This review summarizes the most recent results obtained using molecular biological techniques in the understanding of the arbuscular mycorrhizal symbiosis. On the plant side, cDNA libraries have been established from mycorrhizal RNA using several techniques, such as suppressive subtractive hybridization, and a large number of clones are being sequenced to obtain expressed sequence tags (ESTs). These ESTs can be transferred to solid supports (arrays) and hybridized with cDNA from different sources to obtain RNA accumulation profiles of genes expressed during the mycorrhizal symbiosis. On the fungal side, EST libraries are also being established, both using direct cloning and suppressive subtractive hybridization techniques. For both partners, the next steps are the detailed analysis of some relevant genes concerned with regulation and function, as well as the study of their role in the symbiosis. At the end of this review, we discuss how results about gene expression and functioning from molecular analyses can help in understanding the ecology of the symbiosis and the way in which it can be further exploited for application in plant production systems.
Arbuscular mycorrhizas (AM) are formed between fungi of the order Glomales (Zygomycota) and roots of approximately 80% of the vascular land plants. This mutualistic symbiosis has probably existed for approx. 450 million years since the colonization of the land by the ancient plants and is still a crucial factor for most terrestrial ecosystems. In addition, AM fungi have a high potential or application to plant production (Smith & Read, 1997).
The AM interaction starts after germination of the multinucleated spores, when the emerging hyphae come into the vicinity of the roots, where they show extensive elongation and branching (Giovannetti & Sbrana, 1998). The mode of colonization after contacting the root surface can vary between the so called Paris- or Arum-type (Smith & Smith, 1997). In the latter, the fungi enter the root from an appressorium and subsequently grow intercellularly or cross outer cells with linear or simple coiled hyphae. At the inner cortex, the fungal symbionts penetrate the plant cell wall and form the highly branched arbuscule, the central and name-giving structure of the symbiosis. By contrast, the intercellular phase is absent in the Paris-type, but hyphae grow from cell to cell where they develop large coils with small intercalated arbuscules. In both types, the plant plasma membrane is not disrupted, but invaginates around the developing arbuscules. This results in a new apoplastic compartment, the ‘arbuscular’ interface. The interface is assumed to be the site of nutrient transfer to the plant, while the question of where the carbon is transported to the fungus is still a matter of debate (Harrison, 1999). While AM fungi colonize the root cortex, they also develop mycelium into the surrounding soil where they take up nutrients and form new chlamydospores to fulfil their life cycle.
In the AM symbiosis, both partners possess activities during their interaction which they do not show when they are living alone. This is obvious for the obligate biotrophic AM fungi (Glomales, Zygomycota) which are never able to propagate in pure culture. Increased plant growth, however, is not always obvious, but depends on the genomic background of the fungus and the plant, and how these genomes interact with their environment. In order to use AM fungi efficiently in plant production systems, it is therefore necessary to investigate the molecular biology of the symbiosis. This comprises the genetic basis of both partners, the expression patterns of their genes during the symbiosis and how these patterns are modified and regulated by signals from the respective partner and from the environment (Fig. 1). Such studies were started some 10 years ago and have been reported in several reviews and book chapters (Gianinazzi-Pearson et al., 1996; Harrison, 1999; Franken et al., 2000; Lapopin & Franken, 2001). We therefore only mention some of the recent results and concentrate in this paper on future aspects of molecular AM research.
