Physiological and transcriptomic response of Medicago truncatula to colonization by high- or low-benefit arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal (AM) fungi form a root endosymbiosis with many agronomically important crop species. They enhance the ability of their host to obtain nutrients from the soil and increase the tolerance to biotic and abiotic stressors. However, AM fungal species can differ in the benefits they provide to their host plants. Here, we examined the putative molecular mechanisms involved in the regulation of the physiological response of Medicago truncatula to colonization by Rhizophagus irregularis or Glomus aggregatum, which have previously been characterized as high- and low-benefit AM fungal species, respectively. Colonization with R. irregularis led to greater growth and nutrient uptake than colonization with G. aggregatum. These benefits were linked to an elevated expression in the roots of strigolactone biosynthesis genes (NSP1, NSP2, CCD7, and MAX1a), mycorrhiza-induced phosphate (PT8), ammonium (AMT2;3), and nitrate (NPF4.12) transporters and the putative ammonium transporter NIP1;5. R. irregularis also stimulated the expression of photosynthesis-related genes in the shoot and the upregulation of the sugar transporters SWEET1.2, SWEET3.3, and SWEET 12 and the lipid biosynthesis gene RAM2 in the roots. In contrast, G. aggregatum induced the expression of biotic stress defense response genes in the shoots, and several genes associated with abiotic stress in the roots. This suggests that either the host perceives colonization by G. aggregatum as pathogen attack or that G. aggregatum can prime host defense responses. Our findings highlight molecular mechanisms that host plants may use to regulate their association with high- and low-benefit arbuscular mycorrhizal symbionts.

Nutrient demand and fungal access to resources control the carbon allocation to the symbiotic partners in tripartite interactions of Medicago truncatula.

Legumes form tripartite interactions with arbuscular mycorrhizal fungi and rhizobia, and both root symbionts exchange nutrients against carbon from their host. The carbon costs of these interactions are substantial, but our current understanding of how the host controls its carbon allocation to individual root symbionts is limited. We examined nutrient uptake and carbon allocation in tripartite interactions of Medicago truncatula under different nutrient supply conditions, and when the fungal partner had access to nitrogen, and followed the gene expression of several plant transporters of the Sucrose Uptake Transporter (SUT) and Sugars Will Eventually be Exported Transporter (SWEET) family. Tripartite interactions led to synergistic growth responses and stimulated the phosphate and nitrogen uptake of the plant. Plant nutrient demand but also fungal access to nutrients played an important role for the carbon transport to different root symbionts, and the plant allocated more carbon to rhizobia under nitrogen demand, but more carbon to the fungal partner when nitrogen was available. These changes in carbon allocation were consistent with changes in the SUT and SWEET expression. Our study provides important insights into how the host plant controls its carbon allocation under different nutrient supply conditions and changes its carbon allocation to different root symbionts to maximize its symbiotic benefits.

Fungal nutrient allocation in common mycorrhizal networks is regulated by the carbon source strength of individual host plants.

Common mycorrhizal networks (CMNs) of arbuscular mycorrhizal (AM) fungi in the soil simultaneously provide multiple host plants with nutrients, but the mechanisms by which the nutrient transport to individual host plants within one CMN is controlled are unknown. Using radioactive and stable isotopes, we followed the transport of phosphorus (P) and nitrogen (N) in the CMNs of two fungal species to plants that differed in their carbon (C) source strength, and correlated the transport to the expression of mycorrhiza-inducible plant P (MtPt4) and ammonium (1723.m00046) transporters in mycorrhizal roots. AM fungi discriminated between host plants that shared a CMN and preferentially allocated nutrients to high-quality (nonshaded) hosts. However, the fungus also supplied low-quality (shaded) hosts with nutrients and maintained a high colonization rate in these plants. Fungal P transport was correlated to the expression of MtPt4. The expression of the putative ammonium transporter 1723.m00046 was dependent on the fungal nutrient supply and was induced when the CMN had access to N. Biological market theory has emerged as a tool with which the strategic investment of competing partners in trading networks can be studied. Our work demonstrates how fungal partners are able to retain bargaining power, despite being obligately dependent on their hosts.

The role of carbon in fungal nutrient uptake and transport: implications for resource exchange in the arbuscular mycorrhizal symbiosis.

The arbuscular mycorrhizal (AM) symbiosis, which forms between plant hosts and ubiquitous soil fungi of the phylum Glomeromycota, plays a key role for the nutrient uptake of the majority of land plants, including many economically important crop species. AM fungi take up nutrients from the soil and exchange them for photosynthetically fixed carbon from the host. While our understanding of the exact mechanisms controlling carbon and nutrient exchange is still limited, we recently demonstrated that (i) carbon acts as an important trigger for fungal N uptake and transport, (ii) the fungus changes its strategy in response to an exogenous supply of carbon, and that (iii) both plants and fungi reciprocally reward resources to those partners providing more benefit. Here, we summarize recent research findings and discuss the implications of these results for fungal and plant control of resource exchange in the AM symbiosis.

