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Salinity is an increasing problem in coastal areas affected by saltwater intrusion, with deleterious effects on tree health and forest growth. Ectomycorrhizal (ECM) fungi may improve the salinity tolerance of host trees, but the impact of external potassium (K + ) availability on these effects is still unclear. Here, we performed several experiments with the ECM fungus Paxillus ammoniavirescens and loblolly pine (Pinus taeda L.) in axenic and symbiotic conditions at limited or sufficient K + and increasing sodium (Na + ) concentrations. Growth rate, biomass, nutrient content, and K + transporter expression levels were recorded for the fungus, and the colonization rate, root development parameters, biomass, and shoot nutrient accumulation were determined for mycorrhizal and non-mycorrhizal plants. P. ammoniavirescens was tolerant to high salinity, although growth and nutrient concentrations varied with K + availability and increasing Na + exposure. While loblolly pine root growth and development decreased with increasing salinity, ECM colonization was unaffected by pine response to salinity. The mycorrhizal influence on loblolly pine salinity response was strongly dependent on external K + availability. This study reveals that P. ammoniavirescens can reduce Na + accumulation of salt-exposed loblolly pine, but this effect depends on external K + availability., (© 2024 The Authors. Environmental Microbiology published by John Wiley & Sons Ltd.)

Potassium (K+) acquisition, translocation and cellular homeostasis are mediated by various membrane transport systems in all organisms. We identified and described an ion channel in the ectomycorrhizal fungus Hebeloma cylindrosporum (HcSKC) that harbors features of animal voltage-dependent Shaker-like K+ channels, and investigated its role in both free-living hyphae and symbiotic conditions. RNAi lines affected in the expression of HcSKC were produced and used for in vitro mycorrhizal assays with the maritime pine as host plant, under standard or low K+ conditions. The adaptation of H. cylindrosporum to the downregulation of HcSKC was analyzed by qRT-PCR analyses for other K+-related transport proteins: the transporters HcTrk1, HcTrk2, and HcHAK, and the ion channels HcTOK1, HcTOK2.1, and HcTOK2.2. Downregulated HcSKC transformants displayed greater K+ contents at standard K+ only. In such conditions, plants inoculated with these transgenic lines were impaired in K+ nutrition. Taken together, these results support the hypothesis that the reduced expression of HcSKC modifies the pool of fungal K+ available for the plant and/or affects its symbiotic transfer to the roots. Our study reveals that the maintenance of K+ transport in H. cylindrosporum, through the regulation of HcSKC expression, is required for the K+ nutrition of the host plant., Competing Interests: The authors have declared that no competing interests exist.

While plants mainly rely on the use of inorganic nitrogen sources like ammonium and nitrate, soil-borne microorganisms like the ectomycorrhizal fungus Hebeloma cylindrosporum can also take up soil organic N in the form of amino acids and peptides that they use as nitrogen and carbon sources. Following the previous identification and functional expression in yeast of two PTR-like peptide transporters, the present study details the functions and substrates of HcPTR2A and HcPTR2B by analysing their transport kinetics in Xenopus laevis oocytes. While both transporters mediated high-affinity di- and tripeptide transport, HcPTR2A also showed low-affinity transport of several amino acids-mostly hydrophobic ones with large side chains.

Soil salinity is an environmental condition that is currently increasing worldwide. Plant growth under salinity induces osmotic stress and ion toxicity impairing root water and nutrient absorption, but the association with beneficial soil microorganisms has been linked to an improved adaptation to this constraint. The ectomycorrhizal (ECM) symbiosis has been proposed as a key factor for a better tolerance of woody species to salt stress, thanks to the reduction of sodium (Na + ) uptake towards photosynthetic organs. Although no precise mechanisms for this enhanced plant salt tolerance have been described yet, in this review, we summarize the knowledge accumulated so far on the role of ECM symbiosis. Moreover, we propose several strategies by which ECM fungi might help plants, including restriction of Na + entrance into plant tissues and improvement of mineral nutrition and water balances. This positive effect of ECM fungi has been proven in field assays and the results obtained point to a promising application in forestry cultures and reforestation.

