Frontiers in molecular mycorrhizal research – genes, loci, dots and spins


Functional genomics is revolutionizing our understanding of biological mechanisms, nowhere more than in complex symbiotic systems. Functional genomics entails the analysis of all of the genetic material (the genome) of an organism, and then relating it to the form and function of that organism. Through the application of the cutting-edge tools of genome analysis at several levels – those of the genome, transcriptome, proteome and metabolome – remarkable progress has been made in understanding the mechanisms that control the development and functioning of nitrogen-fixing symbioses (Harrison, 1999; Downie & Bonfante, 2000; Györgyey et al., 2000). A surge of investigations based on these novel approaches (large scale gene sequencing, cDNA array analysis of gene expression, proteomics) have recently allowed an assessment of the development and functioning of arbuscular endomycorrhizal (AM) and ectomycorrhizal (ECM) symbioses (Box 1) on a larger scale (Harrison, 1999; Lapopin et al., 1999; Bago et al., 2001; Voiblet et al., 2001). Similarly, PCR-based detection of genome polymorphisms have revolutionized our perception of the ecology of mycorrhizal fungi (Peter et al., 2001). However, the limitations and ultimate utility of genomics to determine the ecological role of the mycorrhizal symbiosis in the real world remain to be determined. The reviews featured in this issue of New Phytologist have been selected with all these issues in mind. In addition to providing the ‘state-of-the-art’ of current knowledge of molecular mechanisms driving the development, physiology and ecology of the symbiosis, the reviews also provide a glimpse of things to come.

Table Box 1 .  The mycorrhizal symbiosis
The rhizosphere hosts a large and diverse community of microorganisms that compete and interact with each other and with plant roots. Within this community, mycorrhizal fungi are almost ubiquitous. Their vegetative mycelium and root tips form a mutualistic symbiosis. The novel composite organ is the site of nutrient and carbon transfer between the symbionts. The various mycorrhizal associations allow terrestrial plants to colonize and grow efficiently in suboptimal environments. Among the various types of mycorrhizal symbioses, arbuscular endomycorrhiza (AM), ectomycorrhiza (ECM) or ericoid associations are found on most annual and perennial plants (probably > 90%). About two-thirds of these plants are symbiotic with AM glomalean fungi. Ericoid mycorrhizas are ecologically important, but mainly restricted to heathlands. While a relatively small number of plants develop ECM, they dominate forest ecosystems in boreal, temperate and mediterranean regions. In the different mycorrhizal associations, extramatrical and intraradicular hyphal networks are active metabolic entities that provide essential nutrient resources (e.g. phosphate and amino acids) to the host plant. These nutrient contributions are reciprocated by the provision of a stable carbohydrate-rich niche in the roots for the fungal partner, making the relationship a mutualistic symbiosis. The ecological performance of mycorrhizal fungi is a complex phenotype affected by many different genetic traits and by biotic and abiotic environmental factors. Without doubt, anatomical features (e.g. extension of the extramatrical hyphae) resulting from the development of the symbiosis are of paramount importance to the metabolic (and ecophysiological) fitness of the mature mycorrhiza.

A new conceptual framework

Understanding the complexity of the interactions between mycorrhizal symbionts and how these mutualistic associations adapt and respond to changes in the biological, chemical and physical properties of the rhizosphere remains a significant challenge for plant and microbial biologists. Identification of the primary genetic determinants controlling the development of the symbiosis and its metabolic activity (e.g. P and N scavenging) will open the door to understanding the ecological fitness of the mycorrhizal symbiosis. The symbiosis provides an additional layer of complexity to the challenge of designing experimental systems and framing questions, and thus it has been relatively difficult to study these primary genetic determinants. For many years, most physiological analyses were focused on limited sections of the complex metabolic networks (e.g. N and P acquisition and assimilation) taking place in symbionts (Hampp et al., 1995; Harrison, 1999), which limited the degree to which symbiosis behaviour could be understood. Molecular details of mycorrhiza development and functioning are now becoming experimentally tractable owing to simultaneous advances in various fields. The molecular identity of recently discovered symbiosis-regulated (SR) genes, the analysis of mycorrhiza mutants, new experimental approaches, such as cDNA arrays, and innovative applications of older technologies, such as NMR, are together generating a conceptual framework for understanding the formation and physiology of mycorrhizas in which model predictions can now be tested at the molecular level. While familiar with the practicalities of making a PCR or a biochemical analysis, many scientists lack a ‘nuts-and-bolts’ appreciation of the pros and cons of functional genomics. Detailed and thorough understanding of the options available improves the odds that one’s choice will be vindicated; an appreciation of the biological and ecological contexts of genetic (and genomic) questions can also be critical to the success of genome-based analysis.

