Nitrogen and phosphate metabolism in ectomycorrhizas.

1047 I. Introduction 1047 II. Mobilization of soil N/P by ECM fungi 1048 III. N/P uptake 1048 IV. N/P assimilation 1049 V. N/P storage and remobilization 1049 VI. Hyphal N/P efflux at the plant-fungus interface 1052 VII. Conclusion and research needs 1054 Acknowledgements 1055 References 1055 SUMMARY: Nutrient homeostasis is essential for fungal cells and thus tightly adapted to the local demand in a mycelium with hyphal specialization. Based on selected ectomycorrhizal (ECM) fungal models, we outlined current concepts of nitrogen and phosphate nutrition and their limitations, and included knowledge from Baker's yeast when major gaps had to be filled. We covered the entire pathway from nutrient mobilization, import and local storage, distribution within the mycelium and export at the plant-fungus interface. Even when nutrient import and assimilation were broad issues for ECM fungi, we focused mainly on nitrate and organic phosphorus uptake, as other nitrogen/phosphorus (N/P) sources have been covered by recent reviews. Vacuolar N/P storage and mobilization represented another focus point of this review. Vacuoles are integrated into cellular homeostasis and central for an ECM mycelium at two locations: soil-growing hyphae and hyphae of the plant-fungus interface. Vacuoles are also involved in long-distance transport. We further discussed potential mechanisms of bidirectional long-distance nutrient transport (distances from millimetres to metres). A final focus of the review was N/P export at the plant-fungus interface, where we compared potential efflux mechanisms and pathways, and discussed their prerequisites.

Composite poplars: a novel tool for ectomycorrhizal research.

KEY MESSAGE: Composite poplars were used for ectomycorrhiza formation. Structurally normal mycorrhizas of transgenic roots revealed better fungal sugar support. Targeting fluorescent proteins to peroxisomes allowed easy in planta visualization of successful transformation. A bottle neck in ectomycorrhizal research is the time demand for generation of transgenic plants. An alternative strategy for such root-centered research might be the formation of the so-called composite plants, where transgenic roots are formed by non-transgenic shoots. We have developed an Agrobacterium rhizogenes-mediated root transformation protocol using axenic Populus tremula × tremuloides and P. tremula × alba cuttings. When comparing four different bacterial strains, A. rhizogenes K599 turned out to be the most suitable for poplar transformation. Transgenic roots revealed only minor hairy root phenotype when plants were grown on agar plates with synthetic growth medium in the absence of a sugar source. When using different ectomycorrhizal fungi, formation of ectomycorrhizas by transgenic roots of composite poplars was not affected and mycorrhizas were anatomically indistinguishable from mycorrhizas of non-transgenic roots. Elevated trehalose content and marker gene expression, however, pointed towards somewhat better fungal carbon nutrition in ectomycorrhizas of transgenic compared to non-transgenic roots. Cell wall autofluorescence of poplar fine roots is an issue that can limit the use of fluorescent proteins as visual markers for in planta analysis, especially after ectomycorrhiza formation. By targeting marker proteins to peroxisomes, sensitive fluorescence detection, easily distinguishable from cell wall autofluorescence, was obtained for both poplar fine roots and ectomycorrhizas.

Ectomycorrhizal ecology is imprinted in the genome of the dominant symbiotic fungus Cenococcum geophilum.

The most frequently encountered symbiont on tree roots is the ascomycete Cenococcum geophilum, the only mycorrhizal species within the largest fungal class Dothideomycetes, a class known for devastating plant pathogens. Here we show that the symbiotic genomic idiosyncrasies of ectomycorrhizal basidiomycetes are also present in C. geophilum with symbiosis-induced, taxon-specific genes of unknown function and reduced numbers of plant cell wall-degrading enzymes. C. geophilum still holds a significant set of genes in categories known to be involved in pathogenesis and shows an increased genome size due to transposable elements proliferation. Transcript profiling revealed a striking upregulation of membrane transporters, including aquaporin water channels and sugar transporters, and mycorrhiza-induced small secreted proteins (MiSSPs) in ectomycorrhiza compared with free-living mycelium. The frequency with which this symbiont is found on tree roots and its possible role in water and nutrient transport in symbiosis calls for further studies on mechanisms of host and environmental adaptation.

Convergent losses of decay mechanisms and rapid turnover of symbiosis genes in mycorrhizal mutualists.

