1. Best costume design: ‘A formal dress-code: branching out for contact’
The AM symbiosis is the result of a complex exchange of molecular information that starts before the partners engage in physical contact (Bonfante & Requena, 2011). A major breakthrough in elucidating this pre-symbiotic crosstalk relates to the identification of plant root-exuded metabolites, the so-called strigolactones (SLs), which stimulate branching of germinated AM hyphae to encourage host-root colonization (Akiyama et al., 2005). SLs were originally identified as stimulators of the germination of root-parasitic weeds, which are obligate plant biotrophs that threaten resource-limited agriculture (Cook et al., 1966). During the last decade, genes encoding enzymes essential for SL biosynthesis or perception/signalling have been identified through their cloning from a series of mutants displaying increased shoot branching phenotypes, as reviewed in Xie & Yoneyama (2010). Results showed that SLs are synthesized from a carotenoid substrate by sequential cleavages involving two carotenoid cleavage dioxygenases (CCD)7 and 8 and a subsequent oxidation by a cytochrome P450. As illustrated in Fig. 2, three other genes termed D27, D14 and D3 were identified in rice, which encode a plastid-located iron-containing protein involved in SL biosynthesis, a α/β-fold hydrolase and a F-box protein involved in signal perception, respectively (Xie & Yoneyama, 2010).
Figure 2. Schematic overview of the strigolactone (SL) and Myc-LCO-related pathways that mediate arbuscular mycorrhizal (AM) hyphal and root branching responses. Inferred from silencing-based experiments, up- and down-headed arrows indicate plant proteins believed to maximize and minimize plant–fungus contact events, respectively. Because reduced expression of the corresponding genes mostly results into quantitative differences in root colonization without affecting arbuscule morphology, it seems likely that these proteins play roles in AM symbiosis rather through impacting hyphal penetration events than through mediating intra-radical development. Asterisks correspond to plant proteins belonging to the SYM pathway required for fungal colonization. Phosphate regulation events refer to the data provided by: (a) Breuillin et al. (2010) and (b) Liu et al. (2011).
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Initial functional demonstrations for a role of SLs in sustaining the AM symbiotic programme come from SL biosynthesis knockout mutants (ccd8) in tomato and pea, which display a reduction in mycorrhizal colonization of roots (Gomez-Roldan et al., 2008; Koltai et al., 2010). Likewise, significantly decreased concentrations of SL and reduced arbuscule abundance were recorded in tomato plants expressing a cdd7 antisense construct (Vogel et al., 2010). Recently, new insights into the functional characterization of the SL-dependent symbiotic signalling were obtained from silencing-based experiments performed in barrel medic, rice and petunia. In the search for efflux carriers for strigolactones, Kretzschmar et al. (2012) isolated ABC transporters of Petunia hybrida on the basis of their abundance in phosphate-starved or mycorrhizal roots. Using silencing and/or transposon-mediated loss-of-function, the candidate PDR1 was demonstrated to act as a strigolactone export carrier because only extra-radical SL concentrations (orobanchol) were affected in pdr1 mutants, in which root exudates showed a reduced activity for stimulating hyphal branching of AM fungi relative to wild-type. Likewise, petunia pdr1 knockout or knockdown lines displayed a significantly reduced ability to accommodate mycosymbionts without exhibiting defects in arbuscule morphology, suggesting that quantitative differences in colonization were due to decreased hyphal penetration events and retarded intraradical expansion, rather than to defects in intracellular development.
