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Signaling in mycorrhizal symbioses – elegant mutants lead the way

2002; Wiley; Volume: 154; Issue: 3 Linguagem: Inglês

10.1046/j.1469-8137.2002.00434_1.x

1469-8137

Gopi K. Podila,

Plant nutrient uptake and metabolism

Signaling is a critical component of any symbiosis. In this issue, two separate reports detail research on signaling in the two predominant types of mycorrhizal interaction. How does the progress compare? Arbuscular mycorrhizal (AM) fungi are obligate biotrophs, producing spores that germinate in the presence of the host plant and which develop networks of extraradical hyphae. These hyphae are an essential part of mycorrhizal functioning, since they act as a bridge between soil and roots for nutrient transfer (Smith & Read, 1997). Establishment of the symbiosis requires events in the rhizosphere as well as at the surface of and inside the root, and the process of mycorrhizal development is controlled by the host at different levels (checkpoints) during the colonization process. It is these checkpoints that are the focus of the work of Novero et al. (see pp. 741–749 in this issue), and here it is the application of the mutant approach which is providing the answers. The hallmark of ectomycorrhizal symbiosis is the specialized differentiation and morphogenesis of the plant roots, and here it has been suggested that the growth hormones produced by the mycobiont may have a role in this development. However, very little is known about how phytohormones produced by the fungi could actually trigger the necessary cascade of events. The work of Charvet-Candel et al. (see pp. 769–777 in this issue) addresses these triggers. Here, the mutant approach must be the next step forward. ‘One of the most efficient methods to dissect the colonization process and the genetic basis for compatibility is through generation of mutant plants’ The initial stages of mycorrhizal colonization events are dependant on fungal growth and on rhizospheric signals (Giovannetti et al., 1996). However, the development of infection structures at the contact point with the root surface requires signaling between the partners, the determination of compatibility vs. incompatibility, and both the elicitation and suppression of defense reactions. Once the fungus has entered the host, further events inside the root are even more complex, involving fungal morphogenesis as well as cell reorganization and changes in root morphogenesis. It is not known whether the changes in root cell morphogenesis are a direct consequence of the fungal colonization or whether they occur before actual penetration commences. Signal molecules of fungal origin might be involved in this process, the nature of which could be different from the signals involved before the fungus enters the root tissue. Mutants where the fungal colonization is stopped at different steps can offer a good experimental material to compare the events occurring outside with those occurring inside the root. While Arabidopsis is such a good model for studying many plant–microbe interactions, it cannot serve as a model for studying the development of the AM symbiosis. Thus, other models, such as legumes, are needed (Downie & Bonfante, 2000; Stougaard, 2001). One of the most efficient methods to dissect the colonization process and the genetic basis for compatibility is through generation of mutant plants which are defective in one or more steps of the development of the symbiosis, and in recent years many such mutants have been identified among legumes. Particular attention has been given to mutants blocked at early stages of symbiotic interactions as these steps eventually determine the development of a successful symbiosis. Interestingly, many of these legume mutants were blocked for both nodulation by rhizobial strains and mycorrhizal symbioses (Albrecht et al., 1999). The discovery of such mutants suggests an overlap in the genetic basis for these two diverse symbiotic interactions (Duc et al., 1989; Hirsch & Kapulnik, 1998; Albrecht et al., 1999). Analysis of these mutants provides us with a powerful tool to identify genetically defined steps in the development and biology of the symbiotic interaction (Marsh & Schultze, 2001; Stougaard, 2001). Several mutant lines are now available for legumes such as pea, Medicago truncatula and Lotus japonicus as well as other crop plant species (Duc et al., 1989; Gianinazzi-Pearson et al., 1991; Gollotte et al., 1993; Markwei & LaRue, 1992; Barker et al., 1998; Catoira et al., 2000; Gao et al., 