SummaryEctomycorrhizal symbiosis markedly improves plant phosphate uptake, but the molecular mechanisms underlying this benefit are still poorly understood. We identified two ESTs in a cDNA library prepared from the ectomycorrhizal basidiomycete Hebeloma cylindrosporum with significant similarities to phosphate transporters from the endomycorrhizal fungus Glomus versiforme and from non-mycorrhizal fungi. The full-length cDNAs corresponding to these two ESTs complemented a yeast phosphate transport mutant (Dpho84). Measurements of 33 P-phosphate influx into yeast expressing either cDNA demonstrated that the encoded proteins, named HcPT1 and HcPT2, were able to mediate Pi:H + symport with different affinities for Pi (K m values of 55 and 4 lM, respectively). Real-time RT-PCR showed that Pi starvation increased the levels of HcPT1 transcripts in H. cylindrosporum hyphae grown in pure culture. Transcript levels of HcPT2 were less dependent on Pi availability. The two transporters were expressed in H. cylindrosporum associated with its natural host plant, Pinus pinaster, grown under low or high P conditions. The presence of ectomycorrhizae increased net Pi uptake rates into intact Pinus pinaster roots at low or high soil P levels. The expression patterns of HcPT1 and HcPT2 indicate that the two fungal phosphate transporters may be involved in uptake of phosphate from the soil solution under the two soil P availability conditions used.

Ectomycorrhizal symbiosis between fungi and woody plants strongly improves plant mineral nutrition and constitutes a major biological process in natural ecosystems. Molecular identification and functional characterization of fungal transport systems involved in nutrient uptake are crucial steps toward understanding the improvement of plant nutrition and the symbiotic relationship itself. In the present report a transporter belonging to the Trk family is identified in the model ectomycorrhizal fungus Hebeloma cylindrosporum and named HcTrk1. The Trk family is still poorly characterized, although it plays crucial roles in K ؉ transport in yeasts and filamentous fungi. In Saccharomyces cerevisiae K ؉ uptake is mainly dependent on the activity of Trk transporters thought to mediate H ؉ :K ؉ symport. The ectomycorrhizal HcTrk1 transporter was functional when expressed in Xenopus oocytes, enabling the first electrophysiological characterization of a transporter from the Trk family. HcTrk1 mediates instantaneously activating inwardly rectifying currents, is permeable to both K ؉ and Na ؉ , and displays channel-like functional properties. The whole set of data and particularly a phenomenon reminiscent of the anomalous mole fraction effect suggest that the transport does not occur according to the classical alternating access model. Permeation appears to occur through a single-file pore, where interactions between Na ؉ and K ؉ might result in Na ؉ :K ؉ co-transport activity. HcTrk1 is expressed in external hyphae that explore the soil when the fungus grows in symbiotic condition. Thus, it could play a major role in both the K ؉ and Na ؉ nutrition of the fungus (and of the plant) in nutrient-poor soils.

Summary• In an attempt to advance the understanding of ectomycorrhizal symbiosis, which improves mineral nutrition and resistance to abiotic and biotic stresses of most woody plants from temperate and boreal regions, we produced a cDNA library of the ectomycorrhizal fungus Hebeloma cylindrosporum in a yeast expression vector. The library was used for functional complementation cloning and for sequencing of 4036 expressed sequence tags (ESTs).• Expression of the library in a yeast ade2 and trp1 mutant strain allowed cloning of two Hebeloma genes, HcADE2 and HcTRP1 by functional complementation. This experiment highlighted both the efficiency of the complementation strategy and the quality of the library.• All sequence data available on Hebeloma (4596) were analysed and contiged. The whole EST resource is accessible via a web interface, which allows searching and navigation through clusters and tentative contigs (TCs).• Analysis of the database revealed a low level of counterparts already identified in other ectomycorrhizal fungi. For example, it allowed identification of a large set of membrane transporters, such as phosphate, potassium, sulphate or micronutrient transporters. Thus, this EST resource provides a valuable tool for research on ectomycorrhiza and symbiotic interactions.