Dissection of the steps leading to mycorrhizas

At present, we do not know either the molecular mechanisms underlying overall symbiotic tissue patterning nor the specific signals ensuring fungus–root-coordinated development and metabolic continuity between symbiotic partners and between the different hyphal networks (i.e. Hartig net and extraradical hyphal web). Marsh & Schultze (see pp. 525–532, in this issue) emphasize that distinct, but partially overlapping, sets of fungal and plant factors are required and produced at each stage of AM development. Similar developmental stages can be identified during the formation of ECM (Martin & Tagu, 1999). These symbiotic stages are probably controlled by specific signalling pathways and regulatory genes (loci that control the expression of other genes). These genes probably encode sequence-specific DNA-binding transcriptional activators and components of the cell–cell signalling networks. Identification of these symbiosis regulatory factors is a daunting task. It involves the typical gene-to-phenotype approach (i.e. identification of traits through characterization of gene expression and, subsequently, gene inactivation). Although genetic transformation of ECM and AM mycorrhizal fungi has been reported (Bills et al., 1995; Forbes et al., 1998), these species are not yet amenable to gene inactivation. Therefore, a mutant-based genetic approach would allow an unbiased dissection of the steps leading to the symbiosis formation in both AM and ECM. Direct inspection for mycorrhizal mutants in fungi and plants is labour intensive. Genetic analysis is thus scarce and most mutants currently available have been identified in leguminous plants, owing to the fact that the AM symbiosis shares common steps with the plant–Rhizobium symbiosis. Duc et al. (1989) were the first to report that, in pea, some mutants defective in nodulation also did not form the AM symbiosis. Several authors have expanded on that observation (e.g. Albrecht et al., 1998; Resendes et al., see pp. 563–572) and, in the present issue, Marsch & Schultze summarize the current state of knowledge concerning the existing collection of Myc mutants and discuss the future prospects for isolating mycorrhizal specific mutants, as well as the techniques that may be used to generate and identify them. The mutant lines reviewed by these authors represent nearly 40 mutations in at least 7–10 separate loci. Genetic analysis is, however, far from saturation point. Consequently, there is no reliable estimate of the number of genes involved in mycorrhiza development. However, given that mycorrhizal symbioses are widespread, a significant number of mycorrhiza-specific genes must exist. Several of the NodMyc mutants appear to be involved in cell–cell signalling. In view of the observation that some NodMyc mutants induce calcium spiking in root hairs, one of the earliest responses to rhizobial nodulation factors, but others do not, Walker et al. (2000) have speculated that calcium spiking may be common to both mycorrhizal and nodulation signalling and that the pathways could diverge after calcium spiking. Catoira et al. (2000) and Marsch & Schultze proposed a working model with parallel pathways that control different processes required during the development of the complex symbiotic association. Future work on AM leguminous symbioses should concentrate on identification of the components of this signalling network. Direct screens for mycorrhizal mutants in nonleguminous plants are not biased by associated nodule or Nod factor perception defects and might thus identify further signalling mechanisms. The only mutant available so far in a nonleguminous plant was detected in tomato (Barker et al., 1998a). Large collections of transposon insertion lines in Populus spp. (Mathias Faldung, pers. comm.) and UV-generated Hebeloma cylindrosporum mutants (Gilles Gay, pers. comm.) may give a reasonable chance of finding plant and fungal knock-out mutants for ECM development.

Arraying the symbionts to sketch the complexity

For optimal development of the symbiosis, partners need to coordinate complex developmental processes (e.g. the formation of intraradicular symbiotic structures) and, at the same time, sense and respond to novel physiological factors and environmental cues (Barker et al., 1998b; Martin & Tagu, 1999, Nehls et al., 2001a, see pp. 533–542 in this issue). In both AM and ECM symbioses, development involves at least five main events: survival of hyphae in the rhizosphere; primary attachment of hyphal tips to host roots; invasion of root tissues; coordinated construction of symbiotic structures; and bilateral transfer/acquisition of assimilates. All of these steps are dependent of the generation and transduction of rhizospheric and intracellular signals. An understanding of the mechanisms that underlie the temporal and spatial control of genes involved in symbiosis development is now within reach, as more sophisticated techniques of functional genomics are applied to mycorrhizal interactions. In addition to genetic screens for mycorrhiza mutants, Franken & Requena (see pp. 517–523 in this issue) describe how the development of the technology to sequence expressed genes on a large scale and to analyse this DNA through bioinformatics and mRNA profiling will have enormous impacts on the way we think about the biology of mycorrhizal associations. PCR-suppression subtractive hybridizations, transcriptome profilings and proteomics have been used to identify patterns of gene expression underlying the development and physiology of AM and ECM symbioses. Mycorrhiza development affects not only genes involved in cell differentiation and organ development, but also genes controlling nutrient scavenging and assimilation and plant defence reactions.

