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.

Tamara Gómez-Gallego,Karim Benabdellah,Pierre Berthomieu,Ana M Jiménez-Jiménez,Nuria Ferrol,Miguel A Merlos,Carine Alcon

doi:10.3389/fpls.2019.00604

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.

Highlights

The transition metal copper (Cu) is a micronutrient acting as a redox active cofactor of key enzymes involved in a wide array of biochemical processes essential for life, such as respiration, superoxide scavenging and iron mobilization (Linder, 1991; Festa and Thiele, 2011)
Comparisons of the full-length cDNAs with the genomic sequences revealed the presence of three introns in RiCTR1 and two in RiCTR2 and RiCTR3, all of them flanked by the canonical splicing sequences GT and AG at the 5 and 3 ends, respectively (Figure 1A)
Alignment of the RiCTR3A and RiCTR3B transcripts with the RiCTR3 gene sequence indicates that both transcripts are alternatively spliced products of the same gene, as the RiCTR3A and RiCTR3B sequences are contained within the genomic sequence

Summary

Introduction

The transition metal copper (Cu) is a micronutrient acting as a redox active cofactor of key enzymes involved in a wide array of biochemical processes essential for life, such as respiration, superoxide scavenging and iron mobilization (Linder, 1991; Festa and Thiele, 2011). The MetXXXMet motif is located in TM2 and together with a cluster of Met residues in the N terminal domain is involved in Cu sensing and uptake (Puig et al, 2002; Guo et al, 2004), while TM3 harbors the GlyXXXGly motif that is critical for protein folding and oligomerization (Aller et al, 2004). Under Cu toxicity this domain allows protein inactivation through conformational structural changes (Wu et al, 2009). This family of transporters has been widely studied in Saccharomyces cerevisiae, which encodes three members (Ctr, Ctr, and Ctr). Very little is known about the mechanisms of Cu uptake in arbuscular mycorrhizal (AM) fungi, the most ancient and widespread fungal plant symbionts

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Conclusion