To elucidate the genetic bases of mycorrhizal lifestyle evolution, we sequenced new fungal genomes, including 13 ectomycorrhizal (ECM), orchid (ORM) and ericoid (ERM) species, and five saprotrophs, which we analyzed along with other fungal genomes. Ectomycorrhizal fungi have a reduced complement of genes encoding plant cell wall-degrading enzymes (PCWDEs), as compared to their ancestral wood decayers. Nevertheless, they have retained a unique array of PCWDEs, thus suggesting that they possess diverse abilities to decompose lignocellulose. Similar functional categories of nonorthologous genes are induced in symbiosis. Of induced genes, 7-38% are orphan genes, including genes that encode secreted effector-like proteins. Convergent evolution of the mycorrhizal habit in fungi occurred via the repeated evolution of a 'symbiosis toolkit', with reduced numbers of PCWDEs and lineage-specific suites of mycorrhiza-induced genes.

Fungal aquaporins: cellular functions and ecophysiological perspectives.

Three aspects have to be taken into consideration when discussing cellular water and solute permeability of fungal cells: cell wall properties, membrane permeability, and transport through proteinaceous pores (the main focus of this review). Yet, characterized major intrinsic proteins (MIPs) can be grouped into three functional categories: (mainly) water transporting aquaporins, aquaglyceroporins that confer preferentially solute permeability (e.g., glycerol and ammonia), and bifunctional aquaglyceroporins that can facilitate efficient water and solute transfer. Two ancestor proteins, a water (orthodox aquaporin) and a solute facilitator (aquaglyceroporin), are supposed to give rise to today's MIPs. Based on primary sequences of fungal MIPs, orthodox aquaporins/X-intrinsic proteins (XIPs) and FPS1-like/Yfl054-like/other aquaglyceroporins are supposed to be respective sister groups. However, at least within the fungal kingdom, no easy functional conclusion can be drawn from the phylogenetic position of a given protein within the MIP pedigree. In consequence, ecophysiological prediction of MIP relevance is not feasible without detailed functional analysis of the respective protein and expression studies. To illuminate the diverse MIP implications in fungal lifestyle, our current knowledge about protein function in two organisms, baker's yeast and the Basidiomycotic Laccaria bicolor, an ectomycorrhizal model fungus, was exemplarily summarized in this review. MIP function has been investigated in such a depth in Saccharomyces cerevisiae that a system-wide view is possible. Yeast lifestyle, however, is special in many circumstances. Therefore, L. bicolor as filamentous Basidiomycete was added and allows insight into a very different way of life. Special emphasis was laid in this review onto ecophysiological interpretation of MIP function.

8-Hydroxy-efavirenz, the primary metabolite of the antiretroviral drug Efavirenz, stimulates the glycolytic flux in cultured rat astrocytes.

In active antiretroviral therapy antiretroviral drugs are employed for the restoration of a functional immune system in patients suffering from the acquired immunodeficiency syndrome. However, potential adverse effects of such compounds to brain cells are discussed in connection with the development of neurocognitive impairments in patients. To investigate potential effects of antiretroviral drugs on cell viability and the glycolytic flux of brain cells, astrocyte-rich primary cultures were exposed to various antiretroviral compounds, including the non-nucleoside reverse transcriptase inhibitor efavirenz. In a concentration of 10 µM, neither efavirenz nor any of the other investigated antiretroviral compounds acutely compromised the cell viability nor altered glucose consumption or lactate production. In contrast, the primary metabolite of efavirenz, 8-hydroxy-efavirenz, stimulated the glycolytic flux in viable astrocytes in a time- and concentration-dependent manner with half-maximal and maximal effects at concentrations of 5 and 10 µM, respectively. The stimulation of glycolytic flux by 8-hydroxy-efavirenz was not additive to that obtained for astrocytes that were treated with the respiratory chain inhibitor rotenone and was abolished by removal of extracellular 8-hydroxy-efavirenz. In a concentration of 10 µM, 8-hydroxy-efavirenz and efavirenz did not affect mitochondrial respiration, while both compounds lowered in a concentration of 60 µM significantly the oxygen consumption by mitochondria that had been isolated form cultured astrocytes, suggesting that the stimulation of glycolytic flux by 8-hydroxy-efavrienz is not caused by direct inhibition of respiration. The observed alteration of astrocytic glucose metabolism by 8-hydroxy-efavirenz could contribute to the adverse neurological side effects reported for patients that are chronically treated with efavirenz-containing medications.

