Hibbett et al. (2000) suggested that mycorrhizal symbionts have evolved repeatedly from saprotrophic precursors, but also that certain ectomycorrhizal fungi have retained the genes for enzymes that can degrade plant cell walls, which may have facilitated repeated evolutionary reversals to saprotrophy. Sagara et al. (1992) also showed that, in Japan, L. bicolor is a facultative symbiont and may behave as a saprotroph in the soil. One can speculate on the specific aspects of each of these two trophic behaviours (saprobic vs symbiotic). No ectomycorrhiza-specific gene has been found so far (Duplessis et al., 2005), which suggests that ontogenic and metabolic programmes leading to functional symbiosis are driven by changes in the organization of gene networks and in gene expression in both partners (Duplessis et al., 2005). The coevolutionary implication is that the ectomycorrhizal symbiosis is not only a juxtaposition of the genetic potentialities of the two partners but leads indeed to novel metabolic patterns in hyphae and plant cells, based, among others, on increased uptake capacity of the root system and effective exchanges between symbionts (Hibbett et al., 2000; Duplessis et al., 2005).
Recent studies on mycorrhizal interactions have highlighted some key transporters for nutrient uptake or metabolite transfer at the biotrophic interface. New N, phosphorus or carbon transporters have been identified either from mycorrhizal fungi (H. cylindrosporum, A. muscaria, Tuber borchii, Glomus versiforme, Glomus intraradices) or from their host plants (Oryzae sativa, Medicago truncatula, Picea abies, P. trichocarpa) (Harrison et al., 2002; Selle et al., 2005; Chalot et al., 2006; Javot et al., 2007b; Muller et al., 2007). Many of these transporters displayed differential expression depending on nutrient concentration; however, only a few of them (endomycorrhiza-specific Pi transporter (Javot et al., 2007a), A. muscaria hexose transporter (Nehls et al., 1998) and P. trichocarpa ammonium transporters (Selle et al., 2005; Couturier et al., 2007) have been shown to be specifically upregulated in symbiotic tissues, indicating a putative role in symbiotic exchanges at the plant–fungal interface. Precise function and localization of most of these transporters in fungal tissues remain largely unknown and require further investigation. The nature of released nutrients or assimilated metabolites into the apoplast interface by the fungus remains unclear and the answer to whether amino acids or ammonium are the preferentially transferred N form have still not been elucidated. The nature of the N form transferred to plant and mechanisms involved in this transfer through the apoplast will likely arise from molecular approaches coupled with biochemical techniques.
Existing data, mostly deriving from studies based on targeted analytical approaches, lead only to partial views of biotrophic interface functioning. The present analysis aimed at establishing a repertoire of gene models that currently represent the genetic potential for transport of N-related compounds in L. bicolor. We have revealed the presence of at least 128 gene models encoding putative transport protein for N-containing compounds in the genome of L. bicolor. Among them, 118 genes (92%) showed detectable transcript levels. A comprehensive physiological analysis of the current dataset is not within the scope of the present study, but we would like to highlight some of the striking results from this genome survey which would be helpful for ectomycorrhizal and fruiting body characterization of functional compartmentation and key markers identification for exchanges.
In silico analysis of sequenced genomes of symbiotic (L. bicolor), saprobic (P. chrysosporium, C. cinerea) or parasitic (U. maydis and C. neoformans) basidiomycetes have enabled us to point out some remarkable expansions of L. bicolor transporter-encoding genes families (e.g. AMT (Fig. 1), YAT and LAT (Fig. 3), NCS1 (Fig. 7)), mostly by specific expansion of gene model number in several clusters of these families. These members are likely to play a key role in the symbiotic way of life compared with parasitic or saprobic ones. We may also assume that physiological constraints produced by the nature of the available growth substrates may have driven the emergence of paralogue transporters. Furthermore, these paralogues may play an important role in functional specialization of multicellular tissues differentiated in ectomycorrhiza or fruiting body. We also observed some clusters containing a majority of symbiotic and saprobic fungal sequences and only a minority of parasitic fungal ones (e.g. OPT; Fig. 6). Some L. bicolor gene models do not cluster with characterized genes, and could therefore correspond to new molecular determinants. This enrichment in N transporter encoding genes may point out a specialization of L. bicolor during the coevolution with its plant host. Symbiotic relationship is indeed mostly built on the ability of the fungus to transfer nutrient or assimilated metabolites, especially N, to the host.
Expression analyses have allowed us to point out a number of:
constitutively expressed genes, which might play a fundamental role in basal transport processes
upregulated or/and highly expressed genes in ectomycorrhiza, which may be important for mycorrhiza function at either import of N compounds at the soil–fungus interface or export of N compounds at the fungus–plant interface
upregulated or/and highly expressed genes in the fruiting body, which may be important for fruiting body development, functioning and internal nitrogen recycling
repressed genes in the fruiting body compared with free-living mycelium
repressed genes in ectomycorrhiza compared with free-living mycelium. The latter genes may be involved in uptake of N compounds by the free-living mycelium as well as in the prevention of N compound losses by hyphal leakage
We showed that L. bicolor has the genetic potential for both mineral and organic N compound utilization. The expression of a part of this genetic potential will further define the functions of the different mycelia types and will emphasize the dual trophic abilities (symbiotic vs saprobic) of L. bicolor. The expansion of the AMT family, in addition to the putative role of these transporters in the symbiosis, could also be linked to the place of L. bicolor in ecological successions. L. bicolor is indeed described as a member of ‘ammonia fungi’– a group of fungi that occur after decomposition of urine, faeces or dead bodies has occurred, playing thus an important role in waste material removal (Sagara, 1992, 1995). Physiological studies on those fungi suggested that ammonia is the key substance for their growth, even if they readily use nitrate as sole N source (Yamanaka, 1999).
Our results highlighted a wide range of amino acid and peptide transporters involved either in uptake or internal recycling. L. bicolor has been shown to have little/no ability to grow on organic N sources such as amines, proteins or some amino acids (Yamanaka, 1999). However, expression patterns of these transporter genes could indicate that they may play a role in fungal physiology.
Although putative amino acid and ammonium exporters encoding genes (ScATO and ScAQR1 orthologues) did not display mycorrhiza-specific expressions patterns, they were expressed in mycorrhiza and we could not exclude an important role of these exporters at the symbiotic interface. However, vesicle loading proposed by Chalot et al. (2006) could also be an important mechanism in L. bicolor. Direct analysis of apoplast fluid (Solomon & Oliver, 2001) together with tracer experiments could be an appropriate tool to elucidate transfer between fungal and root cells. Detailed analysis of individual compounds using specific isotope analysis may also be a key to further address these important questions (Evans, 2001). Furthermore, the increasing application of molecular and postgenomic methodologies (large-scale oligoarrays), as well as technological improvements with higher spatial resolution (LCM technology), is now available for nutrient, transcript or protein detection within apoplast interfaces (Lohaus et al., 2001; Balestrini et al., 2007). Together with the present gene repertoire, this will allow further establishment of the biotrophic ‘transportome blueprints’ (transport proteins and compounds) at symbiotic interfaces.