INTRARADICES GENES IN ARBUSCULE-CONTAINING CORTICAL CELLS OF THE PSSYM40 MUTANT

Abbreviations

SUMMARY

The arbuscular mycorrhizal (AM) association results from a successful interaction between the genomes of the two symbiotic partners. In this context, the aim of my research was to better characterize the role of the late stage symbiosis-related pea genes PsSym36, PsSym33 and PsSym40 in the functional AM (i) by investigating the effect of mutations in the three genes on fungal and plant gene responses and (ii) by creating conditions for the localization of two of the genes, PsSym36 and PsSym40, on the pea genetic map for future map-based cloning. The expression of a subset of ten fungal and eight plant genes, previously reported to be activated during mycorrhiza development, was compared in Glomus intraradices-inoculated roots of wild type and Pssym36, Pssym33 and Pssym40 mutant pea plants. Most of the fungal genes were down-regulated in roots of the Pssym36 mutant where arbuscule formation is defective, and several were upregulated with more rapid fungal development in roots of the Pssym40 mutant.

Microdissection of mycorrhizal PsSym40 roots corroborated preferential expression of the three G. intraradices genes SOD, DESAT and PEPISOM in arbuscule-containing cells. Inactivation of PsSym36 also resulted in down regulation of plant genes whilst mutation of the PsSym33 and PsSym40 genes affected plant gene responses in a more time-dependent way.

Results thus indicate an implication of the investigated pea SYM genes in the modulation of plant and fungal molecular interactions linked to signaling, nutrient exchange or stress response regulation during AM symbiosis formation and functioning. Conditions for localization of the PsSym36 and PsSym40 genes on the pea genetic map were developed for their future mapbased cloning. Based on the molecular markers obtained, it was possible to conclude that localization of the PsSym40 gene most likely resides outside the linkage groups I, II, III or V of the genetic map of pea.

RESUME

L'association mycorhizienne à arbuscules (AM) est le résultat d'une interaction compatible entre les génomes des deux partenaires symbiotiques. Dans ce contexte, le but de mes recherches a été de mieux caractériser le rôle des gènes de pois liés aux stades tardifs de la symbiose,

PsSym36, PsSym33 and PsSym40, dans le fonctionnement de la symbiose MA (i) en étudiant l'effet des mutations de ces trois gènes sur l'expression des gènes de la plante et du champignon, et (ii) en créant les conditions pour positionner deux de ces gènes, PsSym36 and PsSym40, sur la carte génétique afin d'envisager leur clonage futur. L'expression d'un groupe de dix gènes fongiques et de huit gènes de plante, déjà décrits pour être activés durant le développement de la mycorhize, a été comparée dans les racines de pois inoculées avec G.

intraradices chez les plantes de génotypes sauvages, ou les mutants Pssym36, Pssym33 et

CHAPTER I INTRODUCTION I.1. Arbuscular mycorrhiza, a ubiquitous symbiosis

The arbuscule mycorrhiza (AM) symbiosis is probably the most widespread beneficial interaction between plants and microorganisms. This symbiosis between roots and the fungal phylum of Glomeromycota originated around 450 million years ago and may have been a key event that enabled plants to colonize emerging landscapes [START_REF] Taylor | Four hundred-million-year-old vesicular arbuscular mycorrhizae[END_REF]. Since then, AM associations have become an integral part of most terrestrial plants in different ecosystems.

More than 80% of land plants form AM which play a major role in improving plant nutrient (mainly phosphorus) and water uptake [START_REF] Parniske | Molecular genetics of the arbscular mycorrhizal symbiosis[END_REF]. Colonization of the root system by AM fungi provides an additional pathway for the uptake of nutrients by formation of an extended network of extraradical hyphae and of a system for transfer to the plant by means of specialized intracellular structures, arbuscules (Harrison et al., 2005). More generally, the AM symbiosis may enhance resistance of plants to biotic (root pathogens) and abiotic (heavy metals, salinity, drought) stress (Dumas-Gaudot et al., 2000;[START_REF] Gianinazzi | Contribution of arbuscular mycorrhiza to soil quality and terrestrial ecotoxicology[END_REF]. In return, the plant provides photosynthetic carbon to the fungi which are unable to obtain carbon saprophytically. It has been estimated that worldwide, about 5 billion tons of carbon are transferred from plants to AM fungi each year [START_REF] Parniske | Molecular genetics of the arbscular mycorrhizal symbiosis[END_REF]. The potential agronomic and ecological importance of this unique beneficial fungal/plant association is largely recognized [START_REF] Koide | Effect of mycorrhizal fungi on plant populations[END_REF][START_REF] Ryan | Is there a role for arbuscular mycorrhizal fungi in production agriculture? In: Diversity and Integration in Mycorrhizas[END_REF][START_REF] Johansson | Microbial interactions in the mycorrhizosphere and their significance for sustainable agriculture[END_REF].

I.2. Formation of the AM symbiosis I.2.1. Stages of AM development

AM fungi are obligate biotrophs, their life-cycle depending on the formation of a symbiotic relationship with plant roots. The asymbiotic phase of their life cycle is restricted to spore germination and production of a limited amount of mycelium (Belestrini and Lanfranco, 2006). In the presence of root exudates of a host plant, spores of AM fungi usually show higher germination frequencies, and increased growth and branching of fungal hyphae [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][START_REF] Tamasloukht | Root factors induce mitochondrial-related gene expression and fungal respiration during the developmental switch from asymbiosis to presymbiosis in the arbuscular mycorrhzal fungus Gigaspora rosea[END_REF]. After a series of recognition events between the symbiotic partners involving plant and fungal signals [START_REF] Gianinazzi-Pearson | Plants and arbuscular mycorrhizal fungi: cues and communication in the early steps of symbiotic interactions[END_REF]), the fungi colonize the root surface, form appressoria, and enter the rhizodermis intercellularly or intracellularly depending on the host plant and fungus involved (Dickson, 2004). Fungal hyphae then grow through the outer cell layers to invade cells of the inner cortical parenchyma, where the fungi form highly branched intracellular structures, named arbuscules. Arbuscules, which can occupy the major part of the inner cortical root tissues, are key structures of the AM symbiosis as they are responsible for reciprocal nutrient and signal exchange between partner cells. They are completely surrounded by a periarbuscular membrane (PAM) which is formed de novo from the plant plasma membrane, giving rise to a symbiotic interface (Gianinazzi-Pearson, 1996).

Arbuscules senesce and degrade after 4-10 days of symbiotic activity (Alexander et al. 1988), whereupon the original organization of the plant cells is restored and they can become colonized again by an AM fungus [START_REF] Jacquelinet-Jeanmougin | Endomycorrhizas in the Gentianaceae. II. Ultrastructural aspects of symbiont relationships in Gentiana lutea L[END_REF]Gianinazzi-Pearson, 1996). The life cycle of AM fungi is completed by the formation of extraradical hyphae and new spores [START_REF] Sanders | The development of endomycorrhizal root systems. I. Speed of infection and growth-promoting effects with four species of vesicular-arbuscular endophyte[END_REF][START_REF] Jacquelinet-Jeanmougin | Endomycorrhizas in the Gentianaceae. II. Ultrastructural aspects of symbiont relationships in Gentiana lutea L[END_REF][START_REF] Marsh | Analysis of arbuscular mycorrhizas using symbiosis defective plant mutants[END_REF][START_REF] Hause | Molecular and cell biology of arbuscular mycorrhizal symbiosis[END_REF].

I.2.2. Cytological reorganization of plant cells during AM formation

Upon first contact with the appressoria of AM fungi, the contents of a root epidermal cell undergo an important reorganization to form a specialized membrane-bound cytoplasmic assembly, the prepenetration apparatus (PPA), which directs the penetrating AM hypha through the plant cell. The PPA is a novel structure comprising microtubules, microfilaments and endoplasmic reticulum (ER) assembled within a thin column of cytoplasm, created by repositioning of the plant nucleus directly below the site of appressorium contact, and subsequent migration of the nucleus across the epidermal cell (see figure I.1 A, B; [START_REF] Genre | Arbuscular mycorrhizal fungi elicit a novel intracellular apparatus in Medicago truncatula root epidermal cells before infection[END_REF]. PPA plays an important role in the creation of the membrane/matrix interface surrounding hyphae during AM colonization of the root [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].

A similar penetration mechanism operates during the next steps of AM fungal colonization of outer and inner cortical cells [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][START_REF] Smith | Fast moves in arbuscular mycorrhizal symbiotic signalling[END_REF]. Once the fungus has entered and fully traversed the epidermal cell, a second PPA forms across the adjacent outer cortical cell in advance of the progressing hyphal tip (see figure I.1 A, B). The subsequent pattern of AM fungal colonization of the inner cortical tissues is dependent on the identity of the host plant. In the case of the Medicago truncatula/Gigaspora spp association, intercellular hyphae grow through the apoplastic space of the cortical parenchyma before penetrating individual cells and initiating arbuscule development (Arum-type colonization; Smith and Smith, 1997). Intercellular hyphal growth triggers enlargement and repositioning of the plant nucleus, associated with an accumulation of cytoplasm which aggregates on the inner cell side in contact with the hypha. The subsequent colonization of the individual plant cell is preceded by nuclear movement to the center of the cell and assembly of a cortical PPA. Formation of prebranching cytoplasmic aggregations along the intracellular hypha determines arbuscule branching (see figure I.1 A; [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].

A different pattern of AM development occurs in the inner cortex of Daucus carota roots (see figure I.1 B) which is characterized by an intracellular cell-to-cell colonization (Paris-type of colonization; Smith and Smith, 1997). In this case, there is a linear long-distance coordinated PPA development which is defined by specific nuclear dynamics and the formation of a cytoplasmic bridge in adjacent cortical cells in advance of the approaching hypha [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 presence of the fungus in cells of the inner root cortex induces a decrease in size and fragmentation of the vacuole, disappearance of amyloplasts, nuclear hypertrophy and proliferation of organelles (plastids, mitochondria, dictyosomes) [START_REF] Jacquelinet-Jeanmougin | Endomycorrhizas in the Gentianaceae. II. Ultrastructural aspects of symbiont relationships in Gentiana lutea L[END_REF][START_REF] Bonfante-Fasolo | Plant and endomycorrhizal fungi: the cellular and molecular basis of their interaction[END_REF][START_REF] Balestrini | The plant nucleus in mycorrhizal roots: positional and structural modifications[END_REF]. Mitochondria aggregate in the vicinity of arbuscules and plastids form stromules (stroma filled tubules) leading to extensive network-like structures, both of which may be necessary to support the general increase in metabolic activity of the colonized host cells [START_REF] Lohse | GintAMTi encodes a functional high-affinity ammonium transporter that is expressed in the extraradical mycelium of Glomus intraradices[END_REF]. The nuclear hypertrophy of arbuscule-containing cells [START_REF] Balestrini | The plant nucleus in mycorrhizal roots: positional and structural modifications[END_REF]) is characterized by enhanced fluorochrome accessibility, increased nuclease sensitivity and chromatin dispersion which reflect an increase in chromatin decondensation (Gianinazzi-Pearson, 1996). These modifications indicate a strong activation of host cell transcription in response to fungal colonization.

Formation of the arbuscule results in the genesis of a new interfacial compartment between the fungal wall and PAM which provides the structural basis of the biotrophic interaction between plant and fungus. The presence in this interface of molecules typical of the primary plant cell wall (i.e., cellulose, pectins, hemicellulose, HRGPs) suggests that the PAM retains the enzymatic machinery involved in both synthesis and secretion of cell wall material [START_REF] Bonfante | At the interface between mycorrhizal fungi and plants: the structural organization of cell wall, plasma membrane and cytoskeleton[END_REF]Balestrini and Bonfante, 2005). Specific activation of genes encoding H + -ATPase and a low-affinity phosphate transporter in arbuscule-containing cells, and location of the corresponding protein at the PAM suggest transmembrane transport activities at the symbiotic interface [START_REF] Gianinazzi-Pearson | Differential activation of H + -ATPase genes by an arbuscular mycorrhizal fungus in root cells of transgenic tobacco[END_REF][START_REF] Harrison | A phosphate transporter from Medicago truncatula involved in the acquisition of phosphate released by arbuscular mycorrhizal fungi[END_REF].

When the arbuscule begins to senesce, fibrillar material encapsulates the collapsed fungal structures that are subsequently degraded by the plant cell. Host cell contents regain their original morphology and recolonization by another arbuscule can occur [START_REF] Jacquelinet-Jeanmougin | Endomycorrhizas in the Gentianaceae. II. Ultrastructural aspects of symbiont relationships in Gentiana lutea L[END_REF][START_REF] Strack | Arbuscular mycorriza: biological, chemical, and molecular aspects[END_REF].

I.2.3. Mycorriza-defective pea mutants

Plant mutants are key tools for defining genes involved in distinct morphological stages of AM symbiosis development and for the analysis of specific plant or fungal gene responses during symbiotic interactions. Mutants with a mycorrhiza-defective phenotype were first designated as Myc -by Duc et al. (1989). The majority of such mutants are affected in the earliest stages of symbiosis development, after appressoria formation and preventing penetration of fungi into the root (Myc -1 phenotype). More infrequent are mutants which do not fully block the symbiotic process but are altered for arbuscule formation (Myc -2 phenotype) [START_REF] Gianinazzi-Pearson | Genetic and cellular analysis of resistance to vesicular arbuscular (va) mycorrhizal fungi in pea mutants/ Advances in Molecular Genetics of Pant-Microbe Interactions[END_REF] or in the rate of AM symbiosis development (Rmd -and Rmd ++ phenotypes) (Jacobi et al., 2003a, b).

Mutants having alterations at the appressoria formation stage (App) have been identified for maize and tomato. The maize nope 1 (no perception 1) mutant does not support appressoria formation, suggesting the absence of a plant-encoding function necessary for early recognition prior contact [START_REF] Paszkowski | Maize mutants affected at distinct stages of the arbuscular mycorrhizal symbiosis[END_REF]. In another maize mutant taci 1 (taciturn 1), appressoria form at a reduced frequency but their morphology is normal and leads to penetration of the rhizodermis. However, the majority of hyphae are septate, resulting in limited fungal spread [START_REF] Paszkowski | Maize mutants affected at distinct stages of the arbuscular mycorrhizal symbiosis[END_REF]. Appressorium formation is significantly reduced in roots of tomato pmi (premycorrhizal infection) mutants, M161 and M20 [START_REF] David-Schwartz | Identification of a novel genetically controlled step in mycorrhizal colonization: plant resistance to infection by fungal spores but not extra-radical hyphae[END_REF], 2003).

Enhanced and early fungal invasion was detected in roots of maize pram 1 (Precocious arbuscular mycorrhiza 1) mutant [START_REF] Paszkowski | Maize mutants affected at distinct stages of the arbuscular mycorrhizal symbiosis[END_REF].

The majority of plant mutants block fungal penetration (Pen -) into the root epidermis. These mutants prevent penetration of the root epidermis resulting in the formation of complex hyphal branching, and multiple and usually swollen appressoria [START_REF] Marsh | Analysis of arbuscular mycorrhizas using symbiosis defective plant mutants[END_REF]. A number of Pen -mutants have been identified for Pisum sativum (Duc et al., 1989), Vicia faba (Duc et al., 1989), Medicago truncatula [START_REF] Sagan | Selection of nodulation and mycorrhizal mutants in the model plant Medicago truncatula (Gaertn.) after gamma-ray mutagenesis[END_REF][START_REF] Catoira | Four genes of Medicago truncatula controlling components of a Nod factor transduction pathway[END_REF], Medicago sativa [START_REF] Bradbury | Interactions between three alfalfa nodulation genotypes and two Glomus species[END_REF], Lotus japonicus (Senoo et al., 2000a), Lycopersicon esculentum [START_REF] Barker | A mutant in Lycopersicon esculentum Mill. With highly reduced VA mycorrhizal colonization: isolation and preliminary characterisation[END_REF] and Petunia hybrida [START_REF] Reddy | A petunia mutant affected in intracellular accommodation and morphogenesis of arbuscular mycorrhizal fungi[END_REF]. In pea, mutants having a Pen -phenotype are Pssym8, Pssym9 and Pssym19 (see table I.1). In some Myc -mutants, appressorium formation and epidermal cell penetration can develop normally, but further development into the cortex does not occur (Coi -) [START_REF] Marsh | Analysis of arbuscular mycorrhizas using symbiosis defective plant mutants[END_REF]. Mutations in at least six distinct loci of L. japonicus (SYMRK, CASTOR, POLLUX, LjSym3, LjSym5 and LjSym30) result in the Coi - phenotype [START_REF] Wegel | Mycorrhiza mutants of Lotus japonicus define genetically independent steps during symbiotic infection[END_REF][START_REF] Bonfante | The Lotus japonicus LjSym4 gene is required for the successful symbiotic infection of root epidermal cells[END_REF][START_REF] Kistner | Seven Lotus japonicus genes required for transcriptional reprogramming of the root during fungal and bacterial symbiosis[END_REF].

In addition, mutants in which inner cortex invasion (Ici -) does not occur were identified by Senoo et al. (2000b). They are the L. japonicus mutants mcbee (mycorrhizal colonization blocked between epidermis and exodermis) and mcbex (mycorrhizal colonization blocked in exodermis).

Mutants which allow colonization of the inner cortex but where the fungus cannot form arbuscules are designated Ard -(arbuscule defective). This is the case of the Pssym36 mutant in pea (see table I.1). In contrast, the mutation in the mcbco (mycorrhizal colonization blocked in cortex) gene of L. japonicus results in the formation of normal arbuscules but leads to premature arbuscule senescence (Senoo et al., 2000b). Other plant mutants affected in the rate of AM symbiosis development are found in pea: Pssym33 and Pssym40 (Jacobi et al., 2003a, b). The mutation in the PsSym33 gene decreased mycorrhizal colonization of roots (Rmd - phenotype), whereas the mutation of the PsSym40 gene results in faster arbuscule development and turn-over in comparison with wild-type plants (Rmd ++ phenotype).

P. sativum L. is one of the most developed model plants for genetic studies of the nitrogenfixing symbiosis (NFS) and the AM symbiosis. More than 40 NFS genes have been identified in pea, a part of which are also determinant for the AM symbiosis (Table I. 1;[START_REF] Borisov | Pea (Pisum sativum L.) regulatory genes controlling development of nitrogen-fixing nodule and arbuscular mycorrhiza: fundamentals and application[END_REF]. Duc et al. (1989) were the first to demonstrate that some pea mutants impaired in symbiosis with nitrogen-fixing root nodule bacteria are also defective for AM formation. Gianinazzi-Pearson (1998) and [START_REF] Gherbi | SymRK defines a common genetic basis for plant root endosymbioses with arbuscular mycorrhiza fungi, rhizobia, and Frankiabacteria[END_REF] have hypothesized that at least a part of the genetic machinery necessary for the Rhizobia nodulation symbiosis may have been recruited by legumes from functions in the more ancient plant/AM fungal symbiosis. Since their discovery, the exploitation of mutants defective in both bacterial and fungal symbioses has made an important contribution to investigations of the evolution and fundamental processes involved in plant -microbe interactions. Flavonoids and strigolactones, root secreted molecules, can activate the presymbiotic growth of AM fungi [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][START_REF] Akiyama | Plant sesquiterpenes induce hyphal branching in arbuscular mycorrhizal fungi[END_REF]. Flavonoids are considered signals in symbiotic plant-microbe interactions but a specific role during early stages of AM establishment seems unlikely [START_REF] Steinkellner | Flavonoids and strigolactones in root exudates as signals in symbiotic and pathogenic plant-fungus interactions[END_REF]. Strigolactones are a novel class of plant hormones shown to be active at sub-picomolar concentrations and to stimulate fungal spore germination, branching of germinating hyphae and growth of AM fungi [START_REF] Akiyama | Plant sesquiterpenes induce hyphal branching in arbuscular mycorrhizal fungi[END_REF][START_REF] Besserer | Strigolactones stimulate arbuscular mycorrhiza fungi by activating mitochondria[END_REF]. Previously, they were characterized as germination stimulants of parasitic weeds such as Striga and Orobanche (Bouwmeester et al., 2003;[START_REF] Matusova | The strigolactone germination stimulants of the plant-perasitic Striga and Orobanche spp. are derived from the carotenoid pathway[END_REF]. Chemically, strigolactones are a group of sesquiterpene lactones derived from carotenoid biosynthesis [START_REF] Délano-Frier | Novel developments in the understanding of signaling and nutrient exchange mechanisms in the arbuscular mycorrhizal symbiosis[END_REF]. Treatment of maize seedlings with fluridone, an upstream inhibitor of the carotenoid metabolism, results in a significant reduction in root mycorrhization which can be restored by the addition of GR24, a strigolactone analogue [START_REF] Gomez-Roldan | Strigolactones. Promising plant signals[END_REF]. Similar results were obtained with the y9 mutant of maize defective in an upstream step of carotenoid synthesis, underlining the role of strigolactones as symbiotic signals [START_REF] Gomez-Roldan | Strigolactones. Promising plant signals[END_REF].

Strigolactones induce a rapid (within 1 hour) increase in O 2 consumption by AM fungi and strong changes in the shape, density and motility of mitochondria in hyphae [START_REF] Besserer | Strigolactones stimulate arbuscular mycorrhiza fungi by activating mitochondria[END_REF]. Further investigations [START_REF] Besserer | GR24, a synthetic analog of strigolactones, stimulates the mitosis and growth of the arbuscular mycorrhizal fungus Gigaspora rosea by boosting its energy metabolism[END_REF] have shown that treatment of Gigaspora rosea with a strigolactone analog, GR24, also causes a rapid (within minutes) increase in the NADH concentration, the NADH dehydrogenase activity and the ATP content in the fungal cells, especially in the hyphal tips. A stimulation in the fungal mitotic activity takes place several days after this initial boost to the cellular energy of the fungus. However, the alterations in metabolism do not require new gene expression. The striking stimulation of fungal metabolic activity by strigolactones has led to the elaboration of a model in which mitochondrial or cytoplasmic receptors could be possible strigolactone targets needed to stimulate mitochondrial biogenesis, lipid catabolism and respiration [START_REF] Besserer | GR24, a synthetic analog of strigolactones, stimulates the mitosis and growth of the arbuscular mycorrhizal fungus Gigaspora rosea by boosting its energy metabolism[END_REF][START_REF] Délano-Frier | Novel developments in the understanding of signaling and nutrient exchange mechanisms in the arbuscular mycorrhizal symbiosis[END_REF]. Other models propose the interaction of strigolactones with a hypothetical fungal receptor, followed by the quick inactivation of the lactone ligand through the removal of its D ring [START_REF] Akiyama | Plant sesquiterpenes induce hyphal branching in arbuscular mycorrhizal fungi[END_REF][START_REF] Yoneyama | Strigolactones: structures and biological activities[END_REF], or the response of fungi to strigolactones via an enhancement of endogenous ethylene synthesis which could then stimulate mitochondrial metabolism [START_REF] Besserer | GR24, a synthetic analog of strigolactones, stimulates the mitosis and growth of the arbuscular mycorrhizal fungus Gigaspora rosea by boosting its energy metabolism[END_REF]. In spite of the discovery of strigolactones, only scarce data are available about interactions between strigolactones and fungi other than AM fungi. The mechanism(s) by which AM fungi are able to detect host plant roots therefore still remains a source of speculation [START_REF] Délano-Frier | Novel developments in the understanding of signaling and nutrient exchange mechanisms in the arbuscular mycorrhizal symbiosis[END_REF].

Purification and characterization of active root-exuded molecules are in progress [START_REF] Buée | The pre-symbiotic growth of arbuscular mycorrhizal fungi is induced by a branching factor partially purified from plant root exudates[END_REF]Vierheilig and Piché, 2002;Vierheilig, 2004;Naqahashi and Douds, 2007).

I.3.2. Plant genes known to be activated at the first steps of AM formation

Further establishment of a functional symbiosis is known to require activation of a host plant signaling pathway involving the NORK/SYMRK/DMI2/SYM19 Leu-rich-repeat receptor kinase, the POLLUX/DMI1/SYM8 putative nuclear channel protein and the CCaMK/DMI3/SYM9 calcium and calmodulin-dependent protein kinase (CCaMK) [START_REF] Endre | A receptor kinase gene regulating symbiotic nodule development[END_REF][START_REF] Stracke | A plant receptor-like kinase required for both bacterial and fungal symbiosis[END_REF]Ane et al., 2004;Levy et al., 2004;[START_REF] Mitra | A Ca 2+ /calmodulin-dependent protein kinase required for symbiotic nodule development: gene indentification by transcript-based cloning[END_REF][START_REF] Kistner | Seven Lotus japonicus genes required for transcriptional reprogramming of the root during fungal and bacterial symbiosis[END_REF]. Mutations in the genes encoding these proteins either prevent or reduce entry of the fungus into the root [START_REF] Oldroyd | Peace talks and trade deals. Keys to longterm harmony in legume-microbe symbioses[END_REF]. However, whilst dmi1 mutants are tight and always have a myc -1 phenotype, dmi2 and dmi3 mutants are leaky and can allow fungal colonization under certain conditions [START_REF] Morandi | Characterisation of new symbiotic Medicago truncatula (Gaertn.) mutants, and phenotypic or genotypic complementary information on previously described mutants[END_REF]. Hypothetically, DMI2 is involved in the direct or indirect perception of a fungal signal which is then transduced through the intracellular kinase to activate the ion channel DMI1. Both genes are responsible for induction of oscillations in calcium concentrations to which the DMI3 kinase potentially responds directly [START_REF] Parniske | Molecular genetics of the arbscular mycorrhizal symbiosis[END_REF][START_REF] Navazio | Calcium opens the dialogue between plants and arbuscular mycorrhizal fungi[END_REF]. The implication of Ca 2+ as an intracellular messenger has been predicted by several authors based on similarities with the nitrogen-fixing root nodule symbiosis, and it was recently confirmed that AM fungi can induce Ca 2+ oscillations in plant cell cultures and root epidermal cells (Navazio et al., 2007a, b;[START_REF] Kosuta | Differential and chaotic calcium signatures in the symbiosis signaling pathway of legumes[END_REF].

Several genes are strongly induced in M. truncatula roots in response to unknown signaling molecules exuded by AM fungi prior to and after contact with the host root [START_REF] Kosuta | A diffusible factor from arbuscular mycorrhizal fungi induces symbiosis-related 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]). An essential part of the reported genes are related to signal transduction or to synthesis of the early nodulin MtENOD11. In the case of the signal transduction-related genes, [START_REF] Weidmann | Fungal elicitation of signal transduction-related plant genes precedes mycorrhiza establishment and requires the dmi3 gene in Medicago truncatula[END_REF] showed that their activation is dependent on the DMI3 gene both before contact and with appressorium formation. The MtENOD11 gene, which encodes a repetitive Pro-rich protein, is believed to be a functional component of the plant extracellular matrix. Gigaspora-targeting experiments in root organ cultures systematically revealed P ENOD11 :GFP-HDEL activation in epidermal cells in contact with fungal appressoria as well as in a very limited number of adjacent cells. Observations suggest that the ENOD11 gene is activated in epidermal cells after appressorium differentiation both before and during PPA formation and probably also during hyphal penetration of the root [START_REF] Genre | Arbuscular mycorrhizal fungi elicit a novel intracellular apparatus in Medicago truncatula root epidermal cells before infection[END_REF].

