Doctorate thesis of National Institute of Further Education in Agricultural Science

Friday, November 24, 2017


Impact of the ectomycorrhizal symbiosis for plant adaptation to nutritive, salt and hydric stress: characterization and role of potassium and water transport systems in the model fungus Hebeloma cylindrosporum

Carmen Guerrero-Galán
BPMP, équipe


Jury :
M Roland MARMEISSE, Research Director, CNRS, Université de Lyon 1, Rapporteur
Mme Begoña BENITO, Professor, Universidad Politécnica de Madrid, Espagne, Rapporteur
Mme Claudine FRANCHE, Research Director, IRD, Montpellier, Reviewer
Mme Claire VENEAULT-FOURREY, Associate Professor, Lorraine University, Nancy, Reviewer
M Pierre BERTHOMIEU, Professor, SupAGRO, Montpellier, Reviewer
Mme Sabine D. ZIMMERMANN, Researcher Associate, CNRS, BPMP, Montpellier, Research Director


Abstract :
The ectomycorrhizal symbiosis, widespread in temperate and boreal ecosystems, is based in nutritional exchanges between the host plant and soil-borne fungi. This mutualism improves plant mineral and water nutrition of woody plants through mechanisms that are still largely unknown. This manuscript presents the whole set of membrane transport systems of the ectomycorrhizal fungus Hebeloma cylindrosporum identified from the sequenced genome, with an emphasis on the genes that are up-regulated in symbiosis with its natural host, the maritime pine (Pinus pinaster). These data will help to focalize future research on symbiosis-induced genes. The fungus H. cylindrosporum enhances the potassium (K+) nutrition of P. pinaster under starvation. This study has focused on three ion channels that could transfer the K+ to the plant. These channels belong to the fungal-specific TOK (Tandem-pore Outward-rectifying K+) family and have been characterized using several approaches. In silico analyses have positioned them in two subfamilies, giving them the names HcTOK1, HcTOK2.1 and HcTOK2.2. Their functional activity has been characterized by heterologous expression for two-electrode voltage-clamp measurements and yeast complementation. Localization has been studied by in situ hybridization in mycorrhiza and by expression of gene-eGFP constructs in yeast and H. cylindrosporum. The physiological role of these channels has been tested in pure culture and symbiosis with transgenic fungal lines overexpressing the HcTOK channels. Furthermore, the effects of H. cylindrosporum and K+ nutrition have been tested in P. pinaster seedlings subjected to salt stress. First, the tolerance to salinity of the fungus was analysed in pure culture with different compounds to identify the most toxic component. Second, the fungus was cultured in different NaCl and K+ conditions to know whether it kept the homeostasis and to check the expression of K+ transport systems. Finally, P. pinaster seedlings were cultured inoculated or not in two different K+ nutrition and four salinity conditions. Altogether, analysis of the three HcTOK channels revealed specificities of the TOK1- and TOK2-type and suggested that HcTOK2.2 might be a main player for the K+ transfer from the fungus towards the plant. H. cylindrosporum seems to play a role in the tolerance to salt stress of the maritime pine by reducing the Na+ transfer to the plant and improving K+ nutrition