Plant gene expression profiling
One strategy for the identification of functions involved in the AM symbiosis is the identification of differentially expressed genes. Until now RNA accumulation patterns have been monitored by methods such as differential screening of cDNA libraries, differential RNA display or subtractive hybridization. This has resulted in a number of genes from various plants, which thereafter were further characterized (Lapopin & Franken, 2001). In two projects on Medicago truncatula, a high number of clones were sequence analyzed from nonnormalized cDNA libraries of different sources including mycorrhizas (http://www.TIGR.org; http://www.ncgr.org; http://www.genoscope.cns.fr). In a third project, the cDNA was normalized before cloning by subtractive suppressive hybridization, in order to increase the number of clones from genes with induced RNA accumulation in the symbiosis (V. Gianinazzi-Pearson, pers. comm.). Based on these libraries, inserts with known sequence tags (‘expressed sequence tags’) can be transferred to membranes. These so-called cDNA arrays can be hybridized to labeled cDNA in order to analyze the RNA accumulation of numerous genes in parallel (Duggan et al., 1999). Using this approach, expression patterns of a large number of genes are compared between control roots and mycorrhiza and numerous genes identified which are differentially expressed. This strategy has already been followed in the ectomycorrhizal symbiosis between Eucalyptus globulus and Pisolithus tinctorius (Voiblet et al., 2001). They sequence analyzed 850 cDNA clones and selected 486 for hybridization to cDNA from symbiotic tissue, from roots and from fungal hyphae. Among those, 17% turned out to be > 2.5 times induced or repressed in their RNA accumulation. Once a cDNA array with ESTs is established, it can be used to investigate the expression patterns of the corresponding genes under various conditions. The RNA accumulation of numerous genes of Arabidopsis thaliana has been compared for example in wounding and insect feeding (Reymond et al., 2000) or in response to a fungal pathogen and to salicylic acid, jasmonate or ethylene (Schenk et al., 2000). In the case of AM research it will be interesting to use the cDNA array analyses to find specific and common expression patterns between different plant–microbe interactions as has already been carried out for single genes (Lapopin et al., 1999). In addition it will help to find out whether certain genes directly respond to AM fungal colonization or if they are indirectly regulated via changes in phosphate availability (Burleigh & Harrison, 1999) or hormone concentration. Hybridization of arrays can be also carried out with cDNA from other types of plant tissues or from mycorrhizas which have been developed under various environmental conditions. This will result in the identification of so-called regulons, groups of genes that show the same overlapping expression patterns. Such groupings will be helpful in finding elements of the respective signal transduction pathways and in studying gene regulation in mycorrhizas. It will also be interesting to include plant species that are able to form both ecto- and endomycorrhiza in such analyzes in order to compare the molecular basis of the two symbioses directly .
Although screening of cDNA arrays has a tremendous potential, there are also certain limits to this technology. To concentrate only on genes that show differential RNA accumulation will surely not detect all important functions in AM biology. Firstly, the RNA and protein amounts might differ as has been shown, for example, in yeast (Gygi et al., 1998). Secondly, the function of expressed proteins can be altered by post-translational modifications such as phosphorylation. This is especially true for elements of signal transduction pathways that have important functions in interactions between different organisms. It is therefore necessary to follow in parallel approaches that are directed to the protein level and to compare the results with those from the RNA accumulation analyses.
One important tool for studying gene expression pattern is the analysis of transgenic organisms harbouring promoter–reporter constructs. Recently, a set of H+-ATPase gene promoters was tested in Nicotiana tabaccum (Gianinazzi-Pearson et al., 2000). Two promoters were induced in arbuscule-containing cells, but not in the surrounding cortex of mycorrhizal plants. The advantage of this method is that it is possible to reveal the spatial and temporal pattern of gene activity precisely, but gene cloning, preparing the constructs and transformation of plants is very time consuming. An alternative is in situ hybridization, where RNA accumulation can be directly visualized inside tissues. Results obtained with this technique on the expression of defence-related genes during mycorrhiza development have been reviewed by Blee & Anderson (2000). The high throughput of numerous genes is labour-intensive, but Koltai & Bird (2000) recently described how large numbers of tissue samples and genes could be screened with this technique. Hence, it might be possible to use in situ hybridization with ESTs from cDNA arrays as probes for analysing spatial expression patterns of a high number of genes in mycorrhizas.
Function and role of plant genes in mycorrhiza
The first hypotheses concerning the function of genes are usually obtained from sequence analysis. In the case of significant similarities to genes with known function, the gene product probably has the same function during mycorrhiza development. However, the same function might have a different role in a different context. This is evident for defence- or pathogenesis-related genes, since gene products involved in interactions with pathogens could be needed during different stages of mycorrhizal development. Early and transient accumulation of gene products are often correlated with the first infection stages and are later probably actively repressed by the plant (Gianinazzi-Pearson et al., 1996). Certain genes could also be specifically involved in arbuscule development. Gene products of defence-related genes involved in cell wall metabolism as a hydroxyproline-rich glycoprotein were shown to be expressed in arbuscule-containing cells and localized in the matrix of the symbiotic interface (Balestrini et al., 1997). The gene Prp1 of potato encoding a glutathione-S-transferase has been shown by in situ hybridization to be expressed only in certain arbuscule-containing cells (Franken et al., 2000). Because this enzyme is involved in the removal of toxic material during senescence processes (Hunaiti & Bassam, 1991), it could be involved in the degradation of arbuscules. A number of genes are overexpressed during the whole process of mycorrhiza development. The role of such genes could be the control of hyphal spread in the roots or a reaction to the stress induced by fungal colonization. The latter was discussed for the AM-induced gene Mtaqp1, which encodes an aquaporin localized in the tonoplast and could compensate for decreased vacuole volume in arbuscule cells (Krajinski et al., 2000).