Structure and expression profile of the phosphate Pht1 transporter gene family in mycorrhizal Populus trichocarpa.

Gene networks involved in inorganic phosphate (Pi) acquisition and homeostasis in woody perennial species able to form mycorrhizal symbioses are poorly known. Here, we describe the features of the 12 genes coding for Pi transporters of the Pht1 family in poplar (Populus trichocarpa). Individual Pht1 transporters play distinct roles in acquiring and translocating Pi in different tissues of mycorrhizal and nonmycorrhizal poplar during different growth conditions and developmental stages. Pi starvation triggered the up-regulation of most members of the Pht1 family, especially PtPT9 and PtPT11. PtPT9 and PtPT12 showed a striking up-regulation in ectomycorrhizas and endomycorrhizas, whereas PtPT1 and PtPT11 were strongly down-regulated. PtPT10 transcripts were highly abundant in arbuscular mycorrhiza (AM) roots only. PtPT8 and PtPT10 are phylogenetically associated to the AM-inducible Pht1 subfamily I. The analysis of promoter sequences revealed conserved motifs similar to other AM-inducible orthologs in PtPT10 only. To gain more insight into gene regulatory mechanisms governing the AM symbiosis in woody plant species, the activation of the poplar PtPT10 promoter was investigated and detected in AM of potato (Solanum tuberosum) roots. These results indicated that the regulation of AM-inducible Pi transporter genes is conserved between perennial woody and herbaceous plant species. Moreover, poplar has developed an alternative Pi uptake pathway distinct from AM plants, allowing ectomycorrhizal poplar to recruit PtPT9 and PtPT12 to cope with limiting Pi concentrations in forest soils.

The aquaporin gene family of the ectomycorrhizal fungus Laccaria bicolor: lessons for symbiotic functions.

Soil humidity and bulk water transport are essential for nutrient mobilization. Ectomycorrhizal fungi, bridging soil and fine roots of woody plants, are capable of modulating both by being integrated into water movement driven by plant transpiration and the nocturnal hydraulic lift. Aquaporins are integral membrane proteins that function as gradient-driven water and/or solute channels. Seven aquaporins were identified in the genome of the ectomycorrhizal basidiomycete Laccaria bicolor and their role in fungal transfer processes was analyzed. Heterologous expression in Xenopus laevis oocytes revealed relevant water permeabilities for three aquaporins. In fungal mycelia, expression of the corresponding genes was high compared with other members of the gene family, indicating the significance of the respective proteins for plasma membrane water permeability. As growth temperature and ectomycorrhiza formation modified gene expression profiles of these water-conducting aquaporins, specific roles in those aspects of fungal physiology are suggested. Two aquaporins, which were highly expressed in ectomycorrhizas, conferred plasma membrane ammonia permeability in yeast. This indicates that these proteins are an integral part of ectomycorrhizal fungus-based plant nitrogen nutrition in symbiosis.

Harnessing ectomycorrhizal genomics for ecological insights.

The ectomycorrhizal (ECM) symbiosis is increasingly seen as a crucial component for nutrient cycling in sustainable forest ecosystems. To date, the complete genome sequence of only a single symbiotic fungus, the ECM basidiomycete Laccaria bicolor, has been released. Its analysis revealed unexpected features, including numerous transposons, secreted effector-like proteins and a lack of carbohydrate-hydrolysing enzymes acting on plant cell walls. Genome-wide transcript profilings showed that most symbiosis-induced transcripts code for proteins with unknown function and identifying their role will be challenging. However, these studies confirmed the key role of membrane transporters in the symbiosis. The analysis of additional genomes from ECM fungi will provide further insights into the evolution, development and ecological role of the mycorrhizal symbiosis.

Salt stress affects xylem differentiation of grey poplar (Populus x canescens).

