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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 P 1B1 -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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Arbuscular mycorrhizal (AM) fungi can improve iron (Fe) acquisition of their host plants. Here, we report a characterization of two components of the high-affinity reductive Fe uptake system of Rhizophagus irregularis, the ferric reductase (RiFRE1) and the high affinity Fe permeases (RiFTR1-2). In the extraradical mycelia (ERM), Fe deficiency induced activation of a plasma membrane-localized ferric reductase, an enzyme that reduces Fe(III) sources to the more soluble Fe(II). Yeast mutant complementation assays showed that RiFRE1 encodes a functional ferric reductase and RiFTR1 an iron permease. In the heterologous system, RiFTR1 was expressed in the plasma membrane while RiFTR2 was expressed in the endomembranes. In the ERM, the highest expression levels of RiFTR1 were found in mycelia grown in media with 0.045 mM Fe, while RiFTR2 was upregulated under Fe-deficient conditions. RiFTR2 expression also increased in the intraradical mycelia (IRM) of maize plants grown without Fe. These data indicate that the Fe permease RiFTR1 plays a key role in Fe acquisition and that RiFTR2 is involved in Fe homeostasis under Fe-limiting conditions. RiFTR1 was highly expressed in the (IRM), which suggests that the maintenance of Fe homeostasis in the IRM might be essential for a successful symbiosis., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Arbuscular mycorrhizal fungi (AMF) establish beneficial mutualistic symbioses with land plants, receiving carbon in exchange for mineral nutrients absorbed by the extraradical mycelium (ERM). With the aim of obtaining in vivo produced ERM for gene expression analyses, a whole-plant bi-dimensional experimental system was devised and tested with three host plants and three fungal symbionts. In such a system, Funneliformis mosseae in symbiosis with Cichorium intybus var. foliosum, Lactuca sativa, and Medicago sativa produced ERM whose lengths ranged from 9.8 ± 0.8 to 20.8 ± 1.2 m per plant. Since ERM produced in symbiosis with C. intybus showed the highest values for the different structural parameters assessed, this host was used to test the whole-plant system with F. mosseae, Rhizoglomus irregulare, and Funneliformis coronatus. The whole-plant system yielded 1-7 mg of ERM fresh biomass per plant per harvest, and continued producing new ERM for 6 months. Variable amounts of high-quality and intact total RNA, ranging from 15 to 65 μg RNA/mg ERM fresh weight, were extracted from the ERM of the three AMF isolates. Ammonium transporter gene expression was successfully determined in the cDNAs obtained from ERM of the three fungal symbionts by RT-qPCR using gene-specific primers designed on available (R. irregulare) and new (F. mosseae and F. coronatus) ammonium transporter gene sequences. The whole-plant experimental system represents a useful research tool for large production and easy collection of ERM for morphological, physiological, and biochemical analyses, suitable for a wide variety of AMF species, for a virtually limitless range of host plants and for studies involving diverse symbiotic interactions.

Glutaredoxins (GRXs) are small ubiquitous oxidoreductases involved in the regulation of the redox state in living cells. In an attempt to identify the full complement of GRXs in the arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis, three additional GRX homologs, besides the formerly characterized GintGRX1 (renamed here as RiGRX1), were identified. The three new GRXs (RiGRX4, RiGRX5 and RiGRX6) contain the CXXS domain of monothiol GRXs, but whereas RiGRX4 and RiGRX5 belong to class II GRXs, RiGRX6 belongs to class I together with RiGRX1. By using a yeast expression system, we observed that the newly identified homologs partially reverted sensitivity of the GRX deletion yeast strains to external oxidants. Furthermore, our results indicated that RiGRX4 and RiGRX5 play a role in iron homeostasis in yeast. Gene expression analyses revealed that RiGRX1 and RiGRX6 were more highly expressed in the intraradical (IRM) than in the extraradical mycelium (ERM). Exposure of the ERM to hydrogen peroxide induced up-regulation of RiGRX1, RiGRX4 and RiGRX5 gene expression. RiGRX4 expression was also up-regulated in the ERM when the fungus was grown in media supplemented with a high iron concentration. These data indicate the two monothiol class II GRXs, RiGRX4 and RiGRX5, might be involved in oxidative stress protection and in the regulation of fungal iron homeostasis. Increased expression of RiGRX1 and RiGRX6 in the IRM suggests that these GRXs should play a key role in oxidative stress protection of R. irregularis during its in planta phase.

