The arbuscular mycorrhizal fungus Rhizophagus irregularis uses the copper exporting ATPase RiCRD1 as a major strategy for copper detoxification

Arbuscular mycorrhizal (AM) fungi establish a mutualistic symbiosis with most land plants. AM fungi regulate plant copper (Cu) acquisition both in Cu deficient and polluted soils. Here, we report characterization of RiCRD1, a Rhizophagus irregularis gene putatively encoding a Cu transporting ATPase. Based on its sequence analysis, RiCRD1 was identified as a plasma membrane Cu + efflux protein of the P1B1-ATPase subfamily. As revealed by heterologous complementation assays in yeast, RiCRD1 encodes a functional protein capable of conferring increased tolerance against Cu. In the extraradical mycelium, RiCRD1 expression was highly up-regulated in response to high concentrations of Cu in the medium. Comparison of the expression patterns of different players of metal tolerance in R. irregularis under high Cu levels suggests that this fungus could mainly use a metal efflux based-strategy to cope with Cu toxicity. RiCRD1 was also expressed in the intraradical fungal structures and, more specifically, in the arbuscules, which suggests a role for RiCRD1 in Cu release from the fungus to the symbiotic interface. Overall, our results show that RiCRD1 encodes a protein which could have a pivotal dual role in Cu homeostasis in R. irregularis, playing a role in Cu detoxification in the extraradical mycelium and in Cu transfer to the apoplast of the symbiotic interface in the arbuscules.