A second milestone decisive in understanding the pre-symbiotic crosstalk between mycosymbionts and plants roots was the chemical elucidation of some diffusible AM fungal signalling molecules belonging to the so-called Myc factors. These stimulate root growth and branching through a calcium signal that determines the activation of essential symbiotic genes. It has been shown that the AM fungus R. irregularis secretes symbiotic signals corresponding to a mixture of sulphated and nonsulphated simple lipochitooligosaccharides (Myc-LCOs), thus confirming the working hypothesis that Myc signals are ancestors of the more recent LCO-like Nod factors produced by most rhizobia and required for early steps of legume infection and nodule organogenesis (Maillet et al., 2011). Strikingly, studies also revealed that the intricacy of Nod and Myc signalling was more complex than previously anticipated. Initially, the symbiotic signalling pathway identified in Medicago truncatula included genes coding for Nod factor perception (NFP and LYK3), calcium signalling (DMI1, DMI2 and DMI3), and transcription factors (NSP1 NSP2 and ERN). NFP and LYK3 correspond to Nod factor membrane receptors so far described dispensable for AM fungi signal-induced calcium oscillations in epidermal cells and subsequent mycorrhiza formation. The three transcription factors were thought to be specifically activated upon Nod factor perception, while DMI1, 2 and 3 were required both for nodulation and mycorhization (Maillet et al., 2011 and references therein). However, Op den Camp et al. (2011) provided evidence that in Parasponia, the only nonlegume partner of rhizobia, a single cell surface receptor can recognize both fungal and bacterial signals and induce the common SYM pathway to promote the intracellular accommodation of AM fungi and rhizobia. Actually, silencing of the unique NFP orthologue in Parasponia was found to impair the formation of both symbioses. Likewise, the data obtained by Maillet et al. (2011) suggested that NFP is partly involved in the Myc-signal-elicited root branching response, as inferred from the reduced root branching response observed in nfp mutants relative to the wild-type. Additionally, by reason of a 40% lower colonization level than wild-type plants exhibited in the nsp2 mutant, a NSP2-dependent signalling pathway was found to facilitate mycorrhizal root colonization, thus indicating that the transcriptional activator NSP2 does not function exclusively in rhizobium Nod factor signalling (Maillet et al., 2011). In accordance with this view, recent comparative gene expression studies in symbiotic mutants demonstrated that transcriptional reprogramming by AM fungal LCOs strictly depends on MtNSP and largely requires MtDMI3. It is noteworthy that none of the genes related to arbuscule development were activated by Myc-LCOs, suggesting that the function of Myc-LCOs was restricted to pre-symbiotic AM stages (Czaja et al., 2012). On the basis of transcript profiling experiments in nsp1 and nsp2 knockout mutants, it was also shown that NSP1 and NSP2 are indispensable for strigolactone biosynthesis in M. truncatula and rice (Liu et al., 2011). The disturbed SL biosynthesis in nsp1 nsp2 mutant backgrounds was found to correlate with reduced expression of D27 that encodes the plastid-located iron-containing protein essential for SL biosynthesis. In contrast to nodulation, none of the components of the Nod factor signalling pathway, not even the kinase CCaMK directly active upstream of NSP1 and NSP2, are required for D27 expression. It was thus proposed that the NSP1 and NSP2 proteins fulfil dual regulatory functions to control not only downstream targets after rhizobium-induced signalling, but also SL synthesis in nonsymbiotic conditions. With regard to AM fungal infection, the M. truncatula nsp1 nsp2 double mutant shows a reduction in mycorrhizal root infection but without defects in arbuscule development, suggesting that SLs stimulate root colonization exclusively ex planta (Liu et al., 2011). Finally, mutations in D14 and D3 that encode a α/β-fold hydrolase and an F-box protein, respectively, resulted in rice plants that are insensitive to SL and produce more SLs than the wild-type owing to the lack of feedback suppression. Consistently, hyphae of AM fungi had additional branches when grown in the vicinity of rice roots of d3 and d14 mutants relative to wild-type plants (Yoshida et al., 2012). A dual function for the protein D3 was also proposed by Yoshida and co-workers. Actually, although AM hyphal branching was activated in the vicinity of roots of d3 mutants, hyphae were often unable to extend beyond the epidermal cell layer and progressive internal fungal growth and arbuscule formation were significantly reduced. Once penetration was achieved, arbuscules were shaped similarly to those of controls. On the contrary, d14 RNAi rice lines displayed an increased mycorrhizal colonization, probably reflecting higher fungal activities by reason of a higher SL production (Yoshida et al., 2012). Although it remains unclear whether the AM phenotypes observed in d3 mutants are the result of SL perception/signalling, the physiological role of D3 turns out to be distinct from that of D14.