Arbuscular mycorrhizal (AM) symbioses are mutualistic associations between soil fungi and most vascular plants. Modulation of the hormonal and transcriptional profiles, including changes related to defense signalling, has been reported in many host plants during AM symbioses. These changes have been often related to the improved stress tolerance common in mycorrhizal plants. However, results on the alterations in phytohormones content and their role on the symbiosis are controversial. Here, an integrative analysis of the response of phylogenetically diverse plants (i.e., tomato, soybean, and maize) to two mycorrhizal fungi -Funneliformis mosseae and Rhizophagus irregularis- was performed. The analysis of the defense-related hormones salicylic acid, abscisic acid, and jasmonates, and the expression of marker genes of the pathways they regulate, revealed significant changes in the roots of mycorrhizal plants. These changes depended on both the plant and the AM fungus (AMF) involved. However, general trends can be identified: roots associated with the most effective colonizer R. irregularis showed fewer changes in these defense-related traits, while the colonization by F. mosseae led to significant modifications in all plants tested. The up-regulation of the jasmonate pathway by F. mosseae was found to be highly conserved among the different plant species, suggesting an important role of jasmonates during this AM interaction. Our study evidences a strong influence of the AMF genotype on the modulation of host defense signalling, and offers hints on the role of these changes in the symbiosis.

The role and importance of arbuscular mycorrhizae (AM) in plant nitrogen (N) nutrition is uncertain. We propose that this be clarified by using more integrative experimental designs, with the use of a gradient of N supply and the quantification of an extensive array of plant nutrient contents. Using such an experimental design, we investigated AM effects on plant N nutrition, whether the mycorrhizal N response (MNR) determines the mycorrhizal growth response (MGR), and how MNR influences plants' C economy. Oryza sativa plants were inoculated with Rhizophagus irregularis or Funneliformis mossae. AM effects were studied along a gradient of N supplies. Biomass, photosynthesis, nutrient and starch contents, mycorrhizal colonization and OsPT11 gene expression were measured. C investment in fungal growth was estimated. Results showed that, in rice, MGR was dependent on AM nutrient uptake effects, namely on the synergy between N and Zn, and not on C expenditure. The supply of C to the fungus was dependent on the plant's nutrient demand, indicated by high shoot C/N or low %N. We conclude that one of the real reasons for the negative MGR of rice, Zn deficiency of AMF plants, would have remained hidden without an experimental design allowing the observation of plants' response to AM along gradients of nutrient concentrations. Adopting more integrative and comprehensive experimental approaches in mycorrhizal studies seems therefore essential if we are to achieve a true understanding of AM function, namely of the mechanisms of C/N exchange regulation in AM., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Arbuscular mycorrhizal (AM) symbiosis plays an important role in improving plant fitness and soil quality, particularly in fragile and stressed environments, as those in certain areas of Mediterranean ecosystems. AM fungal communities are usually affected by dynamic factors such as the plant community structure and composition, which in turn are imposed by seasonality. For this reason, a one-year-round time-course trial was performed by sampling the root system of two representative shrubland species (Rosmarinus officinalis and Thymus zygis) within a typical Mediterranean ecosystem from the Southeast of Spain. The 18S rDNA gene, of the AM fungal community in roots, was subjected to PCR-SSCP, sequencing, and phylogenetic analysis. Forty-three different AM fungal sequence types were found which clustered in 16 phylotypes: 14 belonged to the Glomeraceae and two to the Diversisporaceae. Surprisingly, only two of these phylotypes were related with sequences of morphologically defined species: Glomus intraradices and Glomus constrictum. Significant differences were detected for the relative abundance of some phylotypes while no effects were found for the calculated diversity indices. These results may help to design efficient mycorrhizal-based revegetation programs for this type of ecosystems.

The kinetics and energetics of (15)NH (4) (+) uptake by the extraradical mycelium of the arbuscular mycorrhizal fungus Rhizophagus irregularis were investigated. (15)NH (4) (+) uptake increased with increasing substrate concentration over the concentration range of 0.002 to 25 mM. Eadie-Hofstee plots showed that ammonium (NH (4) (+) ) uptake over this range was biphasic. At concentrations below 100 μM, NH (4) (+) uptake fits a Michaelis-Menten curve, typical of the activity of a saturable high-affinity transport system (HATS). At concentrations above 1 mM, NH (4) (+) influx showed a linear response typical of a nonsaturable low-affinity transport system (LATS). Both transport systems were dependent on external pH. The HATS and, to a lesser extent, the LATS were inhibited by the ionophore carbonylcyanide m-chlorophenylhydrazone (CCCP) and the ATP-synthesis inhibitor 2,4-dinitrophenol. These data indicate that the two NH (4) (+) transport systems of R. irregularis are dependent on metabolic energy and on the electrochemical H(+) gradient. The HATS- and the LATS-mediated (15)NH (4) (+) influxes were also regulated by acetate. This first report of the existence of active high- and low-affinity NH4(+) transport systems in the extraradical mycelium of an arbuscular mycorrhizal fungus and provides novel information on the mechanisms underlying mycosymbiont uptake of nitrogen from the soil environment.