Carbon availability triggers fungal nitrogen uptake and transport in arbuscular mycorrhizal symbiosis.

The arbuscular mycorrhizal (AM) symbiosis, formed between the majority of land plants and ubiquitous soil fungi of the phylum Glomeromycota, is responsible for massive nutrient transfer and global carbon sequestration. AM fungi take up nutrients from the soil and exchange them against photosynthetically fixed carbon (C) from the host. Recent studies have demonstrated that reciprocal reward strategies by plant and fungal partners guarantee a "fair trade" of phosphorus against C between partners [Kiers ET, et al. (2011) Science 333:880-882], but whether a similar reward mechanism also controls nitrogen (N) flux in the AM symbiosis is not known. Using mycorrhizal root organ cultures, we manipulated the C supply to the host and fungus and followed the uptake and transport of N sources in the AM symbiosis, the enzymatic activities of arginase and urease, and fungal gene expression in the extraradical and intraradical mycelium. We found that the C supply of the host plant triggers the uptake and transport of N in the symbiosis, and that the increase in N transport is orchestrated by changes in fungal gene expression. N transport in the symbiosis is stimulated only when the C is delivered by the host across the mycorrhizal interface, not when C is supplied directly to the fungal extraradical mycelium in the form of acetate. These findings support the importance of C flux from the root to the fungus as a key trigger for N uptake and transport and provide insight into the N transport regulation in the AM symbiosis.

Germinating spores of Glomus intraradices can use internal and exogenous nitrogen sources for de novo biosynthesis of amino acids.

* Here, nitrogen (N) uptake and metabolism, and related gene expression, were analyzed in germinating spores of Glomus intraradices to examine the mechanisms and the regulation of N handling during presymbiotic growth. * The uptake and incorporation of organic and inorganic N sources into free amino acids were analyzed using stable and radioactive isotope labeling followed by high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS) and liquid scintillation counting and the fungal gene expression was measured by quantitative polymerase chain reaction (Q-PCR). * Quiescent spores store Asp, Ala and Arg and can use these internal N resources during germination. Although not required for presymbiotic growth, exogenous N can also be utilized for the de novo biosynthesis of amino acids. Ammonium and urea are more rapidly assimilated than nitrate and amino acids. Root exudates do not stimulate the uptake and utilization of exogenous ammonium, but the expression of genes encoding a putative glutamate dehydrogenase (GDH), a urease accessory protein (UAP) and an ornithine aminotransferase (OAT) were stimulated by root exudates. The transcript levels of an ammonium transporter (AMT) and a glutamine synthetase (GS) were not affected. * Germinating spores can make effective use of different N sources and the ability to synthesize amino acids does not limit presymbiotic growth of arbuscular mycorrhizal (AM) spores.

Root exudates stimulate the uptake and metabolism of organic carbon in germinating spores of Glomus intraradices.

* Root exudates play a key role during the presymbiotic growth phase and have been shown to stimulate hyphal branching and the catabolic metabolism of arbuscular mycorrhizal (AM) fungal spores. * Here, the effect of root exudates on presymbiotic growth, uptake of exogenous carbon and transcript levels for genes putatively involved in the carbon metabolism of germinating spores were determined. * Crude root exudates led to a slight acceleration of spore germination, increased germ tube branching and stimulated uptake and catabolic metabolism of acetate, and to a greater extent of glucose, but had no effect on gene expression. By contrast, partially purified root exudates increased the transcript levels of acyl-CoA dehydrogenase (ss-oxidation of fatty acids to acetyl-CoA), malate synthase (glyoxylate cycle) and glutamine-fructose-6-phosphate aminotransferase (chitin biosynthesis), but did not differ from crude root exudates in their effect on substrate uptake and respiration. The expression of glycogen synthase (glycogen biosynthesis), glucose-6-phosphate dehydrogenase (pentose phosphate pathway) and neutral trehalase (hydrolysis of trehalose) were only marginally or not affected by root exudates. * Root exudates have an effect on both membrane activity and gene expression and the results are discussed in relation to the catabolic and anabolic metabolism of spores during presymbiotic growth.

Nitrogen transfer in the arbuscular mycorrhizal symbiosis.

Most land plants are symbiotic with arbuscular mycorrhizal fungi (AMF), which take up mineral nutrients from the soil and exchange them with plants for photosynthetically fixed carbon. This exchange is a significant factor in global nutrient cycles as well as in the ecology, evolution and physiology of plants. Despite its importance as a nutrient, very little is known about how AMF take up nitrogen and transfer it to their host plants. Here we report the results of stable isotope labelling experiments showing that inorganic nitrogen taken up by the fungus outside the roots is incorporated into amino acids, translocated from the extraradical to the intraradical mycelium as arginine, but transferred to the plant without carbon. Consistent with this mechanism, the genes of primary nitrogen assimilation are preferentially expressed in the extraradical tissues, whereas genes associated with arginine breakdown are more highly expressed in the intraradical mycelium. Strong changes in the expression of these genes in response to nitrogen availability and form also support the operation of this novel metabolic pathway in the arbuscular mycorrhizal symbiosis.

