Plants and Arbuscular Mycorrhizal Fungi: Cues and Communication in the Early Steps of Symbiotic Interactions

Plants and Arbuscular Mycorrhizal Fungi: Cues and Communication in the Early Steps of Symbiotic Interactions

VIVIENNE GIANINAZZI‐PEARSON,* NATHALIE SE´JALON‐DELMAS,{ ANDREA GENRE,{ SYLVAIN JEANDROZ*,} AND PAOLA BONFANTE{

*UMR INRA 1088/CNRS 5184/Universite´ de Bourgogne Plant‐Microbe‐Environment, INRA, CMSE, BP 86510, Dijon Cedex 21065, France { UMR 5546, Equipe de Mycologie Ve´ge´tale, Poˆle de Biotechnologie Ve´ge´tales, Chemin de Borde‐Rouge, BP 42617, Castanet‐Tolosan 31326, France { Dipartimento di Biologia Vegetale, Universita` di Torino, I.P.P.‐C.N.R., Viale Mattioli 25, Torino 10125, Italy } UMR INRA/UHP 1136 Interactions Arbres‐Microorganismes, Universite´ H. Poincare´ Nancy I, Faculte´ des Sciences, BP 239, Vandœuvre Le`s Nancy 54506, France

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Rhizosphere Signaling in Symbiotic Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . A. Plant Signals in the Presymbiotic Stage: The Case of Flavonoids ..... B. Nonflavonoid Rhizosphere Signals ......................................... C. Identification of the Hyphal Branching Factor: The Strigolactone Story ...................................................... D. Fungal Signaling to Host Roots: Myc Factors ........................... III. Plant Genetic Programs: Mycorrhiza‐Defective Mutants . . . . . . . . . . . . . . . . . IV. Molecular Cross Talk and Signaling Pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Plant Cell Responses to Fungal Colonization: Tissue and Cell Specificity .... VI. Interface Biogenesis: New Facts/New Hypotheses. . . . . . . . . . . . . . . . . . . . . . . . . Advances in Botanical Research, Vol. 46 Incorporating Advances in Plant Pathology Copyright 2008, Elsevier Ltd. All rights reserved.

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0065-2296/08 $35.00 DOI: 10.1016/S0065-2296(07)46005-0

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VII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208

ABSTRACT The ubiquitous nature of arbuscular mycorrhiza (AM) pleads for common molecular and genetic determinants across diVerent plant taxa. The cellular processes determining compatibility in early interactions prior to and during cell contact between arbuscular mycorrhizal fungi and plant roots are starting to be unraveled. The root epidermis is an active checkpoint where signal exchanges and control over root colonization occur. Root‐secreted flavonoids, flavonols, and strigolactones can act as rhizosphere signals in stimulating presymbiotic fungal growth, although their mechanism of action on the fungal cell is as yet unknown. Likewise, fungal signals (Myc factors) activate early plant responses with induction of genes related to signal transduction pathways and biogenesis of a prepenetration apparatus designed to accommodate intracellular fungal growth from appressoria into epidermal cells. Evidence from genetical, transcriptional, and physiological studies points to the implication of calcium as a secondary messenger in signaling pathways leading to early host cell responses. Future challenges for research are to decipher the complexity of symbiosis signaling and to provide new insights into the specificity of the molecular dialogue between AM symbionts.

I. INTRODUCTION Mycorrhizal associations are the root symbioses of the large majority of terrestrial plants, and the beneficial fungi involved represent an important component in the ecology and biology of most soils (Smith and Read, 1997). In the case of arbuscular mycorrhiza (AM), benefits to the plant are multiple but their symbiotic nature is principally characterized by bilateral exchanges where the photosynthetic host receives mineral nutrients and the fungus acquires carbohydrates. Mycorrhizal research has entered the mainstream of biology, thanks mainly to DNA technologies and genomics, which are providing new tools to discover symbiont communication, development, and diversity, and to reveal the contribution of symbiotic partners to the functioning of mycorrhizal associations. Several features of AM associations are considered as landmarks, unanimously accepted by the scientific community. First of all, land plants and AM fungi share a long coevolutionary history, which originated more than 450 million years ago (Remy et al., 1994) and which has ensured the widespread distribution of AM fungi. Reciprocal nutrient exchange within a functional symbiosis requires extensive physical contact between the partners, which results from profound readjustments in the plant and fungal tissues during root colonization (Harrison, 1999). Being asexual, obligately biotrophic, multinucleate, and unculturable microbes,

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AM fungi have been considered intractable organisms from a taxonomic point of view for many years. On the basis of ribosomal gene sequences, they have been moved to a new phylum, the Glomeromycota (Schu¨ßler et al., 2001), and their hierarchical position has been confirmed with the remodeling of the entire phylogenetic tree of fungi (James et al., 2006). More than 150 species have been described so far within the Glomeromycota, but their number and genetic features are a dynamic field of research, as witnessed by debates (reviewed by Pawlowska, 2005). In order to set the scene and provide the background for subsequent discussion, the main aspects of root colonization by the symbiotic fungi are outlined first. Glomeromycota are highly dependent on their hosts for fitness and survival, and hyphae germinating from their large asexual spores can only grow for a few days in the absence of a plant. On recognition of the host plant, these presymbiotic hyphae diVerentiate hyphopodia‐like appressoria on the root epidermis, which in turn form hyphal pegs that cross the root epidermis and initiate infection units to colonize the root cortex. Interactions between the model legume Lotus japonicus and the AM fungus Gigaspora margarita oVer a good example of the diVerent types of root colonization patterns (Arum, Paris) that can occur (Dickson, 2004). The swollen appressorium developing at the root surface gives rise to an intercellular hypha, which usually separates two adjacent epidermal cells to reach their base. Here, it penetrates the radial wall of the epidermal cell and develops into the cell lumen to form the first interface compartment between symbiont cells (Bonfante et al., 2000). This series of events is a crucial step in the interaction, where reciprocal recognition, localized diVerentiation of the appressorium, cell wall breaching, and intracellular accommodation of the fungal symbiont in a novel apoplastic compartment represent the result of complementary, coordinated strategies in both partners, which provide the beneficial fungus access to internal root tissues without causing damage to the plant. Once the fungus has overcome this epidermal checkpoint and reached the inner root layers, it spreads through the parenchymal cortex by hyphal coils and/or intercellular hyphae and eventually forms intracellular, highly branched structures called arbuscules (Bonfante, 1984). These intracellular fungal structures are surrounded by a newly built apoplastic space, which is bordered by a specialized plant membrane (Gianinazzi‐Pearson et al., 2000; Harrison et al., 2002). The construction of this interface compartment, which regulates reciprocal nutrient exchanges between symbiont cells, results from an intense reorganization of plant cell contents and activity, ranging from specific gene activation (Gianinazzi‐Pearson and Brechenmacher, 2004) to localized cell wall and membrane deposition (Balestrini and Bonfante, 2005), cytoskeleton remodeling (Genre and Bonfante, 1997, 1998), organelle mobilization

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(Lohse et al., 2005), and phosphate transport (Karandashov and Bucher, 2005; Liu et al., 1998). Arbuscules are ephemeral structures with a life span of a few days (Toth and Miller, 1984). When host cell reorganization reaches its peak, arbuscule tips, then branches collapse, and finally the host cell contents gradually adopt an aspect similar to that prior to colonization (Jacquelinet‐ Jeanmougin et al., 1987). Interest in this ancient and widespread plant symbiosis has expanded exponentially over the last few years both from a fundamental viewpoint of interorganism interactions and for their potential exploitation in the development of sustainable plant production systems (Science Citation Index, Web of Science, http://scientific.thomson.com/products/wos/). The ubiquitous nature of AM associations, together with the constant structural and functional features characterizing the root–fungal relationships, pleads for common molecular and genetic determinants across diVerent plant taxa. Consequently, recent developments have been toward research that is based on model AM host–fungal combinations (Gianinazzi‐Pearson et al., 2007; Parniske, 2004), which may appear restrictive at first sight but provide a strategy for putting cell programs into a more general context with broader relevance. There are several reviews covering diVerent aspects of the AM symbiosis, including molecular and cell interactions (see citations in Gianinazzi‐Pearson et al., 2007 and Reinhardt, 2007). The purpose of this chapter is not to repeat earlier reviews, but rather to pinpoint events that drive early interactions between AM fungi and plants and propose future lines of research to unravel their role as prerequisites to reciprocal compatibility between the actors of the symbiosis.