Similarities to genes and precise analysis of spatial and temporal expression patterns can be used to formulate a hypothesis for the role of a certain gene for the AM symbiosis. However, such a hypothesis has finally to be confirmed by interruption of the function of the gene. Since it is not possible to delete or interrupt such a gene, antisense-technology has to be followed in plants as an alternative approach which interrupts the expression of the corresponding protein (Kumria et al., 1998). A newly appearing phenotype would point to the function of the gene. The result of such an experiment could answer the question, if the expression of the gene is a prerequisite for a certain function during symbiosis development or if it is just a consequence of, for example, the stress induced by fungal colonization, as discussed previously for the aquaporin. It is, however, clear that this approach can only be carried out for a limited number of genes. Those have to be carefully chosen on the basis of similarity and/or expression data.
The major shortcoming of the analysis of AM fungal gene expression is the obligate symbiotic growth of the fungus. This limits the amount of fungal material available, which can only be obtained either from spores or from extraradical hyphae. In addition, the quantity of living AM fungal material inside the root is very small in comparison with other plant–microbe interactions. Approximately just 1% of the mRNA extracted from highly colonized roots belongs to the fungal partner, which hinders the discovery of low expression fungal genes. With the help of PCR technology many of the limitations have been overcome and minute amounts of RNA have served as templates for PCR-based cloning, RNA accumulation analysis or cDNA library construction.
PCR cloning of fungal genes
Early experiments using the RNA synthesis inhibitor Actinomycin D already showed that imbibed and germinating AM fungal spores contained newly synthesized mRNA (Hepper, 1979). This mRNA was used as a template for RT-PCR cloning reactions of several genes, and among those one codes for a β-tubulin (Franken et al., 1997). Recently β-tubulin gene sequences from several other AM fungi have been isolated (M. Stommel et al., unpublished). On the basis of these sequences a glomalean-specific primer pair was designed to amplify exclusively the β-tubulin gene from AM fungi, and not from other fungi, including zygomycetes, or from plants. Moreover, cDNA and genomic DNA give rise to amplification products of different sizes, because the primers are located left and right of an intron. After simultaneous extraction of DNA and RNA, it is now possible to monitor in parallel the presence and the metabolic activity of an AM fungus both inside and outside the root, even if other fungi are present.
Other targets for PCR cloning were genes involved in nutrient transport as well as in cell wall metabolism. A fragment of the gene coding for the nitrate reductase apoprotein from the AM fungus Glomus intraradices was PCR-amplified and subsequently cloned. In situ hybridization and Northern blot experiments showed that the corresponding mRNA was accumulated in fungal arbuscules and that its expression inversely correlated with the plant nitrate reductase activity. It was hypothesized that the AM fungi could assist their host in nitrate assimilation in this way during symbiosis (Kaldorf et al., 1998). Several copies of chitin synthase genes from Gig. margarita were found after PCR with degenerates primers. Expression analyzes by RT-PCR revealed the induction of two of them during the symbiotic stage (Lanfranco et al., 1999) which might be related to changes in chitin distribution during fungal development.
This PCR cloning strategy appears to be a good tool to identify AM fungal genes, which are highly evolutionarily conserved. Thus, a new interesting target for this PCR cloning could be the enzymes involved in lipid biosynthesis. A recent finding suggests that the failure of AM fungi to complete their life cycle in the absence of the plant could be due to a lack of storage lipid biosynthesis during the asymbiotic growth (Bago et al., 1999). It has therefore been suggested that the switch of catabolism to anabolism of storage lipids is a key step in AM fungal development. Cloning and expression analysis of genes encoding enzymes involved in lipid biosynthesis could possibly provide support for this hypothesis.