Overall, it appears from the above-cited data that at least nine plant proteins (NFP, NSP1, NSP2, CCD7, CCD8, D27, PDR1, D3 and D14) were demonstrated as either directly or indirectly involved in the fungal-root branching-induced crosstalk that takes place during pre-symbiosis, as schematized in Fig. 2.
2. Best stage setting: ‘Fungal entry: the red carpet’
The pre-symbiotic phase of AM symbiosis ends once a hyphal tip has contacted the root epidermis, following exudate-mediated hyphae and root branching events. Unlike aerial pathogens that have to penetrate the leaf cuticle through the differentiation of an appressorium that generates a glycerol-mediated turgor pressure, glomeromycotan fungi develop hyphododia, which are nonmelanized and nonseptate small swellings structures at the hyphal tip that mediate adhesion to epidermal root cells (Genre et al., 2009; Bonfante & Genre, 2010; Heupel et al., 2010). Recently, Gobatto et al. (2012) reported that plants mutated in RAM1, which encodes a GRAS-domain transcription factor specifically required for Myc factor signalling, displayed a defect in hyphopodium formation at the root surface and were unable to be colonized by AM fungi. Because ram1 roots showed a more severe phenotype than plants mutated in components of the symbiosis signalling pathway (dmi1, dmi2 and dmi3), the authors hypothesized that RAM1 functioned downstream of the SYM pathway, but might also act in a manner independent of it. Notably, considering the role played by NSP2 in Myc factor signalling (Maillet et al., 2011), they observed that RAM1 could interact with NSP2, but not with NSP1, suggesting that the SYM pathway might trigger GRAS protein complex formation, with the complex NSP2/RAM1 leading to mycorrhiza-specific responses (Gobatto et al., 2012). Concomitantly, RAM1 was found to regulate the expression of RAM2 that encodes a glycerol-3-phosphate acyl transferase, which promotes cutin biosynthesis and enhances hyphopodium formation (Wang et al., 2012). Considering first that cutin is synthesized as monomers, laid down at the cell surface in an esterified form, and second, that lipid monomers alone could complement the defect in mycorrhizal perception of the ram2 roots, Wang and co-workers favoured for cutin monomers a signalling role in promoting hyphopodium formation rather than a structural function.
Beside signalling events occurring at the root surface, a third outstanding breakthrough in understanding the plant AM symbiotic programme relates to the root responses elicited upon hyphopodium formation, among which belongs the formation of a pre-penetration apparatus (PPA) that outlines the route for hyphal growth across the plant cell lumen (Genre et al., 2005). Following a cytoplasmic aggregation, which consists of a cytoskeleton-driven accumulation of organelles including the plant nucleus, at the contacted epidermal site, the host cell develops a transcellular cytoplasmic column, the PPA, whose elongation follows the migration of the plant nucleus toward the inner cell wall facing the root cortex (Fig. 1). When using roots of M. truncatula expressing a fluorescent tag for the ER, which were challenged with an AM fungus, a necrotrophic pathogen, a hemibiotrophic pathogen, a noncompatible endomycorrhizal fungus, or abiotic stimuli, a correlation was identified between physical stimulation at the cell surface and nuclear repositioning. It was also observed that cytoplasmic aggregation, a cell response shared by the AM fungi and by the two pathogens, is clearlyDMI3/CCaMK-dependent, thus extending the role of this gene during the contact phase from symbiotic to pathogenic interactions (Genre et al., 2009). A key conserved component of the common SYM pathway, CCaMK, is currently believed to decode the Ca2+ spiking that is activated in the host epidermis during initial recognition of endosymbionts and to trigger appropriate downstream signalling pathways leading to gene transcription in association with LjCYCLOPS/MtIPD3. Recently, it has been reported that distinct Ca2+ spiking profiles correlate with specific stages of transcellular