II. RHIZOSPHERE SIGNALING IN SYMBIOTIC INTERACTIONS The rhizosphere is generally considered to be a narrow zone of soil where roots stimulate or inhibit microbial populations and activities in their vicinity. Roots may change the physical and chemical properties of the soil through mucilage production and the excretion of water‐soluble compounds in root exudates. Low‐molecular weight compounds, such as amino acids, organic acids, sugars, hormones, enzymes, secondary metabolites like phenolics and terpenoids, comprise the majority of root exudates (Walker et al., 2003). A large body of knowledge suggests that root exudates act as messengers that communicate and initiate biological and physiological interactions between roots and microorganisms, which in turn will produce signals essential for root colonization. Many studies have shown that root exudates from host

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plants stimulate spore germination, hyphal branching, and growth of AM fungi (Be´card et al., 2004), and mathematical models of the colonization process by Glomus mosseae indicate that the portion of the root where exudation is greatest is most likely to become colonized (Schwab et al., 1991). The phosphate status of host plants can aVect the metabolism of compounds that accelerate AM fungal growth (Elias and Safir, 1987; Nagahashi and Douds, 1996). The application of phosphate to plants can decrease root membrane permeability, and with it alter the production or composition of root exudates (Amijee et al., 1989; Graham, 1982; Graham et al., 1981; Nagahashi and Douds, 2000; Ratnayake et al., 1978; Schwab et al., 1983; Tawaraya et al., 1994, 1996). In addition, phosphate deficiency aVects the exudation of flavonoids and other unidentified compounds (Akiyama et al., 2002; Franken and Gna¨dinger, 1994; Nair et al., 1991; Rossiter and Beck, 1996; Siqueira et al., 1991; Tawaraya et al., 1998; Xie et al., 1995). AM symbiosis establishment can also alter root exudates so that they no longer stimulate fungal growth or even inhibit further colonization (Pinior et al., 1999; Vierheilig et al., 1998b), indicating that in addition to being involved in presymbiotic stages, root exudates also play an important role in regulating root colonization by a negative feedback mechanism allowing the plant to control excessive colonization. The root signal that systemically inhibits further colonization is still unknown. While flavonoids stimulating root colonization can increase under low‐phosphate conditions, flavonoids inhibitory to AM fungi can accumulate under high plant phosphate status following mycorrhiza establishment (Guenoune et al., 2001; Larose et al., 2002). Cytokinin and salicylic acid have also been proposed to be involved in the autoregulation. Cytokinin levels are altered in AM colonized roots (Allen et al., 1980; Shaul‐Keinan et al., 2002), and reduced salicylic acid levels in Nah G tobacco plants increase root mycorrhization (Herrera‐Medina et al., 2003). The role of root exudates in the inability of certain plant species to form mycorrhiza is less clear. Nonmycorrhizal plants arose late in evolution (
100 million years), and their loss of compatibility with AM fungi may be polyphyletic (Brundrett, 2002). Distinction between host and nonhost plants may occur as soon as the presymbiotic stage but the mechanisms by which nonhost exudates influence AM fungal development remain an enigma. It has been suggested that they may simply lack compounds required to stimulate hyphal branching, growth, and/or chemotaxis (Bue´e et al., 2000; Gemma and Koske, 1988; Glenn et al., 1985, 1988), or that they may release inhibitory compounds (Gadkar et al., 2003; Nagahashi and Douds, 2000). Appressorium development is not elicited on nonhost roots, and this lack of appressoria formation is always associated with the absence of diVerential hyphal morphogenesis during the presymbiotic stage (Giovannetti et al., 1994).

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Intergeneric grafts between mycorrhizal pea and nonmycorrhizal Lupinus have provided evidence for a shoot‐produced inhibitor of the symbiosis in Lupinus (Gianinazzi‐Pearson and Gianinazzi, 1992). Lupinus presents high concentrations of alcaloids in leaves, which are translocated throughout the whole plant. However, a role of these compounds in the nonmycorrhizal status of Lupinus seems unlikely as they do not inhibit hyphal growth, suggesting that Lupinus probably also lacks stimulatory compounds (Vierheilig et al., 1995). Glucosinolate that may be metabolized into isothiocyanate, an antifungal compound, has been extracted from roots of nonmycorrhizal Brassica and shown to inhibit spore germination of AM fungi (Schreiner and Koide, 1993; Vierheilig and Ocampo, 1990), but no inhibitory compounds have been found to be representative of other nonmycorrhizal families (Barker et al., 1998a; Schreiner and Koide, 1993). A. PLANT SIGNALS IN THE PRESYMBIOTIC STAGE: THE CASE OF FLAVONOIDS

Evidence that root and fungal signals must be involved in the AM symbiosis came from early observations but the identity of their molecular nature is still at its beginning. Spores of AM fungi can germinate spontaneously in the absence of a host (asymbiotic stage) (Bianciotto et al., 1995; Douds and Schenck, 1991; Gianinazzi‐Pearson et al., 1989; Giovannetti et al., 1993; Mosse, 1959). However, further directional growth and intense branching of the germ tube (presymbiotic stage), which favor fungal contact with the root (appressoria formation) and the establishment of symbiosis, require the presence of root compounds. In the absence of a host plant, spore germination is arrested before complete depletion of carbon resources (Bago et al., 1999; Be´card and Piche´, 1989a). The branching response, resulting in ‘‘fan‐like structures’’ (Powell, 1976) later called ‘‘arbuscule‐like structures’’ (Mosse, 1988), is stimulated by root exudates of host but not nonhost plant species (Be´card and Piche´, 1990; Gianinazzi‐Pearson et al., 1989), showing that an AM fungus senses its host plant and is able to discriminate from nonhost plants. Using mycorrhiza‐defective pea mutants, Balaji et al. (1995) proposed that factors inducing hyphal branching are diVerent from those promoting root penetration and/or colonization. Indeed, the pea mutants were not aVected in stimulatory compounds but rather in root signals inducing appressorium development and root colonization. At present, root factors that aVect spore germination, presymbiotic hyphal proliferation, and appressorium diVerentiation have not been reported. The major influence of root exudates on AM fungi is to stimulate hyphal growth and branching rather than spore germination. Phenolics, including flavonoids, have been proposed as the root molecules that could be involved

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in the stimulation of presymbiotic fungal growth. Flavonoids, which are lipophilic to water‐soluble molecules derived from the phenylpropanoid pathway, play an active role in the regulation of symbiotic and pathogenic interactions (Peters and Verma, 1990). They are widely present in the plant kingdom and more than 4000 thousand have been identified in vascular plants (Harborne, 1988). Their eVects on AM fungi can be (1) stimulation of fungal spore germination, (2) promotion of hyphal growth, and (3) to favor root colonization. A number of isolated flavonoids can activate spore germination of Gigaspora or Glomus species in the micromolar range (Fig. 1) (Gianinazzi‐Pearson et al., 1989; Graham, 1982; Kape et al., 1992; Siqueira et al., 1982; Tsai and Phillips, 1991). The smaller spores of Glomus appear more dependent on an external stimulus for germination than species with large spores (e.g., Gigaspora). Flavonoids or flavonols naturally released from host roots enhance hyphal growth (Be´card et al., 1992; Bel‐ Rhlid et al., 1993; Poulin et al., 1993; Tsai and Phillips, 1991) and promote AM colonization (Nair et al., 1991). AM fungal responses to flavonoids are not uniform and they can diVer between fungi, flavonoid concentration,

B

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Fig. 1. Structure of flavonoid‐related molecules known to influence AM fungal spore germination and/or hyphal growth. A, B, C, and D are flavonol structures: A, quercetin; B, kaempferol; C, apigenin; and D, luteolin. E, F, and G are flavanones: E, hesperitin; F, naringenin; and G, biochanin A. G, H, and I are isoflavonoids: G, biochanin A; H, formononetin; and I, myricetin.