The fungus does not transfer carbon to or between roots in an arbuscular mycorrhizal symbiosis.

• Carbon transfer from fungus to plant in the arbuscular mycorrhizal (AM) symbiosis has been reported, but its significance and even its existence have been called into question and the issue remains controversial. We investigated carbon movement from fungus to plant and from one mycorrhizal root system to another via a common AM fungal network in monoxenic cultures to avoid limitations of some previous studies. • 13 C and 14 C labeled substrates were supplied to functioning in vitro AM mycorrhizas between Ri T-DNA transformed carrot (Daucus carota) roots and Glomus intraradices to follow carbon movement into and between host and fungal metabolite pools. • Fungal triacylglycerol and trehalose were labeled when permeant substrates were supplied to the extraradical mycelium (ERM), but host-specific compounds in the roots did not become labeled. When labeled glucose was provided to a donor root system, label moved to recipient roots via a common AM fungal network but remained in fungal compounds. • We conclude that carbon flow in the AM symbiosis is normally unidirectional from plant to fungus and that while carbon is translocated by the fungus from one metabolically active root system to another, it remains within the intraradical mycelium (IRM).

Carbon export from arbuscular mycorrhizal roots involves the translocation of carbohydrate as well as lipid.

Arbuscular mycorrhizal (AM) fungi take up photosynthetically fixed carbon from plant roots and translocate it to their external mycelium. Previous experiments have shown that fungal lipid synthesized from carbohydrate in the root is one form of exported carbon. In this study, an analysis of the labeling in storage and structural carbohydrates after (13)C(1) glucose was provided to AM roots shows that this is not the only pathway for the flow of carbon from the intraradical to the extraradical mycelium (ERM). Labeling patterns in glycogen, chitin, and trehalose during the development of the symbiosis are consistent with a significant flux of exported glycogen. The identification, among expressed genes, of putative sequences for glycogen synthase, glycogen branching enzyme, chitin synthase, and for the first enzyme in chitin synthesis (glutamine fructose-6-phosphate aminotransferase) is reported. The results of quantifying glycogen synthase gene expression within mycorrhizal roots, germinating spores, and ERM are consistent with labeling observations using (13)C-labeled acetate and glycerol, both of which indicate that glycogen is synthesized by the fungus in germinating spores and during symbiosis. Implications of the labeling analyses and gene sequences for the regulation of carbohydrate metabolism are discussed, and a 4-fold role for glycogen in the AM symbiosis is proposed: sequestration of hexose taken from the host, long-term storage in spores, translocation from intraradical mycelium to ERM, and buffering of intracellular hexose levels throughout the life cycle.

Translocation and utilization of fungal storage lipid in the arbuscular mycorrhizal symbiosis.

The arbuscular mycorrhizal (AM) symbiosis is responsible for huge fluxes of photosynthetically fixed carbon from plants to the soil. Carbon is transferred from the plant to the fungus as hexose, but the main form of carbon stored by the mycobiont at all stages of its life cycle is triacylglycerol. Previous isotopic labeling experiments showed that the fungus exports this storage lipid from the intraradical mycelium (IRM) to the extraradical mycelium (ERM). Here, in vivo multiphoton microscopy was used to observe the movement of lipid bodies through the fungal colony and to determine their sizes, distribution, and velocities. The distribution of lipid bodies along fungal hyphae suggests that they are progressively consumed as they move toward growing tips. We report the isolation and measurements of expression of an AM fungal expressed sequence tag that encodes a putative acyl-coenzyme A dehydrogenase; its deduced amino acid sequence suggests that it may function in the anabolic flux of carbon from lipid to carbohydrate. Time-lapse image sequences show lipid bodies moving in both directions along hyphae and nuclear magnetic resonance analysis of labeling patterns after supplying 13C-labeled glycerol to either extraradical hyphae or colonized roots shows that there is indeed significant bidirectional translocation between IRM and ERM. We conclude that large amounts of lipid are translocated within the AM fungal colony and that, whereas net movement is from the IRM to the ERM, there is also substantial recirculation throughout the fungus.

A 13C-NMR study of exudation and storage of carbohydrates and amino acids in the ectomycorrhizal edible mushroom Cantharellus cibarius.

(13)C-NMR analyses of Cantharellus cibarius growth media were performed. We found exudation of trehalose and mannitol, which may explain the phenomenon of reproducing Pseudomonas bacteria observed inside fruit bodies. Exudation varied with strain and environment. NMR analyses of stored (13)C was also performed. Trehalose, mannitol, and arginine were revealed. The mannitol pathway seems to play an important role for trehalose production in this species. This is the first study of the fate of the photosynthetically derived carbon in the highly appreciated edible ectomycorrhizal mushroom Cantharellus cibarius.