Until recent times, it has been impossible to study more than one, two, or perhaps a handful of genes and how they interact in complex biological systems, such as symbioses. Franken & Requena, and our own investigations on ECM (Voiblet et al., 2001), show that instead of studying one parameter and one gene at a time, we can use cDNA arrays (macro- and microarrays) to study, within a single experiment (Fig. 1), hundreds to thousands of the host genes and those of the fungal partner whose expression is modified during the host–symbiont interaction: the global picture of the dialogue between the mycobiont and the host in one experiment. Transcriptome analyses, based on cDNA arrays, promise great strides in our understanding of gene expression and interactions, and how overlapping signalling networks simultaneously regulate developmental processes in symbiotic partners. Combined studies applying detailed analysis of key genes, such as phosphate and hexose transporters (Harrison, 1999; Nehls et al., 2001b, see pp. 583–589) and comprehensive transcript profilings by cDNA arrays will allow the prospect, in the future, of linking the regulation of a particular metabolic step to the overall physiological complexity of the symbiosis. Applied to expression analysis, this approach facilitates the measurement of RNA levels for the complete set of transcripts of an organism. Arrays containing 1000–10 000 genes are already in common use for Arabidopsis and yeast, and current protocols allow reliable detection of messages present at several copies per cell. Genome-wide expression in Arabidopsis is currently being released at a fast pace and has dealt with circadian rhythms (Schaffer et al., 2001), impact of cold- and drought-stresses (Seki et al., 2001), and both pathogenesis and wounding (Maleck et al., 2000). By differential screening of arrays carrying about 500 ectomycorrhizal cDNAs, we found 65 genes differentially expressed during the formation of the ectomycorrhizal mantle in the Eucalyptus globulus–Pisolithus sp. association (Voiblet et al., 2001). The number of SR genes displaying similarity to genes involved in cell wall and membrane synthesis, stress/defence responses, protein degradation (in plant cells) and protein synthesis (in hyphae) suggested a highly dynamic cellular environment in which both partners are sending and receiving a varied set of cues, including high levels of stress conditions. Hierarchical cluster analysis defines groups of genes having both similar regulation patterns and expression amplitudes (Eisen et al., 1998) (Fig. 1). For example, several genes that clustered with the symbiosis-regulated hydrophobin genes were known cell wall proteins, such as 32 kDa-symbiosis-regulated acidic polypeptides (SRAP32), but also newly identified structural genes, such as proline-rich proteins and proteophosphoglycans (Duplessis & Martin, unpublished). Many sequences in this cluster of coordinately expressed genes (regulon) showed no significant similarity to known genes and are candidates for further biochemical analyses.

Figure 1.

A typical cDNA array experiment to study mycorrhizal development. (a) RNA is prepared from symbionts or free-living partners and converted into radioactive or fluorescent cDNA probes by reverse transcription. (b) Arrays are usually robotically printed by using each of the mycorrhizal genes of known or unknown sequences, which have been previously amplified by PCR. (c) The labelled cDNA probes are used to hybridize cDNA macro- or microarrays containing (mycorrhizal) genes. (d) After hybridization, laser scanning, image detection, and analysis of each array provides a precise indication of the relative expression of the genes present on the arrays. (e) The final results of the comparative analysis are the expression ratio, where a red dot indicates gene activation upon mycorrhiza development and a green dot means downregulation of the gene, whereas a black dot indicates no change in gene expression. Hierachical clustering (e) allows the identification of coordinately expressed genes or regulons (Eisein et al., 1998). In (e), transcripts of Pisolithus sp. coding for hydrophobins, symbiosis-regulated acidic polypeptides (SRAPs), proline-rich proteins, proteophosphoglycans, and membrane proteins are clustered according to their temporal expression patterns (S. Duplessis and F. Martin, unpublished).