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and type of molecule. For example, quercetin, apigenin, and two flavanones (hesperitin and naringenin) all stimulate hyphal growth of G. margarita but the isoflavones biochanin A and formononetin are inhibitory (Be´card et al., 1992; Chabot et al., 1992). Be´card et al. (1992) proposed a possible structure– activity relationship and hypothesized that a hydroxyl group in position 3 is essential to confer activity on hyphal growth of the flavonols quercetin and kaempferol (Fig. 1). However, this could not explain the activity on spore germination of luteolin and apigenin, which do not possess a hydroxyl group in position 3 (Fig. 1). Vierheilig et al. (1998a) suggested that AM fungi might have genus‐, or even species‐, specific requirements during symbiosis, and Larose et al. (2002) proposed that flavonoid patterns of accumulation may be diVerentially modulated depending on the fungal genera. Flavonoid profiles in root exudates diVer considerably between legumes (Phillips, 2000), and this variability is supposed to enable symbiotic Rhizobia to distinguish their hosts from nonlegumes (Mithofer, 2002). This flavonoid‐based host specificity in the nodulation symbiosis may be reminiscent of a preexisting flavonoid‐ fungal specificity in the AM symbiotic program. There is as yet no clue as to the physiological basis of the promoting eVects of flavonoids on AM fungal development. Interestingly, they are known for their estrogenic activity in vertebrates. Poulin et al. (1997) reported that flavonoid activity on AM fungal growth could be blocked by pure antiestrogens and that ‐estradiol stimulates hyphal growth of Glomus intraradices, suggesting a potential estrogen‐like binding site in this fungus. However, the central role of flavonoids in AM interactions was questioned when Be´card et al. (1995) showed that maize mutants deficient in chalcone synthase, an enzyme in the flavonoid synthesis pathway, induced a same hyphal branching activity in AM fungi as compared to wild‐type plants and developed comparable levels of mycorrhization. In addition, carrot roots transformed with Ri T‐DNA used for in vitro culture of AM fungi do not produce flavonoids. Finally, significant amounts of quercetin, kaempferol, and myricetin have been detected in Arabidopsis thaliana, a nonmycorrhizal plant (Burbulis et al., 1996). Taken together, these data suggest that flavonoids are not essential compounds for mycorrhization and that other chemicals may stimulate AM fungal growth, perhaps synergistically with flavonoids.

B. NONFLAVONOID RHIZOSPHERE SIGNALS

Volatiles have been reported as growth stimulants of AM fungi (Saint‐John et al., 1983) but usually in synergy with root exudates. Be´card and Piche´ (1989b) identified the active volatile compound as CO2, assuming that CO2

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serves as carbon source through an active CO2 dark fixation (Bago et al., 1999). However, CO2 and root exudates act synergistically to induce germ tube elongation and each alone has little or no eVect. In contrast, the positive eVect of quercetin is independent of CO2 (Chabot et al., 1992). Consequently, the CO2 eVect on AM fungi may only be visible with other compounds present in root exudates. As mentioned above, AM fungi exhibit a specific pattern of profuse branching in the vicinity of host plant roots (Giovannetti et al., 1993, 1996; Tawaraya et al., 1996). The identity of the branching factor exuded from host roots has been the object of considerable research. Using dialysis membranes and a sandwich system, Giovannetti et al. (1996) determined that the branching factor from Ocimum basilicum had a molecular weight lower than 500 Da. The development of an in vitro bioassay for hyphal branching in germinating spores of the genus Gigaspora has facilitated chemical analysis of the active compound released from roots. Nagahashi and Douds (1999) showed that dilution of crude root exudates reduces branching, while increasing root exudate concentrations induces ‘‘bushier, 3‐D type branching,’’ and finally at higher doses the ‘‘arbuscule‐like structures.’’ The ‘‘bushier branching’’ is normally observed when a hyphal tip grows close to a host root surface while the ‘‘arbuscule‐like structures’’ develop on external hyphae growing in limited nutrient conditions (Be´card and Fortin, 1988; Mosse, 1988; Mosse and Hepper, 1975). The physiological significance of these latter structures has not been determined, but they could mimic the branched absorbing structures (BAS) formed by G. intraradices (Bago et al., 1998), which are supposed to be involved in nutrient uptake by the fungus. A fraction of carrot root exudates prepurified by Bue´e et al. (2000) proved to be active in inducing branching of all AM fungal species tested, contrasting with flavonoids, which display stimulatory, inhibitory, or neutral activity, depending on the AM fungal species. The branching activity was induced also by root exudates of the chalcone synthase maize mutants, excluding once again the hypothesis of flavonoids as the active molecules. The active fraction of root exudates was called ‘‘branching factor,’’ and was supposed to contain several molecules. The branching factor was characterized as being lipophilic and of small molecular weight, in agreement with Giovannetti et al. (1996). It is active in stimulating hyphal branching at very low concentrations and is absent from nonhost plants like A. thaliana or sugar beet (Bue´e et al., 2000). Detailed investigations of the physiological eVects of this ‘‘branching factor’’ have shown that it is accompanied by increased mitotic activity in hyphae (Bue´e et al., 2000) and eVects at the mitochondrial level (Tamasloukht et al., 2003). One to four hours after addition of the branching factor to germinating spores of Gigaspora rosea, biogenesis of mitochondria

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increases and they change in shape and orientation. This cellular response is accompanied by very early induction of several genes related to mitochondrial activity and by an increase in oxygen consumption. These eVects precede the first morphological responses observed 6–24 h after stimulation. The increase in mitochondrial activity can be associated with the increase in phosphate uptake and plasma membrane ATPase activity reported by Lei et al. (1991). Increased transcription in presence of host root exudates has also been reported for a gene encoding a copper–zinc superoxide dismutase (SOD) from G. margarita (Lanfranco et al., 2005). Since this enzyme plays a role in the detoxification of stress‐related molecules, it may be linked to the action of root exudates on mitochondria and on the subsequent increased production of activated oxygen species. Jolicoeur et al. (1998) demonstrated that the cytosolic pH of hyphae was more alkaline when G. rosea spores were germinating in the vicinity of host roots, which could explain observations that transmembrane electric potential becomes more negative after addition of plant root extracts to G. margarita spores (Ayling et al., 2000). These authors concluded to a direct eVect of root compounds on the membrane of G. margarita, rather than an eVect mediated through modified gene expression. There is, as yet, no evidence for the involvement of host root exudates in the regulation of genes involved in AM fungal biotrophy (Tre´panier et al., 2005). In addition, even if the respiratory response to carrot root exudates is conserved in Gigaspora and Glomus (Tamasloukht et al., 2003), there is no clue as to putative diVerential eVects of branching factors on AM fungal species or eventual host specificities. To date, the mechanisms by which root exudates enhance hyphal branching in AM fungi remain obscure but a scenario has been proposed by Be´card et al. (2004) where the ensemble of metabolic changes induced by root factors enables an AM fungus to exploit its own growth potential and so ensure the developmental switch from the asymbiotic stage of spore germination to presymbiotic hyphal branching. C. IDENTIFICATION OF THE HYPHAL BRANCHING FACTOR: THE STRIGOLACTONE STORY

A breakthrough in deciphering plant–AM fungal signaling events was the identification of the L. japonicus branching factor as 5‐deoxy strigol, belonging to the strigolactone family (Akiyama et al., 2005) (Fig. 2). These authors demonstrated activation of branching in G. margarita by a natural strigolactone purified from L. japonicus, as well as by the chemical analogue GR24 (Fig. 2). Strigolactones were previously isolated as seed germination stimulants of the root parasitic weeds Striga and Orobanche (Bouwmeester et al., 2003), which devastate important food crops (maize, sorghum, millet, rice)

