Role of mycorrhiza-induced fungal transporters in ectomycorrhizal symbiosis – MYCOTRANS

To address these objectives, we will use key methodologies which are: (i) heterologous expression in yeast and Xenopus laevis oocytes of the cDNA encoding the metal (putatively Zn, Fe, Mn) transporter of the CDF family, to assess the properties of this transporter, such as its selectivity for several micronutrients; (ii) In situ hybridization and green fluorescent protein (GFP-) fused proteins for cellular and sub-cellular localization of the CDF transporter in yeast and ectomycorrhizae; (iii) production of new CRISPR/Cas vectors and KO fungal mutants to study the role of mycorrhiza-involved fungal genes, as this technique of genome editing will be much more efficient than the RNAi method previously used by Partner 1; (iv) use of an in vitro symbiosis-mimicking system, where the fungus is incubated in a liquid solution either alone or with host plant roots ensuring a cross-talk between both partners of the symbiosis but without the formation of ECM structures on the root; (v) extraction of fungal proteins and separation in three fractions: soluble, microsomal and plasma membrane proteins, to carry out proteome analysis in all protein fractions and phosphoproteome analysis of plasma membrane proteins, as a target of possible post-translational modifications exerted by the host-plant.

The MYCOTRANS project is expecting results concening (i) the physiological function, localization, and regulation of the highly mycorrhiza-induced fungal metal transporter by performing a molecular genetics and functional analysis, (ii) the role of mycorrhiza-induced genes for the fungal symbiosis by developing tools for genome editing (CRISPR/Cas) for the ECM fungus, and (iii) the analysis of the ECM proteome and phosphoproteome.

Establishment of the CRISPR/Cas technique for ECM fungi will lift a technical barrier and provide the scientific community with these missing tools. In addition, the whole set of the expected results should give decisive insights into the actual physiological role of the mycorrhiza-induced genes coding for transport functions, especially those located in the Hartig net that will determine, in turn, the efficiency of the ectomycorrhizal symbiosis. Hence, MYCOTRANS should help us to find true symbiotic marker genes, making it possible to use mycorrhizal interactions for sound management of both croplands and forests taking care of ecosystem services rendered by mycorrhizal fungi.

Ruytinx J., Kafle A., Usman M., Coninx L., Zimmermann S.D., Garcia K. (2020) Micronutrient transport in mycorrhizal symbiosis; zinc steals the show. Fungal Biology Reviews, 34 (1), 1-9. doi.org/10.1016/j.fbr.2019.09.001

MYCOTRANS aims at producing basic knowledge on the functioning of symbiotic exchange between plant roots and fungal symbiont, a beneficial interaction crucial for plant nutrition. MYCOTRANS will focus on the symbiotic ectomycorrhizal (ECM) model association Pinus pinaster – Hebeloma cylindrosporum, because this fungal species is the only one easily transformable with Agrobacterium, enabling genetics studies to be performed. Several transcriptional studies have revealed mycorrhiza-induced fungal membrane transport systems involved in K, N and P nutrition, but surprisingly, a member of the CDF (Cation Diffusion Facilitator) family was identified as the most mycorrhiza-induced transporter. So far, we have studied transport of macronutrients as potassium and phosphate, but we hypothesize that this micronutrient transporter, as other significantly mycorrhiza-induced genes, plays an important role in the development, maintenance or functioning of the ECM association and might provide new keys for understanding the positive effects of mycorrhizal symbiosis on host plant nutrition. However, the molecular function, cellular and subcellular localization, regulation and physiological role in the mycorrhiza of this CDF transporter are unknown yet. Hence, MYCOTRANS objectives will be (i) to decipher physiological function, localization, and regulation of the highly mycorrhiza-induced fungal metal transporter by performing a molecular genetics and functional analysis, (ii) to analyze the role of mycorrhiza-induced genes for the fungal symbiosis by developing tools for genome editing (CRISPR/Cas) for the ECM fungus, and (iii) to discover new aspects of mycorrhizal regulation occurring specifically at the level of proteins by the analysis of the ECM proteome and phosphoproteome. To address these objectives, we will use key methodologies which are: (i) heterologous expression in yeast and Xenopus laevis oocytes of the cDNA encoding the metal (putatively Zn, Fe, Mn) transporter of the CDF family, to assess the properties of this transporter, such as its selectivity for several micronutrients; (ii) In situ hybridization and green fluorescent protein (GFP-) fused proteins for cellular and sub-cellular localization of the CDF transporter in yeast and ectomycorrhizae; (iii) production of new CRISPR/Cas vectors and KO fungal mutants to study the role of mycorrhiza-involved fungal genes, as this technique of genome editing will be much more efficient than the RNAi method previously used by Partner 1; (iv) use of an in vitro symbiosis-mimicking system, where the fungus is incubated in a liquid solution either alone or with host plant roots ensuring a cross-talk between both partners of the symbiosis but without the formation of ECM structures on the root; (v) extraction of fungal proteins and separation in three fractions: soluble, microsomal and plasma membrane proteins, to carry out proteome analysis in all protein fractions and phosphoproteome analysis of plasma membrane proteins, as a target of possible post-translational modifications exerted by the host-plant. Establishment of the CRISPR/Cas technique for ECM fungi will lift a technical barrier and provide the scientific community with these missing tools. In addition, the whole set of the expected results should give decisive insights into the actual physiological role of the mycorrhiza-induced genes coding for transport functions, especially those located in the Hartig net that will determine, in turn, the efficiency of the ectomycorrhizal symbiosis. Hence, MYCOTRANS should help us to find true symbiotic marker genes, making it possible to use mycorrhizal interactions for sound management of both croplands and forests taking care of ecosystem services rendered by mycorrhizal fungi.