Ectomycorrhizas are formed between certain soil fungi and fine roots of predominantly woody plants. An important feature of this symbiosis is the supply of plant-derived carbohydrates to the fungus. As a first step toward a better understanding of the molecular basis of this process, we cloned a monosaccharide transporter from the ectomycorrhizal fungus Amanita muscaria. Degenerate oligonucleotide primers were designed to match conserved regions from known fungal sugar transporters. A cDNA fragment of the transporter was obtained from mycorrhizal mRNA by reverse transcription-polymerase chain reaction. This fragment was used to identify a clone (AmMst1) encoding the entire monosaccharide transporter in a Picea abies/A. muscaria mycorrhizal cDNA library. The cDNA codes for an open reading frame of 520 amino acids, showing best homology to a Neurospora crassa monosaccharide transporter. The function of AmMST1 as monosaccharide transporter was confirmed by heterologous expression of the cDNA in a Schizosaccharomyces pombe mutant lacking a monosaccharide uptake system. AmMst1 was constitutively expressed in fungal hyphae under all growth conditions. Nevertheless, in mycorrhizas as well as in hyphae grown at monosaccharide concentrations above 5 mM, the amount of AmMst1 transcript increased fourfold. We therefore suggest that AmMst1 is upregulated in ectomycorrhizas by a monosaccharide-controlled mechanism.

Enhanced resistance of barley (Hordeum vulgare L. cv. Ingrid) against barley powdery mildew (Blumeria graminis f. sp. hordei race A6) was induced by abiotic stress in a concentration-dependent manner. The papilla-mediated resistance was not only induced by osmotic stress, but also by proton stress. Resistance was directly correlated with increasing concentrations of various salts in the nutrient solution. Resistance induced by proton stress also depended on the stress intensity. Resistance induction occurred even at low stress intensities. Any specific ion toxicity affecting the fungal growth directly, and therefore leading to enhanced pathogen resistance, can be excluded because of the independence of resistance induction of the ion used and of the time course of sodium accumulation in the leaves. BCI-4, a marker for benzo[1,2,3]thiadiazolecarbothioic acid S-methyl ester (BTH)-induced resistance was not induced by these abiotic stresses. However, resistance was induced in the same concentration-dependent manner by the application of the stress hormone ABA to the root medium. During the relief of water stress, resistance did not decrease constantly. On the contrary, after a phase of decreasing resistance for 24 h the pathogen resistance increased again for 48 h before decreasing finally to control levels.

A cDNA coding for a fungal amino acid transporter (AmAAP1) was identified from Amanita muscaria ectomycorrhizas. The transporter gene was expressed at a basal level under all conditions investigated, but its expression was enhanced 10-fold in the absence of a N source utilized by the fungus. Nitrate was not a suitable N source for A. muscaria and resulted in maximal AmAAP1 expression. The expression of AmAAP1 in a yeast mutant revealed its function as a high-affinity amino acid transporter with a broad substrate spectrum. AmAAP1 takes up all investigated amino acids with K m values between 22 µM for histidine and up to 100 µM for proline. Gene expression and amino acid uptake data together indicate two main functions for AmAAP1 : uptake of amino acids from soil for fungal nutrition, and prevention of an amino acid loss by hyphal leakage in the absence of a suitable N source.Key words : fungi, basidiomycete, transport, gene expression, N. Ectomycorrhizas are symbiotic associations of fine roots of mainly woody plants with certain soil fungi (ascomycetes or basidiomycetes). The dominant trees of boreal and temperate forests, members of the Fagaceae and the Pinaceae, are obligate ectomycorrhizal symbionts. It is accepted that in this type of symbiosis, mutual benefit is gained from the exchange of plant-derived carbohydrates for amino acids and mineral nutrients (e.g. phosphate) supplied by the fungus (Marschner & Dell, 1994 ;Smith & Read, 1997). In nutrient-poor soils, ectomycorrhizal fungi, in collaboration with other soil microorganisms, are able to exploit organic debris (e.g. litter) as a nutrient source. Some of these nutrients are transferred to the plant (Smith & Read, 1997).In forest soils, nitrogen (N) is available as nitrate, ammonium or amino acids (in free form or as polypeptides). All ectomycorrhizal fungi investigated so far are able to utilize ammonium as well as free amino acids. The capacity for nitrate uptake is still under discussion (Abuzinadah & Read, 1988 ; Chalot & Brun, 1998).We have identified an A. muscaria cDNA clone with strong homology to fungal amino acid trans-*Author for correspondence (tel j49 7071 2977657 ; fax j49 7071 29 5635 ; e-mail uwe.nehls!uni-tuebingen.de).porters. Here we present its cDNA sequence and an expression analysis of the gene. The kinetic properties of the corresponding transporter were studied by heterologous expression of the entire cDNA in a yeast mutant.    Biological materialAmanita muscaria (L. ex Fr.) Hooker strain CS83 was isolated from a fruiting body (Schaeffer et al., 1995). Mycelia were grown on Petri dishes or in liquid culture for 2-24 d (Nehls et al., 1998) with glucose as C source and various additions of amino acids, nitrate or ammonium as N source. The mycelium was collected by filtration with a Bu$ chner funnel under suction, washed twice with 50 ml of water, frozen in liquid N and stored at k80mC.Mycorrhiza formation was induced with Picea abies (L.) Karsten (Schaeffer et al., 1995) and with Populus tremulaitr...