Through a mutualistic relationship with woody plant roots, ectomycorrhizal fungi provide growth-limiting nutrients, including inorganic phosphate (Pi), to their host. Reciprocal trades occur at the Hartig net, which is the symbiotic interface of ectomycorrhizas where the two partners are symplasmically isolated. Fungal Pi must be exported to the symbiotic interface, but the proteins facilitating this transfer are unknown. In the present study, we combined transcriptomic, microscopy, whole plant physiology, X-ray fluorescence mapping, 32 P labeling and fungal genetic approaches to unravel the role of HcPT2, a fungal Pi transporter, during the Hebeloma cylindrosporum-Pinus pinaster ectomycorrhizal association. We localized HcPT2 in the extra-radical hyphae and the Hartig net and demonstrated its determinant role for both the establishment of ectomycorrhizas and Pi allocation towards P. pinaster. We showed that the host plant induces HcPT2 expression and that the artificial overexpression of HcPT2 is sufficient to significantly enhance Pi export towards the central cylinder. Together, our results reveal that HcPT2 plays an important role in ectomycorrhizal symbiosis, affecting both Pi influx in the mycelium and efflux towards roots under the control of P. pinaster., (© 2018 INRA New Phytologist © 2018 New Phytologist Trust.)

Ectomycorrhizal fungi play an essential role in the ecology of boreal and temperate forests through the improvement of tree mineral nutrition. Potassium (K + ) is an essential nutrient for plants and is needed in high amounts. We recently demonstrated that the ectomycorrhizal fungus Hebeloma cylindrosporum improves the K + nutrition of Pinus pinaster under shortage conditions. Part of the transport systems involved in K + uptake by the fungus has been deciphered, while the molecular players responsible for the transfer of this cation towards the plant remain totally unknown. Analysis of the genome of H. cylindrosporum revealed the presence of three putative tandem-pore outward-rectifying K + (TOK) channels that could contribute to this transfer. Here, we report the functional characterization of these three channels through two-electrode voltage-clamp experiments in oocytes and yeast complementation assays. The expression pattern and physiological role of these channels were analysed in symbiotic interaction with P. pinaster. Pine seedlings colonized by fungal transformants overexpressing two of them displayed a larger accumulation of K + in shoots. This study revealed that TOK channels have distinctive properties and functions in axenic and symbiotic conditions and suggested that HcTOK2.2 is implicated in the symbiotic transfer of K + from the fungus towards the plant., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Soil nutrient acquisition and exchanges through symbiotic plant-fungus interactions in the rhizosphere are key features for the current agricultural and environmental challenges. Improved crop yield and plant mineral nutrition through a fungal symbiont has been widely described. In return, the host plant supplies carbon substrates to its fungal partner. We review here recent progress on molecular players of membrane transport involved in nutritional exchanges between mycorrhizal plants and fungi. We cover the transportome, from the transport proteins involved in sugar fluxes from plants towards fungi, to the uptake from the soil and exchange of nitrogen, phosphate, potassium, sulfate, and water. Together, these advances in the comprehension of the mycorrhizal transportome will help in developing the future engineering of new agro-ecological systems., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Extracellular proteins play crucial roles in the interaction between mycorrhizal fungi and their environment. Computational prediction and experimental detection allowed identification of 869 proteins constituting the exoproteome of Hebeloma cylindrosporum. Small secreted proteins (SSPs) and carbohydrate-active enzymes (CAZymes) were the two major classes of extracellular proteins. Twenty-eight per cent of the SSPs were secreted by free-living mycelia and five of the 10 most abundant extracellular proteins were SSPs. By contrast, 63-75% of enzymes involved in nutrient acquisition were secreted. A total of 150 extracellular protein-coding genes were differentially expressed between mycorrhizas and free-living mycelia. SSPs were the most affected. External environmental conditions also affected expression of 199 exoproteome genes in mycorrhizas. SSPs displayed different patterns of regulation in response to presence of a host plant or other environmental signals. Several of the genes most overexpressed in the presence of organic matter encoded oxidoreductases. Hebeloma cylindrosporum has not fully lost its ancestral saprotrophic capacities but rather adapted them not to harm its hosts and to use soil organic nitrogen. The complex and divergent patterns of regulation of SSPs in response to a symbiotic partner and/or organic matter suggest various roles in the biology of mycorrhizal fungi., (© 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.)