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A

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Fig. 2. Structure of strigolactones that stimulate the hyphal branching response in AM fungi. (A) sorgolactone, (B) GR24 (chemical analogue), and (C) 5‐deoxy strigol.

and legumes. It now turns out that strigolactones are detected as host‐ derived signals both by beneficial fungal symbionts and by parasitic weeds. Strigolactones are active on parasitic weed germination and AM fungal spore germination at very low concentrations (pico to nanomolar). An active component in root exudates of sorghum has also been identified as a sorgolactone (Fig. 2), which stimulates not only Gigaspora branching but also G. intraradices spore germination rates (Besserer et al., 2006). Moreover, strigolactones act on the AM fungus in a way similar to the branching factor from carrot roots, with a rapid increase in respiration rate and in mitochondria biogenesis, morphology, and motility (Besserer et al., 2006). Purification of strigolactones and related compounds has been hampered until now due to their very low concentration in root exudates. This is consistent with a role as a signal molecule and explains why they have been described in few plants since their first discovery (Cook et al., 1966). The chemical lifetime of strigolactones under natural soil conditions may be very short, enabling these chemicals to convey positional information about the roots of living host plants. The presence of strigolactones in both monocots like sorghum and dicots like Lotus suggests a widespread occurrence among angiosperms (Akiyama and Hayashi, 2006; Akiyama et al., 2005; Besserer et al., 2006), which is consistent with the large host spectrum of AM fungi. They have been described by many authors as sesquiterpene lactones, but very little is known about their metabolic pathway or regulation, apart from the involvement of the carotenoid pathway in their biosynthesis (Matusova et al., 2005). The production of parasitic weed stimulants in nonmycorrhizal plant families is largely unreported. The only exception is A. thaliana, for which there are indications of their synthesis but at lower concentrations than in mycorrhizal plants like carrot or tobacco (Goldwasser and Yoder, 2001; Westwood, 2000). However, other classes of active molecules may not be ruled out in AM interactions. Neither Akiyama et al. (2005) nor Besserer et al. (2006) could reproduce the root exudate‐induced branching pattern of

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‘‘arbuscule‐like structures’’ on hyphae using GR24, even at millimolar concentrations. This suggests that strigolactones may act synergistically with other molecules, from the same chemical family or not. The role of strigolactones in fungal appressoria formation has not yet been investigated. In parasitic weeds, after stimulation of seed germination by strigolactones, haustoria development is triggered by other molecules like xenognosin A, phenolics, quinones, and anthocyanidins (Chang and Lynn, 1986; Lynn et al., 1981). It may be hypothesized that flavonoids, which play a role in AM interactions (see above), could also act on appressorium formation and/ or root colonization subsequent to presymbiotic stimulation of hyphal growth and branching by strigolactones. D. FUNGAL SIGNALING TO HOST ROOTS: MYC FACTORS

Microbial symbionts must communicate their presence to host plants (Long, 1996) and plants need to distinguish friends from foes. In the same way that legume root flavonoids may activate nodulation genes in Rhizobia, host root compounds may activate mycorrhization genes in AM fungi. Larose et al. (2002) reported that alfalfa root flavonoid content increased in the presence of mycelium and spores of G. intraradices, before any physical contact between the symbionts, pointing toward the existence of signals derived from the fungus and sensed by the host plant. Hyphae (Gigaspora, Glomus) growing in the vicinity of host–roots but separated by a membrane release a diVusible signal (or signals) of less than 3.5 kDa that induce MtENOD11 gene expression in Medicago truncatula. This expression is synchronous with the induction of hyphal branching, and does not occur with dead spores or fungal pathogens (Kosuta et al., 2003). M. truncatula mutants defective for both nodulation and mycorhization (dmi/Mtsym, Section 3) respond to the AM fungi with the same induction of MtENOD11, whereas they are totally blocked for the Nod factor response (Catoira et al., 2000), suggesting the existence of a signal transduction pathway in presymbiotic AM interactions that is independent of the Nod factor transduction pathway. This conclusion is reinforced by the fact that G. mosseae and Sinorhizobium meliloti activate diVerent sets of signal transduction‐related M. truncatula genes during early interactions with host roots (Sanchez et al., 2005; Weidmann et al., 2004). Furthermore, molecules released by AM spores can induce a transient and rapid calcium response in soybean cells (see Section 3) even after such cells have been challenged with a Nod factor, indicating that the receptors involved in the rhizobial and fungal signal perception are diVerent (Navazio et al., 2007). DiVusible AM fungal signals can also induce lateral root formation in M. truncatula (Ola´h et al., 2005), while root pathogens

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do not have the same eVect. Lateral root formation induced by the fungal signal(s) requires the symbiosis‐related M. truncatula genes DMI1 and DMI2, but not DMI3. Consequently, either the diVusible fungal compound(s) promoting lateral root formation should diVer from the compound(s) inducing MtENOD11 gene expression (Kosuta et al., 2003), or the same compound(s) can activate two diVerent pathways. These diVerent observations, coupled with the chitinous nature of the Nod factor backbone, have led to the speculation that AM fungi (which are much more ancient than Rhizobia) could produce a related ‘‘Myc’’ factor with a role in mycorrhizal signaling similar to that of Nod factors in the nodulation symbiosis. The identification of several plant genes, which are activated during both nodulation and AM interactions in legumes, as well as the discovery of symbiosis‐defective mutants (Section 3), has led to the conclusion that some common mechanisms may regulate root responses to signals from Rhizobia and AM fungi. The next challenge is to identify Myc factors and to describe the Myc factor‐related signaling pathway. Because of the genetic and genomic knowledge accumulated on legumes, this plant family represents a major resource for such investigations.

III. PLANT GENETIC PROGRAMS: MYCORRHIZA‐DEFECTIVE MUTANTS Genetic analyses of interactions between roots and AM fungi have known important advances with the identification of plant mutants (obtained by EMS, gamma irradiation, fast neutron or transposon‐tagging mutagenesis) that are impaired in the development of the symbiosis. These have so far provided evidence for the existence of genes that control root compatibility with the symbiotic fungi in M. truncatula, Pisum sativum, L. japonicus, Vicia faba, Phaseolus vulgaris, Melilotus alba, Zea mays, and Lycopersicon esculentum (Barker et al., 1998b; Borisov et al., 2004; David‐Schwartz et al., 2001, 2003; Duc et al., 1989; Kistner et al., 2005; Lum et al., 2002; Morandi et al., 2005; Oldroyd and Downie, 2004; Paszkowski et al., 2006). Isolation of corresponding genes from mutant backgrounds has been instrumental in identifying some of the plant gene functions essential to the first steps of symbiosis establishment. The cellular and molecular characterization of root interactions with fungal symbionts in mycorrhiza‐defective mutants point to a role of symbiosis‐related plant genes in pathways for specifically sensing and responding to AM fungal signals, and give clues as to how biologically active root or fungal factors may regulate cell functions linked to a successful symbiosis.

