Study of the mechanisms of iron homeostasis in the arbuscular mycorrhizal fungus Rhizophagus irregularis

Zusammenfassung

Arbuscular mycorrhizal (AM) symbioses that involve most plants and Glomeromycota (AM) fungi are integral and functional parts of plant roots. In these associations, the fungi not only colonize the root cortex but also maintain an extensive network of hyphae that extend out of the root into the surrounding environment. These external hyphae contribute to plant uptake of low mobility nutrients, such as P, Fe and Zn. Besides improving plant mineral nutrition, AM fungi can alleviate heavy metal (HM) toxicity to their host plants. The HM Fe plays essential roles in many biological processes but is toxic when present in excess, since it can produce toxic free radicals via the Fenton reaction. This makes its transport and homeostatic control of particular importance to all living organisms. AM fungi play an important role in modulating plant HM acquisition in a wide range of soil metal concentrations and have been considered to be a key element in the improvement of micronutrient concentrations in crops and in the phytoremediation of polluted soils. Although the main benefit of the AM association is an improved P status of the mycorrhizal plant, AM fungi also play a role in Fe nutrition of their host plants, and direct evidence of the capability of the extraradical mycelium (ERM) to take up Fe from the soil and to transfer it to the host plant has been found. Conversely, other studies have shown that AM fungi play a role in reducing Fe uptake when the soil concentration is high. Nevertheless, little is known about the mechanisms of Fe uptake and homeostasis in arbuscular mycorrhizas. Within this PhD thesis, several methods have been used to analyze the mechanisms of Fe homeostasis in the model AM fungus Rhizophagus irregularis, which is easily grown in monoxenic cultures and whose genome has been recently sequenced. Genome-wide analyses were undertaken in order to identify R. irregularis genes involved in Fe transport and homeostasis, by making use of transport databases, genome organism websites and in silico bioinformatics tools for sequence analyses, such as software for protein structure predictions and methods for phylogenetic analyses. For experimental studies, R. irregularis monoxenic cultures were established in order to obtain exclusively fungal material for the subsequent gene isolation, gene expression analyses or enzymatic assays. Since it is not still possible to genetically manipulate AM fungi, functional and localization analyses of the newly identified R. irregularis genes were performed in a heterologous system: the budding yeast Saccharomyces cerevisiae. As a first approach to get some insights into Fe homeostasis mechanisms, a genome-wide analysis of Fe transporters was performed. This in silico analysis allowed the identification of 12 open reading frames in the R. irregularis genome, which potentially encode Fe transporters involved in iron acquisition from the soil or iron use and storage in the different subcellular compartments. Phylogenetic comparisons with the genomes of a set of reference fungi showed an expansion of some Fe transporter families. Analysis of the published transcriptomic profiles of R. irregularis revealed that some genes were up-regulated in mycorrhizal roots compared to germinated spores and ERM, which suggests that Fe is a metal important for plant colonization. Two components of a reductive pathway of high-affinity Fe transport, RiFTR1 and RiFTR2, were found to be within the full complement of genes encoding Fe transporters identified in the R. irregularis genome, showing high homology to the S. cerevisiae Fe permease Ftr1. In the well characterized S. cerevisiae system, the first step of the reductive Fe assimilation system is accomplished by the metalloreductases Fre1 and Fre2. Then, the reduced ferrous Fe is rapidly taken up by a high-affinity ferrous-specific transport complex consisting of a plasma membrane multicopper ferroxidase (Fet3) that oxidizes the Fe, which is then transported to the cytosol by a permease (Ftr1). When the ferric reductase activity was assayed in vivo on R. irregularis mycelia, it was found a basal level of activity in mycelium grown in control plates, which was activated when mycelia was grown in media without Fe. These data indicated that the reductive Fe uptake system is operating in R. irregularis. Only one ferric reductase homolog, RiFRE1, was found in the R. irregularis genome, but it was not able to restore the poor growth of the fre1-2 yeast mutant under low-Fe conditions, probably due to the low sequence homology of RiFRE1 and ScFRE1-2. Protein characterization and gene expression analyses of the two other putative components of the reductive pathway of Fe acquisition in R. irregularis, RiFTR1 and RiFTR2, indicated that RiFTR1 plays an in vivo role in high-affinity reductive Fe acquisition in Fe-limited environments. Nevertheless, the role of RiFTR2 in R. irregularis has not been elucidated in our study, probably because the protein failed to exit the ER, as shown in the yeast localization assays. It was also found that the genes encoding the three components of the reductive Fe assimilation pathway identified (RiFRE1, RiFTR1 and RiFTR2) are responsive to Fe, as shown by the gene expression analyses performed by real time RT-PCR. In order to identify the ferroxidase partner of RiFTR1, a search for candidate genes belonging to the multicopper oxidase (MCO) family was performed. It was concluded that R. irregularis has at least nine MCOs (RiMCO1-9) in its genome. Intron and similarity analyses defined five gene subfamilies. However, a phylogenetic analysis of MCO sequences of different taxonomic groups revealed that all the RiMCOs belong to the ferroxidase/laccase group and none of them clustered with the Fet3-type ferroxidases. RiMCO1 was the only MCO displaying a gene expression pattern typical of a high-affinity Fe transport component. Furthermore, RiMCO1 enables the fet3 yeast mutant to take up Fe. These data suggest that RiMCO1 might have a role in the reductive high-affinity Fe uptake system together with the Fe transporter RiFTR1 at the ERM. However, some other member(s) of the RiMCO family could also have ferroxidase activity. Gene expression analyses also revealed that some transcripts were very abundant in the ERM (RiMCO1 and RiMCO5) and others in the intraradical mycelium (RiMCO2), suggesting the different RiMCOs have specific functions. Finally, as an attempt to identify some other elements involved in the regulation of Fe homeostasis in R. irregularis, and since the yeast glutaredoxins (GRXs) Grx3-4 and Grx5 have been shown to have a role in Fe homeostasis, the full complement of GRXs in R. irregularis (RiGRX1,4,5,6) was identified and characterized (RiGRX1 was previously characterized in our group). Heterologous functional analyses showed that while the four R. irregularis GRXs are involved in oxidative stress protection, RiGRX4 and RiGRX5 (homologues of ScGrx3-4 and ScGrx5, respectively) also play a role in Fe homeostasis in yeast. In addition, gene expression data showed that RiGRX4 was the only R. irregularis GRX gene responsive to Fe, suggesting that it might be involved in Fe uptake regulation by interacting with iron-responsive transcription factors, as previously shown for its homologs in other fungi. Increased expression of RiGRX1 and RiGRX6 in the intraradical mycelium suggests that these GRXs could play a key role in oxidative stress protection of R. irregularis during its in planta phase. Despite in this PhD thesis only the fungal transporters involved in iron uptake from the soil and the glutaredoxins have been characterized, an integrating model is proposed for regulation of iron homeostasis and transport through the mycorrhizal pathway in a mycorrhizal root.