• The arbuscular mycorrhizal symbiosis is arguably the most ecologically important eukaryotic symbiosis, yet it is poorly understood at the molecular level. To provide novel insights into the molecular basis of symbiosis-associated traits, we report the first genome-wide analysis of the transcriptome from Glomus intraradices DAOM 197198. • We generated a set of 25,906 nonredundant virtual transcripts (NRVTs) transcribed in germinated spores, extraradical mycelium and symbiotic roots using Sanger and 454 sequencing. NRVTs were used to construct an oligoarray for investigating gene expression. • We identified transcripts coding for the meiotic recombination machinery, as well as meiosis-specific proteins, suggesting that the lack of a known sexual cycle in G. intraradices is not a result of major deletions of genes essential for sexual reproduction and meiosis. Induced expression of genes encoding membrane transporters and small secreted proteins in intraradical mycelium, together with the lack of expression of hydrolytic enzymes acting on plant cell wall polysaccharides, are all features of G. intraradices that are shared with ectomycorrhizal symbionts and obligate biotrophic pathogens. • Our results illuminate the genetic basis of symbiosis-related traits of the most ancient lineage of plant biotrophs, advancing future research on these agriculturally and ecologically important symbionts., (© 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.)

In the symbiotic association of plants and arbuscular mycorrhizal (AM) fungi, the fungus delivers mineral nutrients, such as phosphate and nitrogen, to the plant while receiving carbon. Previously, we identified an NH(4)(+) transporter in the AM fungus Glomus intraradices (GintAMT1) involved in NH(4)(+) uptake from the soil when preset at low concentrations. Here, we report the isolation and characterization of a new G. intraradicesNH(4)(+) transporter gene (GintAMT2). Yeast mutant complementation assays showed that GintAMT2 encodes a functional NH(4)(+) transporter. The use of an anti-GintAMT2 polyclonal antibody revealed a plasma membrane location of GintAMT2. GintAMT1 and GintAMT2 were differentially expressed during the fungal life cycle and in response to N. In contrast to GintAMT1, GintAMT2 transcript levels were higher in the intraradical than in the extraradical fungal structures. However, transcripts of both genes were detected in arbuscule-colonized cortical cells. GintAMT1 expression was induced under low N conditions. Constitutive expression of GintAMT2 in N-limiting conditions and transitory induction after N re-supply suggests a role for GintAMT2 to retrieve NH(4)(+) leaked out during fungal metabolism., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A new dimorphic fungal species in the arbuscular mycorrhiza-forming Glomeromycota, Ambispora granatensis, was isolated from an agricultural site in the province of Granada (Andalucía, Spain) growing in the rhizosphere of Asparagus officinalis. It was propagated in pot cultures with Trifolium pratense and Sorghum vulgare. The fungus also colonized Ri T-DNA transformed Daucus carota roots but did not form spores in these root organ cultures. The spores of the acaulosporoid morph are 90-150 μm diam and hyaline to white to pale yellow. They have three walls and a papillae-like rough irregular surface on the outer surface of the outer wall. The irregular surface might become difficult to detect within a few hours in lactic acid-based mountings but are clearly visible in water. The structural central wall layer of the outer wall is only 0.8-1.5 μm thick. The glomoid spores are formed singly or in small, loose spore clusters of 2-10 spores. They are hyaline to pale yellow, (25)40-70 μm diam and have a bilayered spore wall without ornamentation. Nearly full length sequences of the 18S and the ITS regions of the ribosomal gene place the new fungus in a separate clade next to Ambispora fennica and Ambispora gerdemannii. The acaulosporoid spores of the new fungus can be distinguished easily from all other spores in genus Ambispora by the conspicuous thin outer wall.

To gain further insights into the mechanisms of redox homeostasis in arbuscular mycorrhizal fungi, we characterized a Glomus intraradices gene (GintSOD1) showing high similarity to previously described genes encoding CuZn superoxide dismutases (SODs). The GintSOD1 gene consists of an open reading frame of 471 bp, predicted to encode a protein of 157 amino acids with an estimated molecular mass of 16.3 kDa. Functional complementation assays in a CuZnSOD-defective yeast mutant showed that GintSOD1 protects the yeast cells from oxygen toxicity and that it, therefore, encodes a protein that scavenges reactive oxygen species (ROS). GintSOD1 transcripts differentially accumulate during the fungal life cycle, reaching the highest expression levels in the intraradical mycelium. GintSOD1 expression is induced by the well known ROS-inducing agents paraquat and copper, and also by fenpropimorph, a sterol biosynthesis inhibitor (SBI) fungicide. These results suggest that GintSOD1 is involved in the detoxification of ROS generated from metabolic processes and by external agents. In particular, our data indicate that the antifungal effects of fenpropimorph might not be only due to the interference with sterol metabolism but also to the perturbation of other biological processes and that ROS production and scavenging systems are involved in the response to SBI fungicides.