2001; Resendes et al., 2001). Of these model plants, M. truncatula and L. japonicus are genetically the most tractable systems (Downie & Bonfante, 2000). Recently many L. japonicus lines have been generated which contain mutations in several loci (Marsh & Schultze, 2001; Stougaard, 2001), and these exhibit blocking of the colonization process at various stages. In some mutants the infection process is stopped following formation of the appressorium and penetration of epidermal cells, but prior to cortex invasion (Wegel et al., 1998; Parniske, 2000; Senoo et al., 2000). However, here it is unclear whether the checkpoint is based on cell type or some other signals. Many papers have been published on mutants, but very often the phenotype description is inaccurate, based largely on observations from stained roots that do not show the details of the colonization. Due to this, the major blocks which have been described so far are located at the surface of the epidermal cells. One of the mutants of L. japonicus, Ljsym4–2, has been found to block the colonization process at the epidermal cell layers and eventually results in death of epidermal cells (Bonfante et al., 2000). Using elegant and detailed cytological studies, Genre & Bonfante (2002) determined that fungal penetration and growth in epidermal cells is a prerequisite for colonization and arbuscule development in cortical cells. Thus in mutant Ljsym4–2, the presence of the fungus in the epidermal cells triggers a cell specific response and eventually leads to death of epidermal cells and functions as a block for further colonization (Fig. 1a). They have also discovered that another allele exists for Ljsym4 where the colonization proceeds beyond epidermal cells but stops at cortical cells. This allele of Ljsym4, identified as Ljsym4–1, allows occasional colonization of the inner cortex (Wegel et al., 1998; Bonfante et al., 2000). Novero et al. performed a detailed cytological and ultrastructural analysis of this allele to verify whether the mutations of the locus LjSym4 are both stage and cell type specific or whether they act at different time points during the infection process leading to pleiotropic effects. They also quantified fungal structures to show that Ljsym4–1 produces a novel mycorrhizal phenotype. Ljsym4–1 shows blockage of fungal growth in the epidermal cells as in Ljsym4–2. However, in Ljsym4- the number of intercellular hyphae in the cortex is significantly higher in comparison with the wild type and limited numbers of hyphae actually penetrate the cortical cell wall and produce arbuscules (Fig. 1b,c). From these results, the authors propose that there is an additional genetically defined checkpoint for mycorrhizal development by Gigaspora margarita in L. japonicus, located at the wall of cortical cells. Colonization patterns for Ljsym4.2 (a) and Ljsym4.1 (b) and wild-type (WT) (c). A large number of ectomycorrhizal fungi produce auxins (Ek et al., 1983; Gay & Debaud, 1987), and these signaling molecules are at the center of the research of Charvet-Candel et al. on pp. 769–777. The initiation, development and functional maintenance of ectomycorrhizas involves significant morphological changes that are mediated by activation and suppression of several fungal and plant genes (Martin & Tagu, 1999, 2001). During the establishment of the partnership, signal exchange must take place between the symbionts to determine their compatibility and to prepare the partners for further physiological and morphological changes essential for formation of the symbiotic organ (Kim et al., 1998, 1999a,b; Martin et al., 2001; Sundaram et al., 2001; Voiblet et al., 2001). In a way this represents a courtship ritual to establish the compatibility between the partners. However, very little is known about the nature of signals and how this process is started. One of the primary distinguishing features in ectomycorrhiza formation is the morphogenetic differences in the plant root which eventually forms a symbiotic organ in association with the fungal partner. It is a well known fact that auxins regulate plant development and morphogenesis. Most of the current knowledge on the molecular basis of how auxins affect plant development comes from studies on above-ground plant development (Napier & Venis, 1995; Sitbon & Perrot-Rechenmann, 1997). Genes primarily regulated by auxins have been found to be transcription factors, which may in turn regulate many target genes (Abel et al., 1994; Kim et al., 1997). Since so many ectomycorrhizal fungi produce auxins, it could be that the fungal auxins