However, the issue about the place of the ENOD11 gene in the signaling pathway between AM symbionts remains open. [START_REF] Kosuta | A diffusible factor from arbuscular mycorrhizal fungi induces symbiosis-related MtENOD11 expression in roots of Medicago truncatula[END_REF] reported that activation of the ENOD11 gene by diffusible fungal signals is not dependent on DMI gene function in M. truncatula, because P ENOD11 :GUS activation by AM fungal exudates was unaltered in mutants of all three DMI genes.

In contrast, ENOD11 gene expression associated with appressorium formation, described by [START_REF] Chabaud | Targeted inoculation of Medicago truncatula in vitro root cultures reveals MtENOD11 expression during early stages of infection by arbuscular mycorrhizal fungi[END_REF] using a P ENOD11 :GUS reporter construct and by [START_REF] Genre | Arbuscular mycorrhizal fungi elicit a novel intracellular apparatus in Medicago truncatula root epidermal cells before infection[END_REF] using a P ENOD11 :GFP-HDEL reporter construct, requires activation of the DMI genes. In experiments by [START_REF] Chabaud | Targeted inoculation of Medicago truncatula in vitro root cultures reveals MtENOD11 expression during early stages of infection by arbuscular mycorrhizal fungi[END_REF], for example, MtENOD11 gene expression was not detectible in a dmi2 mutant background despite the formation of appressoria at the root surface.

Exploiting the Myc -pea mutant P2 (now known to be Pssym9), [START_REF] Roussel | Signalling between arbuscular mycorrhizal fungi and plants: identification of a gene expressed during erly interactions by differential RNA display analysis[END_REF] by RNA display analysis identified a PsAM5 pea gene to be transiently activated at early stages of fungal-root interactions. Deduced PsAM5 protein shares significant similarity with a ClpP serine protease. The authors proposed that this protein may have multiple functions related to the cross-talk between symbiotic partners during early interactions and that in addition to housekeeping functions, it may be involved in controlling levels of key regulatory proteins within a signal-transduction pathway in the AM symbiosis.

I.3.3. Plant and fungal gene expression related to AM establishment and function

Genes associated with plant cell wall and cell matrix modifications

The colonization of a root by AM fungi is accompanied by the reorganization of host cell walls and cell matrix. Most genes associated with these modifications have been described in M. The M. truncatula gene MtCel1, which is predicted to be involved in cell wall modifications, was reported to be specifically induced in mycorrhizal roots by [START_REF] Liu | Transcript profiling coupled with spatial expression analyses reveals genes involved in distinct developmental stages of the arbuscular mycorrhizal symbiosis[END_REF]. In AM roots, expression of the MtCel1 gene was associated with cells containing arbuscules. It was suggested that MtCel1 is located in the periarbuscular membrane and involved in the assembly of the cellulose/hemicellulose matrix of the interface compartment [START_REF] Liu | Transcript profiling coupled with spatial expression analyses reveals genes involved in distinct developmental stages of the arbuscular mycorrhizal symbiosis[END_REF].

A cDNA of M. truncatula gene coding a xyloglucan endotransglucosylase/hydrolase (Mt-XTH) that catalyzes the hydrolysis and transglycosylation of xyloglucan polymers in plant cell walls has been isolated by [START_REF] Van Buuren | Novel genes induced during an arbuscular mycorrhizal (AM) symbiosis between M. truncatula and G. versiforme[END_REF]. Later Maldonado-Mendoza et al. (2005) identified the corresponding XTH genes, Mt-XTH1 and Mt-XTH2. Analysis of transgenic roots expressing an Mt-XTH1 fusion promoter revealed that Mt-XTH1 expression is elevated in cells throughout the root system with significant higher levels of activity in mycorrhizal roots, especially in the mycorrhizal regions. This expression pattern is considered to be consistent with gene activation in response to a systemic signal which may enable fungal penetration of roots (Maldonado-Mendoza et al., 2005).

Expression of M. truncatula and pea genes encoding α-expansin and extensin, respectively, that contribute to plant cell-wall extensibility, has also been detected in mycorrhizal roots [START_REF] Journet | Medicago truncatula ENOD11: A novel RPRP-encoding early nodulin gene expressed during mycorrhization in arbuscule-containing cells[END_REF][START_REF] Liu | Transcript profiling coupled with spatial expression analyses reveals genes involved in distinct developmental stages of the arbuscular mycorrhizal symbiosis[END_REF][START_REF] Grunwald | Identification of mycorrhizaregulated genes with arbuscule development-related expression profile[END_REF]. Such proteins may be involved in the accommodation of the AM fungus by the colonized cortical cells (Balestrini et al., 2005).

Genes encoding putative AGP (arabinogalactan protein) and HRGP (hydroxyproline-rich glycoprotein) are also induced in mycorrhizal roots of M. truncatula and maize, respectively, and transcripts are specifically localized in the cells containing arbuscules [START_REF] Balestrini | Fungal and plant gene expression in arbuscular mycorrhizal symbiosis[END_REF].

Of the genes associated with cell matrix functions, alterations in the microtubular network in cells of AM colonized roots are reflected in AM-specific up-regulation of αand β-tubulin genes, especially in cells containing arbuscules [START_REF] Bonfante | Transcriptional activation of a maize-tubulin gene in mycorrhizal maize and transgenic tobacco plants[END_REF][START_REF] Grunwald | Identification of mycorrhizaregulated genes with arbuscule development-related expression profile[END_REF][START_REF] Hause | Molecular and cell biology of arbuscular mycorrhizal symbiosis[END_REF].

The proliferation and reorganization of root plastids which take place in cortical cells of AMcolonized roots are correlated with the increased transcript levels of the first two enzymes of the plastid-located methylerythritol phosphate (MEP) pathway, 1-deoxy-d-xylulose 5-phosphate synthase (DXS) and 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), and of the carotenoid pathway, phytoene desaturase, ζ-carotene desaturase, carotenoid-cleaving dioxygenase [START_REF] Strack | Isoprenoid metabolism and plastid reorganization in arbuscular mycorrhizal roots[END_REF][START_REF] Walter | Apocarotenoid biosynthesis in arbuscular mycorrhizal roots: contributions from methylerythritol phosphate pathway isogenes and tools for its manipulation[END_REF][START_REF] Floss | Knock-down of the MEP pathway isogene 1-deoxy-D-xylulose 5-phosphate synthase 2 inhibits formation of arbuscular mycorrhiza-induced apocarotenoids, and abolishes normal expression of mycorrhiza-specific plant marker genes[END_REF][START_REF] Sun | Cloning and characterisation of a maize carotenoid cleavage dioxygenase (ZmCCD1) and its involvement in the biosynthesis of apocarotenoids with various roles in mutualistic and parasitic interactions[END_REF].

Genes related to defense and/or stress responses

The colonization of host roots by AM fungi elicits a cascade of general and specific plant defense/stress responses (Dumas-Gaudot et al., 2000). AM-related expression has been reported in different plants for genes encoding proteins of the phenylpropanoid biosynthetic pathway, pathogenesis-related (PR) proteins and enzymes involved in responses against oxidative stress [START_REF] Garcia-Garrido | Regulation of the plant defence response in arbuscular mycorrhizal symbiosis[END_REF][START_REF] Grunwald | Identification of mycorrhizaregulated genes with arbuscule development-related expression profile[END_REF][START_REF] Hause | Molecular and cell biology of arbuscular mycorrhizal symbiosis[END_REF][START_REF] Balestrini | Fungal and plant gene expression in arbuscular mycorrhizal symbiosis[END_REF]. Increased expression of defense and stress-related genes is detected during the early stage interactions between roots and AM fungi, with a subsequent decrease for several gene families as the AM symbiosis develops (Gianinazzi-Pearson, 1996;[START_REF] Liu | Transcript profiling coupled with spatial expression analyses reveals genes involved in distinct developmental stages of the arbuscular mycorrhizal symbiosis[END_REF][START_REF] Deguchi | Transcriptome profiling of Lotus japonicus roots during arbuscular mycorrhiza development and comparison with that of nodulation[END_REF]. However, cytological localization of defense and stress-related gene expression in mycorrhizal roots has shown that in nearly all cases transcripts of the genes accumulate specifically in arbuscule-containing cells (Gianinazzi-Pearson, 1996;[START_REF] Garcia-Garrido | Regulation of the plant defence response in arbuscular mycorrhizal symbiosis[END_REF][START_REF] Liu | Transcript profiling coupled with spatial expression analyses reveals genes involved in distinct developmental stages of the arbuscular mycorrhizal symbiosis[END_REF][START_REF] Wulf | Transcriptional changes in response to arbuscular mycorrhiza development in the model plant Medicago truncatula[END_REF][START_REF] Grunwald | Identification of mycorrhizaregulated genes with arbuscule development-related expression profile[END_REF].

Phenylpropanoid metabolism is activated in AM interactions but to a much lower extent than in plant-pathogen interactions [START_REF] Hause | Molecular and cell biology of arbuscular mycorrhizal symbiosis[END_REF]. Also, the expression of genes encoding enzyme components of the phenylpropanoid pathway is often uncoordinated and can vary during AM establishment. For example, [START_REF] Deguchi | Transcriptome profiling of Lotus japonicus roots during arbuscular mycorrhiza development and comparison with that of nodulation[END_REF] reported that phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) genes are moderately up-regulated on the initial association of L. japonicus with an AM fungus and repressed in the later stages, whilst [START_REF] Harrison | Spatial patterns of expression of flavonoid/isoflavonoid pathway genes during interactions between roots of Medicago truncatula and the mycorrhizal fungus Glomus versiforme[END_REF] described the specific induction of the isoflavone reductaseencoding gene in M. truncatula cells containing arbuscules. The induction of phenylpropanoid biosynthesis during the AM symbiosis has been discussed either in relation to the biosynthesis of antimicrobial phytoalexins and bioprotection against root pathogens or the biosynthesis of flavonoid compounds stimulating the growth of AM fungi during interactions with host roots (Dumas-Gaudot et al., 2000).

PR protein-encoding genes are also more weakly expressed in mycorrhizal roots when compared to plant-pathogen interactions [START_REF] Hause | Molecular and cell biology of arbuscular mycorrhizal symbiosis[END_REF]. PR1 protein (pathogenesisrelated protein 1) was first detected in mycorrhizal tobacco roots in the symbiotic interface between the PAM and the fungal wall [START_REF] Gianinazzi-Pearson | Weak expression of the pathogenesis related PR-b1 gene and localization of related protein during symbiotic endomycorrhizal interactions in tobacco roots[END_REF]. Since then, PR genes and proteins have been studied in roots of a range of mycorrhizal plant species (Dumas-Gaudot et al. 2000). For example in pea, a PR10 gene (pathogenesis-related protein 10) is overexpressed during early interactions with G. mosseae (Ruiz-Lozano et al., 1999) whilst genes encoding members of the protein families PR1 and PR6 (proteinase inhibitor) are up-regulated in colonized roots [START_REF] Grunwald | Identification of mycorrhizaregulated genes with arbuscule development-related expression profile[END_REF]. The activity and transcript accumulation patterns of PR proteins that are hydrolytic enzymes (chitinase, β-1,3-glucanase) suggest that these enzymes could also form part of the plant defense response against the invading AM fungus (Dumas-Gaudot et al. 2000;[START_REF] Garcia-Garrido | Regulation of the plant defence response in arbuscular mycorrhizal symbiosis[END_REF]. It has also been speculated that chitinases may also have a role in the degradation/turnover or formation of arbuscules (Dumas-Gaudot et al. 2000;[START_REF] Hause | Molecular and cell biology of arbuscular mycorrhizal symbiosis[END_REF]. In fact, a class III chitinase gene is expressed specifically in cells of roots M. truncatula containing functionally active arbuscules, suggesting a role of this enzyme in arbuscule turnover [START_REF] Salzer | Differential expression of eight chitinase genes in Medicago truncatula roots during mycorrhiza formation, nodulation and pathogen infection[END_REF].

One of the most rapid reactions in plants to stress situations, including pathogen attack, is the so-called oxidative burst which is marked by the production of reactive oxygen species (ROS) such as superoxide, hydroxyl radicals, and hydrogen peroxide [START_REF] Apel | Reactive oxygen species: metabolism, oxidative stress, and signal transduction[END_REF][START_REF] Garcia-Garrido | Regulation of the plant defence response in arbuscular mycorrhizal symbiosis[END_REF]. Increased production of ROS may result in the activation of various signaling cascades involved in direct toxic effects on a pathogen, in modification of apoplastic structures directed against the pathogen, or in cell senescence [START_REF] Apel | Reactive oxygen species: metabolism, oxidative stress, and signal transduction[END_REF].

It was shown that in AM colonized roots, ROS are accumulated close to fungal structures and that H 2 O 2 accumulation is correlated with arbuscule senescence [START_REF] Fester | Accumulation of reactive oxygen species in arbuscular mycorrhizal roots[END_REF].

Increased ROS production induces the activity of various antioxidative enzymes such as superoxide dismutase (SOD), catalases and peroxidases which play a role in detoxification processes and H 2 O 2 release [START_REF] Apel | Reactive oxygen species: metabolism, oxidative stress, and signal transduction[END_REF]. There are several reports of a general stimulation of SODs by the AM symbiosis in different plants (Dumas-Gaudot et al., 2000), and mycorrhizal protection against oxidative stress caused by drought is associated with increased expression of a Mn-SODII gene in lettuce [START_REF] Ruiz-Lozano | Cloning of cDNAs encoding SODs from lettuce plants which show differential regulation by arbuscular mycorrhizal symbiosis and by drought stress[END_REF]. Gene expression for a germin-like protein, which can posses oxalate oxidase or superoxide dismutase activities, has been repeatedly found to be enhanced in AM roots of M. truncatula [START_REF] Wulf | Transcriptional changes in response to arbuscular mycorrhiza development in the model plant Medicago truncatula[END_REF]Küster et al., 2004;[START_REF] Brechenmacher | Expression profiling of up-regulated plant and fungal genes in early and late stages of Medicago truncatula-Glomus mosseae interactions[END_REF][START_REF] Güimil | Comparative transcriptomics of rice reveals an ancient pattern of response to microbial colonization[END_REF]. On the fungal side, expression of the superoxide dismutase genes of Gig. margarita [START_REF] Lanfranco | The mycorrhizal fungus Gigaspora margarita possesses a CuZn superoxidedismutase which is up-regulated during the symbiosis with legume hosts[END_REF], G. intraradices [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] and G. mosseae [START_REF] Brechenmacher | Expression profiling of up-regulated plant and fungal genes in early and late stages of Medicago truncatula-Glomus mosseae interactions[END_REF], and the thioredoxin peroxidaseencoding gene of G. intraradices [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], have been shown to be up-regulated when the fungi colonize host tissues. Synthesis and activity of corresponding enzymes may help the fungus to contend with ROS production by host roots and deal with plant defense responses during AM symbiosis development.

Genes coding for proteins involved in the enzyme synthesis of nitric oxide (NO), a key signaling molecule crucial to plant defense against pathogens [START_REF] Wendehenne | Nitric oxide: a new player in plant signalling and defence responses[END_REF], are upregulated in mycorrhizal M. truncatula roots [START_REF] Gianinazzi-Pearson | Plants and arbuscular mycorrhizal fungi: cues and communication in the early steps of symbiotic interactions[END_REF]. Plant leghemoglobins are thought to bind NO and enhanced expression of the plant leghemoglobin gene VfLb29 detected in arbuscule-containing cells of V. faba may reflect a role in regulating such a defense response [START_REF] Vieweg | The promoter of the Vicia faba L. leghemoglobin gene VfLb29 is specifically activated in the infected cells of root nodules and in the arbuscule-containing cells of mycorrhizal roots from different legume and non legume plants[END_REF].

Arbuscule-related expression has also been shown for plant glutathione S-transferase (GST)

genes [START_REF] Strittmatter | Infections with various types of organisms stimulate transcription from a short promoter fragment of the potato gst1 gene[END_REF][START_REF] Wulf | Transcriptional changes in response to arbuscular mycorrhiza development in the model plant Medicago truncatula[END_REF][START_REF] Brechenmacher | Expression profiling of up-regulated plant and fungal genes in early and late stages of Medicago truncatula-Glomus mosseae interactions[END_REF] which are known to be activated under various stress conditions, such as pathogen attack, chemical treatments, or changes in environmental conditions [START_REF] Wulf | Transcriptional changes in response to arbuscular mycorrhiza development in the model plant Medicago truncatula[END_REF]. In AM roots of potato, expression of a Gst1 gene was limited to arbuscule-containing cells [START_REF] Strittmatter | Infections with various types of organisms stimulate transcription from a short promoter fragment of the potato gst1 gene[END_REF] whilst in M.

truncatula, the gene MtGst1 was shown to be transcribed in mycorrhizal root tissues, not only in arbuscule-containing cells but also in the direct vicinity of cells containing fungal hyphae [START_REF] Wulf | Transcriptional changes in response to arbuscular mycorrhiza development in the model plant Medicago truncatula[END_REF]. Since glutathione S-transferase is considered to have protective functions and it is induced during programmed cell death, the corresponding gene could be involved in defense responses to the symbiotic fungus but it may also be related to arbuscule senescence [START_REF] Brechenmacher | Expression profiling of up-regulated plant and fungal genes in early and late stages of Medicago truncatula-Glomus mosseae interactions[END_REF].

Pea metallothionein gene expression was detected during the late stage of AM symbiosis development [START_REF] Grunwald | Identification of mycorrhizaregulated genes with arbuscule development-related expression profile[END_REF][START_REF] Rivera-Becerril | Molecular changes in Pisum sativum L. roots during arbuscular mycorrhiza buffering of cadmium stress[END_REF]. In both publications, the gene is not significantly affected by AM. Other genes of M. truncatula known be implicated in plant defense to pests or pathogens and up-regulated in mycorrhizal roots include those encoding a serine protease, a cysteine-rich antifungal protein (defensin), a Kunitz-type trypsin inhibitor and a subtilisin inhibitor [START_REF] Massoumou | Medicago truncatula gene responses specific to arbuscular mycorrhiza interaction with different species and genera of Glomeromycota[END_REF][START_REF] Wulf | Transcriptional changes in response to arbuscular mycorrhiza development in the model plant Medicago truncatula[END_REF]. Several defense or stressrelated genes are down-regulated with establishment of the functional AM symbiosis. In addition to the phenylpropanoid pathway and genes, for example, plant genes encoding WRKY transcription factors, which are mainly involved in tolerance to pathogen-related stress, and BURP domain-like proteins, one of which is a stress-related transcriptional factor, are repressed in AM roots of L. japonicus [START_REF] Deguchi | Transcriptome profiling of Lotus japonicus roots during arbuscular mycorrhiza development and comparison with that of nodulation[END_REF].

In conclusion, a large number of defense-and stress-related genes are known to be expressed during AM symbiosis development. However, the underlying mechanisms of their regulation and the implication for the symbiosis are still mostly unclear.

Genes involved in nutrient exchange between symbiotic partners

Bidirectional nutrient transfer between the plant and the fungus is a key feature of the AM symbiosis. Nutrient exchange takes place across the intraradicular symbiotic interfaces which are bordered by the plant and fungal cells. The major nutrients exchanged are reduced carbon, assimilated through plant photosynthesis and transferred to the fungus, and phosphate, taken up from the soil by the fungal hyphae and transported to the plant cells.

Carbon. Carbon is transferred from the plant to the AM fungus as hexose which is then rapidly converted within hyphae to trehalose and glycogen, the main transient storage carbohydrates (see figure I.2; [START_REF] Bago | Carbon export from arbuscular mycorrhizal roots involves the translocation of carbohydrate as well as lipid[END_REF]. Significant modifications in the host plant's C metabolism are seen at the level of the expression of corresponding genes. In AM colonized roots, up-regulation of gene expression and/or activity of sucrose hydrolyzing enzymes (invertases, sucrose synthase) [START_REF] Ravnskov | Arbuscular mycorrhizal fungi differentially affect expression of genes coding for sucrose synthases in maize roots[END_REF]Hohnject et al., 2003;García-Rodríguez et al., 2005) and sugar transporters [START_REF] Harrison | A sugar transporter from Medicago truncatula: Altered expression pattern in roots during vesicular-arbuscular (VA) mycorrhizal associations[END_REF][START_REF] Hohnjec | Overlaps in the transcriptional profiles of Medicago truncatula roots inoculated with two different Glomus fungi provide insights into the genetic program activated during arbuscular mycorrhiza[END_REF] has been observed. Enhanced expression of a sucrose synthase gene in AM-colonized roots leads to the hypothesis that the enzyme is involved in generating a sink strength [START_REF] Balestrini | Fungal and plant gene expression in arbuscular mycorrhizal symbiosis[END_REF]. Upregulation of a cell wall invertase gene in mycorrhizal roots supports the general assumption that the C transfer across the symbiotic interface requires host sucrose hydrolysis. Transcriptional upregulation of sucrose-splitting enzymes during early root colonization events agrees with the decreased levels of sucrose detected in these roots (García-Rodríguez et al., 2006). It is generally assumed that plant invertase and sucrose synthase are crucial for the delivery of hexoses to the fungal partner [START_REF] Délano-Frier | Novel developments in the understanding of signaling and nutrient exchange mechanisms in the arbuscular mycorrhizal symbiosis[END_REF]. Moreover, on the fungal side, a monosaccharide transporter gene GpMST1 was isolated from Geosiphon pyriformis and characterized [START_REF] Schüßler | Characterization of a carbohydrate transporter from symbiotic glomeromycotan fungi[END_REF]. It was shown that GpMST1 has the highest affinities for glucose then mannose, galactose and fructose. Authors hypothesized that GpMST1 is active at the symbiotic interface and not able to take up glucose through the non-symbiotic [START_REF] Bago | Carbon export from arbuscular mycorrhizal roots involves the translocation of carbohydrate as well as lipid[END_REF]. Carbon taken up from the host in the form of hexose is converted to carbohydrates and storage lipids in the intraradical mycelium.

Lipid and glycogen are translocated from intraradical to extraradical mycelium. Storage (glycogen and trehalose) and structural (chitin) carbohydrates are synthesized in the extraradical mycelium from hexose that is derived from exported carbohydrate as well as from lipid. [START_REF] Bago | Carbon export from arbuscular mycorrhizal roots involves the translocation of carbohydrate as well as lipid[END_REF] [START_REF] Bago | Carbon export from arbuscular mycorrhizal roots involves the translocation of carbohydrate as well as lipid[END_REF]. It has been speculated that a putative acyl-coenzyme A dehydrogenase gene may function in the anabolic flux of C from lipid to carbohydrate [START_REF] Bago | Translocation and utilization of fungal storage lipid in the arbuscular mycorrhizal symbiosis[END_REF]. These expression data confirm that TAGs and glycogen are synthesized by the fungus inside the root and then transferred to the extraradical mycelium. In contrast, genes coding for enzymes of the glycoxylate cycle (isocitrate lyase, malate synthase) are actively expressed in the extraradical mycelium where the fungus, unable to take up hexose, convert lipids into carbohydrates via this metabolic pathway [START_REF] Balestrini | Fungal and plant gene expression in arbuscular mycorrhizal symbiosis[END_REF][START_REF] Lammers | The glyoxylate cycle in an arbuscular mycorrhizal fungus: gene expression and carbon flow[END_REF].

Phosphorus. Phosphate is a key nutrient contributing to the symbiotic functioning of arbuscular mycorrhiza. Once the symbiosis is established, AM fungal hyphae take up inorganic phosphate (Pi) from the soil, accumulate it in the form of vacuolar polyphosphate (polyP) and translocate it along hyphae by means of a motile tubular vacuolar network to the intraradical compartment.

Here, Pi ions resulting from polyP by hydrolysis are assumed to be released by membranepassive carriers into the periarbuscular space and actively taken up into the plant cell via transporters at the PMA [START_REF] Cox | Translocation and transfer of nutrients in vesicular-arbuscular mycorrhizas. 3. Polyphosphate granules and phosphorus translocation[END_REF][START_REF] Rasmussen | 31 P NMR for the study of P metabolism and translocation in arbuscular mycorrhizal fungi[END_REF][START_REF] Uetake | Extensive tubular vacuole system in an arbuscular mycorrhizal fungus, Gigaspora margarita[END_REF][START_REF] Ezawa | P metabolism and transport in AM fungi[END_REF].

The AM fungal-mediated translocation of phosphate from the soil to plant roots requires specific transporters that function in at least two fungal structures: the external hyphae which mediate uptake from the soil and arbuscules within root cortical cells where an efflux transporter or channel is expected (Ferrol et al., 2002). AM fungal phosphate transporter genes have already been isolated from G. versiforme (GvPT), G. intraradices (GiPT) and G. mosseae (GmosPT) [START_REF] Harrison | A phosphate transporter from the mycorrhizal fungus Glomus versiforme[END_REF]Maldonado-Mendoza et al. 2001;[START_REF] Benedetto | Expression profiles of a phosphate transporter gene (GmosPT) from the endomycorrhizal fungus Glomus mosseae[END_REF].

Expression of the GiPT and GmosPT genes in extraradical mycelium is induced in response to phosphate concentrations typical of those found in the soil solution, and modulated by the overall phosphate status of the mycorrhiza (Maldonado-Mendoza et al., 2001;[START_REF] Benedetto | Expression profiles of a phosphate transporter gene (GmosPT) from the endomycorrhizal fungus Glomus mosseae[END_REF]. The intraradical mycelium of G. mosseae also showed a significant level of GmosPT expression that nevertheless was independent of external Pi concentrations [START_REF] Benedetto | Expression profiles of a phosphate transporter gene (GmosPT) from the endomycorrhizal fungus Glomus mosseae[END_REF]. These data suggest that inside the root the fungus may exert a control over the amount of phosphate delivered to the plant [START_REF] Balestrini | Fungal and plant gene expression in arbuscular mycorrhizal symbiosis[END_REF].

It has been proposed that fungal alkaline phosphatases (ALP) may have a role in the transfer of phosphate by hyphae from the soil to the host plant [START_REF] Tisserant | Inplanta histochemical staining of fungal alkaline phosphatase activity for analysis of efficient arbuscular mycorrhizal infections[END_REF]. AM-dependent expression of ALP-encoding genes has been described in G. intraradices and G. mosseae: the levels of GiALP and GmALP transcripts were higher in mycorrhizal roots than in germinating spores and extraradical mycelium, and gene expression in AM roots was irrespective of colonization rates and environmental Pi concentration [START_REF] Aono | Expression of alkaline phosphatase genes in arbuscular mycorrhizas[END_REF]. These results agree with ALP enzyme location and activity data [START_REF] Gianinazzi | Enzymatic studies on the metabolism of vesicular arbuscular mycorrhiza. III. Ultrastructural localization of acid and alkaline phosphatase in onion roots infected by Glomus mosseae (Nicol and Gerd)[END_REF][START_REF] Gianinazzi-Pearson | Cellular and molecular approaches in the characterization of symbiotic events in functional arbuscular mycorrhizal associations[END_REF]. Moreover, [START_REF] Ezawa | Specific inhibitor and substrate specificity of alkaline phosphatase expressed in the symbiotic phase of the arbuscular mycorrhizal fungus, Glomus etunicatum[END_REF] revealed that ALP in the extraradical mycelium has an increased affinity for sugar phosphate. Detailed information on the dynamics of polyP turnover in AM fungi and corresponding gene activity is lacking.

