Overexpression of the HcPT1.1 transporter in Hebeloma cylindrosporum alters the phosphorus accumulation of Pinus pinaster and the distribution of HcPT2 in ectomycorrhizae.

Ectomycorrhizal (ECM) fungi are associated with the roots of woody plants in temperate and boreal forests and help them to acquire water and nutrients, particularly phosphorus (P). However, the molecular mechanisms responsible for the transfer of P from the fungus to the plant in ectomycorrhizae are still poorly understood. In the model association between the ECM fungus Hebeloma cylindrosporum and its host plant Pinus pinaster, we have shown that the fungus, which possesses three H+:Pi symporters (HcPT1.1, HcPT1.2 and HcPT2), expresses mainly HcPT1.1 and HcPT2 in the extraradical and intraradical hyphae of ectomycorrhizae to transport P from the soil to colonized roots. The present study focuses on the role of the HcPT1.1 protein in plant P nutrition, in function of P availability. We artificially overexpressed this P transporter by fungal Agrotransformation and investigated the effect of the different lines, wild-type and transformed ones, on plant P accumulation, the distribution of HcPT1.1 and HcPT2 proteins in ectomycorrhizae by immunolocalization, and 32P efflux in an experimental system mimicking intraradical hyphae. Surprisingly, we showed that plants interacting with transgenic fungal lines overexpressing HcPT1.1 did not accumulate more P in their shoots than plants colonized with the control ones. Although the overexpression of HcPT1.1 did not affect the expression levels of the other two P transporters in pure cultures, it induced a strong reduction in HcPT2 proteins in ectomycorrhizae, particularly in intraradical hyphae, but still improved the P status of host plant shoots compared with non-mycorrhizal plants. Finally, 32P efflux from hyphae was higher in lines overexpressing HcPT1.1 than in the control ones. These results suggest that a tight regulation and/or a functional redundancy between the H+:Pi symporters of H. cylindrosporum might exist to ensure a sustainable P delivery to P. pinaster roots.

The grazing activity of Acrobeloides sp. drives phytate mineralisation within its trophic relationship with bacteria.

The microbial loop has been suggested as an alternative route for better utilization of phytate, a poorly available P source to plants. We hypothesized that bacterial grazer activity might dramatically enhance bacterial migration and proliferation, increasing the probability of phytate hydrolysis by bacterial phytases and, thus, phytate mineralization and release of free phosphate. We tested this hypothesis in a two-compartment system with a solid medium containing phytate or free phosphate as the source of P. Two bacterial species, B. subtilis 168 or Bradyrhizobium sp., with or without bacterial grazing nematodes belonging to Acrobeloides sp. previously fed on each of the bacterial species, were inoculated at a single point in the medium. Whatever the P source, nematode migration within both zones allowed the proliferation of bacteria. However, B. subtilis 168 was more efficient in using phytate than Bradyrhizobium sp. since the highest bacterial cell density and free phosphate concentrations were reached by Acrobeloides sp. fed on B. subtilis 168. The grazer activity seemed to be crucial to enhance phytate mineralization, despite Acrobeloides sp. showing a higher preference to feed on Bradyrhizobium sp. This study provides new insights into the effects of bacterial grazer activity on phytate mineralization.

The Hebeloma cylindrosporum HcPT2 Pi transporter plays a key role in ectomycorrhizal symbiosis.

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.

In vivo and in vitro 31P-NMR Study of the Phosphate Transport and Polyphosphate Metabolism in Hebeloma cylindrosporum in Response to Plant Roots Signals.

We used in vivo and in vitro phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopy to follow the change in transport, compartmentation and metabolism of phosphate in the ectomycorrhizal fungus Hebeloma cylindrosporum in response to root signals originating from host (Pinus pinaster) or non-host (Zea mays) plants. A device was developed for the in vivo studies allowing the circulation of a continuously oxygenated mineral solution in an NMR tube containing the mycelia. The in vitro studies were performed on fungal material after several consecutive treatment steps (freezing in liquid nitrogen; crushing with perchloric acid; elimination of perchloric acid; freeze-drying; dissolution in an appropriate liquid medium).

A Method for Radioactive Labelling of Hebeloma cylindrosporum to Study Plant-fungus Interactions.

In order to quantify P accumulation and P efflux in the ectomycorrhizal basidiomycete fungus Hebeloma cylindrosporum, we supplied 32P to mycelia previously grown in vitro in liquid medium. The culture had four main steps that are 1) growing the mycelium on complete medium with P, 2) transfer the mycelia into new culture solution with or without P, 3) adding a solution containing 32P and 4) rinsing the mycelia before incubation with or without plant. The main point is to rinse very carefully the mycelia after 32P supply in order to avoid overestimation of 32P efflux into the medium.

Establishing a Symbiotic Interface between Cultured Ectomycorrhizal Fungi and Plants to Follow Fungal Phosphate Metabolism.

In ectomycorrhizal plants, the fungal cells colonize the roots of their host plant to create new organs called ectomycorrhizae. In these new organs, the fungal cells colonize the walls of the cortical cells, bathing in the same apoplasm as the plant cells in a space named the 'Hartig net', where exchanges between the two partners take place. Finally, the efficiency of ectomycorrhizal fungi to improve the phosphorus nutrition of their host plants will depend on the regulation of phosphate transfer from the fungal cells to plant cells in the Hartig net through as yet unknown mechanisms. In order to investigate these mechanisms, we developed an in vitro experimental device mimicking the common apoplasm of the ectomycorrhizae (the Hartig net) to study the phosphorus metabolism in the ectomycorrhizal fungus Hebeloma cylindrosporum when the fungal cells are associated or not with the plant cells of the host plant Pinus pinaster. This device can be used to monitor 32Phosphate efflux from the fungus previously incubated with 32P-orthophosphate.