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Few plant mutants have been reported so far that are aVected in the presymbiotic phase of AM interactions prior to fungal contact with the root surface (Table I). Absence of appressoria formation on roots of a maize mutant (nope1) indicates a defect in early plant–fungal communication (Paszkowski et al., 2006), and two nonallelic tomato mutants (pmi1, pmi2) aVect precontact events by reducing spore germination and hyphal growth prior to appressoria formation (David‐Schwartz et al., 2001, 2003). Root exudates alone from one tomato mutant (pmi1) were suYcient to impair hyphal growth, suggesting that the corresponding gene may somehow control the release of inhibitory compounds by the host plant (Gadkar et al., 2003). Other observations that plead for a role of symbiosis‐related plant genes in controlling inhibitory root responses to AM fungi are, on the one hand, that AM fungal gene expression can be significantly downregulated in appressoria formed on roots of the dmi3/Mtsym13 mycorrhiza‐resistant mutant of M. truncatula (P. Seddas, M. C. Arias, D. van Tuinen, and V. Gianinazzi‐Pearson, unpublished data) and, on the other, that AM fungal appressoria elicit defence‐related wall reactions and gene expression in pea mutated for PsSYM9 (DMI3/MtSYM13 homologue) (Gollotte et al., 1993; Ruiz‐Lozano et al., 1999). At the same time, none of the mycorrhiza‐ defective plant mutants tested so far show an alteration in their phenotype vis‐a`‐vis either root or aerial pathogens (Catoira et al., 2000; Gao et al., 2006; Gianinazzi‐Pearson et al., 1994; Mellersh and Parniske, 2006), indicating diVerences in the genetic determinants controlling early processes in symbiotic and pathogenic interactions, even though there exists some overlap in host transcriptional responses at later stages (Gu¨imil et al., 2005). Most of the early stage mutants that have been described support appressoria formation by AM fungi but are aVected in the process of root penetration (Table I). By characterizing cytological and molecular events in mycorrhiza‐defective mutants, it has been possible to start to position the plant processes that are regulated by the corresponding symbiosis‐related genes. In the M. truncatula and pea mutants dmi2/Mtsym2 and Pssym9, AM fungal development is blocked at the junction of epidermal cells where the appressoria induce unusual thickening of subtending walls (Calantzis et al., 2001; Gollotte et al., 1993). The colonization process is also blocked at the surface of the epidermis in the double L. japonicus mutants pollux‐13 har1‐1 and ccamk‐5 har1‐1 (Murray et al., 2006) while inactivation of LjSYM15 aVects the separation of adjacent epidermal cells below appressoria that is required for the passage of hyphae into underlying root layers, and mutations in LjSYM2 (LjSYMRK), LjSYM3 (LjNUP33), and LjSYM4 (LjCASTOR) aVect the intracellular passage of hyphae through neighboring epidermal or hypodermal cells (Bonfante et al., 2000; Demchenko et al., 2004;

TABLE I Plant Mutants AVected in Early Interactions with AM Fungi

Z. mays

L. esculentum

L. japonicus

P. sativum

Mutated gene identity

M. truncatula

Mutant phenotypea


Reference(s)

nope1

–

–

–

–

–

App

–

pmi1, pmi2

–

–

–

–

– –

rmc –

– pollux

– –

– dmi1

– ion channel

Sp/Hyred, Appþ, Pen
Appþ, Penþ/
Appþ, Penþ/


– –

– –

Pssym8 Pssym19

– dmi2/Mtsym2

– receptor‐like kinase

Appþ, Pen
Appþ, Penþ/


–

–

– SymRK‐2 Ljsym2 –

Pssym9

dmi3/Mtdmi13

Appþ, Pen


– –

– –

– –

– –

Appþ, Penþ/
Appþ, Pen


–

–

–

–

–

Appþ, Penþ/


Kistner et al., 2005

–

–

Ljsym3 castor‐2 Ljsym4‐2 castor‐4 Ljsym4‐4 Ljsym15‐2

calcium/calmodulin‐ dependent kinase – ion channel

Paszkowski et al., 2006 David‐Schwartz et al., 2001, 2003 Gao et al., 2001 Kistner et al., 2005; Morandi et al., 2005 Albrecht et al., 1998 Kistner et al., 2005; Morandi et al., 2005 Catoira et al., 2000; Morandi et al., 2005 Kistner et al., 2005 Kistner et al., 2005

–

–

Appþ, Pen


Kistner et al., 2005

–

–

–

–

Appþ, Pen


Murray et al., 2006

–

–

–

–

–

Appþ, Pen


Murray et al., 2006

–

–

pollux‐13 har1‐1 ccamk‐5 har1‐1 castor‐23 har1‐1

calcium/calmodulin‐ dependent kinase –

–

–

–

Appþ, Pen


Murray et al., 2006

a App
: no appressorium formation; Appþ, Pen
: appressoria formed, no root penetration; Sp/Hyred: reduced spore germination and hyphal growth prior to appressorium formation; Appþ, Penþ/
: appressoria formed but root penetration and/or appressorium formation dependent on culture conditions and/or AM fungal isolate.

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Wegel et al., 1998). Inactivation of symbiosis‐related plant genes in L. japonicus also has profound eVects on fungal morphology, inducing extensive branching, swollen or deformed appressoria/hyphopodia on mutant roots (Kistner et al., 2005; Murray et al., 2006). However, detailed observations indicate somatic instability of the mutant phenotype in many cases, with fungal invasion of root tissues occurring as a delayed or rare event depending on plant age, growth conditions, or the AM fungal species involved (Demchenko et al., 2004; Gao et al., 2001; Kistner et al., 2005; Morandi et al., 2005; Paszkowski et al., 2006). Present exceptions appear to be the dmi3/Mtsym13 mutant of M. truncatula and the castor‐2, Ljsym15‐2, pollux‐ 13 har1‐1, ccamk‐5 har1‐1, and castor‐23 har1‐1 mutants of L. japonicus (Table I) (Kistner et al., 2005; Morandi et al., 2005; Murray et al., 2006). The phenotypic modifications in fungal–root interactions in M. truncatula and L. japonicus mutants are accompanied by changes in the molecular dialogue between the symbionts. Inactivation of the symbiosis‐related plant genes alters the perception of fungal signals by roots, including gene responses that are part of signal‐transduction pathways, and abolishes AM‐induced transcriptional activity or protein synthesis (Amiour et al., 2006; Kistner et al., 2005; Sanchez et al., 2005; Weidmann et al., 2004). Legumes are the principle model species for genetic studies of plant determinants regulating early steps in AM formation. This has been largely spurred by the fact that legume mutants impaired in AM are also aVected in nodulation, indicating partially shared genetic programs and setting the scene for a common signal transduction pathway in the two root symbioses (see also Section 2). Early indications that AM fungi and Nod factors share steps in a signal transduction pathway came from the observation that mutation of the pea gene PsSYM8 interferes with ENOD5 and ENOD12A responses to both symbiotic stimuli (Albrecht et al., 1998). Since then, symbiosis‐related gene homologues have been isolated from mutant backgrounds of P. sativum, M. truncatula, Medicago sativa, and L. japonicus. Four sets of genes that are essential for root penetration by AM fungi have been characterized and they encode a leucine‐rich repeat receptor‐like kinase (PsSYM19/MtDMI2/LjSYMRK/MSNORK ) (Endre et al., 2002; Stracke et al., 2002), plastid ion channels (MtDMI1/LjCASTOR/LjPOLLUX) (Ane´ et al., 2004; Imaizumi‐Anraku et al., 2005), a nuclear localized calcium‐ and calmodulin‐binding protein kinase (PsSYM9/MtDMI3/LotusCCamK) (Le´vy et al., 2004; Mitra et al., 2004), and a nucleoporin (LotusNUP33) (Kanamori et al., 2006). These diVerent proteins are also needed for early steps in the Nod factor signaling pathway of the nodulation symbiosis where they participate in intracellular calcium responses. They have been positioned in a model signal transduction pathway where the leucine‐rich repeat

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receptor‐like kinase and plastid ion channel proteins, necessary for Nod factor induction of calcium spiking, are upstream of the calcium‐ and calmodulin‐binding protein kinase, which is assumed to be involved in deciphering the calcium signal (Oldroyd and Downie, 2006). Their participation in the AM symbiosis suggests that calcium should function as a second messenger also in mycorrhizal signaling. The use of soybean cell cultures stably expressing the Ca2þ bioluminescent indicator aequorin has recently provided direct evidence of intracellular Ca2þ changes in response to the culture medium of spores of G. margarita germinating in the absence of the plant partner (Navazio et al., 2007). Rapid and transient elevations in cytosolic free Ca2þ were recorded, indicating that diVusible molecules released by the mycorrhizal fungus are perceived by host plant cells through a Ca2þ‐mediated signaling.