Cloning of fungal genes by comparing expression patterns
Differential expression studies concerning genes from the AM fungal partner were first carried out using presymbiotic mycelium from Glomus mosseae after growth induction by a Bacillus subtilis strain (Requena et al., 1999). A fungal cDNA fragment encoding the homolog of the fatty acid oxidase FOX2 from yeast and human was isolated. The corresponding gene, GmFox2, was found to be down-regulated under the influence of the bacterium. In humans, the homolog protein named 17-hydroxysteroid dehydrogenase IV inactivates estradiol by converting it into estrone. A down-regulation of the gene is known to act as a mitogen by increasing the amount of active estrogens (Carstensen et al., 1996). Interestingly, flavonoids are molecules structurally related to estrogens, whose positive influence on presymbiotic development is well described (Poulin et al., 1997). A possible molecular homolog of estrogens or flavonoids was speculated to be the active molecule produced by the growth-promoting bacterium. Another gene isolated from that study is GmTOR2, encoding a homolog protein of yeast TOR2 involved in control of the cell cycle and actin cytoskeleton (Requena et al., 2000). Experiments using the anti-inflammatory drug Rapamycin (Alexis Biochemicals, Grünberg, Germany), which specifically inhibits the cell cycle controlling activity of TOR2, did not affect spore germination, but further germ tube development. These results support the findings that nuclear replication is not a prerequisite for germination, but it is necessary for presymbiotic hyphal growth (Becard & Pfeffer, 1993; Bianciotto & Bonfante, 1993).
A recent approach used to isolate fungal genes involved in different developmental stages of the fungal life cycle was the construction of a suppressive-subtractive library between presymbiotic and symbiotic mycelium of G. mosseae. Preliminary results have shown transcriptional regulation of some genes such as, for instance, the up-regulation of a gene copy for a P-type H+-ATPase during presymbiotic growth (N. Requena et al., unpublished). Interestingly it was not identical to one of those five members of the ATPase gene family in G. mosseae, which have recently been presented (Ferrol et al., 2000).
Other fungal genes have been isolated by methods that compare RNA accumulation patterns between control roots and mycorrhizas (Delp et al., 2000). These genes might be constitutively expressed, as has been shown for a fungal gene identified in the M. truncatula/G. mosseae mycorrhiza (Franken et al., 2000). Another cDNA fragment from G. mosseae with no similarity to any known sequence was obtained by differential RNA display analyzes of P. sativum wild type mycorrhiza vs a mycorrhiza mutant displaying aborted arbuscule development (Lapopin et al., 1999). RNA accumulation studies by RT-PCR showed that, in this case, the gene is highly expressed in G. mosseae-colonized wild type P. sativum roots, but only weakly induced in the inoculated mutant. Extremely low levels of transcript could be detected in extraradical symbiotic hyphae, dormant spores or presymbiotic mycelium (L. Lapopin et al., unpublished). Current experiments are targeted at the cloning of the entire gene in order to obtain insight into its function. A phosphoglycerate kinase (PGK) cDNA fragment from G. mosseae was identified in tomato mycorrhizas (Harrier et al., 1998). Further studies revealed a significantly higher accumulation of the encoded protein during symbiosis compared with presymbiotic development (Harrier & Sawczak, 2000). Gmpgk is probably regulated by sugar metabolism as it is in other organisms. Accordingly, analysis of the AM fungal promoter has revealed two sequence motifs with homology to carbon source controlled upstream activating elements from Saccharomyces cerevisiae (Harrier, 2001).
EST libraries of AM fungal genes
As described for the plant side of the symbiosis, the construction of cDNA libraries followed by random sequencing to obtain ESTs and screening of cDNA arrays will also be useful tools for the analysis of AM fungal gene expression. RNA accumulation patterns of presymbiotic hyphal development in Gig. rosea using the differential RNA display technique indicated that RNA synthesized during spore activation must be sufficient for further germination and hyphal development, since no significant changes could be observed (Franken et al., 2000). Two EST libraries have been constructed using either activated spores of Gig. rosea (Stommel et al., 2001) or presymbiotic mycelium from Gig. margarita (Lanfranco et al., 2000). In both cases, sequence analyses have revealed similarities to genes coding for proteins involved in multiple cell functions such as translation and protein processing, primary metabolism and transport processes, the cell cycle, DNA replication, chromatin structure, cell structure and signal transduction. Interestingly, in both cases, one of the deduced amino acid sequences showed homology to metallothioneins. These proteins are able to bind metal ions and can play different roles in cellular metabolism (Nordberg, 1998). A tentative speculation here could be that it might be involved in binding of heavy metals and hence the increased tolerance of mycorrhizal plants to soil heavy metal contamination.