In this study the impact of salt stress on the physiology and wood structure of the salt-sensitive Populus x canescens was investigated. Two weeks of salt stress altered wood anatomy significantly. The xylem differentiation zone was reduced and the resulting vessels exhibited reduced lumina. To understand this phenomenon, ion composition, levels of corresponding transcripts and of the stress hormone ABA were analysed. With increasing sodium and chloride concentrations, a general reduction of potassium was found in roots and shoots, but not in leaves. Consequently, the corresponding K+ channel transcripts in roots favoured K+ release. The overall osmolarity in leaves was up to fourfold higher than in roots or shoots. Therefore, adjustment of the K+/Na+ balance seemed not to be required in leaves. Sodium increased gradually from roots to shoots and then to leaves indicating that sodium storage took place first in roots, then in shoots, and finally in leaves to protect photosynthesis from salt effects as long as possible. Since leaf abscisic acid levels markedly increased, stomatal closure seemed to limit CO2 uptake. As a consequence, diminished nutrient supply to the cambium in combination with lowered shoot K+ content led to decreased vessel lumina, and a reduction of the radial cambium was observed. Thus, xylem differentiation was curtailed and the development of full size vessels was impaired.

The sugar porter gene family of Laccaria bicolor: function in ectomycorrhizal symbiosis and soil-growing hyphae.

Formation of ectomycorrhizas, a symbiosis with fine roots of woody plants, is one way for soil fungi to overcome carbohydrate limitation in forest ecosystems. Fifteen potential hexose transporter proteins, of which 10 group within three clusters, are encoded in the genome of the ectomycorrhizal model fungus Laccaria bicolor. For 14 of them, transcripts were detectable. When grown in liquid culture, carbon starvation resulted in at least twofold higher transcript abundances for seven genes. Temporarily elevated transcript abundance after sugar addition was observed for three genes. Compared with the extraradical mycelium, ectomycorrhiza formation resulted in a strongly enhanced expression of six genes, of which four revealed their highest observed transcript abundances in symbiosis. A function as hexose importer was proven for three of them. Only three genes, of which just one was expressed at a considerable level, revealed a reduced transcript content in mycorrhizas. From gene expression patterns and import kinetics, the L. bicolor hexose transporters could be divided into two groups: those responsible for uptake of carbohydrates by soil-growing hyphae, for improved carbon nutrition, and to reduce nutrient uptake competition by other soil microorganisms; and those responsible for efficient hexose uptake at the plant-fungus interface.

Mastering ectomycorrhizal symbiosis: the impact of carbohydrates.

Mycorrhiza formation is the consequence of a mutualistic interaction between certain soil fungi and plant roots that helps to overcome nutritional limitations faced by the respective partners. In symbiosis, fungi contribute to tree nutrition by means of mineral weathering and mobilization of nutrients from organic matter, and obtain plant-derived carbohydrates as a response. Support with easily degradable carbohydrates seems to be the driving force for fungi to undergo this type of interaction. As a consequence, the fungal hexose uptake capacity is strongly increased in Hartig net hyphae of the model fungi Amanita muscaria and Laccaria bicolor. Next to fast carbohydrate uptake and metabolism, storage carbohydrates are of special interest. In functional A. muscaria ectomycorrhizas, expression and activity of proteins involved in trehalose biosynthesis is mainly localized in hyphae of the Hartig net, indicating an important function of trehalose in generation of a strong carbon sink by fungal hyphae. In symbiosis, fungal partners receive up to approximately 19 times more carbohydrates from their hosts than normal leakage of the root system would cause, resulting in a strong carbohydrate demand of infected roots and, as a consequence, a more efficient plant photosynthesis. To avoid fungal parasitism, the plant seems to have developed mechanisms to control carbohydrate drain towards the fungal partner and link it to the fungus-derived mineral nutrition. In this contribution, current knowledge on fungal strategies to obtain carbohydrates from its host and plant strategies to enable, but also to control and restrict (under certain conditions), carbon transfer are summarized.

Interaction with mycorrhiza helper bacterium Streptomyces sp. AcH 505 modifies organisation of actin cytoskeleton in the ectomycorrhizal fungus Amanita muscaria (fly agaric).

The actin cytoskeleton (AC) of fungal hyphae is a major determinant of hyphal shape and morphogenesis, implicated in controlling tip structure and secretory vesicle delivery. Hyphal growth of the ectomycorrhizal fungus Amanita muscaria and symbiosis formation with spruce are promoted by the mycorrhiza helper bacterium Streptomyces sp. AcH 505 (AcH 505). To investigate structural requirements of growth promotion, the effect of AcH 505 on A. muscaria hyphal morphology, AC and actin gene expression were studied. Hyphal diameter and mycelial density decreased during dual culture (DC), and indirect immunofluorescence microscopy revealed that the dense and polarised actin cap in hyphal tips of axenic A. muscaria changes to a loosened and dispersed structure in DC. Supplementation of growth medium with cell-free bacterial supernatant confirmed that reduction in hyphal diameter and AC changes occurred at the same stage of growth. Transcript levels of both actin genes isolated from A. muscaria remained unaltered, indicating that AC changes are regulated by reorganisation of the existing actin pool. In conclusion, the AC reorganisation appears to result in altered hyphal morphology and faster apical extension. The thus improved spreading of hyphae and increased probability to encounter plant roots highlights a mechanism behind the mycorrhiza helper effect.