Active uptake into the plant cell of phosphate released by the fungus across the arbuscule interface requires transporters at the PMA. Plant phosphate transporter genes identified from potato (StPT3), maize (ZEAma) and L. japonicus (LjPT3) show an increased expression in symbiosis [START_REF] Rausch | A phosphate transporter expressed in arbuscule-containing cells in potato[END_REF][START_REF] Karandashov | Evolutionary conservation of a phosphate transporter in the arbuscular mycorrhizal symbiosis[END_REF][START_REF] Nagy | Differential regulation of five Pht1 phosphate transporters from maize (Zea mays L.)[END_REF][START_REF] Maeda | 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], whilst phosphate transporter genes from rice (OsPT11), tomato (LePT4) and M. truncatula (MtPT4) have been identified to be expressed exclusively within the AM symbiosis [START_REF] Paszkowski | Rice phosphate transporters include an evolutionarily divergent gene specifically activated in arbuscular mycorrhizal symbiosis[END_REF][START_REF] Nagy | The characterization of novel mycorrhiza specific phosphate transporters from Lycopersicon esculentum and Solanum tuberosum uncovers functional redundancy in symbiotic phosphate transport in solanaceous species[END_REF][START_REF] Javot | A Medicago truncatula phosphate transporter indispensable for the arbuscular mycorrhizal symbiosis[END_REF]. The MtPT4 gene was shown to be located in the periarbuscular membrane, at the symbiotic interface of arbuscules which is generally assumed to be the preferential site of phosphate transfer from the AM fungus to the host plant [START_REF] Balestrini | Fungal and plant gene expression in arbuscular mycorrhizal symbiosis[END_REF]. [START_REF] Javot | A Medicago truncatula phosphate transporter indispensable for the arbuscular mycorrhizal symbiosis[END_REF] observed that loss of MtPT4 function leads to premature death of the arbuscules, inability of the fungus to proliferate within the root and arrest of the symbiosis.

Other known key enzymes for the AM symbiosis are H + -ATPases. They play a role in establishing the electrochemical gradient required for the transfer of nutrients (either phosphate or carbon) across the plasma membrane in both fungi and plants (Ferrol et al., 2002). Few genes encoding membrane H + -ATPase have been identified in AM fungi. Five were isolated from G. mosseae [START_REF] Ferrol | The plasma membrane H+-ATPase gene family in the arbuscular mycorrhizal fungus Glomus mosseae[END_REF][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] and expression of two of them, GmPMA1 and GmHA5, was investigated during AM formation. It was shown that the GmPMA1 gene is highly expressed during asymbiotic fungal development and that its expression is not affected either by the presence of different nutrients in the growth medium, or by the presence of the host plant. In contrast, the GmHA5 gene is strongly induced once appressoria begin to form, and it remains highly expressed during the in planta phase. Moreover, expression of GmHA5 is induced during symbiotic fungal growth in roots by micromolar amounts of phosphate in the medium [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]. A set of membrane H + -ATPase genes has been identified on the plant side [START_REF] Gianinazzi-Pearson | Differential activation of H + -ATPase genes by an arbuscular mycorrhizal fungus in root cells of transgenic tobacco[END_REF]Ferrol et al., 2002;[START_REF] Krajinski | Mtha1, a plasma membrane H + -ATPase gene from Medicago truncatula, shows arbuscule specific induced expression in mycorrhizal tissue[END_REF][START_REF] Manthey | Transcriptome profiling in root nodules and arbuscular mycorrhiza identifies a collection of novel genes induced during Medicago truncatula root endosymbioses[END_REF]. Up-regulation within the established AM symbiosis and arbusculespecific induction of expression were detected for the tobacco H + -ATPase genes pma2 and pma4, and for Mtha1 from M. truncatula [START_REF] Gianinazzi-Pearson | Differential activation of H + -ATPase genes by an arbuscular mycorrhizal fungus in root cells of transgenic tobacco[END_REF][START_REF] Krajinski | Mtha1, a plasma membrane H + -ATPase gene from Medicago truncatula, shows arbuscule specific induced expression in mycorrhizal tissue[END_REF].

These data indicate that different genes coding for plant and fungal membrane H + -ATPases are differentially regulated during the AM symbiosis. This together with the specific localization of H + -ATPase and phosphate transporter proteins in the PMA underline the complexity of plantfungal interactions and of the mechanisms leading to a functional AM.

Nitrogen. In addition to phosphorus, AM fungi can take up and transfer significant amounts of nitrogen to their host plants [START_REF] Bago | Effect of nikkomycin Z, a chitin-synthase inhibitor, on hyphal growth and cell wall structure of two arbuscular-mycorrhizal fungi[END_REF][START_REF] Johansen | Hyphal transport by a vesiculararbuscular mycorrhizal fungus of N applied to the soil as ammonium or nitrate[END_REF][START_REF] He | Temporal and spatial dynamics of vesicular-arbuscular mycorrhizal fungi under the canopy of Zygophyllum dumosum Boiss. In the: Negev Desert[END_REF]. Stable isotope labeling experiments on G. intraradices/carrot root mycorrhiza [START_REF] Pfeffer | The uptake, metabolism, transport and transfer of nitrogen in an arbuscular mycorrhizal symbiosis[END_REF] showed that inorganic nitrogen taken up by the fungus outside the roots is incorporated into amino acids and translocated from the extraradical to the intraradical mycelium as arginine. N is then probably transferred from the fungus to the plant as ammonium without any loss in the C skeleton (see figure I.3). Data from quantitative real-time PCR are consistent with the described mechanisms. The AM fungal gene of primary nitrogen assimilation (glutamine synthase) is preferentially expressed in the extraradical tissues, whereas genes associated with arginine breakdown (urease accessory protein, ornithine amino transferase) and NH + 4 transfer (ammonium transporter) are more highly expressed in the intraradical mycelium [START_REF] Govindarajulu | Nitrogen transfer in the arbuscular mycorrhizal symbiosis[END_REF]López-Pedrosa et al., 2006).

AM fungi may also be involved in the acquisition of organic N. The activity of an amino acid permease gene (GmosAAP1) from G. mosseae and of a glutamine synthase gene from G.

mosseae and G. intraradices appear to be modulated in AM roots by different N sources [START_REF] Cappellazzo | Characterization of an amino acid permease from the endomycorrhizal fungus Glomus mosseae[END_REF]Breuninger et al., 2004). GmosAAP1, which encodes a functional amino acid transporter, binding nonpolar and hydrophobic amino acids, is expressed in the external mycelium but not in the intraradical fungal structures. It was suggested that GmosAAP1 plays a role in the first steps of amino acid acquisition, allowing direct amino acid uptake by the fungus from the soil [START_REF] Cappellazzo | Characterization of an amino acid permease from the endomycorrhizal fungus Glomus mosseae[END_REF].

On the plant side, induction of genes encoding AM-related nitrate transporters has been reported in M. truncatula and in tomato [START_REF] Hohnjec | Overlaps in the transcriptional profiles of Medicago truncatula roots inoculated with two different Glomus fungi provide insights into the genetic program activated during arbuscular mycorrhiza[END_REF][START_REF] Burleigh | Relative quantitative PCR to study nutrient transport processes in arbuscular mycorrhizas[END_REF][START_REF] Hildebrandt | Expression of nitrate transporter genes in tomato colonized by an arbuscular mycorrhizal fungus[END_REF]. These findings suggest the existence of mechanisms not only supporting increased ammonium uptake but also nitrate acquisition through the AM symbiosis [START_REF] Balestrini | Fungal and plant gene expression in arbuscular mycorrhizal symbiosis[END_REF]. et al., 2005). Inorganic nitrogen is taken up by the fungal extraradical mycelium, assimilated via nitrate reductase and the glutamine synthetase-glutamate synthase cycle and converted into arginine, which is translocated along the fungal hyphae to the intraradical mycelium. Arginine is broken down in the intraradical mycelium, releasing urea and ornithine, which are further broken down by the actions of urease and ornithine aminotransferase (OAT). Ammonia released from arginine breakdown passes to the host via ammonia channels (AMT). Amino acids from ornithine breakdown and/or NH 4 + assimilation in the intraradical mycelium may be catabolized within the intraradical mycelium or translocated to the extraradical mycelium.

I.3.4. High throughput analyses of symbiosis-related transcriptional changes in plant and fungal genes

Targeted approaches based on EST screening by homology with known genes are not sufficient to enable identification of novel genes which may be specific to the AM symbiosis. In order to obtain a comprehensive picture of transcriptional changes during AM symbiosis development, transcriptome-wide studies principally based on large-scale EST sequencing, suppressive subtractive hybridization (SSH) and DNA microarray analysis have been performed.

In the earliest approaches using differential RNA display, only a limited number of novel AMrelated genes were isolated by comparing RNA accumulation patterns in different samples (i.e.

mycorrhizal and non-mycorrhizal) to isolate differentially transcribed mRNAs [START_REF] Martin-Laurent | Differential display analysis of RNA accumulation in arbuscular mycorrhiza of pea and isolation of a novel symbiosis-regulated plant gene[END_REF][START_REF] Lapopin | Comparative di.erential display analysis of arbuscular mycorrhiza in Pisum sativum and a mutant defective in late stage development[END_REF]. A greater number of genes were identified exploiting suppressive subtractive hybridization (SSH) [START_REF] Wulf | Transcriptional changes in response to arbuscular mycorrhiza development in the model plant Medicago truncatula[END_REF][START_REF] Brechenmacher | Expression profiling of up-regulated plant and fungal genes in early and late stages of Medicago truncatula-Glomus mosseae interactions[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]Breuninger and Requena, 2004;[START_REF] Grunwald | Identification of mycorrhizaregulated genes with arbuscule development-related expression profile[END_REF] which consists in a PCR-based cDNA subtraction method allowing selective amplification of target cDNA fragments (differentially expressed) and simultaneous suppression of non target amplified cDNA [START_REF] Diatchenko | Suppression subtractive hybridization: a method for generating differentially regulated or tissuespecific cDNA probes and libraries[END_REF]. SSH analysis of interactions at the appressorium stage and in fully developed mycorrhiza of M. truncatula colonized by G. intraradices or G. mosseae has led to the identification of AM-induced plant genes which are mostly involved in signaling processes, defence/cell-rescue, membrane transport or cell recognition [START_REF] Wulf | Transcriptional changes in response to arbuscular mycorrhiza development in the model plant Medicago truncatula[END_REF][START_REF] Brechenmacher | Expression profiling of up-regulated plant and fungal genes in early and late stages of Medicago truncatula-Glomus mosseae interactions[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]. ESTs from the SSH library obtained by Brechenmacher et al. (2004) were also used as templates to identify plant genes commonly or differentially activated during M. truncatula root colonization by AM fungi (G. mosseae), rhizobacteria (Pseudomonas fluorescens) and nodule bacteria Sinorhizobium meliloti [START_REF] Sanchez | Common gene expression in Medicago truncatula roots response to Pseudomonas fluorescens colonization, mycorrhiza development and nodulation[END_REF].

Application of SSH to specifically target events associated with arbuscule development, by comparing mycorrhizal wild type pea and the Pssym36 mutant, led to the identification of over 20 genes which seem to be induced during this functional stage of AM [START_REF] Grunwald | Identification of mycorrhizaregulated genes with arbuscule development-related expression profile[END_REF].

G. mosseae genes up-regulated during appressoria formation have also been identified using this technique (Breuninger and Requena, 2004;[START_REF] Brechenmacher | Expression profiling of up-regulated plant and fungal genes in early and late stages of Medicago truncatula-Glomus mosseae interactions[END_REF]. Several genes related to Ca 2+ -dependent signaling were found amongst the up-regulated fungal genes, suggesting a role for fungal Ca 2+ in the early stages of mycorrhiza formation (Breuninger and Requena, 2004). In silico analysis of EST data by [START_REF] Journet | Exploring the root symbiotic programs of the model legume Medicago truncatula using EST analysis[END_REF] from three cDNA libraries (MENS database), corresponding to nonmycorrhizal, mycorrhizal and nodulated roots, led to the identification of a large number of novel plant genes predicted to be up-or down-regulated during either symbiotic root interaction. Using a similar strategy based on in silico analysis of M.

A
truncatula random cDNA (MtAmp) and SSH (MtGim) libraries, [START_REF] Frenzel | Combined transcriptome profiling reveals a novel family of arbuscular mycorrhizalspecific Medicago truncatula lectin genes[END_REF] identified additional AM-induced genes with a putative function in transport and signaling processes, as well as a novel family of AM-specific lectin genes. In situ arbuscule-specific expression of two members of this lectin family indicates a role for AM-specific lectins during arbuscule formation or function [START_REF] Frenzel | Combined transcriptome profiling reveals a novel family of arbuscular mycorrhizalspecific Medicago truncatula lectin genes[END_REF].

In silico analysis also formed a useful starting point for cDNA array construction for transcriptome analyses [START_REF] Liu | Transcript profiling coupled with spatial expression analyses reveals genes involved in distinct developmental stages of the arbuscular mycorrhizal symbiosis[END_REF][START_REF] Liu | Arbuscular mycorrhizal symbiosis is accompanied by local and systemic alterations in gene expression and an increase in disease resistance in the shoots[END_REF][START_REF] Küster | Construction and validation of comprehensive cDNAbased macro-and microarrays to explore root endosymbioses in the model legume Medicago truncatula[END_REF][START_REF] Küster | Development of bioinformatic tools to support EST-sequencing, in silico-and microarray-based transcriptome profiling in mycorrhizal symbioses[END_REF]Gianinazzi-Pearson et al., 2004;[START_REF] Manthey | Transcriptome profiling in root nodules and arbuscular mycorrhiza identifies a collection of novel genes induced during Medicago truncatula root endosymbioses[END_REF][START_REF] Hohnjec | Overlaps in the transcriptional profiles of Medicago truncatula roots inoculated with two different Glomus fungi provide insights into the genetic program activated during arbuscular mycorrhiza[END_REF][START_REF] Deguchi | Transcriptome profiling of Lotus japonicus roots during arbuscular mycorrhiza development and comparison with that of nodulation[END_REF]. The construction of M. truncatula cDNA macroarrays for AM studies was first reported by [START_REF] Liu | Transcript profiling coupled with spatial expression analyses reveals genes involved in distinct developmental stages of the arbuscular mycorrhizal symbiosis[END_REF] to examine transcript profiles of M. truncatula roots during the development of AM with G.

versiforme and during growth under different phosphorus nutrient regimes. Using a set of 2268 cDNAs, 67 genes were shown to be differentially regulated during AM symbiosis development and transcript profiling revealed two distinct temporal expression patterns. One group of genes (mostly defense and stress-response related) showed an increase in transcripts during the initial period of contact between symbionts and a subsequent decrease as the symbiosis developed.

A second group (genes linked to signaling transduction pathways) showed a sustained increase in transcript levels that correlated with colonization of the root system [START_REF] Liu | Transcript profiling coupled with spatial expression analyses reveals genes involved in distinct developmental stages of the arbuscular mycorrhizal symbiosis[END_REF]. The first symbiosis-targeted microarray, designated Mt6k-RIT, was constructed using 6359 MtC EST clusters identified by [START_REF] Journet | Exploring the root symbiotic programs of the model legume Medicago truncatula using EST analysis[END_REF] [START_REF] Küster | Construction and validation of comprehensive cDNAbased macro-and microarrays to explore root endosymbioses in the model legume Medicago truncatula[END_REF]. Expression profiling revealed additional marker genes for nodule and arbuscular mycorrhiza development. An extended M. truncatula Mt16kOLI1 microarray, containing 16086 probes, was used to highlight the overlapping genetic program activated by two different AM fungi, G. mosseae and G.

intraradices (201 genes co-induced) and identify plant genes uniquely present in mycorrhizal root cDNA libraries [START_REF] Hohnjec | Overlaps in the transcriptional profiles of Medicago truncatula roots inoculated with two different Glomus fungi provide insights into the genetic program activated during arbuscular mycorrhiza[END_REF].

A contribution to the detection of tissue-or cell-specific gene expression during AM symbiosis has been made by exploiting microdissection techniques. Recently, a number of novel plant and fungal genes specifically induced in colonized cortical cells and in arbuscules were identified using laser microdissection of arbuscule-containing cells of M. truncatula and the Affymetrix Gene Chip®Medicago Genome Array containing 32167 probes designed to sequences from the MtGI database. Within the arbuscule, expression of genes associated with the urea cycle, amino acid biosynthesis and cellular autophagy was detected. Analysis of gene expression in the colonized cortical cell revealed up-regulation of a lysine motif (LysM)-receptor kinase, members of the GRAS transcription factor family and a symbiosis-specific ammonium transporter that is a likely candidate for mediating ammonium transport in the AM symbiosis [START_REF] Gomez | Medicago truncatula and Glomus intraradices gene expression in cortical cells harbouring arbuscules in the arbuscular mycorrhizal symbiosis[END_REF]. Laser microdissection offers a new way to obtain RNA from subsets of cells, and coupled with transcript profiling approaches, it provides an opportunity for monitoring gene expression in an individual cell type.

In conclusion, the large numbers of mycorrhiza-related plant genes identified during different large-scale analyses cover a substantial part of the root endosymbiotic transcriptome. However, further investigations are still necessary to confirm their significance in the fungal-plant interactions and to map their involvement in the molecular pathways driving or regulated by the AM symbiosis.

I.3.5. Influence of SYM gene mutations on AM-related molecular responses

Plant mutants defective for symbiotic AM have been used to obtain information about the role of To identify genes specifically regulated during PPA formation in M. truncatula, [START_REF] Siciliano | Transcriptome analysis of arbuscular mycorrhizal roots during development of the prepenetration apparatus[END_REF] built a PPA-targeted cDNA SSH library and monitored expression profiles of differentially expressed genes in the wild type and dmi3 mutant plant genotypes. This led to the identification of 107 genes potentially involved in early AM interaction. 15 of them were shown to be specifically up-regulated at the time points of appressorium and PPA development, including genes encoding signal responses (receptor-like protein kinase RK20-1), defense responses (resistance protein KR1, PR10 protein, Avr9/Cf-9-rapidly elicited protein 26A ACRE264) and cell wall modifications (cellulase synthase, expansin-like protein 1 precursor, pectinglucuronyltransferase-like, cationic peroxidase). Two novel genes specifically up-regulated during the formation of PPA were identified. One of them encodes an expansin-like protein preferentially expressed in epidermal cells in contact with an appressorium. Another gene encodes a putative Avr9/Cf-9-rapidly elicited protein 26A (ACRE264) found to be up-regulated in the dmi3 mutant, suggesting that it is suppressed by DMI3 to possibly facilitate fungal entry into the host plant. The authors suggest that DMI3 may play a pivotal role in interpreting and modulating plant responses to early AM colonization by boosting the up-regulation of plant genes required for the accommodation of the AM fungus and suppression of the basal defenserelated genes [START_REF] Siciliano | Transcriptome analysis of arbuscular mycorrhizal roots during development of the prepenetration apparatus[END_REF].

SYM
On the fungal side, mutations of the DMI1, DMI2 and DMI3 genes not only block root penetration but also affect fungal gene expression [START_REF] Seddas | Symbiosis-related plant genes modulate molecular responses in an arbuscular mycorrhizal fungus during early root interactions[END_REF]. Transcript accumulation of 14 G.intraradices genes, with predicted functions in transcription, protein synthesis, primary/secondary metabolism or of unknown function, were monitored by real-time RT-PCR in quiescent spores, germination spores and roots of wild type or mutant plants at appressoria formation stage. Tested fungal genes were mostly down-regulated or not expressed in contact with roots of the DMI mutants. Authors suggest that plant SYM genes may regulate AM fungal activity through stimulatory pathways and/or control of inhibitory factors.

In non-targeted approaches, microarray hybridization experiments comparing M. truncatula roots colonized by G. mosseae (Myc + genotype) and its inoculated mycorrhiza defective (Myc -) mutant (dmi3 -) gave consistent and significant differential expression for 72 genes (57 upregulated, 15 down-regulated) from the mycorrhizal wild type genotype compared with 47 genes (37 up-regulated, 10 down-regulated) for the inoculated dmi3 mutant (Gianinazzi-Pearson et al., 2004). The main classes of AM up-regulated genes in the wild type genotype related to protein synthesis/processing (~ 18%), and about 47% were genes with unknown function or homology.

Only two genes of unknown function showed a similar expression pattern in wild type and mutant genotypes, which concords with their very different phenotypes in interactions with AM fungi.

To study arbuscule-related gene expression, differential RNA display [START_REF] Lapopin | Comparative di.erential display analysis of arbuscular mycorrhiza in Pisum sativum and a mutant defective in late stage development[END_REF], SSH and macroarray hybridization [START_REF] Grunwald | Identification of mycorrhizaregulated genes with arbuscule development-related expression profile[END_REF] were performed exploiting the Pssym36 mutant. Comparison of RNA accumulation patterns between controls, Glomus mosseae-colonized Pssym36 mutant and wild type pea [START_REF] Lapopin | Comparative di.erential display analysis of arbuscular mycorrhiza in Pisum sativum and a mutant defective in late stage development[END_REF] resulted in the identification of four differentially occurring cDNA fragments, one from the fungus and three of plant origin. More detailed analysis of the pea Psam4 (Pisum sativum arbuscular mycorrhizaregulated) gene showed that this gene encodes a proline-rich protein and its RNA accumulation is higher in the mutant compared with the wild type. Authors proposed that Psam4 might be itself mutated or belong to a regulon of genes altered in their expression and involved in the greater resistance of the Pssym36 tissues and cortical cells to invasion by the symbiotic microorganisms [START_REF] Lapopin | Comparative di.erential display analysis of arbuscular mycorrhiza in Pisum sativum and a mutant defective in late stage development[END_REF]. In the investigation performed by [START_REF] Grunwald | Identification of mycorrhizaregulated genes with arbuscule development-related expression profile[END_REF], 67 unique fragments presumably up-regulated during the late stage of AM development were identified. Arbuscule-related expression was confirmed by macroarray analysis for 22 of them.

Identified genes belong to different functional groups, part of them being reported to be AMinduced for the first time.

I.3.6. Molecular similarities between legume/AM fungi and legume/Rhizobia symbioses

Although AM fungal and rhizobial symbioses are morphologically distinct, the genetic programs underling them partially overlap in legumes. One of the first evidences for a genetic overlap was the discovery of pea mutants affected in both root symbioses (Duc et al., 1989) indicating the existence of some shared symbiotic steps. Common symbiosis mutants have since been identified in M. truncatula, M. sativa, L. japonicus, Phaseolus vulgaris, Vicia faba and Melilotus alba [START_REF] Bradbury | Interactions between three alfalfa nodulation genotypes and two Glomus species[END_REF][START_REF] Sagan | Selection of nodulation and mycorrhizal mutants in the model plant Medicago truncatula (Gaertn.) after gamma-ray mutagenesis[END_REF][START_REF] Shirtliffe | A nodulation (Nod + /Fix -) mutant of Phaseolus vulgaris L. has nodule-likestructures lacking peripheral vascular bundles (Pvb -) and is resistant to mycorrhizal infection (Myc -)[END_REF][START_REF] Schauser | Symbiotic mutants deficient in nodule establishment identified after T-DNA transformation of Lotus japonicus[END_REF][START_REF] Szczyglowski | Nodule organogenesis and symbiotic mutants of the model legume Lotus japonicus[END_REF][START_REF] Wegel | Mycorrhiza mutants of Lotus japonicus define genetically independent steps during symbiotic infection[END_REF][START_REF] Catoira | Four genes of Medicago truncatula controlling components of a Nod factor transduction pathway[END_REF]Senoo et al., 2000;[START_REF] Kawaguchi | Root, root hair, and symbiotic mutants of the model legume Lotus japonicus[END_REF][START_REF] Lum | Investigation of four classes of non-nodulating white sweetclover (Melilotus alba annua Desr.) mutants and their responses to arbuscular-mycorrhizal fungi[END_REF] truncatula and their orthologs in other plant species) operate downstream of nod factor receptor genes (NFR1, NFR5) in the nitrogen-fixing symbiosis pathway. These SYM genes are known to be required for the Ca 2+ -dependent transduction of both bacterial and fungal signals.

DMI2/PsSYM19/LjSYMRK is involved in the perception of a fungal or bacterial signal and the activation of DMI1/PsSYM8//LjPOLLUX that could function as a calcium channel [START_REF] Oldroyd | Peace talks and trade deals. Keys to longterm harmony in legume-microbe symbioses[END_REF]. dmi1 and dmi2 mutants lack the calcium spiking response. DMI3/PsSYM9/LjCCaMK has been placed downstream of the generated calcium response. The role of DMI3 is considered to be the interpretation of the calcium signals and conversion of them to phosphorylation events [START_REF] Kistner | Seven Lotus japonicus genes required for transcriptional reprogramming of the root during fungal and bacterial symbiosis[END_REF].

However, in spite of the involvement of the same DMI1 and DMI2 proteins, the symbiosis signaling pathway in legumes differentially transduces bacterial and fungal signals to generate distinct calcium responses. Mathematical analyses of calcium oscillations indicate that both the fungal and the bacterial-induced calcium responses are chaotic in nature [START_REF] Kosuta | Differential and chaotic calcium signatures in the symbiosis signaling pathway of legumes[END_REF].

Chaotic systems require minimal energy to produce a wide spectrum of outputs in response to marginally different inputs. Consequently, chaos provides a possible mechanism for flexibility that must be an inherent component of this common SYM pathway [START_REF] Kosuta | Differential and chaotic calcium signatures in the symbiosis signaling pathway of legumes[END_REF].

The common activation of a number of nodulin genes during early nodulation and mycorrhization (MsENOD2, MtENOD11, MtENOD12, MsENOD40, PsENOD5, PsENOD12A, VfLb29) lends support to the existence of common molecular events in the two symbiosis [START_REF] Van Rhijn | Expression of early nodulin genes in alfalfa mycorrhizae indicates that signal transduction pathways used in forming arbuscular mycorrhizae and Rhizobium-induced nodules may be conserved[END_REF][START_REF] Albrecht | Endomycorrhizae and rhizobial Nod factors both require SYM8 to induce the expression of the early nodulin genes PsENOD5 and PsENOD12A[END_REF][START_REF] Journet | Medicago truncatula ENOD11: A novel RPRP-encoding early nodulin gene expressed during mycorrhization in arbuscule-containing cells[END_REF][START_REF] Frühling | The Vicia faba leghemoglobin gene VfLb29 is induced in root nodules and in roots colonized by the arbuscular mycorrhizal fungus Glomus fasciculatum[END_REF]. Existence of plant genes common to both symbioses led to the hypothesis that the evolutionary younger legume root nodule symbiosis might have evolved from the more ancient AM symbiosis (Gianinazzi-Pearson, 1997;[START_REF] Gherbi | SymRK defines a common genetic basis for plant root endosymbioses with arbuscular mycorrhiza fungi, rhizobia, and Frankiabacteria[END_REF].