These libraries are the beginning of more systematic work on gene expression of AM fungi. For this it will be necessary to choose the so-called model isolates. Several studies, however, indicate that different AM fungi might have some specific biological features. Thus, species from the Glomineae vs those from the Gigasporineae differ, for example, in the presence of vesicles inside the root, the absence of auxillary cells accompanying spore germination (Morton et al., 1998), the physiology of phosphate uptake (Smith et al., 2000) and RNA accumulation patterns during germination (Franken et al., 2000). We would therefore recommend considering at least one representative from each suborder for the systematic sequencing and expression analyzes.
Functional analysis of the identified genes
As pointed out for the plant site, identification and cloning of differentially expressed genes has to be followed by studies about the regulation and the function of those genes. First approaches in this direction were done by Forbes et al. (1998) who tried for the first time to transform an AM fungus. In this experiment, the glucuronidase reporter gene under the control of the heterologous promoter of the GAPDH-encoding gene from Aspergillus nidulans was delivered by particle bombardment into spores of Gig. rosea. Results showed that expression was relatively weak, and the authors attributed this to the use of such a heterologous promoter. Further studies from this group are leading to the isolation of promoters from AM fungi for the improvement of the transformation system (Harrier, 2001). Gene inactivation in AM fungi is, however, not a trivial task. In comparison with other filamentous fungi, the spore contain more than two thousand nuclei, and therefore the deletion of a gene of interest is not feasible. Antisense methodology seems therefore to be a more likely approach for inactivating target genes in AM fungi as has been proposed for the plant site of the symbiosis.
Molecular biology for ecological studies and application
There is no doubt that AM fungi play an important role in terrestrial ecosystems, and recent studies have demonstrated that their biodiversity can be crucial for optimal functioning of the ecosystem (Van der Heijden et al., 1998). Up to now this biodiversity has been studied at the molecular level by PCR amplification of regions from the rRNA gene cluster using DNA extracted from mycorrhizas or from soil spores as templates (Franken, 1998). As an alternative step forward we propose following a similar approach, as described previously for the β-tubulin gene. A target fungal gene playing an important role in the symbiosis must be cloned from several isolates of AM fungi, and sequence comparison can result in the design of glomalean-specific primers. After PCR amplification using mycorrhizal roots as templates, similar sized fragments corresponding to different isolates could be distinguished by, for instance, single stranded conformation polymorphism (SSCP) analysis, as has been shown for the AM fungal 18S rRNA (Simon et al., 1993). In this way, functional biodiversity could be measured in ecosystems, because this approach will not only detect the presence of isolates but also their activity, and therefore quantifies their symbiotic competence at least at the time point of sampling. This will also be useful for the formulation and control of inocula optimized for certain conditions such as low nutrient availability, protection against root pathogens or tolerance against abiotic stress. In addition to single genes, it might also be useful to design cDNA arrays with regulons of genes that respond to the different environmental conditions. Hybridization of such arrays with cDNA from a given inoculum could predict its use for certain applications. At the plant site, important genes for mycorrhizal functioning could be used for the improvement of crop plants in sustainable production systems. The green revolution has resulted in varieties that are optimized for mineral nutrition. Resistance against pathogens has already been included in applied research, but it is also important to take into account mycorrhizal competence. Symbiotic genes could, on one hand, be used as markers in breeding programs and on the other hand introduced into cultivars by means of genetic engineering. This should lead to plant lines which in combination with optimized inocula are applicable to micropropagation of cultivars, revegetation of dry landscapes or contaminated soils, or to plant production with reduced fertiliser and pesticide inputs. Besides the utility for applications, molecular studies on arbuscular mycorrhiza are necessary and interesting per se. Sound basic research about their evolution and about the molecular crosstalk between symbiotic partners is the basis for future progresses in the field of plant–microbe interactions.