Increased trehalose biosynthesis in Hartig net hyphae of ectomycorrhizas.

To obtain photoassimilates in ectomycorrhizal symbiosis, the fungus has to create a strong sink, for example, by conversion of plant-derived hexoses into fungus-specific compounds. Trehalose is present in large quantities in Amanita muscaria and may thus constitute an important carbon sink. In Amanita muscaria-poplar (Populus tremula x tremuloides) ectomycorrhizas, the transcript abundances of genes encoding key enzymes of fungal trehalose biosynthesis, namely trehalose-6-phosphate synthase (TPS), trehalose-6-phosphate phosphatase (TPP) and trehalose phosphorylase (TP), were increased. When mycorrhizas were separated into mantle and Hartig net, TPS, TPP and TP expression was specifically enhanced in Hartig net hyphae. Compared with the extraradical mycelium, TPS and TPP expression was only slightly increased in the fungal sheath, while the increase in the expression of TP was more pronounced. TPS enzyme activity was also elevated in Hartig net hyphae, displaying a direct correlation between transcript abundance and turnover rate. In accordance with enhanced gene expression and TPS activity, trehalose content was 2.7 times higher in the Hartig net. The enhanced trehalose biosynthesis at the plant-fungus interface indicates that trehalose is a relevant carbohydrate sink in symbiosis. As sugar and nitrogen supply affected gene expression only slightly, the strongly increased expression of the investigated genes in mycorrhizas is presumably developmentally regulated.

Ectomycorrhiza-mediated repression of the high-affinity ammonium importer gene AmAMT2 in Amanita muscaria.

A main function of ectomycorrhizas, a symbiosis between certain soil fungi and fine roots of woody plants, is the exchange of plant-derived carbohydrates for fungus-derived nutrients. As it is required in large amounts, nitrogen is of special interest. A gene (AmAMT2) coding for a putative fungal ammonium importer was identified in an EST project of functional Amanita muscaria/poplar ectomycorrhizas. Heterologous expression of the entire AmAMT2 coding region in yeast revealed the corresponding protein to be a high-affinity ammonium importer. In axenically grown Amanita hyphae AmAMT2 expression was strongly repressed by nitrogen, independent of whether the offered nitrogen source was transported by AmAMT2 or not. In functional ectomycorrhizas the AmAMT2 transcript level was further decreased in both hyphal networks (sheath and Hartig net), while extraradical hyphae revealed strong gene expression. Together our data suggest that (1) AmAMT2 expression is regulated by the endogenous nitrogen content of hyphae and (2) fungal hyphae in ectomycorrhizas are well supported with nitrogen even when the extraradical mycelium is nitrogen limited. As a consequence of AmAMT2 repression in mycorrhizas, ammonium can be suggested as a potential nitrogen source delivered by fungal hyphae in symbiosis.

Sugar for my honey: carbohydrate partitioning in ectomycorrhizal symbiosis.

Simple, readily utilizable carbohydrates, necessary for growth and maintenance of large numbers of microbes are rare in forest soils. Among other types of mutualistic interactions, the formation of ectomycorrhizas, a symbiosis between tree roots and certain soil fungi, is a way to overcome nutrient and carbohydrate limitations typical for many forest ecosystems. Ectomycorrhiza formation is typical for trees in boreal and temperate forests of the northern hemisphere and alpine regions world-wide. The main function of this symbiosis is the exchange of fungus-derived nutrients for plant-derived carbohydrates, enabling the colonization of mineral nutrient-poor environments. In ectomycorrhizal symbiosis up to 1/3 of plant photoassimilates could be transferred toward the fungal partner. The creation of such a strong sink is directly related to the efficiency of fungal hexose uptake at the plant/fungus interface, a modulated fungal carbohydrate metabolism in the ectomycorrhiza, and the export of carbohydrates towards soil growing hyphae. However, not only the fungus but also the plant partner increase its expression of hexose importer genes at the plant/fungus interface. This increase in hexose uptake capacity of plant roots in combination with an increase in photosynthesis may explain how the plant deals with the growing fungal carbohydrate demand in symbiosis and how it can restrict this loss of carbohydrates under certain conditions to avoid fungal parasitism.