Fungal carbohydrate nutrition is an important aspect of ectomycorrhizal symbiosis. At the plant/fungus interface, fungal and root cortical cells compete for monosaccharides, generated from plant‐derived sucrose. Therefore, the kinetic properties of the monosaccharide uptake systems are decisive for the monosaccharide yield of each partner. For the functional characterization of a hexose transporter (AmMst1) of the ectomycorrhizal fungus Amanita muscaria, the entire cDNA was expressed in a Saccharomyces cerevisiae strain unable to take up hexoses. Uptake experiments with 14C‐labelled monosaccharides resulted in KM values of 0.46 mM for glucose and 4.20 mM for fructose, revealing a strong preference of AmMst1 for glucose as substrate. Glucose uptake by AmMst1 was strongly favoured even in the presence of a large excess of fructose. Comparable affinities of AmMst1 for glucose, 3‐O‐methyl glucose and mannose were obtained. In contrast, AmMst1 imported galactose with a much lower efficiency, revealing that this transporter distinguishes pyranoses by steric hindrance at the C‐4 position. While yeast contains numerous hexose transporter genes, the AmMst1 gene seems to be the main, if not the only, hexose transporter that is expressed in A. muscaria, as concluded from the comparison of hexose import properties of A. muscaria protoplasts and AmMst1 expressed in yeast.

Ectomycorrhizas are formed between certain soil fungi and fine roots of woody plants. An important feature of this symbiosis is the supply of photoassimilates to the fungus. Hexoses, formed from sucrose in the common apoplast at the root/fungus interface, can be taken up by both plant and fungal monosaccharide transporters. Recently we characterised a monosaccharide transporter from the ectomycorrhizal fungus Amanita muscaria. This transporter was up-regulated in mycorrhizas, thus increasing the hexose uptake capacity of the fungal partner in symbiosis. In order to characterise host (Picea abies) root monosaccharide transporters, degenerate oligonucleotide primers, designed to match conserved regions from known plant hexose transporters, were used to isolate a cDNA fragment of a transporter by PCR. This fragment was used to identify a presumably full length clone (PaMST1) in a P. abies/A. muscaria mycorrhizal cDNA library. The entire cDNA code for an open reading frame of 513 amino acids, revealing best homology to H + /monosaccharide transporters from Arabidopsis, Saccharum and Ricinus. PaMST1 was highly expressed in the hypocotyl and in roots of P. abies seedlings, but not in needles. Mycorrhiza formation led to a slight reduction of PaMST1 expression. The results are discussed with special reference to carbon allocation in ectomycorrhizas.