IV. MOLECULAR CROSS TALK AND SIGNALING PATHWAYS Novel avenues of research into gene pathways or networks driving the molecular scenario in AM development have opened up with the advent of transcriptomic and proteomic technologies adapted to the analyses of such complex biological systems. Nevertheless, there is still a paucity of information about molecular responses in roots prior to and during initial contact with AM fungi, mainly due to diYculties in synchronizing developmental events in order to dissect gene expression patterns and correlate them with these early stages of the symbiosis. Time‐course transcript profiling of genes activated in functional AM in M. truncatula has identified a number of plant genes that are already induced in response to appressoria and before intraradical mycelium develops (Brechenmacher et al., 2004; Massoumou et al., 2007). Several belong to protein‐encoding gene families that have been implicated in defence strategies against pests or pathogens (glutathione‐S‐ transferase, PR 10 protein, putative wound‐induced protein, germin‐like protein, serine protease, defensin, Kunitz‐type trypsin inhibitor, subtilisin inhibitor). Specific signal molecules produced by pathogens (elicitors) trigger defence reactions in plant tissues. It has been suggested that wall components of AM fungi may also act as elicitors since extracts from extraradical mycelium of G. intraradices can induce phytoalexin synthesis in soybean cotyledons (Lambais, 2000). Enhanced gene expression, protein synthesis, and/or enzyme activities involved in the synthesis of flavonoid compounds have also been reported during the early stages of AM development (Amiour et al., 2006; Bonanomi et al., 2001; Volpin et al., 1994), and increased expression

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of a disease‐related pea gene (PI206) is directly proportional to the number of appressoria developing on roots (Ruiz‐Lozano et al., 1999). Peroxidase, chitinase, catalase, and 1–3 glucanase activities also increase during early AM plant–fungal interactions then decline strongly as root colonization develops (Blilou et al., 2000; David et al., 1998; Lambais and Medhy, 1993; Spanu and Bonfante‐Fasolo, 1988; Spanu et al., 1989; Vierheilig et al., 1994; Volpin et al., 1994). The fact that plant hydrolases, which can attack fungal cell walls (chitinase, 1–3 glucanase), do not prevent root colonization may be explained by a low aYnity or inaccessibility of these enzymes to chitin or 1–3 glucan of the fungal cell wall (Gianinazzi‐Pearson et al., 1996). In conclusion, the amplitude and kinetics of defence responses to AM development diVer markedly from what is typical of plant invasion by pathogenic microorganisms (Gianinazzi‐Pearson et al., 1996) and mechanisms responsible for the AM‐related control have yet to be elucidated. The possible role of symbiosis‐related plant genes in controlling inhibitory root responses has already been evoked. Alternatively, elicitor degradation or the ability of AM fungi to suppress plant defence responses during symbiotic interactions could explain the balance between induction and suppression of plant defence reactions during development of the AM symbiosis. Other plant genes that are upregulated by appressorium formation have been identified in pea and M. truncatula and several have predicted functions in signal perception, transduction, transcription, and/or translation. It has been speculated that a Clp serine protease gene in pea, which is transiently activated during the appressorium stage of fungal–root interactions, may have multiple functions in the control of key regulatory proteins within a signal transduction pathway in the symbiosis (Roussel et al., 2001). Transcripts of another pea gene, PsENOD12A, accumulate when an AM fungus forms appressoria on pea roots and as the epidermis is penetrated (Albrecht et al., 1998). The function of PsENOD12 is unclear but nucleotide sequences indicate that it may encode cell wall proteins induced by AM fungi, in which case it could contribute to assembling the interface compartment initiated prior to cell penetration (see Section 5). Interestingly, expansins and extensins also appear to be involved in initial cell‐to‐cell contact (Weidmann et al., 2004). This class of extracellular proteins may also be operating during epidermal penetration in construction of the interface surrounding the penetrating hypha (Section 5) and/or be involved in producing signaling molecules from the plant cell wall. By targeting gene expression in root systems of M. truncatula where appressorium formation by G. mosseae was synchronized, 21 genes associated with putative signal transduction events have been identified among plant genes activated during this early event in mycorrhiza development (Sanchez et al., 2005; Weidmann et al., 2004).

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These M. truncatula genes encode such proteins as a receptor kinase, calcium lipid‐binding protein, casein kinase, 14.3.3 protein that can interact with calcium‐dependent kinases (Camoni et al., 1998), and annexin that belongs to a family of calcium‐binding proteins (Morgan and Pilar Fernandez, 1997). Proteins that change conformation or catalytic activity on binding calcium allow the cellular perception and transduction of the signal generated by intracellular calcium oscillations (Oldroyd and Downie, 2006). The fact that AM fungal molecules can induce a calcium response in soybean cell cultures (see Section 3) highlights the implication of such a calcium‐modulated signaling pathway in early host cell responses to AM fungi. Nitric oxide (NO) is another well‐known signal molecule in animal systems and it has been identified as a key signaling molecule in plants, where it has diverse functions in a broad spectrum of pathophysiological and developmental processes (Wendehenne et al., 2004). It has been hypothesized that NO may also be involved in AM symbiosis (Vieweg et al., 2005; Weidmann et al., 2004). Nitrate reductase (NR) is a central enzyme in nitrogen assimilation in plants (reducing nitrate to nitrite) that can also catalyze the reduction of nitrite, in high concentrations, to NO (Yamasaki and Sakihama, 2000). Plant NR gene expression and protein activity are reduced or not aVected in functional AM (Hildebrandt et al., 2002; Kaldorf et al., 1998; M. Massoumou, S. Jeandroz, and V. Gianinazzi‐Pearson, unpublished data). However, M. truncatula NR gene expression is, in contrast, enhanced in response to appressorium formation by G. mosseae or G. intraradices (Weidmann et al., 2004; Fig. 3) where it seems more likely to be related to NO metabolism

J5 I

6d NI

TRV25 I

10 d NI

T

GSNO

NR NIR Mtgapdh1

Fig. 3. Plant gene activation related to nitric oxide (NO) metabolism in roots of wild‐type (J5) and myc‐mutant (TRV25) Medicago truncatula. Transcript profiling by RT‐PCR of nitrate reductase (NR) and nitrite reductase (NIR) gene expression in roots inoculated (I) or not (NI) by Glomus intraradices BEG141, and after 4 h treatment or not (T) with an NO donor (GSNO). At 6 days (6 d) after inoculation, 3.5 appressoria/cm root developed on wild‐type (J5) plants; 7 appressoria/cm root developed on dmi3/Mtsym3 mutant roots (TRV25) 10 days (10 d) after inoculation. Mtgapdh1 is a constitutively expressed plant gene encoding a glyceraldehyde phosphate dehydrogenase. (M. Massoumou and S. Jeandroz, unpublished data).

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rather than nitrate assimilation. Nitrite reductase (NiR) is likewise suspected to participate in NO production but through another enzymatic pathway (Sto¨hr and Stremlau, 2006), and recent results have shown that NiR gene expression is also stimulated in response to appressorium formation by G. intraradices on M. truncatula roots (Fig. 3). To test the hypothesis that M. truncatula NR and NiR may be related to NO metabolism, noninoculated roots were treated with NO donors (NONOate and GSNO). Monitoring of NR and NiR gene expression showed that, as in the case of appressorium formation, GSNO treatment activates both plant genes (Fig. 1). The mobile nature of NO, and its chemical reactivity with various cell targets, makes it a potentially important molecule in cell responses. The downstream eVects of NO may be induced directly by its interactions with, for example, ion channel proteins or proteins that regulate gene expression, or indirectly by interactions with signaling proteins such as protein kinases or with secondary messenger‐generative enzymes (Neill et al., 2003). In this context, GSNO treatment also increases transcript levels of a M. truncatula MAPK gene (M. Massoumou, S. Jeandroz, and V. Gianinazzi‐Pearson, unpublished data), which is activated by appressorium formation (Weidmann et al., 2004). Finally, plant NR and NiR transcript accumulation is not aVected when appressoria develop on roots of the mycorrhiza‐deficient dmi3/Mtsym13 mutant of M. truncatula (Fig. 1), indicating that NR and NiR gene expression may be regulated in vivo by a complex signal transduction pathway. NO appears to act through cGMP and cADPR to activate intracellular Ca2þ‐ permeable channels, and also plays a role in elevating free cytosolic Ca2þ (Wendehenne et al., 2004). This is particularly relevant in relation to the fact that DMI3 encodes a Ca2þ and calmodulin‐dependent protein kinase in M. truncatula. NO production during AM interactions has to be verified in situ, using fluorescent specific probes like DAF‐2A, and its role in the symbiosis elucidated. Interestingly, NO production occurs in functional nodules in Rhizobium interactions where it appears to be unrelated to defence or cell death activation (Baudouin et al., 2006). Considerably less is known about fungal processes involved in plant recognition and signal transduction prior to root penetration, or those active in regulating hyphal growth arrest and cytoplasm retraction that occurs after spore germination in the absence of a host root. A gene of G. mosseae encoding a putative hedgehog protein with GTPase activity (GmGIN1) is mainly expressed during spore germination prior to contact with the plant and completely shut down during symbiosis (Requena et al., 2002). Such a protein could be involved in the signaling cascade controlling growth arrest and further programmed cell death of hyphae in the absence of a signal from the host plant. An analysis of transcriptome modifications in germinated