Global comparisons of the complex root symbiotic programs activated during host plant interactions with AM fungi and nitrogen-fixing bacteria have been performed using in silico comparisons in the MENS database, targeted expression analyses and the microarray tools described above [START_REF] Journet | Exploring the root symbiotic programs of the model legume Medicago truncatula using EST analysis[END_REF][START_REF] Manthey | Transcriptome profiling in root nodules and arbuscular mycorrhiza identifies a collection of novel genes induced during Medicago truncatula root endosymbioses[END_REF][START_REF] Sanchez | Common gene expression in Medicago truncatula roots response to Pseudomonas fluorescens colonization, mycorrhiza development and nodulation[END_REF][START_REF] Sanchez | Pseudomonas fluorescens and Glomus mosseae trigger DMI3dependent activation of genes related to a signal transduction pathway in roots of Medicago truncatula[END_REF][START_REF] Küster | Construction and validation of comprehensive cDNAbased macro-and microarrays to explore root endosymbioses in the model legume Medicago truncatula[END_REF][START_REF] Kistner | Seven Lotus japonicus genes required for transcriptional reprogramming of the root during fungal and bacterial symbiosis[END_REF][START_REF] Deguchi | Transcriptome profiling of Lotus japonicus roots during arbuscular mycorrhiza development and comparison with that of nodulation[END_REF]. A considerable number of M. truncatula and L. japonicus genes are reported as being modulated in root nodules as well as in mycorrhizal roots, and a significant proportion is involved in build-up of cell walls, metabolism, transport, protein processing or signal transduction. [START_REF] Deguchi | Transcriptome profiling of Lotus japonicus roots during arbuscular mycorrhiza development and comparison with that of nodulation[END_REF] reported that many of the plant genes repressed during both nodule and AM symbioses are involved in host plant defense and stress responses, suggesting that host plants accept AM fungi and compatible rhizobia in similar manners, their defense mechanisms being suppressed. However if all the published data are taken together, the overall overlapping of induced or repressed genes is limited in nodules and AM tissues, suggesting that co-regulated genetic networks will not be such a common feature of the established symbioses as so far supposed.

I.4. Genetic mapping of symbiosis-related plant genes for map-based cloning I.4.1. Cloning of pea symbiotic genes

Gene cloning is an essential step for the determination of the molecular basis of mutant phenotypes. The first cloned SYM gene of legumes was the Nin gene from L. japonicus [START_REF] Schauser | A plant regulator controlling development of symbiotic root nodules[END_REF]. The LjNin mutant was identified in screening of symbiotic mutants carrying the autonomous maize transposon Ac and isolated using a transposon tag. It is one of the possible ways for gene cloning. Briefly, the transposon insertion knocks out the gene and is used for the gene isolation at the same time. A few years later, the comparison of the phenotype of Ljnin with known pea mutants facilitated the identification of a possible Nin orthologue in pea, the Pssym35 gene [START_REF] Borisov | The Sym35 gene required for root nodule development in pea is an ortholog of Nin from Lotus japonicus[END_REF]. Synteny between genomes of legume species allows isolation of orthologous genes based on the nucleotide sequence of the identified gene in other legume species (Table I .4.1). Primer design and PCR amplification of the pea Nin orthologue were performed as well as subsequent co-segregation analysis and sequencing of the three independent Pssym35 mutant alleles [START_REF] Borisov | The Sym35 gene required for root nodule development in pea is an ortholog of Nin from Lotus japonicus[END_REF]. The same approach was later applied to isolate the M.truncatula Nin orthologue, MtNin [START_REF] Marsh | Medicago truncatula NIN is essential for Rhizobial-independent nodule organogenesis induced by autoactive aalcium/calmodulindependent protein kinase[END_REF].

A modification of this approach was applied to clone the PsSym19 gene [START_REF] Stracke | A plant receptor-like kinase required for both bacterial and fungal symbiosis[END_REF][START_REF] Endre | A receptor kinase gene regulating symbiotic nodule development[END_REF], which differed by the initial isolation from L. japonicus of the orthologous gene SYMRK by map-based cloning. The LjSYMRK gene was positioned on the bottom arm of chromosome 2 between the molecular markers AUX and SHMT. A contig of transformationcompetent artificial chromosome (TAC) clones, comprising the SYMRK region and flanking markers, was assembled and then sequenced in the context of the L. japonicus genome sequencing project. Eighteen putative genes were predicted within the targeted region. Among them, a receptor-like kinase-encoding gene (RLK) was suggested as an obvious candidate for a signal transduction component, a function compatible with the SYMRK mutant phenotype and which was confirmed by subsequent sequence analysis [START_REF] Stracke | A plant receptor-like kinase required for both bacterial and fungal symbiosis[END_REF]. [START_REF] Vos | AFLP: a new technique for DNA fingerprinting[END_REF][START_REF] Lacou | Genetic mapping in pea. 1-RAPD-based genetic linkage map of Pisum sativum[END_REF][START_REF] Flavell | Retrotransposon-based insertion polymorphisms (RBIP) for high throughput marker analysis[END_REF]. The subsequent integration of generated molecular markers into previous classical genetic and cytogenetic maps has resulted in the development of consensus genetic maps containing different types of markers [START_REF] Weeden | A consensus linkage map for Pisum staivum[END_REF][START_REF] Ellis | An integrated and comparative view of pea genetic and cytogenetic maps[END_REF]. However, random DNA markers are not universal tools for genetic mapping because the majority of them cannot be transferred from one cross to another or from one legume species to another.

In this context, expressed sequence tag (EST)-based DNA markers appear more promising.

This approach is based on the exploitation of information about conserved DNA sequences like those found in exons of genes. Such information is available in electronic databases (NCBI, different EST databases). For EST polymorphism detection, PCR-based methods using Cleaved Amplified Polymorphic Sequences (CAPS), Derived Cleaved Amplified Polymorphic Sequences (dCAPS) or size markers are usually used. These methods reveal differences in DNA sequence between individuals (polymorphism) by cutting of amplified PCR products with a restriction endonuclease (CAPS and dCAPS) or by differences in the size of amplified PCR products. The first pea map containing 18 EST-based DNA markers was created by [START_REF] Gilpin | A linkage map of the pea Pisum sativum L. genome containing cloned sequences of known function and expressed tags ESTs[END_REF]. They converted sequenced-characterized pea cDNAs into CAPS markers. This linkage map consists of 209 markers, covers 1330 cM and also includes RFLP, RAPD and AFLP markers. A number of pea genes were afterwards successfully located on the pea linkage map as EST (mostly as CAPS) markers using nucleotide sequence polymorphisms [START_REF] Brauner | STS markers for comparative mapping in legumes[END_REF]Irzykowska and Wolko, 2004;Konovalon et al., 2005;[START_REF] Morin | Functional mapping in pea, as an aid to the candidate gene selection and for investigating synteny with the model legume Medicago truncatula[END_REF]. In fine, the EST approach has proved to be most effective for genetic mapping and for comparative genome studies.

I.4.3. Exploitation of synteny between legume genomes

The development of genetic, cytogenetic and physical maps of legume species combined with data from large-scale M. truncatula and L. japonicus genome sequencing has facilitated the understanding of conservation (synteny) of genome structure among legumes. Macrosynteny generally refers to conserved gene order between species (an example is loci found on corresponding chromosomes), whilst microsynteny applies to conserved gene content and order over a short, physically defined DNA contig (an example is loci found on a corresponding bacterial artificial chromosome). The degree of synteny is correlated with the phylogenetic distance of the investigated species.

Several comparative genome studies were performed recently to evaluate synteny between legume species based on mapping of common DNA markers or in silico mapping of homologous sequences [START_REF] Brauner | STS markers for comparative mapping in legumes[END_REF][START_REF] Kaló | Comparative mapping between Medicago sativa and Pisum sativum[END_REF][START_REF] Choi | Estimating genome conservation between crop and model legume species[END_REF][START_REF] Zhu | Syntenic relationships between Medicago truncatula and Arabidopsis reveal extensive divergence of genome organization[END_REF][START_REF] Zhu | Bridging model and crop legumes through comparative genomics[END_REF]Moffet et al., 2006;[START_REF] Hougaard | Legume anchor markers link syntenic regions between Phaseolus vulgaris, Lotus japonicus, Medicago truncatula and arachis[END_REF]. The comparative mapping of M. truncatula and P. sativum genomes shows that, despite some major differences (the pea genome is approximately 10 times larger than that of M. truncatula and has one less chromosome), the co-linearity of genes is remarkably conserved between the two species, with main evident differences being inferred from inter-chromosomal rearrangements [START_REF] Choi | Estimating genome conservation between crop and model legume species[END_REF][START_REF] Zhu | Bridging model and crop legumes through comparative genomics[END_REF]. Much of the expansion in the genome size of P. sativum may be due to retro-elements [START_REF] Choi | Estimating genome conservation between crop and model legume species[END_REF]). The difference in chromosomes number between pea and Medicago has been suggested to result from rearrangements involving the Medicago chromosome 6 [START_REF] Zhu | Bridging model and crop legumes through comparative genomics[END_REF].

The conserved genome structure between M. truncatula and P. sativum has allowed for mapbased cloning of genes (homology-based gene cloning; see I.4.1.) and creation of EST-based DNA markers, using M. truncatula as a surrogate genome. For example, [START_REF] Morin | Functional mapping in pea, as an aid to the candidate gene selection and for investigating synteny with the model legume Medicago truncatula[END_REF] used syntenic relationships between pea and M. truncatula to infer gene position in the pea genome from M. truncatula gene position, and provided a functional pea map containing 111 genes and mutations. EST-based cross-species genetic markers have also been used for comparative mapping between P. sativum and M. sativa [START_REF] Kaló | Comparative mapping between Medicago sativa and Pisum sativum[END_REF]) and between P.

sativum and M. truncatula [START_REF] Choi | Estimating genome conservation between crop and model legume species[END_REF].

The location of each new gene identified using synteny between genomes provides an additional tool for future studies. Moreover, the idea that conserved genome structure can facilitate transfer of knowledge among related plant species may also be applicable to the mapbased gene cloning for the creation of molecular tools for the fine mapping of genes of interest. truncatula [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] Seddas | Symbiosis-related plant genes modulate molecular responses in an arbuscular mycorrhizal fungus during early root interactions[END_REF]. Plant genes were selected among genes reported as being modulated during plant-fungal interactions in the established AM symbiosis [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 interaction with different species and genera of Glomeromycota[END_REF].

I.5. Thesis objectives

The second part of my research focused on the creation of conditions for localization of pea symbiotic genes PsSym36 and PsSym40 on the pea genetic map was performed in the Laboratory of Genetics of Plant-Microbe Interactions, ARRIAM, St. Petersburg. For this aim, a set of morphological and molecular markers was used for PsSym36 and PsSym40 gene mapping.

II.1. Biological material

Pea genotypes: Throughout this study the following genotypes of Pisum sativum L. were used:

♦ cv Finale and its isogenic mutant

II.2. Growth conditions and sampling of plant material

For gene expression analysis

Seeds were surface-disinfected by 10-min treatment with a hypochlorite solution (4%) at room temperature for the genotypes cv Finale and RisNod24, and by a 25-min treatment with 98 % sulphuric acid at room temperature for lines SGE, SGEFix-1, SGEFix-2, and washed with distilled water. The method of surface-disinfection was chosen based on seed coat properties.

Seeds were germinated in Petri dishes (12-cm diameter) on humid filter paper at room temperature in the dark for 3 days.

Seedlings were planted into pots containing 200 ml of a Plants of F2 populations were inoculated by Rhizobium leguminasarum bv. vicia strain CIAM1026 known to have high symbiotic capacity [START_REF] Safronova | Comparison of two methods for root nodule bacteria preservation: lyophilization and liquid nitrogen freezing[END_REF]. Bacteria were cultured on solid nutrient medium №79 [START_REF] Sambrook | Molecular cloning: a laboratory manual[END_REF] with streptomycin (600 μg/l) at 28°C for 3 days. Plants were inoculated with an aqueous suspension of bacterial cells (10 7 -10 8 cells per plant) at sowing, and fertilized with 250ml per pot of a nutrient solution (tab. II.2.) every 2 weeks [START_REF] Borisov | Sequential functioning of Sym13 and Sym31, two genes effecting symbiosome development in root nodules of pea (Pisum sativum L.)[END_REF].

Tab.II.

II.3. Estimation of mycorrhizal root colonization

Mycorrhization parameters were determined at 3 and 4 weeks after inoculation with G. intraradices. To estimate total root colonization, staining with black ink (Noir de Jais of Shaeffer, Sheaffer Manufacturing Co., USA) was performed. Roots were digested in 10% KOH at 90°C for 80 min, rinsed several times with distilled water, stained for 8 min in 5% ink solution in 8% acetic acid and destained in 0.8% acetic acid for 25 min (Vierheilig et al., 1998).

Detection of fungal alkaline phosphatase activity in colonized roots was used to evaluate presence of a functionally active symbiosis [START_REF] Tisserant | Inplanta histochemical staining of fungal alkaline phosphatase activity for analysis of efficient arbuscular mycorrhizal infections[END_REF]. A 0.5 g root sample was incubated in 20 ml of 0.05 M Tris/citric acid, pH 9.2, containing sorbitol (50 mg/ml), cellulose (15 units/ml) and pectinase (15 units/ml) for 2 h at room temperature, rinsed with distilled water several times and left overnight at room temperature in 20 ml of the staining solution (0.05 mol/l

Tris/citric acid pH 9.2; 1 mg/ml α-naphthyl acid phosphate; 1mg/ml Fast Blue RR salt; 0.5 mg/ml MgCl 2 ; 0.

II.5. Plant hybridization analysis

Parental pea lines were crossed when plants were budding or started flowering. Immature stamens in maternal flowers were cut out using sterile tweezers. Mature pollen from paternal flowers was transferred to pistil stigma of maternal plants with another set of sterile tweezers.

Plants of F2 populations were obtained from self-fertilization of plants of F1 population.

Goodness-of-fit for the co-dominant 1:2:1 or dominant 1:3 ratios in F2 populations were tested by χ 2 analysis using the computer program MapL98.

II.6. Nucleic acid preparation

II.6.1. RNA extraction from plant roots

Total RNA was isolated from roots stored in liquid nitrogen following the method of [START_REF] Logemann | Improved method for the isolation of RNA from plant tissues[END_REF], modified by Franken and Gnadinger (1994). Samples were ground into a fine powder were used to remove any DNA contamination. RNA quantity, quality and integrity were checked again.

II.6.2. RNA extraction from laser-dissected arbuscule-containing cells

RNA was extracted from dissected cells using the PicoPure RNA Isolation kit (Arcturus)

according to the manufacturer's protocol. CapSure HS Caps (Arcturus) containing dissected cells were incubated with extraction buffer for 30 min at 42°C and then cells were collected in microcentrifuge tubes by centrifugation for 2 min at 800 g. Cell extract containing RNA was stored at -80°C and RNA isolation was continued after grouping together extracts from a series of cell sections. Cell extract was loaded onto a preconditioned purification column (600-700 sections/column) to bind RNA to the column membrane and washed with washing buffer. After removing DNA by DNase treatment (RNase-Free DNase Set, Qiagen), the membrane was washed twice with washing buffer and the RNA was eluted into a fresh centrifuge tube using a low ionic strength buffer. RNA was stored at -80°C. Quality and quantity of the RNA were estimated using a Bio-Rad Experion System and Bio-Rad Experion HighSens Analysis kit following the manufacturer's protocol.

II.6.3. DNA extraction from plant shoots

DNA was extracted using CTAB (Cetyl trimethyl ammonium bromide) buffer using the modified protocol of [START_REF] Rogers | Extraction of DNA from milligram amount of fresh, herbarium and mummified plant tissue[END_REF]. Plant tissue (approximately 70 mg) was ground in 1.5 ml tubes containing 700 μl of 2×CTAB buffer and approximately 30 μg of Al 2 O 3 powder, and incubated for 1 h at 65°C.

After incubation, an equal volume of phenol/chloroform solution (1/1, vol/vol) was added, tubes were inverted several times to mix, and centrifuged for 15 min at 14000 rpm. The upper phase was transferred into a new 1.5 ml eppendorf tube, and the procedure was repeated twice with phenol/chloroform solution and once with pure chloroform. DNA was precipitated from the final solution by adding an equal volume of 96% ethanol, transfering into new 1.5 ml eppendorf tubes containing 1 ml of 75 % ethanol, and centrifuging 5 min at 14000 rpm. After centrifugation, the liquid phase was removed, and the pellet was air-dried then diluted in distilled DNase free water.

Composition of 2×CTAB buffer: 2% CTAB (m/V), 1.4 M/l NaCl, 0.1 M/l Tris-HCl pH 8.0, 20 mM/l EDTA, H 2 O.

DNA quantity and quality were checked using Beckman Coulter DU800 Spectrophotometer.

II.6.4. Plasmid DNA preparation

Amplified parts of genes were cloned into a plasmid vector using the TOPO TA Cloning kit (Invitrogen) (see II.9). After plasmids were recuperated by white-blue selection and PCR (see II.9). Recombinants (white colonies) were multiplied in liquid LB culture containing ampicillin (100 μg/ml), and plasmid DNA was isolated using the NucleoSpin®Plasmid kit (Machrey-Nagel)

following the manufacturer's instructions. PCR of cDNA was carried out in 20 µl of PCR mix (Invitrogen) supplemented with 1.5 mM MgCl 2 , containing 0.5 U of Taq polymerase (Invitrogen), 125 μM dNTP, and 0.5 μM of each primer using 1 µl of diluted 1:5 cDNA for both fungal and plant genes. PCR was performed in a Biometra thermocycler (Göttingen, Germany) at 94°C for 1 min, followed by 30 cycles at 94°C for 45 s, the appropriate primer annealing temperature for 45 s, 72°C for 45 s, and a final extension at 72°C for 1 min.

Amplification products were analyzed by 1.4 % (wt/vol) agarose gel electrophoresis in 0.1 Trisacetate-EDTA (4 mM Tris, 0.1 mM EDTA, 2 mM acetic acid) (Fluka, St Gallen, Switzerland), stained with ethidium bromide for 10 min and observed under UV light using GelDoc EQ apparatus [START_REF] Weidmann | Fungal elicitation of signal transduction-related plant genes precedes mycorrhiza establishment and requires the dmi3 gene in Medicago truncatula[END_REF].

II.8.2. Primer design

For trancriptome studies Primers for real-time RT-PCR were designed to amplify PCR product with a size of 120-350 bp.

The protein-encoding genes from G. intraradices genes chosen for this study have predicted functions in transcription, protein turnover, primary or secondary metabolism (table II.4), and are known to be active in G. intraradices at the appressoria stage of root colonization [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] Seddas | Symbiosis-related plant genes modulate molecular responses in an arbuscular mycorrhizal fungus during early root interactions[END_REF]. The primers used had previously been designed by [START_REF] Seddas | Symbiosis-related plant genes modulate molecular responses in an arbuscular mycorrhizal fungus during early root interactions[END_REF]. Primer sequences, melting temperature and expected amplicon size are presented in Table II.4.

Table II Protein-encoding plant genes were selected amongst those reported in the literature to be specifically up-regulated during AM interactions or in arbuscule-containing cells of P. sativum or M. truncatula (table II .5). A part of the chosen P. sativum genes had been studied before [START_REF] Grunwald | Identification of mycorrhizaregulated genes with arbuscule development-related expression profile[END_REF]; these encode a disease resistance protein pI230, a trypsin inhibitor and a MAP kinase. Since nucleotide sequences were not known for the other genes in pea, primers were designed for specific amplification of those genes in pea based on nucleotide sequences of conserved regions of homologous genes of M. truncatula and some other plant species (annex 1). Amplicons from RT-PCR were cloned and sequenced to confirm the identity of the amplicon and to verify primer specificity. Using the obtained sequences, primers were then designed for amplification of 130-300 bp size PCR products in real-time RT-PCR experiment (table II.5.). The first reverse transcription reaction was primed with a synthetic T7-oligo(dT) containing a phage T7 RNA Polymerase promoter sequence at its 5'-end. The primer was annealed to the RNA template at 65°C for 5 min then chilled on ice for 1 min. First-strand cDNA synthesis was performed at 50°C for 30 min using a premix containing primer and RNA, 50 U of SuperScript III reverse transcriptase (Invitrogen), reverse transcription premix (supplied with the amplification kit) and DTT (dithiothreitol). The RNA component of the resulting cDNA:RNA hybrid was digested by RNase H into small RNA fragments that were primed for second-strand cDNA synthesis. For this purpose DNA polymerase and DNA polymerase mix from the amplification kit was added to the reaction mix and incubated for 10 min at 65°C and for 3 min at 80°C. The reaction was stopped by adding finishing solution from the amplification kit. In vitro production of anti-sense RNA (aRNA) was obtained by adding T7 RNA polymerase, T7 transcriptional buffer,

In Vitro transcriptional premix and DTT. The reaction was performed at 42°C for 4 hours followed by a DNase I treated at 37°C for 15 min. Qiagen RNeasy MinElute Cleanup kit was used to purify aRNA according to the manufacturer's protocol. A second round of reverse transcription was catalyzed by SuperScript II reverse transcriptase (Invitrogen). After adding random sequence hexamer primers, the volume mix was adjusted to 3 μl by speed vacuum centrifugation without heating and the reaction was incubated at 65°C for 5 min. The first-strand cDNA synthesis mix was combined with the RNA-primer mix, 50U of SuperScript II reverse transcriptase (Invitrogen), reverse transcription premix and DTT. Reaction was incubated for 10 min at room temperature and for 1 h at 37°C. The RNA component of the cDNA:RNA hybrid was digested with RNase H, as previously described. Double-stranded cDNA was produced using a T7-Oligo(dT) primer, DNA polymerase and DNA polymerase premix supplied by the amplification kit. The reaction was incubated for 10 min at 37°C, for 3 min at 80°C, chilled on ice and stored at -20°C. The obtained cDNA was used for semi-quantitative and real-time PCR.

II.9. DNA fragment sequencing

Sequencing was performed using CEQ™8000 Genetic Analysis System (Beckman Coulter) and

II.10. Cloning and sequencing of PCR products

To prepare standard plasmid dilutions for real-time RT-PCR, PCR fragments of cDNA were cloned into a plasmid vector using the TOPO TA Cloning kit for sequencing (Invitrogen). 1 μl of salt solution (supplied by the kit) and 1 μl of Vector (supplied by the kit) were added to 4 μl of PCR product; the mix was incubated for 30 min at room temperature and chilled on ice. To transform 1 aliquot of Escherichia coli competent cells (supplied by the kit), 2 μl of the previously prepared mix was added and transformation was performed by a heat shock at 42°C for 30 sec followed by cooling on ice.

To increase transformation efficiency, bacteria were cultured with 250 μl of SOC medium (Super were used for the analysis. Reactions were performed according to conditions recommended by producer for each enzyme and analyzed by electrophoresis in agarose gel (see II.8.1.).

II.12. Molecular marker creation

In this study two types of EST (Expressed Sequence Targ) molecular markers were used. They are CAPS (Cleavage Amplified Polymorphic Sequences) and ALP (Amplified Length Polymorphic sequences). CAPS marker creation was done in three steps: specific amplification of the part of the gene, search for polymorphic restriction sites and checking of the size of PCR products after restriction by electrophoresis in agarose gel. To check specific amplification, PCR products were sequenced (see II.10.). Analysis of obtained nucleotide sequences was done using online computer programme SDSC Biology Workbench (http://workbench.sdsc.edu/). In process of sequences analysis for some genes, polymorphism was detected by the size of PCR products which can be visualized by electrophoresis in agarose gel. It became a base for ALP marker creation. Pssym36 (line RisNod24) was first reported as a nitrogen fixation symbiosis (NFS) mutant by [START_REF] Engvild | Nodulation and nitrogen fixation mutants of pea (Pisum sativum)[END_REF]. Later, more detailed investigations of the process of NFS development showed that the mutation in the PsSym36 gene blocks infection thread growth in the root hair and nodule primordium development [START_REF] Sagan | Phenotypic characterization and classification of nodulation mutants of pea (Pisum sativum L.)[END_REF]. Pssym36 is also an AM-defective mutant where the stages of appressorium formation, root penetration and cortex colonization by intercellular hyphae are completed, but arbuscule formation is reduced to a few stumpy branches [START_REF] Gianinazzi-Pearson | Genetic and cellular analysis of resistance to vesicular arbuscular (va) mycorrhizal fungi in pea mutants/ Advances in Molecular Genetics of Pant-Microbe Interactions[END_REF]Gianinazzi-Pearson, 1996), leading to a lower level of root colonization than in the wild type genotype [START_REF] Lapopin | Comparative di.erential display analysis of arbuscular mycorrhiza in Pisum sativum and a mutant defective in late stage development[END_REF][START_REF] Grunwald | Identification of mycorrhizaregulated genes with arbuscule development-related expression profile[END_REF].

The pea symbiotic mutants Pssym40 and Pssym33 were identified after a complementation analysis in SGEFix --1 and SGEFix --2 lines, respectively, and were shown to control stages in symbiotic root nodule development by [START_REF] Tsyganov | The pea (Pisum sativum L.) genes sym33 and sym40 control infection thread formation and root nodule function[END_REF]. SGEFix --1 (Pssym40) is blocked at the stage of infection droplet formation during nodule organogenesis, whilst SGEFix --2 (Pssym33) is impaired in the process of endocytosis and rhizobia are not properly released into the nodule cells. These mutants were later shown to affect AM formation (Jacobi et al., 2003 a,b). Mycorrhizal colonization of the Pssym40 roots is significantly increased but only at the Both Pssym40 and Pssym33 mutants showed differences in plant development compared with the wild type plants (Jacobi et al., 2003 a). Analysis of rate of biomass accumulation and P accumulation was performed under bioavailable P fertilization, full NPK nutrition, AM inoculation with G. intraradices and control conditions without inoculation and without additional mineral nutrition. Pssym40 mutant had improved root biomass accumulation and P accumulation in roots in the presence of AM fungi. Mutation in PsSym33 gene resulted in increased biomass accumulation in both shoots and roots and P accumulation in roots for all experimental treatments (addition of bioavailable P fertilization, full NPK nutrition and inoculation with G. intraradices). An increased rate of P accumulation in shoots was observed with the addition of biovailable P and NPK. The mutant plants were characterized by a delayed timing of flowering and pod ripening. These observations suggest that mutations in the PsSym40 and PsSym33 genes have pleiotropic effects and induce slight systemic changes in whole plant development (Jacobi et al, 2003 a). [START_REF] Gianinazzi-Pearson | Cellular and molecular defence-related root responses to invasion by arbuscular mycorrhizal fungi[END_REF] No vesicles were observed within roots. The proportion of overall alkaline phosphatase-active compared to total (ink-stained) mycelium in roots remained low in either cv Finale (6%) or Pssym36 (7%) roots at 21 dai but increased to 19% in cv Finale and 51% in Pssym36 at 28 dai.