The challenge is no longer in the use of expression arrays themselves, but in developing data mining tools to exploit the full power of a global perspective (e.g. identifying the coregulation of N, P and C metabolic pathways). Some of the most important biological applications involve studying very small target tissues – for example, the endomycorrhizal arbuscules or different regions of ectomycorrhizal tissues; or a particular class of cells in the root cortex. It is hoped that studies based on functional genomics (cDNA arrays and proteomics) will be carried out in the near future on a wider range of mycorrhizal symbioses. In an optimistic scenario, identification of several alternative functional symbiotic patterns generated in a given genetic background (i.e. same host interacting with different AM and ECM fungi, or same mycobiont colonizing different hosts) could soon provide a molecular understanding of both the evolution and development of mycorrhizal symbioses.

A maze of metabolic pathways

The nutritional benefits that mycorrhizal symbioses confer on each partner hinge on the structural and physiological intricacy between the symbionts, and therefore detailed information on metabolism, transport (Nehls et al., 2001a) and functional anatomy of intact systems is required. In addition, elucidation of the function of the hundreds of known and unknown genes analysed by transcript profilings and proteomics may be nicely supplemented by the simultaneous analysis of the metabolome, the latter changing according to the physiological and developmental state of a cell, tissue, organ, or organism. Although metabolic profiling using gas chromatography-mass spectrometry (GC-MS) technologies (Roessner et al., 2001) can give valuable information and is often more sensitive than NMR, it yields much less quantitative information on the position of isotopic enrichments within each molecule. In their review, Pfeffer et al. (see pp. 543–553 in this issue) demonstrate that the NMR approach has been used to trace, in detail, the biochemical pathways by which a given molecule was made in AM and ECM symbioses. NMR spectroscopy yields labelling information directly and quantitatively and this is a significant advantage for metabolic studies. The potential to differentiate, spectroscopically, host from fungal metabolites in vivo or in crude extracts without the need for separation or chemical derivatization is another useful aspect of NMR measurements in studying the biochemistry of mycorrhizas. NMR has therefore contributed significantly to such questions as the biochemistry of polyphosphates and the pathways and regulation of C and N metabolism in mycorrhizas, as well as the identification of secondary metabolites made in response to the synthesis of mycorrhizas and/or to xenobiotics (Bago et al., 2001; Pfeffer et al., 2001). High throughput techniques, such as high-resolution NMR and GC-MS, are already used for comprehensive phenotyping of model systems in conjunction with transcriptome analysis (Roessner etal., 2001; Raamsdonk et al., 2001). They may be equally useful in analysing known AM and ECM mutants and future genetically modified plant and fungal systems.

Bridging genomics and molecular ecology

During the past decade, many PCR-based molecular methods have been developed to identify mycorrhizal fungi, and their potential for mycorrhizal ecology has been proven. Dahlberg (see pp. 555–562 in this issue) emphasizes the fact that the application of these molecular methods has provided detailed insights into the complexity of ECM fungal communities and offers exciting prospects to elucidate processes that structure ECM fungal communities. They will improve our understanding of plant ecology, such as plant interactions and ecosystem processes. About 50 such ECM community studies have been published over the past five years. Molecular studies have shown the following:

  •  Any single mycorrhiza can potentially be identified to species either by PCR-RFLP of the nuclear ribosomal DNA internal transcribed spacers or by DNA sequencing.
  •  Sporocarp production is unlikely to reflect below-ground symbiont communities. Not all ectomycorrhizal fungi produce conspicuous epigeous sporocarps and of those fungi that do produce conspicuous sporocarps, a species’ sporocarp production does not necessarily reflect its below-ground abundance.
  •  A few fungal ECM taxa account for most of the mycorrhizal abundance and are widely spread, whereas the majority of species are only rarely encountered.
  •  The spatial variation of ECM fungi is very high and most species show aggregated distributions. As stressed by Dahlberg, little is known about the relative importance of vegetative spread and longevity of genotypes vs novel colonization from meiospores for any ECM fungal species: this deserves attention if we want to understand the dynamics and structure of ECM communities/populations. Similar advances are being made in our understanding of AM communities.