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sporocarps of G. mosseae, triggered in synchrony with appressorium formation on a host root, identified 27 upregulated genes putatively encoding proteins with functions in signaling, transduction, general cell metabolism, defence/stress responses, or of unknown function (Breuninger and Requena, 2004). Some proteins have a potential role in Ca2þ‐based signaling pathways (calmodulin, leucine zipper protein, Ca2þ‐induced Ras inactivator, Ca2þ‐ATPase) that may be an indication of Ca2þ as a second messenger in the fungal perception of a plant signal leading to appressorium formation. Calcium‐ and calmodulin‐dependent signaling during appressorium formation has been demonstrated in pathogenic fungi.

V. PLANT CELL RESPONSES TO FUNGAL COLONIZATION: TISSUE AND CELL SPECIFICITY Morphological observations dating back to the seventies carefully demonstrated that an AM fungus is always surrounded by a membrane of host origin, enclosed in a newly built apoplastic compartment, when it develops within root cells (Scannerini and Bonfante, 1983). The development of in situ protocols (immunolabeling and in situ hybridization), new technological platforms (confocal microscopy and in vivo imaging), the availability of transformed plants with GUS or GFP marker genes and of mutants impaired in their colonization capabilities, as well as the improvement of in vitro mycorrhization systems, have all contributed to the achievement of new insights into the understanding of plant cell responses to root colonization. Liverwort thalli and fern gametophytes are examples of AM‐like associations that do not take place in root organs (Bonfante, 1984), demonstrating that not only the root organ architecture, but even the diploid stage, is not a strict prerequisite to successful colonization by symbiotic fungi. These observations may also have some relevance from an evolutionary point of view. First land plants known to host AM‐like fungi, like Aglaophyton, did not possess true roots (Remy et al., 1994) and the colonized tissues were derived from a vegetative meristem. Strictly speaking, adventitious roots have the same origin and a large number of experiments have demonstrated that these roots are highly susceptible to AM colonization. Therefore, it can be concluded that the first symbiotic niche of AM fungi was epigeous tissues and that even today they do not always require a root for the colonization of plant tissues. Interestingly, rhizobial colonization of stem tissues has been reported in the literature (Goormachtig et al., 2004), although this is limited to tropical legumes and often let aside by mainstream research on nodulation. This suggests that both so‐called root symbioses can be considered as

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present forms of previous interactions where organ specificity was not a rule. On the other hand, it is known that leaf pathogens like Magnaporthe may colonize roots (Sesma and Osbourn, 2004) with a developmental program largely overlapping their better known behavior above ground. Altogether, fungi interacting with plant tissues may be more versatile than generally considered with regards to the host organs they target. In contrast, multicellular tissue organization seems to be a mandatory requirement for fungal colonization: isolated or cultured cells are never colonized, even when put under strong fungal pressure, as in laboratory experiments (Fig. 4A and B). For example, when soybean cells are cocultured with germinating spores of G. margarita, a dense hyphal web develops to surround the individual cells, which often become attached to the hyphae. However, specific adhesion structures such as appressoria are never observed (B. Baldan, L. Navazio, and P. Mariani, unpublished data), suggesting that signaling events triggered by appressorium induction are inoperative in such cell cultures. That said, not all cell types in an organized tissue can be colonized: only epidermal and cortical cells apparently represent a convenient niche for AM fungi. Root meristems and diVerentiating tissues are never colonized, neither are the endodermis, the vascular tissues, or specialized cortical cells, such as idioblasts or those containing raphides or accumulating phenols. To our knowledge, evidence‐based explanations for such a selective colonization pattern have not yet been proposed. Existence of a plant control over AM fungal colonization is obvious from cellular and genetic evidence. It can be hypothesized that plant accommodation

Fig. 4. Gigaspora margarita–Glycine max interactions. (A) Dark field microscopy image showing contacts among hyphae and cultured soybean cells. No appressoria or cell penetration events are present. Bar ¼ 15 mm. (B. Baldan, unpublished data). (B) Brightfield image showing cotton blue‐stained arbuscules in the cortex of a colonized root. Bar ¼ 30 mm.

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responses are only programmed in epidermal and cortical tissues, although the mechanisms involved remain obscure. Root responses to AM fungi can therefore be described on a spatial and temporal scale, depending on the cell type involved (epidermis, cortex) and the corresponding step in root colonization. While investigations of expression profiles for AM‐regulated plant genes have demonstrated profound diVerences with respect to the timescale (Liu et al., 2003), the notion that epidermal and cortical cells may be programmed diVerently is a relatively new concept (Genre and Bonfante, 2005). AM colonization is normally the result of multiple, isolated infection points scattered on the root surface, the development of which is not usually synchronized. In addition, it is diYcult to observe the colonization process in vivo without inactivating the symbiosis and destroying tissue vitality. It is only with the achievement of in vitro mycorrhization of root organ cultures (Chabaud et al., 2002) that critical advances have been possible toward the direct observation of the living interaction. This has also opened the way to studying plant responses even before fungal contact, highlighting the presence of a molecular dialogue between the partners, until then completely unexplored (Kosuta et al., 2003). The root epidermis is the first barrier to all soil microorganisms during their colonization process and it is therefore likely to be the site of recognition mechanisms as well as accommodation/defence responses (Parniske, 2004). As already mentioned, mycorrhizal mutant plants have pointed to the importance of epidermal cells in early steps in the establishment of the AM symbiosis and, as for the Nod factor response in nodule interactions (Oldroyd and Downie, 2006), the epidermis appears to be the site of the earliest plant responses to the still evanescent Myc factor of AM fungi (see Section 2). Rather than being a passive barrier, epidermal cells are an active checkpoint where signal exchanges and a strong control over root colonization occur (Demchenko et al., 2004; Novero et al., 2002). Direct evidence for this has come from the recent description of prepenetration responses designing, a few hours before cell penetration, the track that the AM fungus will subsequently follow in the host cell. In creating an intracellular niche to host another organism within living cells, the AM interaction resembles all other cases of endosymbioses, where the guest organisms are confined into specialized membrane‐bordered spaces. The impact of AM fungi on root cell contents has promoted investigations of the role of the host cytoskeleton as a key structure permitting root cell colonization (Takemoto and Hardham, 2004). While increases in the complexity of microtubule (Blancaflor et al., 2001; Bonfante et al., 1996; Genre and Bonfante, 1997) and actin microfilament (Genre and Bonfante, 1998) organization are striking in arbuscule‐colonized cortical cells, evidence for

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the pivotal role of the cytoskeleton also in fungal accommodation during early stages of root colonization was first provided by studies of the L. japonicus Ljsym4‐2 mutant. When an AM fungus achieves epidermal cell penetration of mutant roots, the absence of a correct cytoskeletal response leads to plant cell death and colonization arrest (Genre and Bonfante, 2002). Taken as a whole, host cell reorganization in response to the symbiotic fungus can be explained by the need for preserving cell integrity, on the one hand, and for optimizing reciprocal compatibility, on the other. In particular, the repositioning of cytoskeleton, as well as endoplasmic reticulum (ER) and Golgi bodies mediating localized membrane proliferation and cell wall deposition, is most likely involved in the construction between symbiont cells of the novel interface compartment.