III.2. Results

III.2.1. Mycorrhiza development

wild type cv Finale

Pssym36 phenotype

III.3. Conclusions

Estimation of AM development confirmed previous observations [START_REF] Gianinazzi-Pearson | Weak expression of the pathogenesis related PR-b1 gene and localization of related protein during symbiotic endomycorrhizal interactions in tobacco roots[END_REF]Jacobi et al., 2003 a, b) that the Pssym36, Pssym33 and Pssym40 mutants have modified AM phenotypes as compared to the wild type parent genotypes:

Pssym36 is blocked at the arbuscule developmental stage. Mutation in the PsSym36 gene also leads to a reduction in mycorrhizal colonization of roots.

Mutation in the PsSym33 gene decreases the rate of mycorrhizal colonization but does not influence development of an effective AM symbiosis.

Mutation in the Pssym40 results in an early increased rate of AM development.

Mutations in both of the PsSym33 and PsSym40 genes influence plant development (shoot and root biomass accumulation), suggesting a pleiotropic effect of these mutations. Mutation in the PsSym36 gene affected plant development in the absence of an AM fungus and G. intraradices colonization of the Pssym36 roots had a negative influence on both wild type and mutant plant growth at 28 dai.

An increased rate of AM development and a higher proportion of alkaline phosphatase-active mycelium in roots of the SGE line and the corresponding mutants Pssym33 and Pssym40, as compared to the cv Finale genotype, suggests an influence of the genetic background of these genotypes on the ability of pea to form a successful AM symbiosis.

IV.1. Introduction

Identification of AM symbiosis-related plant and fungal genes has progressed rapidly over the last decade due to the realization of sequencing projects and transcriptome-wide studies [START_REF] Journet | Exploring the root symbiotic programs of the model legume Medicago truncatula using EST analysis[END_REF][START_REF] Liu | Transcript profiling coupled with spatial expression analyses reveals genes involved in distinct developmental stages of the arbuscular mycorrhizal symbiosis[END_REF][START_REF] Liu | Arbuscular mycorrhizal symbiosis is accompanied by local and systemic alterations in gene expression and an increase in disease resistance in the shoots[END_REF][START_REF] Wulf | Transcriptional changes in response to arbuscular mycorrhiza development in the model plant Medicago truncatula[END_REF][START_REF] Brechenmacher | Expression profiling of up-regulated plant and fungal genes in early and late stages of Medicago truncatula-Glomus mosseae interactions[END_REF]Gianinazzi-Pearson et al., 2004;[START_REF] Küster | Construction and validation of comprehensive cDNAbased macro-and microarrays to explore root endosymbioses in the model legume Medicago truncatula[END_REF][START_REF] Küster | Development of bioinformatic tools to support EST-sequencing, in silico-and microarray-based transcriptome profiling in mycorrhizal symbioses[END_REF][START_REF] Sanchez | Pseudomonas fluorescens and Glomus mosseae trigger DMI3dependent activation of genes related to a signal transduction pathway in roots of Medicago truncatula[END_REF]Weidmann et al., 2004;[START_REF] Frenzel | Combined transcriptome profiling reveals a novel family of arbuscular mycorrhizalspecific Medicago truncatula lectin genes[END_REF][START_REF] Deguchi | Transcriptome profiling of Lotus japonicus roots during arbuscular mycorrhiza development and comparison with that of nodulation[END_REF][START_REF] Massoumou | Medicago truncatula gene responses specific to arbuscular mycorrhiza interaction with different species and genera of Glomeromycota[END_REF][START_REF] Seddas | Symbiosis-related plant genes modulate molecular responses in an arbuscular mycorrhizal fungus during early root interactions[END_REF]. However, despite the extensive list of plant and fungal genes identified, information about molecular events related to defined steps of AM symbiosis development is still poor. In this context, previous research of AM fungal behavior has been limited to the use of plant mutants affected in the early steps of appressoria formation and root penetration [START_REF] Seddas | Symbiosis-related plant genes modulate molecular responses in an arbuscular mycorrhizal fungus during early root interactions[END_REF]. For this reason, we chose to use the pea mutants Pssym36, Pssym33 and Pssym40 to study how corresponding SYM genes affect fungal gene expression in the late stage of AM formation or functioning. G. intraradices genes were selected from a recent investigation showing their expression during early and late stages of AM symbiosis development in M. truncatula [START_REF] Seddas | Symbiosis-related plant genes modulate molecular responses in an arbuscular mycorrhizal fungus during early root interactions[END_REF]. These genes are predicted to have functions in transcription, protein synthesis, primary/secondary metabolism or have unknown function. They encode for a plasma membrane H + -ATPase (H + -ATPase), an alkaline phosphatase (ALP), a putative Rho/GDP dissociation inhibitor (RHO), a peptidylprolyl isomerase (PEPISOM), a 26S proteasome subunit 2 (26S), a 26S proteasome regulatory subunit (26SREG), a stearoyl-CoA desaturase (DESAT), a thioredoxin peroxidase (THIO), a superoxide dismutase (SOD) and a hypothetical vacuolar import protein (VACU). Expression of these fungal genes was compared at 21 and 28 dai in G. intraradices-colonized roots of Pssym36, Pssym33 and Pssym40 mutants and the corresponding wild type pea genotypes cv. Finale and SGE, by real-time RT-PCR.

Expression was quantified as transcript levels and normalized against the expression of the house-keeping TEF gene of G. intraradices.

IV.2. Results

To amplify the G. intraradices genes RHO, PEPISOM, 26S, 26SREG, DESAT, THIO, SOD and VACU, the primers previously designed by [START_REF] Seddas | Symbiosis-related plant genes modulate molecular responses in an arbuscular mycorrhizal fungus during early root interactions[END_REF] were used. Nucleotide sequences of amplified products were identical to those present in the MENS database. For amplification of H + ATPase and ALP transcripts, primers were newly designed; the sequences of the amplified products showed high similarity with those of GiHA5 (AF420481, 1e-48) and GiALP (AB114298.1, 6e-70) genes, respectively. 26SREG tended to be also down-regulated in Pssym36 roots (P=0.1) and 26S and ALP tended to be up-regulated (P=0.1) as compared to cv Finale roots. No difference was observed in expression of the fungal H + ATPase, 26S, 26SREG and ALP genes. The fungal genes generally had lower expression levels in roots of both pea genotypes at 28 dai than at 21 dai, except for the DESAT gene. The DESAT gene continued to be significantly down-regulated in roots of the Pssym36 mutant at 28 dai. Transcripts of the 26S gene were not detected at 28 dai whilst transcript accumulation of fungal H + ATPase and 26SREG was significantly decreased in Pssym36 roots by 3.2 and 10 fold, respectively. ALP was the only gene of G. intraradices to show significantly enhanced expression (4.5 fold) in mutant as compared to wild type colonized roots at 28 dai. None of the other fungal genes showed significant differences in expression levels between mutant and wild type-colonized roots at 28 dai.

IV.2.1. Interactions with the

IV.2.2 Interactions with the Pssym33 mutant

The PsSym33 gene mutation had less pronounced effects on G. intraradices gene expression than mutations in the PsSym36 and PsSym40 genes (see figure IV In this study, we used the late stage symbiotic mutants Pssym36, Pssym33 and Pssym40 of pea to gain insight into AM fungal molecular responses linked to AM formation and functioning.

Modifications in transcript accumulation of G. intraradices genes resulting from mutations in the Pssym36, Pssym33 and Pssym40 genes gave an opportunity to better define mutant phenotypes and the role of the selected fungal genes in the symbiosis. The mutation in the PsSym36 gene not only led to a lack of arbuscule as was shown before [START_REF] Gianinazzi-Pearson | Genetic and cellular analysis of resistance to vesicular arbuscular (va) mycorrhizal fungi in pea mutants/ Advances in Molecular Genetics of Pant-Microbe Interactions[END_REF] but also to a considerably lower expression of most of the monitored G. intraradices genes, suggesting that sub-optimal conditions are created in the Pssym36 mutant for fungal development and activity inside the root. Lower levels of expression were detected for genes involved in fungal membrane transport, transcription, protein synthesis, primary/secondary metabolism.

The decreased expression of the fungal membrane H + -ATPase gene when arbuscule formation is defective in Pssym36 roots correlates with the lack of enzyme activity on the fungal plasma membrane bordering aborted arbuscules branches, shown before in the same pea genotype [START_REF] Gianinazzi-Pearson | Cellular and molecular approaches in the characterization of symbiotic events in functional arbuscular mycorrhizal associations[END_REF]. H + -ATPase enzymes are known to generate the electrochemical gradient of protons that drives active secondary transport system across membrane. It can therefore be hypothesized that in the AM symbiosis the fungal H + -ATPase plays an important role in the active transport of nutrients across the plasma membrane, and that absence of the enzyme activity will limit fungal uptake of photosynthetic carbon at the plantfungal interface in Pssym36 roots. This could explain the strong down-regulation of the DESAT gene of G. intraradices which reflects modifications in fungal lipid metabolism, particularly reduction in biosynthesis of fatty acids due to carbon limitation. Down-regulation of this gene was also recently shown when G. intraradices develops on the roots of the M. truncatula mutants dmi1, dmi2/Mtsym2 and dmi3/Mtsym13 which block AM symbiosis development at early stages after appressoria formation [START_REF] Seddas | Symbiosis-related plant genes modulate molecular responses in an arbuscular mycorrhizal fungus during early root interactions[END_REF]. In contrast, expression of fungal H + -ATPase and DESAT genes in mycorrhizal roots of the Pssym33 and Pssym40 mutants was maintained at a similar level or increased as compared mycorrhizal wild type roots. Increased expression of these G. intraradices genes in Pssym40 mutant may be connected with greater carbon acquisition by the fungus linked to the more rapid root colonization, arbuscule formation and turn-over typical for this mutant (Jacobi et al. 2003 b). Transcript analysis of microdissected arbuscule-containing cells from Pssym40 mutant performed in this study (see chapter VI) confirmed arbuscule-related expression of the DESAT gene.

The weaker expression of the fungal RHO gene, encoding a putative Rho/GDP dissociation inhibitor, in G. intraradices-colonized Pssym36 roots could indicate effects on cell signaling events at the fungal-plant interface, as was suggested for mutations in the DMI2 and DMI3/MtSym13 genes of M. truncatula which similarly repressed RHO expression when appressoria are formed [START_REF] Seddas | Symbiosis-related plant genes modulate molecular responses in an arbuscular mycorrhizal fungus during early root interactions[END_REF]. Such inhibitors regulate GTP-GDP conversion on Rho/GTPase proteins which are essential to basic cell functions including metabolism, proliferation and differentiation [START_REF] Groysman | Vav1 and Ly-GDI Two regulators of Rho GTPases, function cooperatively as signal transducers in T cell antigen receptorinduced pathways[END_REF]. Lower transcript levels in colonized Pssym36 roots of the PEPISOM and 26SREG genes may contribute to, or result from, defective arbuscule development in the pea mutant. PEPISOM plays a role in cellular regulation as a chaperon protein that activates or inhibits regulatory proteins by catalyzing proline isomerization [START_REF] Miele | A peptidylprolyl cis/trans isomerase from Xenopus laevis skin: cloning, biochemical characterization and putative role in the secretion[END_REF]. The 26SREG-regulated proteasome is a proteasome complex that is responsible for selective degradation of abnormal proteins and naturally short-lived proteins related to metabolic regulation and cell cycle progression [START_REF] Murray | Cyclin ubiquitination : the destructive end of mitosis[END_REF]. Inactivation of the Pssym33 gene was not detrimental to the expression of these three genes whilst mutation of Pssym40 significantly up-regulated the RHO and 26SREG genes, which may be explained by quicker arbuscule turn-over in roots of this mutant (Jacobi et al., 2003 b).

Expression of the ALP gene, considered to have a role in phosphate transfer within AM fungi to the host plant, unexpectedly increased in the G. intraradices-colonized roots of Pssym36 and Pssym33, as compared to mycorrhizal roots of the corresponding wild type pea genotypes. This correlated with an increased proportion of ALP-active mycelium in the two mutants although intraradical development of G. intraradices was lowest in their root systems. Mutation in the Pssym40 gene did not affect ALP transcript accumulation. Because purification of the ALP enzyme from AM fungi has so far been unsuccessful, little is known about its enzymatic characteristics or its function in the AM symbiosis [START_REF] Kojima | Electrophoretic detection and partial purification of the phosphatase specific for arbuscular mycorrhizal symbiosis[END_REF]. Microorganisms produce alkaline phosphatases in response to various environmental stresses [START_REF] Lim | The effects of osmotic stress on survival and alkaline phosphatase activity of Aeromonas hydrophila[END_REF] and the up-regulation of G. intraradices ALP expression in Pssym36 and Pssym33 roots may be indicative of the perception of a stress by the fungus in these genotypes.

Transcript accumulation of the fungal genes of anti-oxidative stress metabolism, THIO and SOD, in the mycorrhizal roots of wild type, Pssym33 and Pssym40 pea genotypes is typical for AM symbiosis [START_REF] Balestrini | Fungal and plant gene expression in arbuscular mycorrhizal symbiosis[END_REF]. Such gene activation may enable the AM fungus to contend with plant defense/stress responses which are usually activated in mycorrhizal roots, especially within arbuscule-containing cells [START_REF] Gianinazzi-Pearson | Cellular and molecular defence-related root responses to invasion by arbuscular mycorrhizal fungi[END_REF].

Activation of these two genes has been reported previously for mycorrhiza of other plant/fungal combinations [START_REF] Brechenmacher | Expression profiling of up-regulated plant and fungal genes in early and late stages of Medicago truncatula-Glomus mosseae interactions[END_REF][START_REF] Seddas | Symbiosis-related plant genes modulate molecular responses in an arbuscular mycorrhizal fungus during early root interactions[END_REF]. In this context, expression of

V.2 Results

Nucleotide sequences were known for two of the eight analyzed pea genes (kunitz-like trypsin inhibitor (TI) and disease resistance response protein pI230 (Drp)) and primers published by [START_REF] Grunwald | Identification of mycorrhizaregulated genes with arbuscule development-related expression profile[END_REF] were used to amplify them. For the other genes, primer design was performed based on conserved regions of amino acid and nucleotide sequences of homologous genes of different plant species. Obtained pea nucleotide sequences showed high homology to

Vicia faba H + -ATPase vha4 (AJ310523. Mutations in the PsSym36, PsSym33 and PsSym40 genes had a less pronounced effect on expression of the pea genes monitored in this study to reflect AM symbiosis development and functioning than on G. intraradices genes reported in the previous chapter.

The pea H + -ATPase gene identified here was active in non-mycorrhizal roots of wild type pea genotypes and of all three Pssym36, Pssym33 and Pssym40 mutant plants, but its expression was not affected by G. intraradices colonization of root in any of the genotypes. This contrasts with reported up-regulation of an H + -ATPase-encoding gene in mycorrhizal roots of tobacco and M. truncatula (Strittmater et al., 1996;[START_REF] Krajinski | Mtha1, a plasma membrane H + -ATPase gene from Medicago truncatula, shows arbuscule specific induced expression in mycorrhizal tissue[END_REF]. Likewise, expression of the pea BCOP gene selected for this study was also not influenced by AM formation, contrary to a M. truncatula gene [START_REF] Vieweg | The promoter of the Vicia faba L. leghemoglobin gene VfLb29 is specifically activated in the infected cells of root nodules and in the arbuscule-containing cells of mycorrhizal roots from different legume and non legume plants[END_REF]. Both H + -ATPase and blue copper proteins belong to large gene families and members may behave differently in response to signals or external stimuli in different plant species.

The pea phosphate transporter gene (PsPT4) which was identified in this study shows a high homology with the MtPT4 reported to be induced during the AM symbiosis in M. truncatula.

There is recent evidence that MtPT4 transporter activity is essential for the maintenance of arbuscules [START_REF] Javot | A Medicago truncatula phosphate transporter indispensable for the arbuscular mycorrhizal symbiosis[END_REF], and lyso-phosphatidylcholine has been proposed as a possible signal molecule at the symbiotic interface to maintain this activity [START_REF] Drissner | Lyso-phosphatidylcholine is a signal in the arbuscular mycorrhizal symbiosis[END_REF].

Expression of the PsPT4 gene was strongly up-regulated in fully mycorrhizal roots of wild type pea genotypes, as well as in those of the Pssym33 and Pssym40 mutants but it did not respond to G. intraradices colonization of roots in the Pssym36 mutant, which concords with a role of PsPT4 in P i transport at the symbiotic interface of arbuscules [START_REF] Harrison | A phosphate transporter from Medicago truncatula involved in the acquisition of phosphate released by arbuscular mycorrhizal fungi[END_REF][START_REF] Javot | A Medicago truncatula phosphate transporter indispensable for the arbuscular mycorrhizal symbiosis[END_REF].

Up-regulation of the expression of genes encoding for the pI230 disease resistance response protein (DRP), a trypsin inhibitor (TI) and glutathione-S-transferase (GST) in mycorrhizal roots of wild type, Pssym33 and Pssym40 genotypes may reflect activation of pea defence/stress responses to the AM fungal development. Glutathione-S-transferases are enzymes that catalyze the conjugation of the tri-peptide glutathione which is known to play an important role in plant defence mechanisms [START_REF] Anderson | Abiotic stress alters transcript profiles and activity of glutathione S-transferase, glutathione peroxidase, and glutathione reductasein Euphorbia esula[END_REF]. Glutathione-S-transferase transcripts have been found to accumulate in the arbuscule stage of mycorrhiza [START_REF] Wulf | Transcriptional changes in response to arbuscular mycorrhiza development in the model plant Medicago truncatula[END_REF][START_REF] Brechenmacher | Expression profiling of up-regulated plant and fungal genes in early and late stages of Medicago truncatula-Glomus mosseae interactions[END_REF]). Activation of the DRP and TI genes may be involved in the control of fungal development within host plant roots. The disease resistance response protein pI230 and the trypsin inhibitor have been described as having an antifungal activity [START_REF] Chiang | The Fusarium solaniinduced expression of a pea gene family encoding high cysteine content proteins[END_REF]Blee, 1998;Griudici et al., 2000). Plant DRP and TI genes were not activated by G. intraradices colonization of roots of the arbuscule-defective Pssym36 mutant, suggesting a role of these genes at the arbuscule development stage. These observations on DRP and TI gene expression in roots of the Pssym36 mutant are in agreement with data reported previously by [START_REF] Grunwald | Identification of mycorrhizaregulated genes with arbuscule development-related expression profile[END_REF]. It is interesting that lack of activation of these two plant defencerelated genes in Pssym36 roots coincides with a lack of up-regulation of the G. intraradices THIO and SOD genes implemented in anti-oxidative stress metabolism, which may reflect arbuscule formation as a stress situation for the fungus.

The MAP kinase-encoding pea gene was consistently down-regulated in mycorrhizal roots of the wild type pea genotypes but expression was not significantly affected in the three mutants. [START_REF] Liu | Transcript profiling coupled with spatial expression analyses reveals genes involved in distinct developmental stages of the arbuscular mycorrhizal symbiosis[END_REF] reported down regulation of two M. truncatula MAP kinase genes as G. versiforme developed within roots, although the genes were transiently up-regulated during the initial stages of symbiosis establishment, whilst [START_REF] Grunwald | Identification of mycorrhizaregulated genes with arbuscule development-related expression profile[END_REF] found that a MAP kinase gene was up-regulated in mycorrhizal cv Finale roots and unaffected in Pssym36. MAP kinases belong to a gene family which interacts with a large number of receptors making it difficult to hypothesize as to a specific function in mycorrhizal interactions since distinct MAP kinase pathways are involved in a wide variety of biological processes including cell growth and death, differentiation, the cell cycle and stress responses [START_REF] Jonak | MAP kinases in plant signal transduction[END_REF].

The pea serine protease (SERPROT) gene identified here shows a high homology with a previously reported serine protease gene activated in mycorrhizal roots of M. truncatula [START_REF] Gianinazzi-Pearson | Cellular and molecular defence-related root responses to invasion by arbuscular mycorrhizal fungi[END_REF][START_REF] Garcia-Garrido | Regulation of the plant defence response in arbuscular mycorrhizal symbiosis[END_REF]Massoumou et al., 2004).

Likewise, expression of pea SERPROT was also up-regulated by AM development.

In summary, expression of seven of the eight selected plant genes (except the H + -ATPase ). This approach has previously been applied to monitor plant and fungal gene expression within arbuscule-containing cells of mycorrhizal roots of Lycopersicon esculentum [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] and M. truncatula [START_REF] Gomez | Medicago truncatula and Glomus intraradices gene expression in cortical cells harbouring arbuscules in the arbuscular mycorrhizal symbiosis[END_REF]. Transcript levels of the three selected G. intraradices genes were quantified by absolute quantitative real-time RT-PCR and normalized against the expression of the house-keeping TEF gene, as reported in chapter IV.

VI.2. Results

RNA for fungal gene expression analysis

VI.3. Discussion and conclusions

It was previously shown that expression of G. intraradices SOD, PEPISOM and DESAT genes is strongly related with the arbuscule stage of AM symbiosis development (see chapter IV).

These genes had an important level of transcripts in dissected arbuscule-containing cells of the Pssym40 mutant which was chosen for this study because it has a more rapid arbuscule development. Transcript levels of SOD, DESAT and PEPISOM genes were higher by 60, 6 and 2117 times, respectively, in dissected arbuscule-containing cells than in the pool of all cells of a mycorrhizal root. The significant up-regulation of these genes observed in mycorrhizal roots of wild type SGE, Pssym33 and Pssym40 mutants, but not in roots of the arbuscule-defective Pssym36 mutant, supports the hypothesis of their arbuscule-related expression.

As was discussed previously (chapter IV), activation of the anti-oxidative SOD gene may result from a reaction of G. intraradices to stress/defense responses generated in roots by the AM fungus. These responses, especially localized in arbuscule-containing cells [START_REF] Gianinazzi-Pearson | Cellular and molecular defence-related root responses to invasion by arbuscular mycorrhizal fungi[END_REF], probably explain up-regulation of the SOD gene in that the fungus has to SOD DESAT PEPISOM overcome the oxidative defense system of the plant for its establishment and functioning in the root.

The DESAT gene which is also significantly activated in dissected arbuscule-containing cells, has a function in lipid metabolism. This gene may be involved in G. intraradices lipid metabolism necessary for fungal plasma membrane modifications during symbiosis development, especially during symbiotic interface formation. Its activity may also be related to photosynthetic carbon uptake at the arbuscule interface and subsequent storage as lipid globules in mycelium because we observed low expression level of the DESAT gene in G. intraradices-colonized roots of the arbuscule-defective Pssym36 mutant.

PEPISOM is also a fungal gene highly expressed in dissected arbuscule-containing cells.

PEPISOM encodes a chaperon protein that activates or inhibits regulatory proteins by catalyzing proline isomerization [START_REF] Miele | A peptidylprolyl cis/trans isomerase from Xenopus laevis skin: cloning, biochemical characterization and putative role in the secretion[END_REF]. This gene may play a role in protein turnover during cellular regulation of arbuscule formation and/or functioning, which would explain the lack of expression in G. intraradices-colonized roots of the Pssym36 mutant.

In conclusion, the expression analysis of SOD, DESAT and PEPISOM genes in dissected arbuscule-containing cells provides strong evidence that the up-regulation observed by qRT-PCR monitoring of transcripts in mycorrhizal roots preferentially occurs in the fungal arbuscules.

VII.1. Introduction

Genetic mapping, as a tool for gene cloning, is a necessary step in studies of the functioning and maintenance of different living processes, including interactions between plants and microbes during beneficial symbioses. One way to define the molecular basis of a mutant phenotype is by map-based cloning which first necessitates localization of the corresponding gene on the genetic map. In this context, the objective of this study was to create conditions for genetic mapping of the mutated PsSym36 and PsSym40 genes.

Neither PsSym36 nor PsSym40 genes have been localized on the pea genetic map so far.

Previous attempts to localize the PsSym40 gene using a set of 10 morphological markers (A, b, d, Fs, gp, k, r, s, tl w ,wb ) were not successful (Dr. V.Tsyganov, personal communication). For PsSym36, weak linkage to the morphological marker wb of linkage group (LG) II was observed in a small mapping population of 50 F2 plants (Dr. V. Zhukov, personal communication). This makes it necessary to resort to the use of DNA-based molecular markers for mapping PsSym36

and PsSym40 in the pea genome.

Recent efforts to construct pea genetic maps have produced an extensive list of molecular markers based on genes of known function and known position in pea genome (so called anchor markers) [START_REF] Gilpin | A linkage map of the pea Pisum sativum L. genome containing cloned sequences of known function and expressed tags ESTs[END_REF][START_REF] Konovalov | A CAPS marker set for mapping in linkage group III of pea (Pisum sativum L.)[END_REF][START_REF] Morin | Functional mapping in pea, as an aid to the candidate gene selection and for investigating synteny with the model legume Medicago truncatula[END_REF]. This information provides a tool for mapping new pea mutations in relation to the anchor markers.

Most of the protein encoding genes used in the present study to create molecular markers for mapping of the PsSym40 gene were localized on the pea genetic map by [START_REF] Morin | Functional mapping in pea, as an aid to the candidate gene selection and for investigating synteny with the model legume Medicago truncatula[END_REF].

In the present study, molecular markers (mostly CAPS -Cleaved Amplified Polymorphic

Sequences [START_REF] Konieczny | A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based markers[END_REF])) were firstly developed on the basis of gene nucleotide sequence polymorphisms which were identified between parental lines (one of which was the Pssym40 mutant) by sequencing of PCR products, and afterwards they were localized on the pea genetic map. The position of the PsSym36 gene in the linkage group II close to the morphological marker Wb was also investigated.

VII.2. Results

VII.2.1. Creation and screening of F2 populations

For mapping of the PsSym36 and PsSym40 genes, F1 and F2 populations were produced (see Material and Methods II.2, II.5) by the following crosses:

RisNod24 (Pssym36) mutant pea line with:

• morphologically mapped laboratory lines: NGB 1238, NGB 851, NGB 2715,

• lines from the N.I. Vavilov Institute of Plant Industry (VIR) collection: K 3034, K 1693, K 7128.

SGEFix --1 (Pssym40) mutant pea line with:

• laboratory line NGB 1238

VII.3. Discussion and conclusions

The main aim of the present study was to develop conditions for the localization of the PsSym36 and PsSym40 genes without creating a highly saturated pea genetic map. After generating the necessary plant material, the possible linkage between the PsSym36 gene and the morphological marker Wb shown in a previous study was examined. No significant linkage between these two genes was detected in the F2 population. However, plant material developed for this analysis will be useful for future experiments based on molecular markers to localize the position of the PsSym36 gene on the pea genetic map.