Despite the fact that mycorrhizal fungi play an important role in N, P and C cycling in ecosystems in decomposing organic materials, the detailed function of fungi in nutrient dynamics is still unknown. Mycorrhizal fungi differ in their functional abilities and the different mycorrhizas they establish thus offer distinct benefits to the host plant. Some fungi may be particularly effective in scavenging organic N, and may associate with plants for which acquisition of N is crucial (Peter et al., 2001); others may be more effective at P uptake and transport. An important goal is therefore to develop approaches by which the functional abilities of the symbiotic guilds are assessed in the field. In any case, it is necessary to look below ground to see what is really happening (Lilleskov & Bruns, 2001). Analyses of 13C and 15N isotopic signatures have a significant potential to provide information in this area (Hobbie et al., see pp. 601–610), although further investigation is required to understand the isotopic enrichment phenomenon (Henn & Chapela, 2000). Combined community/population structure and function studies applying genomics may, in the future, significantly promote our understanding of the interactions between mycorrhizal fungal species with their hosts and with their biotic and abiotic environment. A first step toward this environmental genomics is to explore fungal community functioning under simulated forest conditions using microcosm systems (Timonen et al., 1997). In these systems, intact mycorrhizal root systems comprising individual species (e.g. abundant taxa likely to be functionally important) or natural communities can be manipulated and analysed to determine, for example, C and N relations and host root–fungus metabolic activities that contribute to plant growth or plant community productivity. As a consequence, within the next decade, determining large sets of gene sequences of symbionts will become a prerequisite for truly detailed analyses of gene expression and its regulation in response to environmental changes.

What can we expect to dig up next?

The genomics era for mycorrhizas is not yet in full swing, but it is clear from recent studies, highlighted in the reviews in this issue, that functional genomics and molecular ecology have already made significant contributions to our understanding of developmental and metabolic mechanisms leading to the formation and functioning of mycorrhizas. What will the future bring? We are likely to see imminent advances in understanding of the molecular and cellular mechanisms of the coordinated regulation of developmental and metabolic gene expression in symbiotic partners, although the equally important mechanisms that modulate cell growth rates and shape during mycorrhiza development have not generated a similar intensity of interest. The current compendium of mycorrhiza-regulated genes will provide the basis for a more precise molecular dissection of the complex genetic networks that control symbiosis development and function. The symbiosis regulated-genes identified might be especially interesting targets for future gene disruption technology. Further studies are now needed to delineate the functions of both the known and novel genes that are differentially expressed during mycorrhiza development. Understanding how mycorrhiza regulated-genes exert their developmental effects at the cellular and supracellular levels will be a difficult challenge. In this regard, one anticipates that increased attention will be given to the role of N, P and C transporters (Harrison, 1999; Nehls et al., 2001a) in mycorrhiza function. There will certainly be automation of hybridization assays based on microarray/DNA chip technology.

It will be far harder to define genes that influences symbiont fitness. Any gene that provides the mycobiont with a growth advantage could easily influence the benefits of a particular strain within a given host. Therefore, dissecting the molecular mechanisms of symbiosis requires both identification of the functions of individual genes as well as knowledge of how genes interact to form complex traits such as those expressed in a mutualistic symbiosis. There are more than 5000 different ECM associations and 150 species described in the Glomales, and each and every mycorrhizal type may express a specific set of genes. Despite this, a variety of morphological and molecular parameters can be used to classify mycorrhizal symbioses into discrete molecular classes for further investigation. Symbiotic molecular phenotypes may in the future be correlated to ecological phenotypes. It will be rewarding to compare plant and fungal gene expression profiles among different AM and ECM associations, mycorrhizal symbioses and other plant–fungus interactions. This will certainly identify overlaps in the genetic make up of plant–fungus interactions.

Environmental samples will soon be probed with hundreds to thousands of different DNA probes to tackle ecological questions. Single nucleotide polymorphisms (SNPs) and high-density DNA arrays usher in the possibility of determining allelic imbalance at hundreds of thousands of loci from hundreds of DNA samples, allowing the contemplation of whole genome association studies to determine the genetic contribution to complex polygenic processes (Rothberg et al., 2000). Applied to screening of genetic variation (allelic heterogeneity), the use of microarrays is likely to bring typing of individuals, or even entire populations, into the realm of practical reality (Mei et al., 2000). A combination of the analysis of community/population composition of mycorrhizal fungi by DNA probing (e.g. Bruns & Gardes, 1993) with in situ assessment of function by transcript profiling (environmental genomics) will have a substantial effect on the kinds of questions that can be addressed, particularly for model symbionts where genome analyses have already been initiated (e.g. Pisolithus, Hebeloma, Glomus, Medicago and Populus).


I would like to thank Denis Tagu and Sébastien Duplessis (INRA-Nancy) for valuable discussions on functional genomics of ectomycorrhiza. Genomics studies carried out in my laboratory were supported by grants from the INRA (Programmes ‘Functional Genomics of Poplar’ and ‘LIGNOME’).