VI. INTERFACE BIOGENESIS: NEW FACTS/NEW HYPOTHESES The above‐mentioned technological advances have made live cell imaging possible, especially of the epidermal tissue, which is most easily accessible to direct microscopic observation. An important advance in understanding the mechanisms of interface construction in AM interactions has come from the combined application of several up‐to‐date techniques. The first description of in vivo epidermal cell responses to fungal contact was possible through in vivo imaging of GFP‐labeled cell components in mycorrhizal root organ cultures using confocal microscopy (Genre et al., 2005). The main discovery from this research stands in the observation of a novel, ephemeral apparatus, the prepenetration apparatus (PPA), which is assembled from the first moment of surface contact between an AM appressorium and the root, and which is supposed to be responsible for the assembly of the interface compartment prior to cell penetration. The PPA is organized in the epidermal cell cytoplasm below the appressorium as soon as this fungal structure develops at the root surface and remains visible for a few hours, until fungal penetration occurs (Fig. 5). This short life span, together with the possible diYculties in preserving such a membrane structure, accounts for the lack of earlier observations of the PPA. In detail, appressorium contact with the outer epidermal cell triggers the repositioning of the plant nucleus in vicinity of the contact site (Fig. 5A and B). This repositioning, which occurs in a couple of hours, is accompanied by the assembly of localized patches of ER, reorganization of cytoskeleton, and the subsequent appearance of a polarized array of microfilaments radiating from the contact area. After this first step, the nucleus starts to migrate anew,

CUES AND COMMUNICATION IN ARBUSCULAR MYCORRHIZA

A

B

C

205

D

Fig. 5. Scheme of the prepenetration apparatus (PPA) development in a root epidermal cell of Medicago truncatula. Upon contact with the appressorium of an arbuscular mycorrhizal fungus (A), the plant nucleus repositions underneath the contact site (B). A second nuclear migration toward the inner tangential wall coordinates the assembly of a transcellular column of cytoplasm, containing a central membranous thread (C). Finally, a penetration hypha crosses the cell wall and grows within the new‐built interface compartment as the PPA disassembles (D).

toward the cell wall that faces the cortex. This movement is accomplished in 2–4 h and corresponds to complete development of the PPA. It results in a column of cytoplasm being assembled between the nucleus and the contact site, containing a very high density of cytoskeletal fibers and ER cisternae. When nuclear migration terminates, a fine thread of plasma membrane has been laid down in the middle of the PPA to form an apoplastic tunnel (Fig. 5C). Only then fungal penetration occurs and, interestingly, the hypha grows exactly along the route traced by the PPA (Fig. 5D), which then starts to disassemble about 6–8 h after appressorium contact. These observations unambiguously demonstrate an active control by the plant cell over the infection process: fungal development is arrested at the stage of appressorium until the apoplastic tunnel—the future interface—is completed. In addition, intracellular fungal growth is confined to the new compartment, indicating that the epidermal cell has traced in advance a route that the hyphal tip will follow in order to access the root inner tissues. The homology between the PPA and the Rhizobium‐induced infection thread in nodule symbioses is a visible sign of the evolutionary relationship between the two symbioses. The existence of the PPA also opens several new questions about AM interactions (Smith et al., 2006), especially concerning the specificity of such a response to AM fungi or any root‐penetrating microorganism, or the nature of the local signal(s) triggering cell polarization and PPA orientation. In an attempt to address these questions, experiments where GFP‐transformed roots are challenged with diVerent fungi or by mere physical stimulations using a micromanipulator have been designed (A. Genre and coworkers, unpublished data). Initial observations from these experiments suggest that nuclear repositioning can be triggered by physical contact, while

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the assembly of ER patches only occurs in the presence of a living fungus. Furthermore, a completely assembled PPA has only been observed in the presence of AM fungi. Altogether, these observations suggest the existence of a specific fungal signal that orientates plant responses, shifting them toward fungal accommodation and, as a first requirement, interface assembly. As a counterpart of the plant strigolactones, perceived by AM fungi as signals of the presence of a potential host (see Section 2), such fungal signals or Myc factors are supposed to be the main actors of the signaling dialogue that precedes and accompanies root colonization. The molecular programs that regulate early signal exchange and transduction between AM symbionts is of uttermost importance for the mycorrhizal scientific community and one of the key questions in deciphering communication in the symbiosis.

VII. CONCLUSIONS Many of the compounds found in root exudates display both antifungal and antibacterial activities. Thus, it is likely that several secreted compounds act synergistically. This may be true also for stimulatory compounds. Flavonoids have been shown to either stimulate or inhibit AM establishment, depending of the stage of the symbiosis considered and the nutrient status of the plant. Strigolactones have recently been described as new rhizosphere signals involved in early interactions between AM symbionts. The role of these new molecules in the presymbiotic stage is quite well described now, even if the mechanism of action on the fungal cell is still not elucidated. However, research is still in its infancy and their potential role in other stages of the symbiosis is not known. Likewise, the combined eVects with flavonoids or other unknown active molecules are not yet described. Their role as essential molecules for AM symbiosis establishment will be assessed only when plant mutants unable to produce strigolactones will be available. The AM symbiosis is considered nonspecific, but some diVerences in functional compatibility are becoming apparent. Multiple stimulatory molecules capable of eliciting diVerential growth responses in AM fungi may provide the mechanisms by which plants favor interactions with a given symbiont. Root exudate profiles from model plants would be necessary to describe these molecules. The low concentration of active molecules, which hampered characterization of the branching factor for some years, is no longer a problem, thanks to improved isolation protocols and mass spectrometry. The coming years should see an impetus in this new field of plant research.

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The process of compatibility between plants and AM fungi requires a complex cascade of events, some of which are starting to be deciphered. Pathways underlying the early cell processes leading to successful establishment of an AM symbiosis must require activation of specific signal transduction‐related events in both partners. Although the structure of AM fungal signals has yet to be identified, it is already evident that some steps are shared in legume perception of symbiotic Nod and Myc factors. Convergence of genetical, transcriptional, and physiological evidence points to calcium as a secondary messenger in AM interactions and to the involvement of a calcium‐modulated signaling pathway in early host cell responses to AM fungi. The implication of plant NO in the initial molecular dialogue between AM symbionts is also an emerging possibility. A challenge for future research will be to unravel the complexity of such symbiosis signaling and to provide new insights into the specificity of the molecular dialogue of host roots with AM fungi (Harrison, 2005). The discovery of new symbiosis‐defective plant mutants and further exploitation of the genomic and cDNA resources available for model legumes like M. truncatula and L. japonicus (www.noble.org/MedicagoHandbook/; www.lotusjaponicus.org/) will be instrumental for the identification of the host genes involved. The molecular forces governing the role played by the fungus in the development of the AM symbiosis are more diYcult to define. While it is well acknowledged that plant SYM genes control the accommodation process leading to root colonization, fungal genes that could be involved in such a process are completely unknown. Similarly, the morphogenetic mechanisms determining appressorium diVerentiation, intraradical, and intracellular growth are at least as obscure. Advances are hampered by the limited amount of sequence information presently accessible in public databases for AM fungi (e.g., ESTs in NCBI ¼ 10,000 for Glomus and 1100 for Gigaspora). However, breakthroughs are expected within the near future from the ongoing international eVort to sequence the first genome of an AM fungus, G. intraradices, and to expand on the number of available EST sequences (http://darwin.nmsu.edu/~fungi/).

ACKNOWLEDGMENTS Contributions to this chapter were partly funded by MIUR projects (FIRB and PRIN), CEVIOBEM as well as by CNR grants (P.B.), and by the Conseil Re´gional de Bourgogne (V.G.‐P. and S.J.). We thank M. Massoumou (INRA), P. Seddas (INRA), and B. Baldan (CNR) for access to unpublished data, and D. van Tuinen for useful discussion. We apologize to all those researchers whose work could not be included due to page limitations.

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