Development of bioinformatic tools to support EST-sequencing, in silico- and microarray-based transcriptome profiling in mycorrhizal symbioses.

The great majority of terrestrial plants enters a beneficial arbuscular mycorrhiza (AM) or ectomycorrhiza (ECM) symbiosis with soil fungi. In the SPP 1084 "MolMyk: Molecular Basics of Mycorrhizal Symbioses", high-throughput EST-sequencing was performed to obtain snapshots of the plant and fungal transcriptome in mycorrhizal roots and in extraradical hyphae. To focus activities, the interactions between Medicago truncatula and Glomus intraradices as well as Populus tremula and Amanita muscaria were selected as models for AM and ECM symbioses, respectively. Together, almost, 20.000 expressed sequence tags (ESTs) were generated from different random and suppressive subtractive hybridization (SSH) cDNA libraries, providing a comprehensive overview of the mycorrhizal transcriptome. To automatically cluster and annotate EST-sequences, the BioMake and SAMS software tools were developed. In connection with the eNorthern software SteN, plant genes with a predicted mycorrhiza-induced expression were identified. To support experimental transcriptome profiling, macro- and microarray tools have been constructed for the two model mycorrhizae, based either on PCR-amplified cDNAs or 70mer oligonucleotides. These arrays were used to profile the transcriptome of AM and ECM roots under different conditions, and the data obtained were uploaded to the ArrayLIMS and EMMA databases that are designed to store and evaluate expression profiles from DNA arrays. Together, the EST- and transcriptome databases can be mined to identify candidate genes for targeted functional studies.

The alpha-tubulin gene AmTuba1: a marker for rapid mycelial growth in the ectomycorrhizal basidiomycete Amanita muscaria.

The apical extension of hyphae is of central importance for extensive spread of fungal mycelium in forest soils and for effective ectomycorrhiza development. Since the tubulin cytoskeleton is known to be important for fungal tip growth, we have investigated the expression of an alpha-tubulin gene from the ectomycorrhizal basidiomycete Amanita muscaria (AmTuba1). The phylogenetic analysis of protein sequences revealed the existence of two subgroups of alpha-tubulins in homobasidiomycetes, clearly distinguishable by defined amino acids. AmTuba1 belongs to subgroup1. The AmTuba1 transcript level is related to mycelial growth rate. Growth induction of carbohydrate starved (non-growing) hyphae resulted in an enhanced AmTuba1 expression as soon as hyphal growth started, reaching a maximum at highest mycelial growth rate. Bacterium-induced hyphal elongation also leads to increased AmTuba1 transcript levels. In mature A. muscaria/P. abies ectomycorrhizas, where fungal hyphae are highly branched, and slowly growing, AmTuba1 expression were even lower than in carbohydrate-starved mycelium, indicating a further down-regulation of gene expression in symbiosis. In conclusion, our analyses show that the AmTuba1 gene can be used as a marker for active apical extension in fly agaric, and that alpha-tubulin proteins are promising tools for the classification of fungi.

The high-affinity poplar ammonium importer PttAMT1.2 and its role in ectomycorrhizal symbiosis.

One way to elucidate whether ammonium could act as a nitrogen (N) source delivered by the fungus in ectomycorrhizal symbiosis is to investigate plant ammonium importers. Expression analysis of a high-affinity ammonium importer from Populus tremulax tremuloides (PttAMT1.2) and of known members of the AMT1 gene family from Populus trichocarpa was performed. In addition, PttAMT1.2 function was studied in detail by heterologous expression in yeast. PttAMT1.2 expression proved to be root-specific, affected by N nutrition, and strongly increased in a N-independent manner upon ectomycorrhiza formation. The corresponding protein had a K(M) value for ammonium of c. 52 microm. From the seven members of the AMT1 gene family, one gene was exclusively expressed in roots while four genes were detectable in all poplar organs but with varying degrees of expression. Ectomycorrhiza formation resulted in a strong upregulation of three of these genes. Our results indicate an increased ammonium uptake capacity of mycorrhized poplar roots and suggest, together with the expression of putative ammonium exporter genes in the ectomycorrhizal fungus Amanita muscaria, that ammonium could be a major N source delivered from the fungus towards the plant in symbiosis.