The Rhizophagus irregularis Genome Encodes Two CTR Copper Transporters That Mediate Cu Import Into the Cytosol and a CTR-Like Protein Likely Involved in Copper Tolerance

Abstract

Arbuscular mycorrhizal fungi increase fitness of their host plants under Cu deficient and toxic conditions. In this study, we have characterized two Cu transporters of the CTR family (RiCTR1 and RiCTR2) and a CTR-like protein (RiCTR3A) of Rhizophagus irregularis. Functional analyses in yeast revealed that RiCTR1 encodes a plasma membrane Cu transporter, RiCTR2 a vacuolar Cu transporter and RiCTR3A a plasma membrane protein involved in Cu tolerance. RiCTR1 was more highly expressed in the extraradical mycelia (ERM) and RiCTR2 in the intraradical mycelia (IRM). In the ERM, RiCTR1 expression was up-regulated by Cu deficiency and down-regulated by Cu toxicity. RiCTR2 expression increased only in the ERM grown under severe Cu-deficient conditions. These data suggest that RiCTR1 is involved in Cu uptake by the ERM and RiCTR2 in mobilization of vacuolar Cu stores. Cu deficiency decreased mycorrhizal colonization and arbuscule frequency, but increased RiCTR1 and RiCTR2 expression in the IRM, which suggest that the IRM has a high Cu demand. The two alternatively spliced products of RiCTR3, RiCTR3A and RiCTR3B, were more highly expressed in the ERM. Up-regulation of RiCTR3A by Cu toxicity and the yeast complementation assays suggest that RiCTR3A might function as a Cu receptor involved in Cu tolerance.

Keywords: CTR family; Rhizophagus irregularis; arbuscular mycorrhizal fungi; copper homeostasis; copper transporters; symbiosis.

FIGURE 1

Structure of the R. irregularis CTR proteins. (A) Exon/intron organization of the R. irregularis CTR genes. Exons (E) and introns are represented by gray lines and dashed boxes, respectively. The two transcript variants of RiCTR3 are illustrated at the right. The coding regions of the two spliced variants of RiCTR3 are shown in gray and the non-coding in red. The start and stop codons are indicated. (B) Typical topological model of a CTR transporter. (C) Schematic representation of the structure of the R. irregularis CTR transporters. Orange boxes illustrate transmembrane domains, blue boxes Met motifs and green boxes Cys and His motifs. Red diamonds show the positions of the MetXXXMet and the GlyXXXGly motives of the CTRs master signature and gray diamonds the positions of the C-terminal His/Cys residues. Amino acid lengths are also indicated.

FIGURE 2

Analysis of the Cu transport activity and subcellular localization of the R. irregularis CTRs in yeast. (A) ctr1Δctr3Δ and ctr1Δctr2Δctr3Δ yeast cells transformed with the empty vector or expressing RiCTR1, RiCTR2, RiCTR3A or RiCTR3B were plated on YPEG media supplemented with Cu (0, 10, or 20 μM CuSO₄) or on SD medium without uracil. ctr1Δctr3Δ and ctr1Δctr2Δctr3Δ plated cells were incubated at 30°C for 4 and 7 days, respectively. RiCTR3A and RiCTR3B had the same effect (see Supplementary Figure S5), only the result with RiCTR3A is illustrated. (B) ctr1Δctr2Δctr3Δ yeast cells expressing GFP (empty vector), N-terminal (upper panel) or C-terminal (lower panel) GFP-tagged versions of RiCTR1, RiCTR2 and RiCTR3A were plated on YPEG media supplemented with Cu (0, 10, or 20 μM CuSO₄) or on SD medium without uracil. Plates were incubated at 30°C for 7 days. (C) ctr1Δctr2Δctr3Δ cells expressing GFP (the empty vector pFGWDR196) and GFP::RiCTR1 were visualized with a confocal microscope. eGFP, enhanced GFP fluorescence; BF, bright field.

FIGURE 3

RiCTRs expression levels in the R. irregularis ERM and IRM. (A) R. irregularis ERM and mycorrhizal carrot roots (IRM) were grown in compartmented monoxenic cultures (in vitro culture system). (B) R. irregularis ERM and mycorrhizal chicory roots (IRM) were grown in the whole plant bidimensional experimental system (in vivo culture system). Relative gene expression levels were calculated by the 2^-ΔCT method using RiEF1α as a normalizer. Bars represent standard error. Asterisks show statistically significant differences (P < 0.05; n = 4) between ERM and IRM.