apoplastic infection (Sieberer et al., 2012). Low-frequency spiking cells are characterized by nuclear migration to the site of future cell infection associated with cytoplasmic reorganization in the vicinity of the nucleus. By contrast, there is an increase in the frequency of Ca2+ spiking just before and during initial cortical cell entry by both bacterial and fungal symbionts, which involves an irreversible cell wall disassembly and de novo interface synthesis linked to membrane invagination. It has been proposed that the protein Vapyrin could mediate Ca2+-mediated membrane and cytoskeleton rearrangements during initial stages of root cell infection by rhizobia and AM fungi (Ercolin & Reinhardt, 2011; Sieberer et al., 2012). Actually, in M. truncatula, Vapyrin RNAi roots show a high frequency of hyphopodia that attempt but fail to penetrate the epidermal cells (Pumplin et al., 2010). In contrast to common sym mutants impaired in initial endosymbiotic signalling (Parniske, 2008), in Vapyrin RNAi roots, the fungus attempts to penetrate the cells as exemplified by the hyphal projections existing below hyphopodia. Consequently, Pumplin et al. (2010) hypothesized that the signalling process necessary to induce fungal penetration was not affected in vapyrin knockdown lines, but that the cellular machinery supporting fungal entry and membrane invagination was impaired. Recently, MtVapyrin was reported to be induced upon Myc-LCOs application, suggesting that the encoded protein is accumulated in anticipation of colonization (Czaja et al., 2012). Interestingly, the Vapyrin mutant hyphopodia phenotype was also observed in transgenic rice lines upon silencing of the D3 gene, which results in an increased strigolactone production, suggesting a functional relationship between D3 and Vapyrin that remains to be characterized (Yoshida et al., 2012). A critical role for membrane reorganization for fungal accommodation at the early stage of AM symbiosis was also reported by Kuhn et al. (2010) who showed that downregulation of the membrane steroid-binding protein 1 MtMSBP1 through RNAi led to an aberrant mycorrhizal phenotype characterized by thick and septate hyphopodia with aborted penetration attempts. Because MtMSBP1 encodes a membrane steroid-binding protein involved in sterol homeostasis, it has been proposed that alteration of lipid metabolism is required to sustain membrane invagination and intracellular accommodation of symbionts (Kuhn et al., 2010).
Aside from cellular remodelling events in response to AM fungus sensing, it was recently shown that silencing of the Rac1 GTPase MtROP9 triggered the stimulation of early root colonization by the AM fungus R. irregularis coupled to an inhibition of reactive oxygen species (ROS) and anti-oxidative compounds production in M. truncatula roots (Kiirika et al., 2012). RAC proteins are plant-specific small GTPases that function as molecular switches within elementary signal transduction pathways, including the regulation of ROS generation via activation of plasma membrane-associated NADPH oxidases. It was thus concluded that the oxidative burst that occurs 20 min after AM spore inoculation, somehow concomitantly with the calcium spiking response (Chabaud et al., 2011), did play a role in mounting an early defence barrier against mycorrhizal colonization. Interestingly, the PPA triggered by AM fungi was found to elicit a specific transcriptome response in epidermal cells, including the DMI3-dependent upregulation of an expansin-like gene having a role in cell wall plasticity, and the downregulation of the defence-related gene ACRE264 (Siciliano et al., 2007a,b). ACRE264 is known to encode the protein Avr9/Cf-9 that is required for full resistance to Cladosporium fulvum strains expressing the Avr9 gene. Consequently, the DMI3-mediated suppression of defence-related genes like ACRE264 after physical contact with the hyphopodium led Siciliano et al. (2007b) hypothesize that plant–AM fungus compatibility requires basal defence responses to be kept under control as observed in compatible plant–pathogen interactions.