A new fungal species in the arbuscular mycorrhiza-forming Glomeromycetes, Entrophospora nevadensis, was isolated from soil near the roots of several endemic and endangered plant species (e.g. Plantago nivalis and Alchemilla fontqueri) growing in Sierra Nevada National Park (Granada, Andalucia, Spain). The fungus was propagated in trap cultures on Plantago nivalis and Sorbus hybrida and in pure cultures on Trifolium pratense and Sorghum vulgare. Spores are yellow brown to brown, 90-115 .m diam and form singly in soil, in the neck of adherent sporiferous saccules that form either terminally or intercalary on mycelial hyphae. Spores have two three-layered walls and conspicuous, 6-12 microm long, spiny, thorn-like projections on the outer wall consisting of hyaline to subhyaline, evanescent tips and yellow brown to brown, persistent bases. In aging spores these projections are usually shorter (1-2.8 microm) and dome-shaped or rounded, sometimes with a central pit on top where the evanescent tip has sloughed off. Molecular analysis with partial sequences of the 18S ribosomal gene places the fungus within the Diversisporales. The new fungus was found in soil near plants with different living strategies but growing in high altitude soils with acidic pH, high soil moisture and organic carbon content, and close to streams.

A full-length cDNA sequence putatively encoding an ATP-binding cassette (ABC) transporter (GintABC1) was isolated from the extraradical mycelia of the arbuscular mycorrhizal fungus Glomus intraradices. Bioinformatic analysis of the sequence indicated that GintABC1 encodes a 1513 amino acid polypeptide, containing two six-transmembrane clusters (TMD) intercalated with sequences characteristics of the nucleotide binding domains (NBD) and an extra N-terminus extension (TMD0). GintABC1 presents a predicted TMD0-(TMD-NBD)(2) topology, typical of the multidrug resistance-associated protein subfamily of ABC transporters. Gene expression analyses revealed no difference in the expression levels of GintABC1 in the extra- vs the intraradical mycelia. GintABC1 was up-regulated by Cd and Cu, but not by Zn, suggesting that this transporter might be involved in Cu and Cd detoxification. Paraquat, an oxidative agent, also induced the transcription of GintABC1. These data suggest that redox changes may be involved in the transcriptional regulation of GintABC1 by Cd and Cu.

Vitamin B6 is an essential metabolite that has recently been implicated in defense against cellular oxidative stress. In fungi, the de novo biosynthetic pathway of vitamin B6 involves two genes, PDX1 and PDX2. Here, we report a component of the PDX1/PDX2 vitamin B6 biosynthetic pathway in an arbuscular mycorrhizal (AM) fungus. Using rapid amplification of cDNA ends, we isolated the full-length cDNA of a PDX-like gene, GintPDX1, from Glomus intraradices. GintPDX1 expression was analysed by real-time reverse transcription-polymerase chain reaction (RT-PCR). GintPDX1 activity and function were investigated by heterologous complementation of the yeast strainDeltasnz1, which is deficient in vitamin B6 biosynthesis. Sequence data revealed that GintPDX1 is highly homologous to other identified PDX1 proteins. GintPDX1 restores prototrophy to the vitamin B6 auxotrophic yeast mutant and reverts its superoxide sensitivity. GintPDX1 is expressed throughout the fungal life cycle, with the highest transcription levels found in the intraradical fungal structures. GintPDX1 expression was induced in response to hydrogen peroxide, paraquat and copper. The results demonstrate that AM fungi possess at least one component of the machinery necessary for vitamin B6 biosynthesis. Transcriptional regulation of GintPDX1 suggests a role for vitamin B6 as an antioxidant and modulator of reactive oxygen species in G. intraradices.

Glutaredoxins (GRXs) are small proteins with glutathione-dependent disulfide oxidoreductase activity involved in cellular defense against oxidative stress. This work reports the identification and characterization of the first glomeromycotan dithiol glutaredoxin gene from the fungus Glomus intraradices. The corresponding gene, named GintGRX1, shares high sequence similarity with previously described fungal GRXs. GintGRX1 contains the characteristic dithiol active site CPYC. By using a yeast expression system, we found that GintGRX1 encodes a multifunctional protein with oxidoreductase, peroxidase and glutathione S-transferase activity. GintGRX1 partially reverted sensitivity to superoxide radicals of the Deltagrx1Deltagrx2Saccharomyces cerevisiae strain. GintGRX1 was transcriptionally regulated by paraquat but not by hydrogen peroxide. Copper induced an accumulation of reactive oxygen species in the extraradical mycelium of G. intraradices and up-regulation of GintGRX1 transcript levels. These data suggest a role for GintGRX1 in protecting the fungus against the oxidative damage induced directly by the superoxide anion or indirectly by copper.