A total of 34 protein encoding genes with known function and position on the pea genetic map were selected for molecular marker creation to determine the position of the PsSym40 gene, and 17 molecular markers were developed on the basis of the nucleotide sequence polymorphism identified between parental lines in amplified parts of the chosen genes. The segregation data obtained did not reveal any significant linkage of the developed molecular markers with the PsSym40 gene. Thus, the three molecular markers ) is an important legume crop in French and in Russian agriculture and it is one of the most developed model plants for genetic studies of symbiotic root interactions with beneficial soil microorganisms [START_REF] Zhukov | Molecular Genetic Mechanisms Used by Legumes to Control Early Stages of Mutually Beneficial (Mutualistic) Symbiosis[END_REF]. Phenotypic characterization of pea mutants has led to the identification of plant genes that are implicated in the establishment or in the functioning of nodulation and AM symbioses [START_REF] Borisov | Pea (Pisum sativum L.) regulatory genes controlling development of nitrogen-fixing nodule and arbuscular mycorrhiza: fundamentals and application[END_REF]. Such mutants provide tools for genomic and molecular studies to identify plant SYM genes and to gain insight into their control of the metabolic circuits driving symbiotic interactions. In AM associations, the symbiotic fungi facilitate uptake of mineral nutrients (especially Pi) from the soil to plant roots and, in return, they receive photosynthetically fixed carbon [START_REF] Smith | Mycorrhizal symbiosis[END_REF]. The principal site of Pi and carbon exchange between plant and fungus occurs across a symbiotic interface formed between specific intracellular fungal structures, arbuscules, and cortical root cells. Whilst plant genes involved in the early steps of AM formation are a focus of research [START_REF] Gherbi | SymRK defines a common genetic basis for plant root endosymbioses with arbuscular mycorrhiza fungi, rhizobia, and Frankiabacteria[END_REF], little is known about the genetic programme controlling the formation and function of arbuscules which are the key symbiotic structures in the established mycorrhiza.

The aim of my research was therefore to better characterize the late stage AM symbiosisrelated pea genes PsSym36, PsSym33 and PsSym40 by investigating the effect of mutations in the three genes on fungal and plant gene responses and by creating conditions for the localization of two of the genes, PsSym36 and PsSym40, on the pea genetic map for future map-based cloning. Mutation in the PsSym36 gene results in incomplete, aborted arbuscule development [START_REF] Gianinazzi-Pearson | Weak expression of the pathogenesis related PR-b1 gene and localization of related protein during symbiotic endomycorrhizal interactions in tobacco roots[END_REF] whilst mutations in the PsSym33 and PsSym40

genes induce alterations in the rate of AM development (Jacobi et al., 2003 a, b). The study focusing on the genetic localization of PsSym36 and PsSym40 was performed in the Laboratory of Genetics of Plant-Microbe Interactions, ARRIAM. During this research period, production of necessary plant material was achieved and 34 protein encoding genes with known location in the linkage groups LGI, LGII, LGIII and LGV of the pea genetic map were selected. 17 Observed down-regulation of the G. intraradices H + -ATPase gene when arbuscule formation is defective in Pssym36 roots correlates with the lack of enzyme activity on the fungal plasma membrane bordering aborted arbuscules branches, shown before in the same pea genotype [START_REF] Gianinazzi-Pearson | Cellular and molecular approaches in the characterization of symbiotic events in functional arbuscular mycorrhizal associations[END_REF]. It can be hypothesized that this H + -ATPase enzyme plays a role in the active fungal uptake of photosynthetic carbon at the plant-fungal interface in wild type roots. If so, limited carbon uptake of plant-derived carbon could be expected in Pssym36 roots and this could explain the strong down-regulation of the fungal DESAT gene which suggests a reduced biosynthesis of fatty acids as a result of reduced carbon acquisition. In the Pssym33 and Pssym40 pea genotypes, where arbuscules are fully developed, expression of fungal transporter (PsPT4) gene was not activated in G. intraradices-colonized roots of the Pssym36 mutant, in contrast to its up regulation in mycorrhizal roots of the wild type, Pssym33 and Pssym40 pea genotypes. Observed expression of the PsPT4 gene is in agreement with a role of PT4 in P i transport only at the symbiotic interface of fully developed, functional arbuscules in mycorrhizal pea roots, as was reported for M. truncatula mycorrhiza [START_REF] Harrison | A phosphate transporter from Medicago truncatula involved in the acquisition of phosphate released by arbuscular mycorrhizal fungi[END_REF][START_REF] Javot | A Medicago truncatula phosphate transporter indispensable for the arbuscular mycorrhizal symbiosis[END_REF].

The lack of activation of the DRP and TI plant defence-related genes in Pssym36 roots coincides with a lack of up-regulation of the G. intraradices THIO and SOD genes implemented in anti-oxidative stress metabolism. This may reflect absence of a stress situation for the fungus when arbuscule formation is inhibited. In fact, fungal development and arbuscule formation appears to result in the activation of defence/stress responses, with up-regulation of the expression of pea genes encoding for the pI230 disease resistance response protein (DRP), a trypsin inhibitor (TI) and glutathione-S-transferase (GST) in mycorrhizal roots of wild type, Pssym33 and Pssym40 genotypes. In this context, the activation of the fungal THIO and SOD genes in the mycorrhizal roots of wild type, Pssym33 and Pssym40 pea genotypes may enable the AM fungus to contend with plant defense/stress responses which are usually activated in mycorrhizal roots, especially within arbuscule-containing cells [START_REF] Gianinazzi-Pearson | Cellular and molecular defence-related root responses to invasion by arbuscular mycorrhizal fungi[END_REF].

The weaker expression of the G. intraradices RHO gene, encoding a putative Rho/GDP dissociation inhibitor, in Pssym36 roots could indicate effects on cell signaling events at the fungal-plant interface, as was suggested for mutations in the DMI2 and DMI3/MtSym13 genes of M. truncatula [START_REF] Seddas | Symbiosis-related plant genes modulate molecular responses in an arbuscular mycorrhizal fungus during early root interactions[END_REF]). The lower transcript levels in colonized Pssym36 roots of the PEPISOM and 26SREG genes which are implicated in cellular recognition [START_REF] Miele | A peptidylprolyl cis/trans isomerase from Xenopus laevis skin: cloning, biochemical characterization and putative role in the secretion[END_REF] and metabolic regulation/cell cycle progression [START_REF] Murray | Cyclin ubiquitination : the destructive end of mitosis[END_REF], respectively, may also contribute to or result from defective arbuscule development in the pea mutant. In this context, inactivation of either PsSym33 or PsSym40 was not detrimental to the expression of these three pea genes or to arbuscule development.

Exploitation of the microdissection technique confirmed that expression of the three G.

intraradices genes SOD, DESAT and PEPISOM does occur preferentially in arbuscule containing cells, supporting the above conclusions as to their eventual function in fungal interactions with the plant cells.

In conclusion, the present study contributes to a better understanding of the role of the PsSym36, PsSym33 and PsSym40 genes in the AM symbiosis and of the implication of molecular responses in the two partners during mycorrhiza formation and functioning, as well as progressing knowledge about PsSym36 and PsSym40 gene positioning on the pea genetic map. The most significant results obtained can be resumed as follows:

the Pssym36, Pssym33 and Pssym40 mutants have modified AM phenotypes which is in agreement with previous observations [START_REF] Gianinazzi-Pearson | Weak expression of the pathogenesis related PR-b1 gene and localization of related protein during symbiotic endomycorrhizal interactions in tobacco roots[END_REF], Jacobi et al., 2003 a, b): mutation in the PsSym36 gene affects arbuscule formation whilst the

  Pssym40. L'expression de la plupart des gènes fongiques a été inhibée dans les racines du mutant Pssym36 où la formation des arbuscules est avortée, tandis que l'expression de plusieurs d'entre eux a été activée lorsqu'il existe un développement plus rapide du champignon dans les racines du mutant Pssym40. Des microdisséquats obtenus à partir de racines mycorhizées du mutant PsSym40 confirment l'expression préférentielle de trois gènes de G. intraradices (SOD, DESAT et PEPISOM) dans les cellules contenant les arbuscules. L'inactivation du gène PsSym36 provoque également une inhibition des gènes de plante alors que la mutation des gènes PsSym33 and PsSym40 affecte l'expression des gènes de plante plutôt de façon temporelle. Les résultats indiquent ainsi une implication des gènes SYM de pois dans la modulation des interactions moléculaires entre la plante et le champignon impliquées au niveau de la signalisation, des échanges nutritifs ou de la régulation des réponses au stress durant la formation et/ou le fonctionnement de la symbiose AM. Les conditions pour la localisation des gènes PsSym36 and PsSym40 sur la carte génétique du pois ont été développées pour leur clonage basé sur la cartographie. En utilisant les marqueurs moléculaires obtenus, il a été possible de conclure que la localisation du gène PsSym40 réside vraisemblablement à l'extérieur des groupes de liaison I, II, III ou V de la carte génétique du pois.РЕЗЮМЕ Формирование арбускулярной микоризы (АМ) является результатом успешного взаимодействия между геномами двух симбиотических партнёров. Целью моего исследования являлось изучение роли поздних симбиотических генов гороха PsSym36, PsSym33 и PsSym40 в формировании функционального АМ симбиоза. Для этого было проведено исследование эффекта мутаций в генах PsSym36, PsSym33 и PsSym40 на экспрессию грибных и растительных генов, предположительно (по литературным данным) вовлечённых в процессы формирования АМ, а так же проведена работа по локализации генов PsSym36 и PsSym40 на генетической карте гороха для последующего более точного картирования и позиционного клонирования данных генов. Экспрессия десяти грибных и восьми растительных генов была определена в корнях растений дикого типа и PsSym36, PsSym33 и PsSym40 мутантов, инокулированных G. intraradices. В корнях PsSym36 мутанта, имеющего дефект развития арбускул, большая часть грибных генов была супрессирована, в то время как в корнях PsSym40 мутанта, для которого характерна более быстрая по сравнению с диким типом микоризация, был отмечен более высокий уровень экспрессии грибных генов. Использование метода микродиссекций позволило выделить клетки, содержащие арбускулы, из микоризованных корней мутанта PsSym40 и подтвердить, что гены G. intraradices SOD, DESAT и PEPISOM преимущественно экспрессируются в клетках, содержащих арбускулы. Мутация в гене PsSym36 также привела к подавлению экспрессии большинства вовлечённых в анализ растительных генов, тогда как мутации в генах PsSym33 и PsSym40 оказали влияние на эксперессию растительных генов в меньшей степени. Полученные результаты свидетельствуют о роли исследуемых SYM генов гороха в контролировании растительно-грибных молекулярных взаимодействий, связанных с сигналингом, обменом питательными веществами и стрессовыми реакциями в процессе формирования и функционирования АМ симбиоза. Проведённое генетическое картирование не привело к локализации генов PsSym36 и PsSym40 на генетической карте гороха. Однако разработка и использование молекулярных маркеров для картирования позволили исключить локализацию гена PsSym40 в I, II, III и V группах сцепления с высокой долей вероятности.

Figure I. 1 .

 1 Figure I.1. Schematic summarizing the different AM colonization patterns and associated

  truncatula. A set of genes associated with cell wall degradation and modification during AM symbiosis was identified by[START_REF] Hohnjec | Overlaps in the transcriptional profiles of Medicago truncatula roots inoculated with two different Glomus fungi provide insights into the genetic program activated during arbuscular mycorrhiza[END_REF] through a microarray analysis. Activation of genes coding for cell wall-degrading enzymes ((endo)-glucanases, pectinesterase, polygalacturonase, pectate lyase, α-D-xylosidase) was detected in the AM symbiosis. α-Dxylosidase is involved in the degradation of complex carbohydrates and a suggested function is to modify the extracellular matrix during fungal spread into the root and the formation of the PAM. Up-regulation of genes encoding proline-rich proteins, extensins or arabinogalactans points to cell wall modifications by the incorporation of structural or glycosylated proteins(Honhject et al., 2005).

  plasma membranes. Nevertheless, the question about the site of carbon transfer in the AM symbiosis remains open. A substantial amount of the hexose C transferred from plant to AM fungal cells can be transformed to storage lipids, mostly in the form of triacylglyceroles (TAGs). Most of the carbohydrates are known to be metabolized in the intraradical mycelium, and transport of C to the extraradical mycelium is performed in the form of TAGs and glycogen (see figure I.2; Bagoet al., 2003;[START_REF] Délano-Frier | Novel developments in the understanding of signaling and nutrient exchange mechanisms in the arbuscular mycorrhizal symbiosis[END_REF].

Figure I. 2 .

 2 Figure I.2. Biochemical pathways of carbon metabolism active in symbiotic intraradical and

Figure I. 3 .

 3 Figure I.3. Model of nitrogen movement in the arbuscular mycorrhizal symbiosis (Govindarajulu

  genes in plant and fungal gene regulation associated with the different stages of symbiosis development. Responses of plant genes associated with signaling pathways at the appressorium stage were compared byWeidman et al. (2004) and[START_REF] Sanchez | Pseudomonas fluorescens and Glomus mosseae trigger DMI3dependent activation of genes related to a signal transduction pathway in roots of Medicago truncatula[END_REF] in wild type and Myc -dmi3 roots of M. truncatula roots inoculated with G. mosseae. Weidman et al.(2004) identified 11 plant genes activated from the appressorium stage up to the fully established AM symbiosis. Expression of these genes in roots of the dmi3 mutant was not activated by appressorium formation or diffusible fungal molecules, indicating lack of recognition of AM fungus by the dmi3 mutant. Likewise, none of the plant genes identified by[START_REF] Sanchez | Pseudomonas fluorescens and Glomus mosseae trigger DMI3dependent activation of genes related to a signal transduction pathway in roots of Medicago truncatula[END_REF], coding for proteins associated with a putative signal transduction pathway and induced during early stages of AM colonization, were responded to inoculation of roots of the dmi3 mutant.

Figure I. 4 .

 4 Figure I.4. Signaling pathways of arbuscular mycorrhizal and nitrogen fixing symbioses (Smith

  Such a strategy based on the positional cloning of a gene of interest in model legume species and subsequent homology-based cloning of the orthologous gene in pea has been applied quite often. Up to day, artificial chromosomes available in the model legume species L. japonicus and M. truncatula significantly simplify the process of map-based (or positional-based) gene cloning.

  [START_REF] Zhu | Bridging model and crop legumes through comparative genomics[END_REF] demonstrated macrosyntenic relationships between legume species in a simplified consensus map(Figure I.5).

Figure I. 5 .

 5 Figure I.5. A simplified consensus comparative map of legume species[START_REF] Zhu | Bridging model and crop legumes through comparative genomics[END_REF].

  My thesis work has been performed within the framework of a co-operation programme between the Laboratory of Genetics of Plant-Microbe Interactions (ARRIAM, St. Petersburg, Russia) and the Plant-Microbe-Environment Research Unit (UMR INRA 1088/CNRS 5184/Université de Bourgogne PME, France). The research focuses on the characterization of the late symbiotic pea (Pisum sativum L.) genes PsSym36, PsSym33 and PsSym40 in arbuscular mycorrhiza formation and functioning. In this context, the objectives of my thesis were: to monitor effects of PsSym36, PsSym33 and PsSym40 gene mutation on G.intraradices gene expression, to monitor effects of PsSym36, PsSym33 and PsSym40 gene mutation on plant gene expression in order to identify those involved in late stage of symbiotic interactions, to determine G.intraradices genes expression related to arbuscule formation stage of AM symbiosis, to localize the symbiotic pea genes PsSym36 and PsSym40 on the pea genetic map in order to create conditions for future map-based cloning of theses genes. The research focuses on the characterization of fungal and plant gene responses during AM formation and functioning was performed in 'Plant-Microbe-Environment' Mixed Research Unit, INRA Dijon. In this part of my study, the pea symbiotic mutants Pssym36, defective for arbuscule formation[START_REF] Engvild | Nodulation and nitrogen fixation mutants of pea (Pisum sativum)[END_REF], Pssym40, and Pssym33 having alterations in rate of AM development(Jacobi et al., 2003 a, b) and their corresponding wild type genotypes were used as genetic background for investigations of plant and fungal (Glomus intraradices) gene expression. The aim of exploiting the symbiotic pea mutants was to gain insight into the possible symbiotic function of the investigated genes in the AM symbiosis. G. intraradices genes were chosen amongst those that have been previously shown to be specifically upregulated during AM symbiosis establishment by exploitation of early symbiotic mutants of M.

  RisNod24 (Pssym36) (provided by Gerard Duc, UMR Légumineuses à Graines, INRA, Dijon, France) ♦ line SGE and its corresponding mutant lines SGEFix -2 (Pssym33), SGEFix -1 (Pssym40) (provided by Alex Y. Borisov, Laboratory of Plant-Microbial Interactions, ARRIAM, Saint-Petersburg, Russia) ♦ laboratory lines NGB1238, NGB851 and NGB2715 (provided by Stig Blixt, Nordic Gene Bank, Alnarp, Sweden) ♦ lines K3034, K1693, K7128 (from the N. I. Vavilov Institute of Plant Industry (VIR) collection) AM fungal isolate: The arbuscular mycorrhizal fungus Glomus intraradices Schenck & Smith (BEG 141) was supplied by the International Bank for Glomeromycota (IBG, http://www.ukc.ac.uk/bio/beg/) (INRA/Dijon) where it was produced on Allium porrum L. in pot cultures in neutral γ-irradiated Epoisses clay-loam soil. Root nodule bacteria: Rhizobium leguminosarum bv. vicia strain CIAM1026 supplied by All-Russia Collection of Nonpathogenic Microorganism for Agriculture (ARRIAM, Saint-Petersburg, Russia).

  in ceramic mortars in liquid nitrogen and transferred into plastic 50 ml tubes containing NTES extraction buffer (50 mM Tris/HCl, pH 9; 150 mM NaCl; 5 mM EDTA; 5% SDS; 1% βmercaptoethanol; 1.6 buffer volume (ml) per mg sample). An equal volume of phenol:chloroform:isoamyl alcohol solution (mixed by volume as 25:24:1, correspondingly) was added to remove proteins. Tubes were inverted several times to mix, then centrifuged 15 minutes at 14500 g at room temperature, and the upper aqueous phase transferred into new 1.5 ml eppendorf tubes. This procedure was repeated twice with the phenol:chloroform:isoamyl alcohol solution and once with pure chloroform. To precipitate RNA and eliminate polysaccharides, 0.7 vol of 96% ethanol (from -20°C) and 0.05 vol of 1 M acetic acid were added to the aqueous phase and left overnight at -20°C. The next day, tubes were centrifuged for 35 min at 10000 g at 4°C and the liquid phase was discarded. The pellet was resuspended in DEPC water (vol=vol NTES/4) and RNA was specifically precipitated from the solution by 4 M LiCl (1 vol) treatment for 4 h at 4°C. Tubes were centrifuged for 35 min at 10000 g at 4°C, the aqueous phase was discarded, the pellet was resuspended in DEPC water and another RNA precipitation was performed overnight with 3 M NaAc (0.1 vol) and 96 % ethanol (3 vol) at -20°C. The final pellet was air-dried at room temperature and dissolved in 40 μl RNase-free water. Quantity and quality of RNA were checked in an Eppendorf BioPhotometer and RNA integrity was checked by electrophoresis in a denaturing agarose gel (seeII.8.2). The DNasecontaining columns of the Promega kit for total RNA isolation (SV Total RNA Isolation System)

. 4 .

 4 Specific primers for G. intraradices gene expression

  primer and dNTPs were preheated at 70 0 C in a thermocycler for 5 min and cooled on ice for 3 min. Reverse transcriptase, reaction buffer, RNasine and distilled water were then added to obtain a final mix volume of 25 μl and the reaction was performed under conditions recommended by[START_REF] Weidmann | Fungal elicitation of signal transduction-related plant genes precedes mycorrhiza establishment and requires the dmi3 gene in Medicago truncatula[END_REF] (25 0 C for 15 min, 42 0 C for 1 h and 96 0 C for 2 min).cDNA was stored at -20°C. aRNA amplification. RNA extracted from laser microdissected arbuscular-containing cells (see II.6.4) was amplified using TargetAmp™2-Round aRNA amplification kit (Epicentre Biotechnologies, Madison,WI) and 40 pg -200 pg of total RNA in the following protocol.

  GenomeLab™ DTCS Quick Start Kit (Beckman Coulter). To purify PCR products for DNA sequencing, 40 μl of PCR reaction was loaded into 1% agarose gel. PCR products were cut from the gel and dissolved in solution A (1 μl of solution A to 1 μg of gel) for 5 min at 65°C. 50 μl of solution B containing silica was added to each reaction, tubes were vortex well and centrifuged for 1 min at 3000 rpm. The liquid phase was discarded then the pellet was rinsed sequentially with 100 μl of solution A, 100 μl of solution C, 100 μl of 70% ethanol and dried for 10 min at 37°C. DNA was washed from the silica particle using 10 μl of 10 mM Tris-HCl pH 8.0 and stored at -20°C. Composition of solution A: 3 M guanidine(iso)tiocianat, 20 mM Na 2 EDTA, 10 mM Tris-HCl pH=6.8, 40 mg/ml Triton X-100, 20 mg/ml β-mercaptoethanol Composition of solution B: 40 mg/ml Silica in solution A Composition of solution C: 25% ethanol, 25% isopropanol, 100 mM NaCl, 10 mM Tris-HCl pH=8.0 Reaction was performed in 5 μl full mix containing 25 fmol PCR product, 1 μl of DTCS mix (supplied by the kit), 0.25 μl of reaction buffer (supplied by the kit) and 1.6 pmol of original primer following the manufacturer's protocol.

Figure♦

  Figure II.2. Molecular marker development

  early time-points of development. The mutation in the gene PsSym33 results in decreased mycorrhizal colonization of roots but does not affect arbuscule abundance in the mycorrhizal root fragments. Transmission electron microscopy of arbuscule-containing cells revealed differences in the dynamics of arbuscule development between mutant and wild type plants in fully-developed mycorrhiza at an early flowering stage (Jacobi et al, 2003 b). The majority of cells of the Pssym40 mutant contained arbuscules in an almost fully-degraded state. In contrast, the large majority of cells in roots of the Pssym33 contained arbuscules without signs of degradation. In comparison, roots of the wild type contained cells belonging to all defined groups: cells with arbuscules without signs of degradation, cells with degrading and degraded arbuscules (see figure III.1.). Based on these data, Jacobi et al. (2003 b) concluded that the Pssym40 mutant accelerated mycorrhiza development and increased rate of arbuscule turnover, whilst Pssym33 has a slower rate of AM development than wild type plants.

Figure III. 1 .

 1 Figure III.1. Proportion of cells containing arbuscules in different developmental stages in roots

  Mycorrhization parameters were estimated by ink staining and alkaline phosphatase-active fungal structures at 21 and 28 days after inoculation (dai) with G. intraradices (see Material and Methods II.3.). The high frequency of mycorrhiza development in colonized roots of the wild type, Pssym33 and Pssym40 mutant genotypes (parameter F) indicated high inoculum infectivity (table III.1 -4). Pssym36 mutant versus wild type cv Finale The mycorrhization of Pssym36 mutant roots was much lower than wild type cv Finale, as indicated by all mycorrhization parameters (tables III.1 and III.2). Only aborted arbuscules with stumpy branches were observed within root cortical cells (see figure III.2).

Figure III. 2 .

 2 Figure III.2. Light microscopy of mycorrhizal roots of wild type and Pssym36 mutant plants

Figure III. 3 .Figure III. 4 .Figure III. 5 .Figure III. 6 . 2 (

 34562 Figure III.3. Shoot biomass accumulation of wild type (wt) cv Finale and the RisNod24

  Pssym36 mutant Mutation in the PsSym36 gene affected expression of all tested G. intaradices genes. At 21 dai, RHO, PEPISOM, DESAT, THIO, SOD and VACU genes were significantly down-regulated in colonized roots of the Pssym36 mutant in comparison with wild type cv Finale roots (see figure IV.1). Differences in fungal gene expression during colonization of cv Finale and Pssym36 roots were greatest for the PEPISOM (4.7 fold), DESAT (39 fold), THIO (5.6 fold) and SOD (8.3 fold).

Figure IV. 1 .

 1 Figure IV.1. G. intraradices gene expression in colonized roots of wild type cv Finale ( ) and mutant Pssym36 ( ). Transcript abundance of each gene, measured by real-time RT-PCR, is expressed relative to that of the house-keeping fungal TEF gene. Letters indicate significant differences between treatments (P≤0.05, n=3).

Figure IV. 2 .

 2 Figure IV.2. G. intraradices gene expression in colonized roots of wild type SGE ( ) and mutant Pssym33 ( ). Transcript abundance of each gene, measured by real-time RT-PCR, is expressed relative to that of the house-keeping fungal TEF gene. Letters indicate significant differences between treatments (P≤0.05, n=3).

Figure IV. 3 .

 3 Figure IV.3. G. intraradices gene expression in colonized roots of wild type SGE ( ) and mutant Pssym40 ( ). Transcript abundance of each gene, measured by real-time RT-PCR, is expressed relative to that of the house-keeping fungal TEF gene. Letters indicate significant differences between treatments (P≤0.05, n=3).

  THIO and SOD genes was significantly decreased in G. intraradices-colonized roots of the Pssym36 mutant in the absence of full arbuscule development. In conclusions, mutation of the PsSym36 gene had a stronger effect on G. intraradces gene expression, as compared with the Pssym40 and Pssym33 mutants. Expression of most of the tested fungal genes was considerably repressed in G. intraradices-colonized roots of the Pssym36 mutant as compared with those of mycorrhizal wild type cv Finale. This could be the result of incomplete arbuscule development and the impossibility for nutrient exchanges between the fungal and plant cells in the absence of a functional symbiotic interface. Mutation in the PsSym40 gene resulted in an increase in the expression of most of the investigated G. intraradices genes, which could be associated with the more rapid AM symbiosis development in roots of the Pssym40 mutant. Of the three tested pea mutants, Pssym33 had less pronounced effects on G. intraradices gene expression as well as on AM formation and function (see chapter III).

Figure V. 1 .

 1 Figure V.1. Expression of pea genes in mycorrhizal ( ) and non-mycorrhizal ( ) roots of wild type cv Finale and in colonized ( ) and non-colonized ( ) roots of the mutant Pssym36, 21 and 28 days after inoculation with G. intraradices. Transcript abundance of each gene, measured by real-time RT-PCR, is expressed relative to that of the house-keeping pea GAPDH gene. Different letters indicate significant differences between inoculated and non-inoculated plants for each pea genotype (P≤0.05, n=3).

Figure V. 2 .

 2 Figure V.2. Expression of pea genes in mycorrhizal and non-mycorrhizal roots of the wild type SGE genotype ( , ) and of the Pssym33 mutant ( , ), 21 and 28 days after inoculation with G. intraradices. Transcript abundance of each gene, measured by real-time RT-PCR, is expressed relative to that of the house-keeping pea GAPDH gene. Different letters indicate significant differences between inoculated and non-inoculated plants for each pea genotype (P≤0.05, n=3).

Figure V. 3 .