In this line of reasoning, a fourth significant recent advance in understanding the early steps mediating symbiont accommodation by plant cells was the discovery that AM fungi do use effector proteins to short-circuit the plant defence programme (Kloppholz et al., 2011). Plants are known to have a basal defence system trained to recognize conserved traits of microbial pathogens termed MAMPS (microbial-associated molecular patterns). Recognition of these epitopes by pattern recognition receptors induces MAMP-triggered immunity, a first line of plant defence to prevent further colonization of the host. In return, microbial colonizers have evolved the capacity to deliver effector proteins inside host cells to cope with MAMP-triggered immunity, often through suppression of host defences (de Jonge et al., 2011; Zamioudis & Pieterse, 2012). When investigating whether AM fungi use effector proteins to short-circuit the plant defence programme, Kloppholz et al. (2011) showed that R. irregularis secreted a protein, SP7, which can cross plant membranes to interact with the defence-related ethylene-responsive factor ERF19 in the plant nucleus to block the ERF19-mediated transcriptional programme, including the expression of target defence proteins. The constitutive expression of SP7 in roots was reported to lead to higher mycorrhization while reducing the levels of the fungal pathogen Colletotrichum trifolii-mediated defence responses, as assessed by the PR10 marker. Noteworthy, SP7 expression in the rice blast fungus Magnaporthe oryzae, a hemibiotrophic pathogen that can infect both leaves and roots of host plants, results in the reduced expression of defence genes encoding PR10 proteins in rice roots and extends the length of the biotrophic phase, delaying the root decay that characterized the necrotrophic phase. Overall, the results obtained by Kloppholz et al. (2011) support the view that SP7 acts as a broad spectrum effector to promote the biotrophic phase of a fungus inside a plant. Aside from this report, the use of transformable hemibiotroph fungal pathogens such as Colletotrichum spp. and M. oryzae whose development share common features with the AM fungal lifecycle (spore, appressoria and/or hyphopodia, haustoria), coupled to comparative analyses of genome sequences from plant-infecting fungi, also succeeded in revealing AM fungal proteins mediating plant cell entry. Tollot et al. (2009) demonstrated as essential the transcription factor STE12 in mediating hyphal penetration of leaf surfaces from appressoria and in some cases for subsequent invasive growth by hemibiotrophic plant pathogens. Introduction of GinSTE, a STE12 homologue, isolated from R. irregularis, into a noninvasive mutant of Colletotrichum lindemuthianum was found to restore penetration and infectivity of the fungal pathogen in Phaseolus vulgaris leaves. Likewise, Erl1, a Ras-like GTPase from the rice blast M. oryzae was found to be homologous to the mature amino terminal part of the Gin1 protein of R. irregularis. Deletion of ERL1 in M. oryzae resulted in delayed appressorium formation, slow growth in planta and reduced intracellular colonization without defect in the necrotic ability of the fungus, indicating that ERL1 is required for invasive growth of root tissues. Because the root browning defect of ∆erl1 strains could be complemented by the AM fungus gene, it was suggested that Erl1 and Gin-N are orthologues and might be involved in the control of polar hyphal growth in planta, thus extending the hypothesis of common genetic features underlying plant colonization strategies among different fungi (Heupel et al., 2010).
Besides the common SYM pathway, the identification of twelve additional proteins involved in the early intracellular accommodation of AM fungi allows us to propose a hypothetical model illustrating the protein pattern associated with AM fungal entry into plant cells at the early stages of symbiosis, as schematized in Fig. 3.
Figure 3. Schematic representation of a model protein pattern associated with arbuscular mycorrhizal (AM) fungal entry into plant cells at the early stages of symbiosis. (a) The perception by host plants of Myc factors produced by arbuscular mycorrhizal fungi (AMF) induces the symbiotic programme through the SYM pathway (DMI1, DMI2, DMI3) that triggers a NSP2/RAM1/RAM2-mediated signal at the cell surface via the production of cutin monomers, and the activation of cellular remodelling events in both plant (MSP1, Vapyrin, D3, Expansin) and fungal (GinSTE12) partners. (b) Host plants are able to recognize AMF as potential colonizers through pattern-recognition receptors (PRR) that perceive microbe-associated molecular patterns (MAMPs). As a result, a signalling cascade probably involving Rac1-mediated reactive oxygen species (ROS) production (Kiirika et al., 2012) is induced, which results in MAMP-triggered immunity (MTI) through the production of defence-related compounds. In response, AMF have developed the capacity to secrete the SP7 protein effector into the plant cytosol, which upon targeting the nucleus, interacts with the defence-related transcription factor ERF19 to block the ERF19-mediated transcriptional programme. The SYM pathway is also possibly involved in the suppression of MTI, as exemplified by the DMI3-dependent downregulation of the defence-related protein ACRE264 (Siciliano et al., 2007a,b). White and blue colours refer to fungal and plant structures/metabolites/proteins, respectively.
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