporting P-type ATPases, including those that transport H + , Na + ,
	Ca 2+ , Na + /K + , and H + /K +
	2) an E1-E2_ATPase (PFAM PF00122.15) which catalyzes auto-(or self-)
	phosphorylation of a key conserved aspartate residue within the pump; in
	addition, they all appear to interconvert between at least two different
	conformations, denoted by E1 and E2;
	3) the hydrolase domain (HAD_like; PFAM PF00702.21) of the haloacid
	dehalogenase-like (HAD) superfamily which includes L-2-haloacid
	dehalogenase, epoxide hydrolase, phosphoserine phosphatase, P-type ATPase
	and many others, which all use a nucleophilic aspartate in their phosphoryl
	transfer reaction;
	4) the C terminus of the cation ATPase (Cation_ATPase_C; PFAM PF00689)
	involved in Na + /K + , H + /K + , Ca 2+ and Mg 2+ transport.
	(BEG141_c17763) in Pssym36 roots may reflect the perception of a stress by the AM
	fungus in the mutant, as was suggested to explain the over-expression of the fungal
	alkaline phosphatase gene in the same pea genotype (Kuznetsova et al., 2010). Three of the
	G. intraradices genes, BEG141_c2781 (endoplasmic reticulum P-type Ca 2+ ATPase),
	BEG141_c10704 (Golgi reticulum P-type Ca 2+ ATPase) and BEG141_lrc 995 (nuclear
	CCaMK), were expressed in both arbuscules and intercellular hyphae; no significant effect
	was observed of the Pssym36 mutation in pea roots, suggesting a possible function in the
	general development of the fungus within host tissues. In contrast, transcripts of the
	plasma membrane/vacuolar Ca 2+ -transporting ATPase (BEG141_c3514) were only weakly
	detected in intercellular hyphae in mycorrhizal M. truncatula roots, and not in colonized
	pea roots, indicating no function in arbuscule formation or function.

Acknowledgements

This thesis was completed within the context of a postgraduate education and training agreement between Huazhong Agricultural University, Wuhan, China (President Professor Deng Xiu Xin), and the University of Burgundy, Dijon, France (President Professor Sophie Béjean). I am grateful to the National Natural Science Foundation of China for financial support of the 1 st year research period in Wuhan, and to the Chinese Ministry of Education (State Scholarship) for travelling costs and living expenses during the 2 nd and 3 rd year period in Dijon. I am greatly indebted to Professor Bin Zhao (State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University), and Dr. Vivienne Gianinazzi-Pearson (UMR INRA/CNRS/Burgundy University, INRA centre, Dijon) who jointly supervised my research work. My sincere thanks go to Professor Bin Zhao for his valuable suggestions and comments on my work, the organization of the research in China, and for a chance to do further study in France. Many thanks also go to Ms. Hui Lin, Nianwu Tang and all the colleagues of the State Key Laboratory of Agricultural Microbiology, for their help and concern. I am particularly grateful to Drs. Vivienne Gianinazzi-Pearson and Diederik van Tuinen who supervised my scientific progress, greatly helped me to organize and conduct research in France, and advised me in the English writing of the thesis and articles. I express my thanks for their continual guidance, encouragement and support both in solving different problems of work organization and life in a different country. I express my gratitude particularly to Ms. Christine Arnould (Microscope Centre and Pôle IPM, Dijon) for her help and guidance in the research with laser cell microdissection and in situ RT-PCR method, and to Ms. Anne Colombet and Ms. Valérie Monfort-Pimet, members of the Dijon laboratory, for their help in conducting experiments with mycorrhiza. Most sincere thanks to all the colleagues of the Pôle IPM and UMR PME: Dr. Silvio Gianinazzi, Prof. Daniel Wipf, Ms. Odile Chatagnier, Dr. Eliane Dumas-Gaudot, Joan Doidy, Soukayna Hayek, Virginie Molinier, Ivan Sanchez-Castro, Léonardo Casieri, Herbert Stockinger and all the other members for the chance to work together, their friendly support and concern.