 3 Figure V.3. Expression of pea genes in mycorrhizal and non-mycorrhizal roots of the wild type SGE genotype ( , ) and of the Pssym40 mutant ( , ), 21 and 28 days after inoculation with G. intraradices. Transcript abundance of each gene, measured by real-time RT-PCR, is expressed relative to that of the house-keeping pea GAPDH gene. Different letters indicate significant differences between inoculated and non-inoculated plants for each pea genotype (P≤0.05, n=3).

  gene) was affected by mycorrhiza formation and functioning in wild type cv Finale and SGE roots, compared to non inoculated roots. At 21 dai in cv Finale roots colonized by G. intraradices, TI and GST genes were up-regulated, whilst the DRP, MAPK and SERPROT genes were all down-regulated. Similar trends persisted for the TI, GST and MAPK genes at 28 dai, whilst DRP became up-regulated in mycorrhizal roots. At this time point, development of G. intraradices within cv Finale roots significantly repressed expression of the BCOP gene, and activated the PT4 gene. In roots of the mutant Pssym36, only GST expression was significantly modified (up-regulated) by G. intraradices colonization at 21 dai; no significant responses were observed for any of the other seven plant genes at either time point which could reflect absence of signalling responses from the plant side to AM fungal colonization in roots of the Pssym36 mutant. When plant gene responses to G. intraradices in Pssym33 and Pssym40 were compared to those in the wild type SGE line, modifications in gene expression were of the same tendency as for the wild type genotype in the two analyses: PT4, TI, GST and SERPROT were up-regulated, and BCOP and MAPK down-regulated in mycorrhizal roots. In conclusions, mutations of pea PsSym36, PsSym33 and PsSym40 genes modify both G. intraradices and pea gene expression during AM interactions. Mutation of the PsSym36 pea gene results in strong alterations in the process of AM symbiosis development that are reflected not only in defects in fungal morphogenesis but also in reduced expression of plant genes which may be connected with arbuscule formation and/or functioning. Inactivation of the PsSym40 and PsSym33 genes has less pronounced effects on the AM symbiosis. Modifications in pea gene expression in roots of the Pssym33 and Pssym40 mutants were less significant and more time dependent. Chapter VI. Transcriptome profiling of G. intraradices genes in arbusculecontaining cortical cells of the Pssym40 mutant VI.1. IntroductionOne of the objectives of this study was to verify expression of some investigated genes during arbuscule development in the pea AM symbiosis. For this, three G. intraradices genes SOD, PEPISOM and DESAT were chosen as candidate genes expressed in arbuscules. These fungal genes were active in mycorrhizal roots of both cv Finale and SGE wild type genotypes and in Pssym33 and Pssym40 mutants. At the same time, their expression was significantly lower in the arbuscule-defective Pssym36 mutant, suggesting a role in arbuscule development and/or functioning. To localize their expression, arbuscule-containing cells from mycorrhizal roots of the Pssym40 mutant were dissected using a laser capture microdissection system (see Material and Methods II.5.

Figure VI. 2 .

 2 Figure VI.2. G. intraradices gene expression in whole sections of colonized Pssym40 roots ( )

. 2 )

 2 and continuation of the investigations is necessary. In conclusion, the performed study has permitted some advance towards defining the localization of the PsSym40 gene; the plant material and data obtained will facilitate further investigations.

Figure VII. 2 .

 2 Figure VII.2. Location of gene-anchored markers on the pea genetic map with regions of the

  molecular markers were developed from these and segregation analysis in the F2 population from a SGEFix --1(Pssym40)×NGB1238 cross was performed. Whilst the position of some of the molecular markers was located on the pea genetic map, neither the PsSym36 nor the PsSym40 genes could be localized. There is a high probability of the PsSym40 gene being located outside the molecular marker regions of LGI, LGII, LGIII or LGV. Symbiosis-associated fungal and plant molecular responses were monitored to study the effect of inactivation of late stage symbiosis-related pea genes PsSym36, PsSym33 and PsSym40 on AM functioning. This part of my research work was performed in the Plant-Microbe-Environment Research Unit, Dijon. Expression of fungal and plant genes was analyzed in roots of the Pssym36, Pssym33 and Pssym40 mutants and corresponding wild type cv Finale and SGE plants at 21 and 28 day after inoculation with G. intraradices by real-time RT-PCR. It was shown that mutations in the PsSym36, PsSym33 and PsSym40 genes modify both fungal and plant responses during AM establishment but mutation of the PsSym36 gene had a greater impact than inactivation of the PsSym33 and PsSym40 genes. All the studied G. intraradices genes were expressed in roots of the mycorrhizal wild type and mutant pea genotypes. Most of them were strongly down-regulated in roots of the Pssym36 mutant and up-regulated in roots of the Pssym40 mutant, which could be associated with the absence of full arbuscule development in roots of the Pssym36 mutant and the more rapid AM symbiosis development in roots of the Pssym40 mutant. Of the three tested pea mutants, Pssym33 had less pronounced effects on G. intraradices gene expression as well as on AM formation and function. Expression of selected pea genes for this study was less affected by Pssym36, Pssym33 and Pssym40 mutations than fungal gene responses.

Pssym33

  and Pssym40 mutants have alterations in rate of AM development, the pea genes PsSym36, PsSym33 and PsSym40 not only control root colonization and morphological differentiation of an AM fungus but they also influence plant and fungal gene responses linked to symbiotic interactions between the partners, mutation in the PsSym36 gene results in strong alterations in G. intraradices and P. sativum gene expression, mostly down-regulation, which is coherent with a role of the PsSym36 gene in the coordinated cellular development in plant and fungus to elaborate a functional interface in arbuscule-containing cells, mutation in the PsSym33 gene has less pronounced effects on plant and AM fungal gene expression as well as on AM formation and functioning than mutations in PsSym36 or PsSym40, mutation in the PsSym40 gene results in an increased expression of most of the investigated G. intraradices genes and some of the plant genes which may be associated with the more rapid mycorrhiza development in Pssym40 roots, observed up-regulation of G. intraradices SOD, DESAT and PEPISON genes in microdissected arbuscule-containing cells provides strong evidence of the arbusculerelated expression of these fungal genes, 123 localization of the PsSym40 gene in linkage group I, II, III or V of the genetic map of pea can be almost excluded. The methodology and molecular tools developed in the present study will be useful for further research activities with respect to other plant genes controlling development and functioning of the AM symbiosis, including those yet to be discovered. The plant material (mapping populations), molecular markers and data obtained will facilitate future experiments to localize the PsSym36 and PsSym40 genes on the pea genetic map and create conditions for mapbased cloning of these genes. The gene expression data obtained in the present study contribute to the accumulating knowledge about plant, fungal and bacterial molecular responses to interactions between symbiotic partners in legumes. They provide a basis for further research to discover the role of the pea genes PsSym36, PsSym33 and PsSym40 (and their orthologs in other legume species) in the development of both the fungal and bacterial symbioses of legume plants, to decipher interrelated signal transduction pathways during development of such complex plant-microbe systems and to facilitate creation of approaches to manage such systems in sustainable agriculture.

  The author thanks Ms. Anne Colombet and Ms. Valérie Monfort-Pimet, members of the International Bank of Glomeromycota, INRA, Dijon, France, for their help in conducting experiments with mycorhiza. Great thanks to all the members of the Laboratory of Genetics of Plant-microbe Interactions ARRIAM: O. Y. S(h)tark, G. A. Akhtemova, V. Zhukov, A. Zhernakov, T. Nemankin, E. Ovchinnikova, L. Dvoryaninova, T. Rychagova for a chance to work together, their support and concern. Sincere thanks to all the colleges of PME INRA Mr. Diederik van Tuinen, Ms. Odile Chatagnier, Marie Tollot, Zeina Daher, Aloui Achref, Zhipeng Hao and all the rest for a chance to work together, their support and concern.

Table I .

 I

	Gene symbol NFS phenotype I.3AM phenotype

. Molecular background of the AM symbiosis I.3.1. Early signaling events

  Successful root colonization and a functional interaction between host plant and AM fungi are based on exchange of signaling molecules at different stages of symbiosis development.

  . In pea, SYM genes common to both symbioses are essential for comparable stages in partner interactions: fungal and bacterial root penetration

	(PsSYM8, PsSYM9/PsSYM30, PsSYM19), arbuscule development and infection thread growth
	inside root hairs (PsSYM36), rate of mycorhization/arbuscule turnover and infection thread
	growth or infection droplet differentiation (PsSYM14, PsSYM33, PsSYM40, PsSym41) (see
	table I.1).
	Researchers have paid special attention to early steps of symbioses establishment. Analysis of
	signaling pathways taking place during root colonization by AM fungi and rhizobial bacteria have
	revealed that a legume host plant utilizes a partially common Ca 2+ -dependent pathway for both

symbioses (see figure I.4).

Table I

 I

						Radutoiu et al., 2003
	PsSym37	MtHcl	LjNfr1	Myc + Nod -	receptor-like	Limpens et al., 2003
		(MtLyk3)			kinase	Smit et al., 2007
						Zhukov et al., 2008
	Myc + -wild type AM formation, Myc --blocked AM formation, Myc ++ -increased AM formation,
	Nod + -wild type root nodule formation, Nod --blocked root nodule formation, Nod ++ -increased
	root nodule formation.
	.4.1. Cloned symbiotic genes of pea and their orthologues
	I.4.2.
		Gene name		Mutant
					Encoding protein	References
	P. sativum M. truncatula	L. japonicus	phenotype
					transcriptional	Kaló et al., 2005
	PsSym7	MtNsp2	LjNsp2	Myc + Nod -	regulator	Murakami et al., 2006
					GRAS family
						Ané et al., 2004
	PsSym8	MtDMI1	LjCastor,	Myc -Nod -	multimeric	Imaizumi-Anraku et al.,
			LjPollux		ion channel
						2005
					Ca 2+ /calmodulin-	Mitra et al., 2004
	PsSym9	MtDMI3	LjCCaMK	Myc -Nod -	dependent	Lévy et al., 2004
					protein kinase	Tirichine et al., 2006
						Radutoiu et al., 2003
	PsSym10	MtNfp	LjNfr5	Myc + Nod -	receptor-like	Madsen et al., 2003
					kinase
						Arrighi et al., 2006
	PsSym19	MtDMI2	LjSYMRK	Myc -Nod -	receptor-like	Stracke et al., 2002
					kinase	Endre et al., 2002
						Krusell et al., 2002
	PsSym29	MtSunn	LjHar1	Myc ++ Nod ++	receptor-like	Nishimura et al., 2002
					kinase
						Schnabel et al., 2005
						Schauser et al., 1999
	PsSym35	MtNin	LjNin	Myc + Nod -	transcriptional	Borisov et al., 2003
					regulator
						Marsh et al., 2007

Expressed sequence tag-based molecular markers for gene mapping

The genetic map of pea has been developed gradually, starting with the first version by Lamprecht in 1948 up to the significant advance in the last years resulting from the development of DNA techniques for molecular marker creation.

A number of methods allowing detection of random nucleotide sequence polymorphism, such as RAPD (Random Amplified Polymorphic DNA), AFLP (Amplified Fragment Length Polymorphism) and RFLP (Restriction Fragment Length Polymorphism), have led to the creation of moderate density pea linkage maps

  1:1 mixture of quartz sand and mycorrhizal soil-based inoculum produced in pot cultures, or quartz sand and gamma-irradiated and heat-sterilized Epoisses soil for controls. Non-inoculated controls received a filtered (Whatman n°2) water suspension of inoculum to introduce the associated microflora. Plants were raised in a growth cabinet under control conditions (24/19 0 C, 16 h light, 420 μmol m -2 s -1 , 70 % relative humidity), fertilized with 10 ml/pot of a modified Long Ashton solution (TableII.1, Hewitt, 1966) without phosphorus three times a week, or 10 ml of distilled water on other days.Plants were harvested at 7, 14, 21 or 28 after inoculation with G. intraradices and weighed fresh, after washing the root systems in ice-cold water. Root samples were taken for RNA extraction and stored in liquid nitrogen.

	Tab.II.1. Modified Long Ashton solution
	Component	Concentration, mg/l	Component	Concentration, mg/l
	KNO 3	808	ZnSO 4 × 7H 2 O	0.29
	Ca(NO 3 ) 2 × 4H 2 O	1890	H 3 BO 3	3.10
	MgSO 4 × 7H 2 O	368	NaCl	5.90
	MnSO 4 × H 2 O	2.23	H 24 Mo 7 N 5 O 24 × 4H 2 O	0.088
	CuSO 4 × 5H 2 O	0.25	FeNa-EDTA	22
	For genetic crossing experiments
	Seeds were surface-disinfected by a 25-min treatment with 98% sulphuric acid, washed with
	distilled water and directly soaked in distilled water for 2 h before sowing. Plants were grown in

4l pots in a sod-podzol sandy soil (pH KCl 5.5, P 2 O 5 18.5 mg/100g soil, K 2 O 4.3 mg/100g soil) (parental lines for crossing, F1 population of plants) or sterile quartz send (F2 population plants), 4 plants per pot, in greenhouse conditions in ARRIAM.

4. Laser Capture Microdissection (LCM) of arbuscule-containing cortical root cells II.4.1. Tissue preparation

  8 mg/ml MnCl 2 × 4H 2 O). The next day, root samples were washed with distilled water, destained in hypochlorite solution (containing 1% active chlorine) for 5 min at room temperature, and rinsed with water again. with 100% ethanol for 1 hour. After the last ethanol step, root tissues were treated at room temperature with a 100% ethanol (VWR ref: 370720601)/histoclear II solution series (3v/1v, 1v/1v and 1v/3v) for 30 min each. They were then embedded in paraffin (paraplast plus SIGMA réf P 3683) through a series of histoclear II /paraffin solutions, in which the histoclear II was gradually replaced by paraffin (15 pastilles of paraffin/25 ml of histoclear II, 30 pastilles of paraffin/25 ml of histoclear II, 45 pastilles of paraffin/25 ml of histoclear II and pure paraffin). Roots were incubated at 55°C for 1h in each paraffin-containing solution, and subsequently overnight in pure paraffin. Embedded samples were stored in pure paraffin at 4°C in small Petri dishes until use. Selected cells were marked using graphical software, cut twice with an IR laser (810 nm, laser spot size of 10 µm, laser Power of 69 mw, laser pulse duration of 25 ms), and captured automatically into CapSure HS LCM Caps (Arcturus). Harvested cells were transferred into RNA extraction buffer (Picopure Kit Arcturus) within a maximum of 30 min after dissection had begun. RNA was frozen at -80°C and stored until extraction (seeII.7.4).

	Mycorrhizal roots were cut into 1-cm pieces and fixed in freshly prepared Farmer's fixative
	solution (acetic acid/absolute ethanol [1v/3v]) overnight at 4°C. The next day, the liquid was

For light microscopy, 30 1cm-long root pieces were mounted on glass slides, observed according to

Trouvelot et al. (1986)

, and colonization estimated using the Mycocalc programm (http://www2.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 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).

II.

discarded and root tissues were rinsed twice with 96% ethanol, incubated for 15 min with 96% ethanol at 4°C, then incubated for 15 min with ethanol/eosin (SIGMA ALDRICH 861006 solution (0.1% in DEPC (diethyl pyrocarbonate) water) at room temperature, rinsed with 100% ethanol, and incubated Sectioning (10-μm) was performed at room temperature using a Jung RM 2065 (Leica) microtome and RNAse-free tools. Sections were placed on a slide, stretched with few drops of distilled RNase-DNase-free water and kept in a slide warmer at 42°C overnight. Just before use, sections were deparaffinized by treating slides twice with histoclear II for 5 min, histoclear II/100%ethanol solution (1v/1v) for 2 min, 100 % ethanol for 2 min, and fresh 100% ethanol for 1 min. Deparaffinized slides were placed in a desiccator for 1 hour before dissection.

II.4.2. Dissection of arbuscule-containing cells

Dissection was performed using an Arcturus™Microdissection instrument (ARCTURUS NIKON inverse XT100). Slides with sections were visualized on a computer monitor through a video camera.

Table II

 II

					60
	Glutathione-S-transferase	ACCAAAAGGTGCCTGTGTTC
				133	58
	(GST)		CTAGCTTGTGCGCGATCATA
			GCACTGGCACACCCTTACTTG
	MAP kinase (MAPK)		154	60
			GGGGTTAAATGCTAGAGCTTCTCTG
			AGAGTAGCCAAAGGTCAAGCAGT
	Serine protease (SERPROT)		253	58
			GACCTCTTGATGAAAATGAAGCA
	Glyceraldehyde	phosphate	AAGAACGACGAACTCACCG
				187	55 to 60
	dehydrogenase (GAPDH)	TTGGCACCACCCTTCAAATG
	For molecular marker creation
	Primers were designed based on information about EST nucleotide sequences of genes for
	.5. Specific primers for P. sativum gene expression analysis
	Gene		Primer sequences	Expected size, bp Tm, °C
	Plasma membrane ATPase	TACAGGTGAGTCCCTGCCG
	(H + ATPase)		CCCAATTGCAGTCAAGACCT	252	60
	Inorganic phosphate	CTTCACGTGCCATGTTCATC
				273	58
	transporter (PT4)		GCGTCGGAAACAGCTCC
	Blue copper binding protein	GTTGGGTGATTGGTGGTGA
				187	55
	(BCOP)		AAGAGGAATGGTTGTCGCAC
	Disease resistance protein	GCGGCCGAGGTCAAGAAATAG
				278	58
	pI230 (DRP)		CCGGGCAGGTACAAATACTGC

P. sativum. For some genes, primer sequence information was taken from the literature

[START_REF] Morin | Functional mapping in pea, as an aid to the candidate gene selection and for investigating synteny with the model legume Medicago truncatula[END_REF]

. The list of ESTs and primers used is presented in table VII.1 and annex 2, respectively.

II.8.3. cDNA synthesis

Reverse transcription-PCR (RT-PCR). cDNA was synthesized from 1 μg DNA-free RNA using 300 U M-MLV Reverse transcriptase RNase H Minus (Promega), 1 μg oligo(dT) 15 primer (Promega), 40 U RNasine RNA inhibitor (Promega) and 7.5 mM dNTPs. RNA, oligo(dT)

[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]

  three technical replicates and analyzed by the SDS 2.2 program (Applied Biosystems, Foster City, USA). Expression analyses for each gene were performed in replicate RNA batches (three biologically independent experiments) from G. intraradices-inoculated or non-inoculated roots of each wild type pea genotype and the corresponding mutants.

	To calculate the absolute amount of transcripts present in original samples, plasmid DNA
	containing each amplicon was prepared (see II.7.3.), quantified by UV absorbance
	spectroscopy, and linearized by restriction enzyme digestion (see II.11). Standard amplification
	curves were determined from duplicate samples of plasmid DNA at dilutions of 10 2 , 10 3 , 10 4 ,
	10 5 , 10 6 , and 10 7 copies for each assay. Relative amounts of transcripts for each gene were

calculated as a ratio to the amount of translation elongation factor 1, α-subunit (TEF) transcripts as the fungal reference gene

[START_REF] Seddas | Symbiosis-related plant genes modulate molecular responses in an arbuscular mycorrhizal fungus during early root interactions[END_REF]

or of glyceraldehyde phosphate dehydrogenase (GAPDH) transcripts as the plant reference gene

[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 DMI3dependent activation of genes related to a signal transduction pathway in roots of Medicago truncatula[END_REF]

.

11. Digestion of DNA fragments by restriction enzymes

	Plasmid DNA digestion
	Reactions were performed in 50 μl final volume mix containing 300 ng of plasmide DNA, 1 unit
	of restriction enzyme NotI, ×1 reaction buffer (corresponding to this restriction enzyme) and
	sterile water (qsp 50 μl) and incubated for 3 hours at 37°C. Effectiveness of digestion was
	checked by electrophoresis in 1% agarose gel (see II.7.1.).
	PCR fragment digestion
	PCR fragment digestion was used for CAPS (Cleavage Amplified Polymorphic Sequences)
	Optimal broth with Catabolite repression: 2% [m/V] bacto-tryptone, 0.5% yeast extract, 10 mM
	NaCl, 2.5 mM KCl, 20 mM Mg [MgCl 2 ×6H 2 0/MgSO 4 ×7H 2 O], 20 mM glucose) for 1 hour at 37°C.
	200 μl of the cell culture were transferred into Petri dishes containing LB medium (Luria Broth:
	1% [m/vol] bacto-tryptone, 0.5% [m/vol] yeast extract, 5% [m/vol] NaCl pH 7.0, 1.5% [m/vol]
	bacto-agar) containing ampicillin (50 μg/ml), IPTG (20 μg/ml), X-Gal (20 μg/ml) and incubated
	overnight at 37°C. Recombination was checked by white-blue selection (recombinant colonies
	are white) and by PCR. Recombinant colonies were transferred into liquid LB medium
	containing ampicillin (50 μg/ml) and grown overnight at 37°C with shaking. Plasmid DNA was
	extracted as described in chapter II.7.3.
	Cloned PCR products were sent for sequencing to NWG Biotech (Ebersberg, Germany).

II.

marker analysis. The reaction mix contained 10 μl of PCR product, 1 unit of restriction enzyme, ×1 reaction buffer (recommended to be used with restriction enzyme) and sterile water (qps 20 μl). A set of restriction enzymes provided by Fermentas (Lithuania) and CибЭнзим (Russia)

Table III .

 III 1. Mycorrhization parameters of the wild type (wt) cv Finale line and mutant RisNod24 (Pssym36) line at 21 days after inoculation with G. intraradices. F -frequency of mycorrhiza in the root system; M -intensity of mycorrhizal colonization in the root system; m -intensity of mycorrhizal colonization in mycorrhizal root fragments; A -arbuscule abundance in the root system; a -arbuscule abundance in mycorrhizal root fragments. mutants and wild type became less significant (see tableIII.4). The rate of root colonization (M, m) of Pssym33 was still significantly less but arbuscule abundance rose to wild type levels. Colonization of Pssym40 roots slowed down. No significant differences in mycorrhizal colonization and arbuscule abundance between wild type and Pssym40 roots were detected at 28 dai. Table III.3. Mycorrhization parameters of the wild type (wt) SGE line and mutant SGEFix --2 (Pssym33) and SGEFix --1 (Pssym40) lines at 21 days after inoculation with G. intraradices. Table III.4. Mycorrhization parameters of the wild type (wt) SGE line and mutant SGEFix --2 (Pssym33) and SGEFix --1 (Pssym40) lines at 28 days after inoculation with G. intraradices. Mycorrhiza parameter abbreviations are those defined in table III.1. at 28 dai both parameters were significantly lower in inoculated roots than in nonmycorrhizal ones. Wild type cv Finale shoot and root biomass accumulation was not modified by root colonization by G. intraradices at 21 dai but at 28 dai, similar to Pssym36 mutant, shoot and root biomass of wild type plants was significantly decreased in comparison to nonmycorrhizal plants (see figures III.3, III.4). At 28 dai, the root/shoot biomass ratio for wild type cv Finale was equal to 2.02 in mycorrhizal plants and 2.12 in non-mycorrhizal ones. For the Pssym36 mutant it was equal to 1.29 and 1.55 in G. intraradices-inoculated and non-inoculated plants, respectively.

	Plant line	F %	M %	m %	a %	А %
			Ink staining
	Plant line	F %	M %	m%	a %	А %
	SGE (wt)	99.1 ± 0.9	36.3 ± 5.6	36.5 ± 5.5	21.5 ± 3.6	9.1 ± 3.9
	SGEFix --2 (Pssym33)	99.1 ± 0.9	Ink staining 27.7 ± 3.0	27.9 ± 3.0	24.8 ± 1.9	7.0 ± 1.1
	Finale (wt) SGEFix --1 (Pssym40)	78.3 ± 5.5 99.2 ± 0.8	19.7 ± 3.6 29.3 ± 1.9	24.7 ± 3.1 29.6 ± 2.1	19.5 ± 2.3 25.1 ± 3.5	3.9 ± 0.9 7.5 ± 1.4
	Mycorrhiza parameter abbreviations are those defined in table III.1.
	RisNod24 (Pssym36)	48.9 ± 3.9 Alkaline phosphatase-active fungal structures 5.6 ± 1.8 11.3 ± 3.7	1.6 ± 1.0	0.2 ± 0.1
	Plant line SGE (wt)	Alkaline phosphatase-active fungal structures F % M % m % 93. ± 3.71 15.0 ± 3.2 15.9 ± 3.0	a % 22.5 ± 4.8	А % 3.8 ± 1.3
	Finale (wt) SGEFix --2 (Pssym33)	51.0 ± 5.9 94.1 ± 2.8	1.2 ± 0.4 Ink staining 11.6 ± 1.5	2.2 ± 0.5 11.8 ± 1.6	9.4 ± 1.6 23.2 ± 5.1	0.1 ± 0.1 2.7 ± 0.8
	RisNod24 (Pssym36) SGE (wt) SGEFix --1 (Pssym40)	24.8 ± 2.2 94.1 ± 3.9 95.7 ± 2.3	0.4 ± 0.1 34.5 ± 4.8 29.5 ± 4.2	1.4 ± 0.2 36.2 ± 3.9 30.5 ± 3.9	4.1 ± 1.2 30.9 ± 5.7 24.5 ± 3.7	0.01 ± 0.01 10.8 ± 2.9 7.3 ± 1.6
	SGEFix --2 (Pssym33)	99.2 ± 0.8	24.1 ± 1.1	24.3 ± 1.1	21.3 ± 2.2	5.1 ± 0.5
	SGEFix --1 (Pssym40)	100 ± 0	44.2 ± 9.9	42.7 ± 16.9	33.1 ± 8.1	16.2 ± 6.5
		Alkaline phosphatase-active fungal structures
	SGE (wt)	96.5 ± 0.1	16.8 ± 2.1	17.4 ± 2.2	30.6 ± 5.0	5.3 ± 1.3
	SGEFix --2 (Pssym33)	94.9 ± 3.2	10.9 ± 1.6	11.4 ± 1.4	27.5 ± 4.3	3.1 ± 0.7
	Plant line SGEFix --1 (Pssym40)	F % 93.3 ± 2.7	M % 24.5 ± 5.7	m % 26.1 ± 5.9	a % 30.8 ± 3.9	А % 8.0 ± 2.9
			Ink staining
	Finale (wt)	83.8 ± 5.3	47.6 ± 7.0	55.6 ± 5.4	19.9 ± 3.1	9.8 ± 2.1
	RisNod24 (Pssym36)	61.2 ± 6.8	11.6 ± 3.0	18.4 ± 3.6	4.6 ± 0.7	0.6 ± 0.2
		Alkaline phosphatase-active fungal structures
	Finale (wt) III.2.2. Plant growth	78.5 ± 3.7	9.1 ± 2.7	11.1 ± 2.8	19.0 ± 3.8	2.0 ± 1.0
	RisNod24 (Pssym36)	69.8 ± 6.1	6.0 ± 0.9	8.7 ± 1.1	13.8 ± 2.0	0.8 ± 0.1
	Pssym33 and Pssym40 mutants versus the wild type SGE line Pssym36 mutant versus the wild type cv Finale line
	Mutation in the Pssym36 gene did not affect the shoot biomass accumulation but root biomass

Table III.2. Mycorrhization parameters of the wild type (wt) Finale line and mutant RisNod24 (Pssym36) line at 28 days after inoculation with G. intraradices. Mycorrhiza parameter abbreviations are those defined in table III.1.

