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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.

Nutrient acquisition and transfer are essential steps in the arbuscular mycorrhizal (AM) symbiosis, which is formed by the majority of land plants. Mineral nutrients are taken up by AM fungi from the soil and transferred to the plant partner. Within the cortical plant root cells the fungal hyphae form tree-like structures (arbuscules) where the nutrients are released to the plant-fungal interface, i.e., to the periarbuscular space, before being taken up by the plant. In exchange, the AM fungi receive carbohydrates from the plant host. Besides the well-studied uptake of phosphorus (P), the uptake and transfer of nitrogen (N) plays a crucial role in this mutualistic interaction. In the AM fungus Rhizophagus irregularis (formerly called Glomus intraradices), two ammonium transporters (AMT) were previously described, namely GintAMT1 and GintAMT2. Here, we report the identification and characterization of a newly identified R. irregularis AMT, GintAMT3. Phylogenetic analyses revealed high sequence similarity to previously identified AM fungal AMTs and a clear separation from other fungal AMTs. Topological analysis indicated GintAMT3 to be a membrane bound pore forming protein, and GFP tagging showed it to be highly expressed in the intraradical mycelium of a fully established AM symbiosis. Expression of GintAMT3 in yeast successfully complemented the yeast AMT triple deletion mutant (MATa ura3 mep1Δ mep2Δ::LEU2 mep3Δ::KanMX2). GintAMT3 is characterized as a low affinity transport system with an apparent Km of 1.8 mM and a V max of 240 nmol(-1) min(-1) 10(8) cells(-1), which is regulated by substrate concentration and carbon supply.

Arbuscular mycorrhizal fungi (AMF), belonging to the Glomeromycota, are soil microorganisms that establish mutualistic symbioses with the majority of higher plants. The efficient uptake of low mobility mineral nutrients by the fungal symbiont and their further transfer to the plant is a major feature of this symbiosis. Besides improving plant mineral nutrition, AMF can alleviate heavy metal toxicity to their host plants and are able to tolerate high metal concentrations in the soil. Nevertheless, we are far from understanding the key molecular determinants of metal homeostasis in these organisms. To get some insights into these mechanisms, a genome-wide analysis of Cu, Fe and Zn transporters was undertaken, making use of the recently published whole genome of the AMF Rhizophagus irregularis. This in silico analysis allowed identification of 30 open reading frames in the R. irregularis genome, which potentially encode metal transporters. Phylogenetic comparisons with the genomes of a set of reference fungi showed an expansion of some metal transporter families. Analysis of the published transcriptomic profiles of R. irregularis revealed that a set of genes were up-regulated in mycorrhizal roots compared to germinated spores and extraradical mycelium, which suggests that metals are important for plant colonization.

Arbuscular mycorrhizal fungi (AMF), which form an ancient and widespread mutualistic symbiosis with plants, are a crucial but still enigmatic component of the plant micro biome. Nutrient exchange has probably been at the heart of the success of this plant-fungus interaction since the earliest days of plants on land. To characterize genes from the fungal partner involved in nutrient exchange, and presumably important for the functioning of the AM symbiosis, genome-wide transcriptomic data obtained from the AMF Rhizophagus irregularis were exploited. A gene sequence, showing amino acid sequence and transmembrane domains profile similar to members of the PTR2 family of fungal oligopeptide transporters, was identified and called RiPTR2. The functional properties of RiPTR2 were investigated by means of heterologous expression in Saccharomyces cerevisiae mutants defective in either one or both of its di/tripeptide transporter genes PTR2 and DAL5. These assays showed that RiPTR2 can transport dipeptides such as Ala-Leu, Ala-Tyr or Tyr-Ala. From the gene expression analyses it seems that RiPTR2 responds to different environmental clues when the fungus grows inside the root and in the extraradical phase.