serve as an initial signaling molecule involved in mycorrhiza formation. Studies by Gay et al. (1994) and Gea et al. (1994) using IAA-overproducing mutants of Hebeloma cylindrosporum suggests that fungal produced IAA might trigger morphogenetic changes in host roots during mycorrhiza formation. They further suggested that the fungal auxin could change the physiology of the host root in preparation for the interaction with the fungus. After the initial IAA signals from the fungus, further regulated release of hormones within the plant could then be involved in the morphogenesis of the plant roots and eventual formation of mycorrhiza. Charvet-Candela et al. identified host plant (Pinus pinaster) genes up-regulated by auxins and other signals from H. cylindrosporium, from early stages of interaction through to the formation of ectomycorrhizas. One of the cDNAs identified, Pp-iaa88, encodes a polypeptide with high similarity to angiosperm Aux/IAA transcription factors induced by auxin treatment (Oeller et al., 1993; Abel et al., 1995; Abel & Theologis, 1996). Pp-iaa88 is the first reported gymnosperm homolog of Aux/IAA gene. The high sequence similarity Ppiaa88 with other known IAA/Aux genes from various angiosperms shows that these genes may have evolutionarily conserved functions in the development and morphogenesis of shoot and root. Aux/IAA genes have been postulated to be transcriptional regulators of secondary auxin response genes (Abel et al., 1994; Abel & Theologis, 1996). The fact that fungal IAA could trigger transcription of Ppiaa88 in the absence of protein synthesis (Charvet-Candela et al.) indicates that IAA can function as a primary signal to induce the transcription of target genes such as Ppiaa88. In turn, Ppiaa88 may have a potential role in triggering a cascade of molecular events in the pine roots leading to the formation of mycorrhizas, similar to expression of EgHypar, another auxin-inducible gene, which is also up-regulated during the differentiation of Eucalyptus globulus/Pisolithus tinctorius ectomycorrhizas (Nehls et al., 1998). Charvet-Candela et al. also made the interesting observation that in addition to fungal IAA, other molecules such as quercetin, a flavanoid involved in plant–microbe signaling, could also trigger induction of Ppiaa88 transcription. It is likely that quercetin can induce other pathways that in turn regulate Ppiaa88 transcription. This observation suggests that there may be redundant mechanisms through which Ppiaa88 can be triggered, which in turn regulate target genes involved in root development and morphogenesis. The work by Novero et al. shows that it is possible to dissect the colonization process of AM fungi as a multistep process and that the plant host has many checkpoints to make sure that the fungal partner is the right partner to establish mycorrhizas (Fig. 2). Use of mutants greatly facilitates identification of the genetic controls for these checkpoints. The elegance of this work comes from the fact that the authors were able to do a thorough and accurate quantitative analysis associated with different morphological observations using more refined cytological and ultrastructural analyses. These strategies allowed them to establish that there are many levels of scrutiny in the colonization process and success at each checkpoint is a prerequisite for the progression of colonization and development of the mycorrhizal symbiosis. These studies and others (Genre & Bonfante, 2002) demonstrate that the epidermis is an active check point for the AM symbiosis in a similar way to the situation for Rhizobium legume−1 interactions. Future studies involving identification of the gene products for Ljsym4 (Checkpoint Charlie) and the basis for its activities will further help understand this fascinating courtship of mycorrhizal symbiosis. Ljsym4 based checkpoints for colonization by Gigaspora margarita in Lotus japonicus. Left panel outer checkpoint (epidermis specific) and right panel inner checkpoint (cortical cell specific). Based on the work of Charvet-Candela et al. it appears that Ppiaa88 may serve as an important marker for early stages of ectomycorrhizal symbiosis in pine roots. Future studies using IAA defective mutants of H. cylindrosporum and P. pinaster will no doubt yield useful information on the role of IAA and other fungal derived signals in the induction of Ppiaa88. These types of studies are critical to understand the nature of signals and the mechanism of fungal hormone based signal transduction pathways that form the basis for ectomycorrhiza formation.