SlZRT2 Encodes a ZIP Family Zn Transporter With Dual Localization in the Ectomycorrhizal Fungus Suillus luteus

Abstract

Ectomycorrhizal (ECM) fungi are important root symbionts of trees, as they can have significant effects on the nutrient status of plants. In polluted environments, particular ECM fungi can protect their host tree from Zn toxicity by restricting the transfer of Zn while securing supply of essential nutrients. However, mechanisms and regulation of cellular Zn homeostasis in ECM fungi are largely unknown, and it remains unclear how ECM fungi affect the Zn status of their host plants. This study focuses on the characterization of a ZIP (Zrt/IrtT-like protein) transporter, SlZRT2, in the ECM fungus Suillus luteus, a common root symbiont of young pine trees. SlZRT2 is predicted to encode a plasma membrane-located Zn importer. Heterologous expression of SlZRT2 in yeast mutants with impaired Zn uptake resulted in a minor impact on cellular Zn accumulation and growth. The SlZRT2 gene product showed a dual localization and was detected at the plasma membrane and perinuclear region. S. luteus ZIP-family Zn uptake transporters did not show the potential to induce trehalase activity in yeast and to function as Zn sensors. In response to excess environmental Zn, gene expression analysis demonstrated a rapid but minor and transient decrease in SlZRT2 transcript level. In ECM root tips, the gene is upregulated. Whether this regulation is due to limited Zn availability at the fungal-plant interface or to developmental processes is unclear. Altogether, our results suggest a function for SlZRT2 in cellular Zn redistribution from the ER next to a putative role in Zn uptake in S. luteus.

Keywords: Mycorrhiza; Suillus luteus; ZIP; zinc homeostasis; zinc transporter.

FIGURE 1

SlZRT1, SlZRT2, ScZRT1, and ScZRT2 protein sequence. Transmembrane domains (TMDs) as predicted by TMHMM2.0, are indicated in green, putative histidine-rich metal binding domains (MBDs) in blue, and the ZIP signature sequence (Eng et al., 1998) in red. Two among ZIP proteins conserved histidines (located in TMD4 and TMD5) are indicated with an asterisk.

FIGURE 2

Functional complementation assays of Zn, Fe, and Mn uptake-deficient yeast strains Δzrt1Δzrt2 (A), Δftr1 (B), and Δsmf1 (C). Cultures of wild-type (WT) and mutant yeast cells (OD₆₀₀ = 1) were 10-fold serial diluted (10⁰, 10^–1, 10^–2, and 10^–3) and spotted on control (first column) or selection SD medium (second and third column). Control medium was supplemented with Zn, Fe, or Mn and selection medium with different concentrations of citrate, EDTA, or EGTA. WT cells were transformed with the empty vector (EV, pYES-DEST52; Invitrogen). Yeast mutants were transformed with the EV or the vector containing SlZRT2 or previously characterized SlZRT1. Pictures were taken after 3 days of growth and experiments were carried out for three independent clones.

FIGURE 3

Localization of the SlZRT2:EGFP fusion protein in yeast. (A) FM4-64 plasma membrane staining on ice to avoid endocytosis of the dye and (B) Hoechst 33324 nuclear staining. From left to right, pictures visualize the EGFP (green) fusion protein (left), FM4-64 (red) or Hoechst (blue) staining (middle), and the merged image (right).

FIGURE 4

Zn content of WT and Δzrt1Δzrt2 transformed yeast cells transformed with the EV (pYES-DEST52; Invitrogen) or the vector containing SlZRT2. Data are the average ± standard error (SE) of five replicates; significant differences (p < 0.05) are indicated by different letters.

FIGURE 5

Trehalase activity in Zn-depleted yeast cells after the re-supplementation of Zn. Trehalase activity was assessed in yeast cells maintained on Zn starvation medium (in gray; negative control) and after the addition of 5 mM ZnCl₂ (in black). (A) WT cells transformed with the EV (pYES-DEST52; Invitrogen). Data are the mean ± SE of three biological replicates. Cells were cultured for 2 days in SD medium supplemented with 10 mM citrate and 1 mM EDTA to trigger Zn starvation. (B,C) Trehalase activity of Δzrt1Δzrt2 yeast mutant cells. The trehalase assay with Δzrt1Δzrt2 cells was performed three times, each time under a different Zn starvation regime. Representative results from one starvation regime are shown. Cells were cultured for 2 h in SD medium supplemented with 10 mM citrate and 1 mM EDTA to trigger Zn starvation. Δzrt1Δzrt2 cells were transformed with the EV (B; pYES-DEST52; Invitrogen), SlZRT1 (C), or SlZRT2 (D). (Results from the two other Zn starvation regimes are shown in Supplementary Figure S5.)

FIGURE 6

SlZRT2 expression (A) and Zn content (B) in S. luteus mycelium after 0-, 1-, 2-, 4-, 8-, and 24-h exposure to different concentrations of Zn. Significant differences within each time point are indicated by different letters (p < 0.05). (A) SlZRT2 expression data are the average ± SE of three biological replicates and expressed relative in log2(fold change) to the control condition (20 μM Zn) within each time point. (B) Mycelial Zn content data are the average ± standard error (SE) of four biological replicates.