FIGURE 4

Effect of Cu deficiency on mycorrhizal colonization. (A) Mycorrhizal colonization of carrot roots grown in monoxenic cultures in M media (control, 0.5 μM Cu) or in M media lacking Cu in plates started either with roots and inoculum previously grown in M media containing 0.5 μM CuSO₄ (moderate Cu deficiency) or without Cu (severe Cu deficiency). (B) Mycorrhizal colonization of chicory roots grown in the whole plant bidimensional experimental system fertilized with half-strength Hoagland solution (control, 0.16 μM Cu) or with a modified nutrient solution without Cu. Colonization rates were determined by using the Trouvelot method after histochemical staining and by determining the expression level of the R. irregularis elongation factor 1α (RiEF1α). The relative expression of RiEF1α was calculated using the 2^-ΔCT method with EF1α of the corresponding host plant as internal control. Bars represent standard error. Different letters indicate significant differences (P < 0.05; n = 4) between treatments and asterisks statistically significant differences (P < 0.05; n = 4) in comparison with the control. F%, frequency of mycorrhiza in the root system; M%, intensity of the mycorrhizal colonization in the root system; m%, intensity of the mycorrhizal colonization in the root fragments; a%, arbuscule abundance in mycorrhizal parts of root fragments; A%, arbuscule abundance in the root system. Scale bar of the left panels: 100 μm.

FIGURE 5

Effect of Cu deficiency on RiCTR1 and RiCTR2 IRM expression. (A) RiCTR1 and (B) RiCTR2 expression in mycorrhizal carrot roots developed in monoxenic cultures in M media (control, 0.5 μM Cu) or in M media lacking Cu in plates started either with roots and inoculum previously grown in M media containing 0.5 μM CuSO₄ (moderate Cu deficiency) or without Cu (severe Cu deficiency) (left panel) and in mycorrhizal chicory roots grown in the whole plant bidimensional experimental system and fertilized with half-strength Hoagland solution (control, 0.16 μM Cu) or with a modified nutrient solution without Cu (right panel). Relative gene expression levels were calculated by the 2^-ΔΔCT method using RiEF1α as a normalizer. Bars represent standard error. Asterisks show statistically significant differences (P < 0.05; n = 4) in comparison to the corresponding control value.

FIGURE 6

Regulation of RiCTRs expression by Cu availability. R. irregularis ERM was grown in monoxenic cultures in M media containing 0.5 μM CuSO₄ (control) or in M media lacking Cu in plates started with roots and inoculum previously grown either in M media containing 0.5 μM CuSO₄ (moderate Cu deficiency) or in M media without Cu (severe Cu deficiency). For the Cu toxicity treatments, the ERM grown in optimal M media was exposed for 1, 2, and 7 days to 250 μM CuSO₄ or for 1 and 2 days to 500 μM CuSO₄. (A) RiCTR1, (B) RiCTR2, (C) RiCTR3A and (D) RiCTR3B gene expression. Relative expression levels were calculated by the 2^-ΔΔCT method using RiEF1α as a normalizer. Bars represent standard error. Asterisks show statistically significant differences (P < 0.05; n = 3) in comparison to the corresponding control value.

FIGURE 7

Regulation of RiCTRs expression by oxidative stress. R. irregularis ERM grown in monoxenic cultures in M-C medium was exposed or not (Control) for 1 h to 1 mM H₂O₂. (A) RiCTR1, (B) RiCTR2, (C) RiCTR3A (D) RiCTR3B, and (E) RiSOD1 gene expression. Relative expression levels were calculated by the 2^-ΔΔCT method using RiEF1α as a normalizer. Bars represent standard error. Asterisks show statistically significant differences (P < 0.05; n = 3) in comparison to the control value.

FIGURE 8

Functional analysis of RiCTR3A and RiCTR3B in the Δ-yap1 S. cerevisiae mutant. (A) Δ-yap1 cells transformed with the empty vector or expressing RiCTR3A or RiCTR3B were plated on SD media supplemented or not with 1.5 mM CuSO₄ or with 0.5 mM H₂O₂. (B) Δ-yap1 cells transformed with the corresponding empty vector expressing GFP or N-terminal or C-terminal GFP-tagged versions of RiCTR3A were plated on SD media supplemented or not with 1.5 mM CuSO₄. Plates were incubated 4 days at 30°C. (C) Δ-yap1 cells expressing GFP (the empty vector pFGWDR196) and GFP::RiCTR3A were visualized with a confocal microscope. eGFP, enhanced GFP fluorescence; BF, bright field.