Mycorrhizal associations are known to improve the hydro-mineral nutrition of their host plants. However, the importance of mycorrhizal symbiosis for plant potassium nutrition has so far been poorly studied. We therefore investigated the impact of the ectomycorrhizal fungus Hebeloma cylindrosporum on the potassium nutrition of Pinus pinaster and examined the involvement of the fungal potassium transporter HcTrk1. HcTrk1 transcripts and proteins were localized in ectomycorrhizas using in situ hybridization and EGFP translational fusion constructs. Importantly, an overexpression strategy was performed on a H. cylindrosporum endogenous gene in order to dissect the role of this transporter. The potassium nutrition of mycorrhizal pine plants was significantly improved under potassium-limiting conditions. Fungal strains overexpressing HcTrk1 reduced the translocation of potassium and phosphorus from the roots to the shoots of inoculated plants in mycorrhizal experiments. Furthermore, expression of HcTrk1 and the phosphate transporter HcPT1.1 were reciprocally linked to the external inorganic phosphate and potassium availability. The development of these approaches provides a deeper insight into the role of ectomycorrhizal symbiosis on host plant K(+) nutrition and in particular, the K(+) transporter HcTrk1. The work augments our knowledge of the link between potassium and phosphorus nutrition via the mycorrhizal pathway., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)

Mycorrhizal exchange of nutrients between fungi and host plants involves a specialization and polarization of the fungal plasma membrane adapted for the uptake from the soil and for secretion of nutrient ions towards root cells. In addition to the current progress in identification of membrane transport systems of both symbiotic partners, data concerning the transcriptional and translational regulation of these proteins are needed to elucidate their role for symbiotic functions. To answer whether the formerly described Pi-dependent expression of the phosphate transporter HcPT1.1 from Hebeloma cylindrosporum is the result of its promoter activity, we introduced promoter-EGFP fusion constructs in the fungus by Agrotransformation. Indeed, HcPT1.1 expression in pure fungal cultures quantified and visualized by EGFP under control of the HcPT1.1 promoter was dependent on external Pi concentrations, low Pi stimulating the expression. Furthermore, to study expression and localization of the phosphate transporter HcPT1.1 in symbiotic conditions, presence of transcripts and proteins was analyzed by the in situ hybridization technique as well as by immunostaining of proteins. In ectomycorrhiza, expression of the phosphate transporter was clearly enhanced by Pi-shortage indicating its role in Pi nutrition in the symbiotic association. Transcripts were detected in external hyphae and in the hyphal mantle, proteins in addition also within the Hartig net. Exploiting the transformable fungus H. cylindrosporum, Pi-dependent expression of the fungal transporter HcPT1.1 as result from its promoter activity as well as transcript and protein localization in ectomycorrhizal symbiosis are shown., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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.

Constraints on plant growth imposed by low availability of nitrogen are a characteristic feature of ecosystems dominated by ectomycorrhizal plants. Ectomycorrhizal fungi play a key role in the N nutrition of plants, allowing their host plants to access decomposition products of dead plant and animal materials. Ectomycorrhizal plants are thus able to compensate for the low availability of inorganic N in forest ecosystems. The capacity to take up peptides, as well as the transport mechanisms involved, were analysed in the ectomycorrhizal fungus Hebeloma cylindrosporum. The present study demonstrated that H. cylindrosporum mycelium was able to take up di- and tripeptides and use them as sole N source. Two peptide transporters (HcPTR2A and B) were isolated by yeast functional complementation using an H. cylindrosporum cDNA library, and were shown to mediate dipeptide uptake. Uptake capacities and expression regulation of both genes were analysed, indicating that HcPTR2A was involved in the high-efficiency peptide uptake under conditions of limited N availability, whereas HcPTR2B was expressed constitutively.