Doctorate thesis

Friday, December 13, 2013


Trophic exchanges between Hebeloma cylindrosporum and Pinus pinaster: Analysis of fungal potassium and inorganic phosphate transport systems involved in ectomycorrhizal symbiosis

Kévin Garcia
BPMP, “Ion Channels” team


Jury :
Gilles GAY, Pr/Université Claude Bernard, Lyon, Rapporteur
Uwe NEHLS, Pr/Universität Bremen, Allemagne, Rapporteur
Michel LEBRUN, Pr/Université Montpellier II, Examinateur
Daniel Wipf, Pr/Université de Bourgogne, Dijon, Examinateur
Sabine ZIMMERMANN, CR/CNRS, Montpellier, Directrice de thèse


Abstract :
Ectomycorrhizal symbiosis is defined as a mutual association between the roots of woody plants and the mycelium of soil fungi. This symbiosis is widespread in northern forests and plays a major role in nutrient and water uptake of woody plants, especially when resources become scarce. Transcriptomic and genomic data of the ectomycorrhizal fungus Hebeloma cylindrosporum allowed the identification of several genes coding for nutrient transport proteins. Their putative involvement in ectomycorrhiza-dependent potassium and phosphate nutrition of the host plant Pinus pinaster needs to be assessed. In this study, two candidate genes coding for potassium (K+) transport systems, HcTrk1 and HcSKC, and two other genes coding for inorganic phosphate (Pi) transporters, HcPT1.1 and HcPT2, were analyzed. Molecular approaches allowing the localization of transcripts (in situ hybridization) and proteins (translational fusion) of these candidates in ectomycorrhiza were obtained. These tools allowed us to show that the HcTrk1 transporter and the HcSKC channel were localized in K+ uptake and release sites of the ectomycorrhiza, respectively. In order to know whether these proteins play a role in plant K+ and Pi nutrition, H. cylindrosporum transgenic lines with up- and/or down-regulated expression of candidate genes were produced. In mycorrhizal assays, the use of fungal strains with up- or down- regulated expression of HcTrk1 and HcSKC, respectively, affects the K+ nutrition and phosphorus homeostasis of the host plant. The same approaches were used for HcPT1.1 and HcPT2 Pi transporters. Therefore, using transgenic strategies, we demonstrated that the previously shown up-regulation of HcPT1.1 expression under Pi shortage is related to its promoter activity. Concerning HcPT2, preliminary localization analysis suggested that this transporter might be involved in Pi uptake from soil and in release in the Hartig net. However, complementary studies are needed. Five and three novel K+ and Pi transport systems, respectively, were identified from the recent genome accession of H. cylindrosporum, opening the way to a fine dissection of molecular mechanisms controlling the ectomycorrhiza-dependent K+ and phosphate nutrition of Pinus pinaster.