Mycorrhizal colonization (M, m) and arbuscule abundance (A, a) of Pssym33 mutant roots were significantly lower in comparison with wild type roots at 21 dai whereas mycorrhizal levels in roots of Pssym40 mutant were higher than wild type ones (see table

III.3

.). At 28 dai, differences between

The AM fungus developed rapidly in the root systems of the SGE line and corresponding mutants Pssym33 and Pssym40, which all showed consistently higher values for overall root colonization (M%) and arbuscule abundance (A%) at 21 dai than in either cv Finale or Pssym36. The proportion of overall alkaline phosphatase-active compared to total (ink-stained) mycelium in roots at 21 dai was also considerably higher in parent and mutant SGE lines (45-55% of ink-stained structures) than in either cv Finale or Pssym36 roots. The proportion of alkaline phosphatase-active/total (ink-stained) mycelium remained fairly constant up to 28 dai for SGE and Pssym33, and increased in Pssym40.

of non-inoculated roots of Pssym36 mutant was marginally decreased as compared with wild type cv Finale plants under the same conditions (see figures

III.3, III.4)

. Pssym36 root colonization by G. intraradices slightly increased the shoot and root biomass accumulation at 21 dai but

  At 21 dai, significant enhancement was observed in the expression of TI and GST pea genes in wild type mycorrhizal roots compared to non-mycorrhizal ones; the same tendency persisted in roots of the Pssym36 mutant but only the expression of GST-encoding gene was increased significantly. Expression of the DRP, MAPK and SERPROT genes was, on the contrary, downregulated in colonized roots of wild type plants (see tableV.1, figure V.1.). No significant differences were detected in expression levels of these genes in mycorrhizal and nonmycorrhizal roots of the Pssym36 mutant. Neither G. intraradices root colonization nor mutation in the PsSym36 gene significantly affected the expression of H + -ATPase, PT4 and BCOP-At 28 dai, expression of genes encoding PT4, DRP and GST was significantly up-regulated in AM-colonized wild type roots. No significant responses to G. intraradices root colonization were observed in roots of the Pssym36 mutant. Mycorrhiza development strongly repressed expression of the BCOP gene in both wild type and mutant plants but differences were only marginally significant for the mutant genotype (P=0.1). Expression of the pea genes H + -ATPase, TI, and SERPROT, was not significantly modified by G. intraradices development at 28 dai in wild type or in Pssym36 mutant roots, whilst MAPK gene expression followed the same reduced trend (P=0.1) in cv Finale mycorrhizal roots. No significant mycorrhiza-mutant interactions were observed at this time point (see table V.1).Expression of PT4, TI, GST and SERPROT genes was significantly up-regulated in response to G. intraradices colonization of wild type and Pssym33 mutant roots at 21 dai (see table V.1, figure V.2). Transcript abundance of the MAPK gene was significantly lower in mycorrhizal roots of wild type SGE plants than in non-mycorrhizal ones, but mutation of the PsSym33 gene reduced this effect of G. intraradices colonization on MAPK gene expression so that no significant differences were detected between mycorrhizal and non-mycorrhizal mutant roots.No significant differences in H + -ATPase gene expression were detected in all roots whatever the treatment at 21 and 28 dai, although it tended to be lower in mycorrhizal roots at 28 dai.At 28 dai, only expression of the GST gene was significantly up-regulated in mycorrhizal roots of wild type in comparison with non-mycorrhizal roots. Increases in PT4 and TI gene expression also persisted in mycorrhizal SGE roots (P=0.1, P=0.08). Expression of all other genes was not affected by G.intraradices colonization either of wild type or Pssym33 mutant roots. Expression of the DRP gene was not detected in any roots of either genotypes 21 and 28 dai. Significant mycorrhiza-mutant interactions were observed for GST gene at both time points (see table V.1).It was observed that, at 21 dai, G. intraradices colonization results in significant up-regulation of the expression of the genes PT4, TI, GST and SERPROT in wild type and Pssym40 mutant plants and down-regulation of the MAPK gene in mycorrhizal roots of the SGE line (see tableV.1, figure V.3). Mutation in the PsSym40 gene did not affect transcript accumulation of these genes in mutant compared to wild type roots. Transcript accumulation of the DRP gene was only detected at 28 dai when expression was reduced in response to AM colonization although differences were only significant for the wild type SGE line. No significant modifications in expression of H + -ATPase or BCOP genes were observed in mycorrhizal wild type and PsSym40 mutant roots at 21 dai. At 28 dai, significant increases in gene expression in response to AM formation were detected for PT4 and GST genes only in mutant roots, although marginally significant (P=0.06, P=0.1) differences in the wild type genotype were observed. Expression of the H + -ATPase, BCOP, TI, MAPK and SERPROT genes was not significantly modified by G. intraradices colonization of roots in either wild type SGE or Pssym40 mutant roots 28 dai, although MAPK expression tended to remain higher in mycorrhizal wild type plants. Table V.1. Two-way ANOVA for influence of the factors mycorrhiza (Myc), pea genotype (mutant) and an interaction between both factors (Myc -mutant) on plant gene expression 21 and 28 dai by G. intraradices in roots of cv Finale vs Pssym36, line SGE vs Pssym33 and line SGE vs Pssym40. Values in bold indicate significant P-values (P<0.05)

	V.2.2 Pssym33 mutant
				1, 1e-79), M. truncatula phosphate transporter MtPt4
	(QY116211, 9e-35), P. sativum blue copper protein (Z25471, 1e-37), M. truncatula glutathione-
	S-transferase (AY134608, 2e-15), P. sativum MAP kinase homologue (X70703, 8e-28) and a
	putative M. truncatula serine protease (AL382601, 7e-40). The new pea sequences have been
	deposited in EMBL database under the accession numbers FN554871-76.
	V.2.1. Pssym36 mutant
	Plant gene:	H+ATPase	PT4	BCOP	Drp	TI	GST	MAPK	SERPROT
	Finale/sym36
	21 dai
	Myc	0.678	0.098	0.815	0.092 0.001 <0.001	0.001	0.049
	Mutant	0.348	0.077	0.114	0.770 0.002	0.502	0.063	0.849
	Myc*mutant	0.027	0.317	0.036	0.206 0.006	0.175	0.014	0.120
	28 dai
	Myc	0.594	0.050	0.003	0.027 0.348	0.100	0.110	0.302
	Mutant	0.115	0.184	0.266	0.577 0.086	0.096	0.903	0.497
	Myc*mutant	0.988	0.098	0.373	0.205 0.240	0.063	0.311	0.299
	SGE/sym33
	21 dai
	Myc	0.402	0.003	0.270	-	0.010 <0.001	0.025	0.031
	Mutant	0.260	0.467	0.010	-	0.219	0.059	0.542	0.339
	Myc*mutant	0.667	0.435	0.987	-	0.208	0.046	0.254	0.399
	28 dai
	Myc	0.081	0.060	0.139	-	0.065	0.021	0.182	0.251
	encoding genes at the 3 weeks time point, although significant mycorrhiza-mutant interactions Mutant 0.491 0.606 0.128 -0.377 0.140 0.215 0.495 V.2.3 Pssym40 mutant Myc*mutant 0.660 0.231 0.751 -0.096 0.026 0.220 0.221
	SGE/sym40 were observed (see table V.1). 21 dai
	Myc	0.114	0.007	0.873	-	0.002	0.031	0.049	0.033
	Mutant	0.593	0.411	0.001	-	0.994	0.308	0.158	0.562
	Myc*mutant	0.787	0.387	0.431	-	0.954	0.821	0.034	0.824
	28 dai
	Myc	0.326	0.028	0.422	0.027 0.165	0.012	0.238	0.384
	Mutant	0.669	0.410	0.048	0.509 0.597	0.074	0.324	0.309
	Myc*mutant	0.708	0.496	0.660	0.663 0.233	0.361	0.317	0.125

  ACCox and Sym10) and one allele-specific (NIN) molecular marker are known to be positioned in LGI. Significant linkage was detected between the NIN, PsSym37 and ACCox markers (see tableVII.3). No linkage of the PsSym10 gene with PsSym37, ACCox or NIN was observed, probably because PsSym10 lies quite far from these. No significant linkage of the PsSym40 gene with the tested molecular markers was detected.Four CAPs markers Enol, IGN1, Met2 and UDPgd were developed based on sequences of genes localized in LGV. Significant linkage was detected between Met2 and UDPgd molecular markers only. Markers Enol, IGN1 and Met2 are positioned too far from each other to detect any linkage (see figureVII.1). No significant linkage of the PsSym40 gene with the tested molecular markers was detected.

	UDP-glucose dehydrogenase Linkage group I Table VII.4. Segregation analysis data (linkage group II) UDPgd Linkage group V		V		CAPs
	VII.2.3. Genetic mapping PsSym36 The F2 population obtained from the RisNod24(Pssym36)×NGB1238 cross was analyzed (see Figure VII.1. Location of gene-anchored markers on the pea genetic map LGI LGII LGIII LGV NIN Sym37 ACCox ThiolP PepTrans Agpl PsAAT1 Enol Number of progeny Gene pair PP PH PQ HP HH HQ QP QH QQ Total Rec. S.E. LOD Joint χ Sym40 -ThiolP 11 17 7 0 0 0 5 14 3 57 0.512 0.069 0.01 2.16 Sym40-PepTrans 12 19 6 0 0 0 5 16 3 61 0.500 0.067 0.00 2.89 Sym40-PPIlike 6 21 10 0 0 0 3 14 6 60 0.467 0.071 0.05 0.30 Sym40-pPgm 9 21 7 0 0 0 5 13 6 61 0.525 0.068 0.03 0.22 ThiolP-PepTrans 12 4 0 3 28 0 0 1 9 57 0.073 0.026 13.38 60.44 Three CAPs (Sym37, Table VII.3. Segregation analysis data (linkage group I) Table VII.6. Segregation analysis data (linkage group V)	2
	VII.1) in order to verify whether PsSym36 is linked to morphological marker Wb of linkage group PepTrans-PPIlike 2 10 5 7 17 10 0 8 1 60 0.538 0.068 0.07 2.50
	II. PPIlike-pPgm Gene pair Gene pair		5		4		0		NIP Number of progeny 8 22 5 Number of progeny 1	7	8	IGN1 60	Rec. 0.255 0.050 S.E. Rec. S.E.	LOD Joint χ 3.11 12.87 LOD Joint χ	2 2
	DiPept HQ QP Abbreviations are those defined in table VII.2. PP PH PQ HP HH PP PH PQ HP HH HQ QP	QH QH QQ Total QQ Total
	Table VII.2. Segregation data in the F2 progeny of the RisNod24(Pssym36)×NGB1238 cross Sym40 -NIN 10 15 7 0 0 0 6 12 4 54 0.519 0.069 0.02 Sym40 -Enol 4 15 16 0 0 0 4 10 5 54 0.560 0.070 0.16	0,51 1.40
	Sym40-Sym37 10 Sym40-IGN1	1	19	3	7	1	0	0	0 0	0 0	2 3	6 11	0 5		44 24	0.545 0.104 0.443 0.145	0.04 0.03	0.89 0.11
	• lines from VIR collection: K 3034, K 1693, K 7128, K 8274. Number of progeny Rec. S.E. PPIlike Sym40-ACCox 10 Gene pare 18 9 0 0 0 6 12 5 60 0.512 0.067 LOD Sym40-Met2 9 24 4 0 0 0 8 10 5 60 0.530 0.075	Joint χ 0.01 2 0.03	0.13 2.40
	Sym40-Sym10 Sym40-UDPgd	PP 6 11	PH 15 20	PQ 9 6	QP 0 0	QH 0 0	QQ Total 0 0 0 10	0 12	0 2		30 61	0.637 0.162 0.658 0.106	0.13 0.51	0.20 2.23
	A total of 217 individual F2 plants from the RisNod24(Pssym36)×NGB1238 cross and 158 Sym36 -Wb 34 0 10 135 0 38 217 0.513 0.056 0.01 Met2 0.04 PsSym40 A joint segregation analysis was performed using the F2 population from the SGEFix --1 (Pssym40) × NGB1238 cross. 17 molecular gene-based markers positioned in LGI, LGII, LGIII NIN-Sym37 5 6 0 6 15 1 0 2 5 40 0.213 0.054 3.19 14.55 Enol-IGN1 1 1 2 2 7 2 1 4 2 22 0.500 0.125 0.00 1.91 Sym10 pPgm Eno12B NIN-ACCox 7 7 2 7 17 3 0 4 7 54 0.269 0.052 2.78 14.70 Sym37-ACCox 12 0 0 0 24 1 0 0 7 44 0.011 0.012 16.89 75.68 Abbreviations are those defined in table VII.2. Linkage group II 11 protein encoding genes with known location in LG II (see table VII.1) and 4 molecular markers ThiolP, PepTrans, PPIlike and pPgm, developed on basis of nucleotide sequences Table VII.5. Segregation analysis data (linkage group III) Number of progeny Gene pair PP PH PQ HP HH HQ QP QH QQ Total Rec. S.E. LOD Joint χ Sym40 -Agpl 7 24 6 0 0 0 8 10 6 61 0.536 0.073 0.05 3.16 Sym40-PsAAT1 5 20 12 0 0 0 5 13 6 61 0.525 0.068 0.03 0.22 IGN1-Met2 2 1 1 5 8 1 1 3 2 24 0.386 0.107 0.21 3.00 Met2-UDPgd 14 2 1 6 25 3 0 5 4 60 0.207 0.081 2.05 52.83 individual F2 plants from the SGEFix VII.2.2. EST-based molecular marker development UDPgd ACCox-Sym10 3 4 2 3 7 5 0 4 2 30 0.409 0.084 0.24 2.00 Abbreviations are those defined in table VII.2.	2
	and LGV were adopted for PsSym40 gene localization (see figure VII.1). polymorphism, were selected for segregation analysis. Significant linkage was detected Sym40-NIP 6 0 31 0 0 0 5 0 19 61 0.503 0.088 0.00 0.00
	For PsSym40 gene localization, 34 protein encoding genes known to be localized in the pea between the pairs of molecular markers ThiolP-PepTrans (7.3 cM; LOD=13.38) and PPIlike-Sym40-DiPept 5 20 12 0 0 0 1 15 6 59 0.447 0.070 0.12 3.21
	linkage groups LGI, LGII, LGIII, LGIV and LGV were selected (see table VII.1). 18 molecular pPgm (25.5 cM; LOD=3.11). No linkage was observed between PepTrans and PPIlike markers, Sym40-Eno12B 8 16 12 0 0 0 6 10 8 60 0.489 0.065 0.01 0.40
	Agpl-PsAAT1		6		8		1		4	23	7	0	2	10	61	0.215 0.044	4.76	23.46
	probably due to the long distance between them. Again, no significant linkage of the PsSym40 markers were identified between parental lines, based on nucleotide sequence differences of PsAAT1-NIP4 4 0 6 6 0 27 1 0 17 61 0.290 0.074 1.31 4.38
	gene with the tested molecular markers was detected. PsAAT1-DiPept 3 7 0 3 18 10 0	10	8		59	0.302 0.054	2.10	8.49
	DiPept-Eno12B		3		3		0		8	15	11	3	8	7		58	0.382 0.060	0.75	3.72

--1(Pssym40)×NGB1238 cross were used for Pssym36 and Pssym40 gene localization, respectively. Pssym36 and Pssym40 mutant phenotypes in corresponding F2 plant populations were observed by alterations in root nodule formation in quartz sand after inoculation with Rhizobium leguminosarum bv. viciae (strain CIAM 1026; see Material and Methods II.2). Pssym36 mutant plants do not form root nodules. Pssym40 mutant plants form small white root nodules whilst wild type nodules are larger and pink. amplified parts of the chosen genes. The origin of primers, primer sequences, melting temperature of primers and restriction enzymes used for CAPs (Cleaved Amplified Polymorphic sequences) polymorphisms detection are presented in annex 2. Computational analysis of P-allele 1, Q -allele 2, H-heterozygote; Rec. -recombination; S.E. -standard error No significant linkage between the two genes was detected. The plant material obtained will be used in the future for PsSym36 gene localization in relation to gene-based molecular markers.

Linkage group III

Molecular markers were developed for 5 of the 13 selected ESTs positioned in LGIII. Significant linkage was detected in the Agpl-PsAAT1 pair of molecular markers. Linkage in pairs of markers PsAAT1-DiPept and DiPepet-Eno12B were marginally significant and no significant linkage of the PsSym40 gene with the tested molecular markers was detected.

Abbreviations are those defined in table VII.2.

  NIN, PsSym37 and ACCox localized in linkage group I are significantly linked to each other but do not link with PsSym40 which excludes localization of this gene in this part of the pea genome. Another molecular marker PsSym10, which is positioned lower in LGI, does not link with the NIN, PsSym37, ACCox markers or the PsSym40 gene, which suggests that the PsSym40 gene is also not positioned in the region around PsSym10 marker. Similar conclusions can be drawn about the possible absence of PsSym40 from LGII, LGIII and LGV. Overall, the performed analyses make it improbable that the PsSym40 gene lies in one of these four linkage groups, although this can not be absolutely confirmed at present. However, it does appear likely that PsSym40 lies outside regions close to the molecular marker locations (see figureVII

  The completion of the research with the use of laser cell microdissection method was possible due to the help and guidance of Ms. Christine Arnould, member of PME INRA, whose work the author greatly appreciates. To work with her interesting and exciting.Most sincere thanks to Mr. Pinayev, member of the Laboratory of Genetics of Plant-microbe Interactions, for his scientific advice, inimitable humour and moral support.

	Gene symbol NIN Cwi1 MDHc Pip1 PPIlike TE002L09	GenBank accession № Ps-AJ493066 Ps-Z83747 Ps-CD858827 Ps-AJ548795 Ps-CD860684 Ps-CD860523	Primer sequences GAAAGAGGAAGCGGACTTGT TGGTCTTCTCCGCCTTGG GTGTCATTGATGTTGTTATCGCA TCCCAAAGGAGCTGTTTGGGG GAATCGGGTGCTTGGCCCG CTTATCACTCTGCTTGGTTCTGTGG mATGCTGCTTTCCCTCTTTTG TCTAGGGAATCCACCAACCA CATTCGGTTTGTTCTTGGCGAGG GAGTCTCTGGCACTACGTTTGGC hTTTTGAGCATGGCGAATG GCTTGCTTGTCTCCTGTGATT hAACGGCCACTTCTCCAATC TCAACACCACCAACACGAAC GCATCCATCACGTCCGTGTGACG	Tm, °C 60 60 58 60 58 60	Restriction enzyme ----HhaI -	Reference Borisov et al., 2003 Aubert et al., Aubert et al., 2006 Aubert et al., Aubert et al., 2006 Aubert et al.,
	PsSym37 Gigantea PsAS1 ACCox cOMT Gpt PsSym10 pPgm Eno12B ThiolP Sut1 PepC PepTrans AgplI PGK1 PsAAT1 Bfruct	EU564102 AY826734 Ps-Y13321 Ps-CD859427 Ps-CD858528 Ps-AF020814 AJ575252 Ps-AJ250770 Ps-X57232 Ps-X66061 Ps-AF109922 Ps-D64037 Ps-CD860432 Ps-X96766 Ps-CD858627 Ms-L25334 Ps-X85327	GATGATGACCTGCGTCCACCA ATGCAAGCTTAACAACTGTTGAG GAATGCCCGACCATTTGTGCC TCCTCGTGGAAGCCCGT CCGATTCTAGACCATTCTGC CGATGTCACTACGATAAGGGCTGC CATCAAAGGCCTTTCTTAGAATCC AGGTGTCATGGTCATGAACA ATGGTCGTCGCTCCAGTC TGCACACACAATTGGACTTGTCG CTTACCTCAAGCTTCTACCTTGCC GAATCATGGCATACCTCATGACC GAGAAGGATGATCCCGCCAG CAAGGCTAATATAGATGGTAGAG GCTAATACCAATTATAAAAGCAGG AACCTGCACCAGAATCCATC CACCATCACCATCACTAGCG GAGGATCCTTACTAGTATAAAACC CCTGAAACACATATACCAAAAGAC GTCTATCTTTTTTGTCCCCG GTCTTCTACACAACGGCTTC CCGTACCGATGAAATCAAGAGG GCATCCATCACGTCCGTGTGACG CCGAAGAGGATTACCCCTAYCGTGC GCTTCTCCCCAGCTACCACCCC CGTCGAGAATGTTATTGATCTTGG CAATCCATAATCAGATGCGCGGC mCCATGGGAGTGTTCGAGTTT CGACGGAATCTCCTCCTACA GCCGTGATTCGGATCTGATGG CGGTCGTATAAAGGAATGACTAC GTCAACTGGCAACCCAATCT CAGTTTCACAAGGAGTGTCGC CATAGCCTATGAGTTCTTAACCAG GGACCAATGAGATATGGAGG CTTGGTTGGCCAATTTACCGG	60 60 55 58 62 58 58 58 55 55 62 58 58 60 58 60 55	--RsaI --HinfI RsaI NlaIII PstI -BstBI -AvaII -NlaIII --	Aubert et al., 2006 Aubert et al., Zhukov, personal communication Aubert et al., Aubert et al., 2006 Aubert et al., 2006 Aubert et al., Aubert et al., 2006 Zhukov, personal communication Aubert et al., Aubert et al., 2006 Aubert et al., 2006 Aubert et al., Aubert et al., 2006 Aubert et al., 2006 Aubert et al., Aubert et al., 2006
	Gbsts1 NIP Xyft	Ps-X88789 Ps-U15036 Ps-AF223643	CACTGTAAGTCCACATTATGCC GACATGGAACATAGAAACTGA AGTTGAGTTTTTCTGTTCTAGGCCATAGTAC CTGCATGTCAGAGGTCCATAGG GTGTTCCCAAACGATAAACCAATT CTTAAATGGCCAGAATTGAGATCC	55 64 55	---	Aubert et al., 2006 Aubert et al., Aubert et al., 2006
	P54 PsAAP2 Cwi2 DiPept IV IGN1	Ps-Y11207 -Ps-Z8339 Ps-CD859478 Lj-AB251640	AAAATGACAGTTTTGATGAACAC TTTGGACCATATATGGCATATGC CAATAAAATGCAGCAATCACAGCC CTGATCTTGTTAACTGGACCCC TAATCCATTCCCTTAGAAAAGGGCC CTATGCTCAAACTTAGCCGTAGGC CAGAACCAGAAGAAGGTGGC CATACAGCTGTGGATGGTTACCC WGTRGTGAARTTGCTTCTTGMTGC	55 58 55 58 58	---NlaIII AluI	Aubert et al., Aubert et al., 2006 Aubert et al., 2006 Aubert et al., Kumagai et al., 2007

Acknowledgements

The accomplishment of this thesis was made possible due to the agreement about joint postgraduate scientific work supervision between the Universite de Bourgogne and its president Mr.

CHAPTER II MATERIAL AND METHODS

Absolute real-time RT-PCR was carried out using the Absolute QPCR SYBR Green kit (ABgene, UK) and fluorescence was quantified using an ABI PRISM 7900 real-time cycler (Applied BioSystems, Foster City, CA, USA). Reactions were performed in 20 μl full mix volume containing tested cDNA, 10 μl of reaction mix (from Absolute QPCR SYBR Green kit), 2 pM of each primer and sterile water. After 10 min denaturation at 95 °C, 40 amplification cycles (95 °C for 15 s, primer annealing temperature for 30 s and 72 °C for 30 s) were followed by a melting curve program included at the end of each PCR run according to the thermal profile suggested by the manufacturer (95 °C partners at the molecular level. Any alteration in one symbiotic partner will lead to modifications in the other. Most of studies of the effect of mutations in SYM genes on plant gene responses to AM fungi have focused on early steps of arbuscular mycorrhiza formation [START_REF] Kistner | Seven Lotus japonicus genes required for transcriptional reprogramming of the root during fungal and bacterial symbiosis[END_REF][START_REF] Sanchez | Pseudomonas fluorescens and Glomus mosseae trigger DMI3dependent activation of genes related to a signal transduction pathway in roots of Medicago truncatula[END_REF][START_REF] Seddas | Symbiosis-related plant genes modulate molecular responses in an arbuscular mycorrhizal fungus during early root interactions[END_REF]. Only [START_REF] Lapopin | Comparative di.erential display analysis of arbuscular mycorrhiza in Pisum sativum and a mutant defective in late stage development[END_REF] and [START_REF] Grunwald | Identification of mycorrhizaregulated genes with arbuscule development-related expression profile[END_REF] have reported plant gene expression in a mutant affected in the late stages of mycorrhiza development (Pssym36). In order to further investigate a role of SYM genes in late stages of the AM symbiosis, we studied the effect of mutations in the pea PsSym36, PsSym33 and PsSym40 genes on symbiosis-associated plant molecular responses. Eight proteinencoding plant genes were selected amongst those reported in the literature to be up-regulated during AM interactions or in arbuscule-containing cells of P. sativum or M. truncatula [START_REF] Krajinski | Mtha1, a plasma membrane H + -ATPase gene from Medicago truncatula, shows arbuscule specific induced expression in mycorrhizal tissue[END_REF][START_REF] Wulf | Transcriptional changes in response to arbuscular mycorrhiza development in the model plant Medicago truncatula[END_REF][START_REF] Liu | Transcript profiling coupled with spatial expression analyses reveals genes involved in distinct developmental stages of the arbuscular mycorrhizal symbiosis[END_REF][START_REF] Brechenmacher | Expression profiling of up-regulated plant and fungal genes in early and late stages of Medicago truncatula-Glomus mosseae interactions[END_REF][START_REF] Grunwald | Identification of mycorrhizaregulated genes with arbuscule development-related expression profile[END_REF][START_REF] Massoumou | Medicago truncatula gene responses specific to arbuscular mycorrhiza interaction with different species and genera of Glomeromycota[END_REF]. The chosen plant genes are putatively involved in processes of signaling and gene regulation (MAP kinase (MAPK)), membrane transport (plasma membrane H + -ATPase (H + -ATPase), inorganic phosphate transport (PT4), blue copper protein (BCOP), protein synthesis and processing (serine protease (SERPROT)), defense and cell rescue (pathogenesis-related protein pI230 (DRP), trypsin inhibitor (TI), glutathione-S-transferase (GST)). Expression of pea genes was analyzed at 21 and 28 dai with the G. intraradices in mycorrhizal and non-inoculated roots of Pssym36, Pssym33 and Pssym40 mutants and their corresponding wild type genotypes cv. Finale and SGE by real-time RT-PCR. Expression was quantified as transcript levels related to those of the pea house-keeping GAPDH gene. The author heartily thanks the guidance and founders of this research project for the chance to participate in it, and also thanks the French Embassy, the Conseil Regional de Bourgogne and the Russian Foundation for Basic Research for the financial aid.

CHAPTER VI TRANSCRIPTOME PROFILING OF G. INTRARADICES

GENES IN ARBUSCULE-CONTAINING CORTICAL CELLS OF THE PSSYM40 MUTANT

The author is especially thankful to Pr. Tikhonovich for moral support and moneyed assistance during research and thesis completion. The author expresses her heartiest thanks to her Russian supervisor Dr. Borisov, Head of the Laboratory of Genetics of Plant-microbe Interactions, for a chance to participate in this international project, for research organisation and his help in the thesis completion.

The author thanks the co-supervisor of her research Ms. Pascale Dozolme for her invaluable help in research, advice on the methodology, her help in articles and thesis writing and for friendly attitude and concern.