La surexpression du transporteur de phosphate HcPT1.1 chez Hebeloma cylindrosporum confirme sa capacité de transfert de P dans les ectomycorhizes

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The Hebeloma cylindrosporum phosphate transporter HcPT2 was involved in phosphate efflux at the fungus-plant interface

Ectomycorrhizal (ECM) symbiosis is defined as a mutual association between fungal symbionts and roots of woody plants. This symbiosis is strongly represented in temperate and boreal forests and is considered to be the most widespread means of increasing P acquisition by trees. Despite the importance of the ECM association, current knowledge about molecular mechanisms involved in membrane transport between the two partners is still limited. Therefore, the identification of the mechanisms essential for the symbiosis is a major issue. To better understand these mechanisms, we used a model association between the ECM basidiomycete Hebeloma cylindrosporum and the maritime pine Pinus pinaster. The fungal genome contains three transporters of Pi (HcPT1.1, HcPT1.2, and HcPT2), which have been already characterized as H+ Pi carriers. We hypothesized that one of these carriers could be responsible for both influx and efflux of Pi from the soil to the fungus and from the fungus to the plant. Our results suggest that HcPT2 would be the best candidate to fill both roles. An in vitro ectomycorrhizal system was used to investigate phosphate flux from H. cylindrosporum toward a liquid compartment containing pine roots. Interestingly, the expression pattern of HcPT2 was correlated to fungal phosphate efflux. Moreover, immuno-localization experiments showed that the protein is located (i) in the extraradical hyphae and the fungal sheath, compatible with Pi uptake, and (ii) within the Hartig net, which is the place of exchange between fungal and host cells. In order to study more specifically the role of HcPT2 at the fungus-plant interface, overexpressing and knock-down transgenic H. cylindrosporum strains were produced. Fungal strains with up regulated expression of HcPT2 increased phosphate efflux within the interaction medium, supporting a role of HcPT2 in phosphate transfer to the plant. In addition, the modification of HcPT2 expression strongly affects Pi nutrition of P. pinaster

Fluorescent in situ RT-PCR to visualise the expression of a phosphate transporter gene from an ectomycorrhizal fungus.

Adresse actuelle: I.M. van Aarle Unité de Microbiologie Université catholique de Louvain Place croix du Sud, 3, 1348 Louvain-la-Neuve BelgiqueExpression of a mycorrhizal fungal-specific phosphate (P) transporter gene (HcPT1) was studied in mycelium of the ectomycorrhizal fungus Hebeloma cylindrosporum, by in situ reverse transcriptase polymerase chain reaction using amplification of complementary DNA sequences. The expression of HcPT1 was visualised under two different P treatments. Mycelium was transferred to liquid medium with or without P and incubated for 5 days. Under P starvation, mycelium growth and vitality was reduced and the expression of HcPT1 up regulated. Enzyme-labelled fluorescent substrate was used to detect gene expression in situ with epi-fluorescence microscopy and to visualise it at the level of the individual hyphae both in starved and non-starved hyphae. Up-regulation of HcPT1 was observed as a more intense fluorescent signal and from the larger proportion of hyphae that showed expression