Full length gene sequences and analyses

A total 7498bp genomic sequence of Gm152 was obtained by PCR amplification of G. mosseae genomic DNA using TAIL-PCR, and the upstream regulating sequence was obtained by Inverse PCR. It has 22 exons and 21 introns (Fig. 3.2); a promoter region was predicted in 1484 to 1734bp

(http://www.ncbi.nlm.nih.gov/Class/NAWBIS/Modules/DNA/dna21b.html). The final full-length cDNA, named Gm152, covers 3907bp and includes an open reading frame encoding 1188 amino acids, a 160 bp 3'-untranslated region (UTR), and a polypeptide with a predicted molecular weight of 134.81kDa. It was predicted to be a P-ATPase, member of the E1-E2_ATPase superfamily. A hydrolase domain, a protein tyrosine phosphatase and twelve transmembrane regions were also predicted (http://www.isrec.isb-sib.ch/ftp-server/tmpred/www/TMPRED_form.html) (Fig. 3.3).

Multiple sequence alignments with amino acid sequences in databases showed that Gm152 is closely related to various fungal P-type ATPases (Fig. 3.4): Ajellomyces dermatitidis (54% identities), Aspergillus nidulans (56% identities), Pichia stipitis (52% identities),

Schizosaccaromyces pombe (50% identities). In particular, it shows 58% identity to an endoplasmic reticulum Ca 2+ -transporting P-type ATPase from the ectomycorrhizal fungus Laccaria bicolor (XP_001889526). (PMCA). In addition, mitochondrial and nucleus communicate through a Ca 2+ signal [START_REF] Szabadkai | Mitochondria: The hub of cellular Ca 2+ signaling[END_REF].

Expression of the G. intraradices nuclear CCaMK was unaffected during presymbiotic fungal development; the gene was active during root colonization in the symbiotic stage suggesting a possible role in Ca 2+ perception related to this process.

CCaMK proteins are involved in signalling pathways leading to gene regulation. The calmodulin-binding domain and calcium-binding EF hand motifs of CCaMK allow the protein to sense calcium [START_REF] Oldroyd | Nuclear calcium changes at the core of symbiosis signalling[END_REF]. A calcium/calmodulin-dependent signaling system is involved in appressorium formation in the plant pathogenic fungus

Magnaporthe grisea and calmodulin gene expression is induced concomitantly with appressorium formation [START_REF] Ebbole | Magnaporthe as a model for understanding host-pathogen interactions[END_REF]. Moreover, a plant CCaMK is a prime candidate for the response to calcium signatures that are induced by AM fungi or Nod factors [START_REF] Lévy | A putative Ca 2+ and calmodulin-dependent protein kinase required for bacterial and fungal symbioses[END_REF][START_REF] Mitra | A Ca 2+ /calmodulin-dependent protein kinase required for symbiotic nodule development: Gene identification by transcript-based cloning[END_REF]. signal or an important background. However, the DESAT gene has previously been detected in arbuscules of G. intraradices BEG141 by in situ RT-PCR [START_REF] Seddas | Spatial monitoring of gene activity in extraradical and intraradical developmental stages of arbuscular mycorrhizal fungi by direct fluorescent in situ RT-PCR[END_REF].

Several experiments performed to improve the experimental conditions did not give satisfactory results. It was therefore decided to use targeted cryo-microdissection to localize fungal gene expression. Finally, the nuclear CCaMK from G. intraradices includes an open reading frame encoding for 440 amino acids and shows similarity (48 %) to a calcium/calmodulindependent protein kinase of Capsaspora owczarzaki (EFW47030). It contains a protein kinase, catalytic domain (PKc_like super family, cl09925). The protein kinase superfamily is mainly composed of the catalytic domains of serine/threonine-specific and tyrosinespecific protein kinases which catalyze the transfer of the gamma-phosphoryl group from ATP to hydroxyl groups in specific substrates such as serine, threonine, or tyrosine residues of proteins.

G. intraradices gene expression in

Yeast complementation assays

Growth tests

For phenotypic characterization, the wild-type yeast (W303), knockout (YDL128W, K667 and YGL167C), and complemented transformations (with gene or empty vector) strains were grown on YPD or SD medium (+/-uracil) to a cell density of OD 600 =0.6-0.8. The cell suspensions were serially diluted 10 fold, and 2.5 μl of each dilution was spotted on YPD or SD agar supplemented with CaCl 2 (50, 100, 200, 400, and/or 600, 800mM).