Heike Bücking

Reciprocal Rewards Stabilize Cooperation in the Mycorrhizal Symbiosis

Plants and their associated fungi reward partners that offer the best resources to sustain mutualism in complex systems. Plants and their arbuscular mycorrhizal fungal symbionts interact in complex underground networks involving multiple partners. This increases the potential for exploitation and defection by individuals, raising the question of how partners maintain a fair, two-way transfer of resources. We manipulated cooperation in plants and fungal partners to show that plants can detect, discriminate, and reward the best fungal partners with more carbohydrates. In turn, their fungal partners enforce cooperation by increasing nutrient transfer only to those roots providing more carbohydrates. On the basis of these observations we conclude that, unlike many other mutualisms, the symbiont cannot be “enslaved.” Rather, the mutualism is evolutionarily stable because control is bidirectional, and partners offering the best rate of exchange are rewarded.

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.

Regulation of the Nitrogen Transfer Pathway in the Arbuscular Mycorrhizal Symbiosis: Gene Characterization and the Coordination of Expression with Nitrogen Flux

The arbuscular mycorrhiza (AM) brings together the roots of over 80% of land plant species and fungi of the phylum Glomeromycota and greatly benefits plants through improved uptake of mineral nutrients. AM fungi can take up both nitrate and ammonium from the soil and transfer nitrogen (N) to host roots in nutritionally substantial quantities. The current model of N handling in the AM symbiosis includes the synthesis of arginine in the extraradical mycelium and the transfer of arginine to the intraradical mycelium, where it is broken down to release N for transfer to the host plant. To understand the mechanisms and regulation of N transfer from the fungus to the plant, 11 fungal genes putatively involved in the pathway were identified from Glomus intraradices, and for six of them the full-length coding sequence was functionally characterized by yeast complementation. Two glutamine synthetase isoforms were found to have different substrate affinities and expression patterns, suggesting different roles in N assimilation. The spatial and temporal expression of plant and fungal N metabolism genes were followed after nitrate was added to the extraradical mycelium under N-limited growth conditions using hairy root cultures. In parallel experiments with 15N, the levels and labeling of free amino acids were measured to follow transport and metabolism. The gene expression pattern and profiling of metabolites involved in the N pathway support the idea that the rapid uptake, translocation, and transfer of N by the fungus successively trigger metabolic gene expression responses in the extraradical mycelium, intraradical mycelium, and host plant.

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.

Hiding in a crowd—does diversity facilitate persistence of a low-quality fungal partner in the mycorrhizal symbiosis?

Given that arbuscular mycorrhizal (AM) fungi are not consistently beneficial to their host plants, it is difficult to explain the evolutionary persistence of this relationship. We tested the hypothesis that increasing either fungal or host biodiversity allows an AM fungus to persist on a host where it shows little benefit. We found that growing such a fungus (an isolate of Glomus custos associating with Plantago laceolata) in combination with certain fungi improved its success as measured by mtLSU DNA abundance. Increasing plant species richness facilitated the spread of this fungus as measured by spore density and fungal colonization; the role of host species richness was not as clear when looking at measures of root abundance. These results indicate that diversity in the AM symbiosis, both plant and fungal, can promote the persistence of low-quality fungi. By existing within a complex mycelial network fungal strains that show little growth benefit to their hosts have a better chance of persisting on that same host. This has the potential to promote selection for heterogeneous AM fungal communities on a small spatial scale.

Triacylglyceride Metabolism by Fusarium graminearum During Colonization and Sexual Development on Wheat

Fusarium graminearum, a devastating pathogen of small grains, overwinters on crop residues and produces ephemeral perithecia. Accumulation of lipids in overwintering hyphae would provide reserves for overwinter survival and perithecium development. Fatty acid composition of cultures during perithecium development indicated a drop in neutral lipid levels during development but little change in fatty acid composition across stages. Microscopic examination of cultures early in sexual development revealed hyphal cells engorged with lipid bodies. In comparison, vegetative hyphae contained few lipid bodies. Microarray analysis was performed on wheat stems at stages of colonization through perithecium development. Gene expression analysis during stages of perithecium development both in planta and in vitro (previously published) supports the view that lipid biosynthesis occurs during early stages of wheat colonization leading to sexual development and that lipid oxidation occurs as perithecia are developing. Analysis of gene expression during the stages of wheat stem colonization also revealed sets of genes unique to these stages. These results support the view that lipids accumulate in hyphae colonizing wheat stalks and are subsequently used in perithecium formation on stalk tissue. These results indicate that extensive colonization of plant tissue prior to harvest is essential for subsequent sporulation on crop residues and, thus, has important implications for inoculum reduction.

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.

Do fungivores trigger the transfer of protective metabolites from host plants to arbuscular mycorrhizal hyphae

A key objective in ecology is to understand how cooperative strategies evolve and are maintained in species networks. Here, we focus on the tri-trophic relationship between arbuscular mycorrhizal (AM) fungi, host plants, and fungivores to ask if host plants are able to protect their mutualistic mycorrhizal partners from being grazed. Specifically, we test whether secondary metabolites are transferred from hosts to fungal partners to increase their defense against fungivores. We grew Plantago lanceolata hosts with and without mycorrhizal inoculum, and in the presence or absence of fungivorous springtails. We then measured fungivore effects on host biomass and mycorrhizal abundance (using quantitative PCR) in roots and soil. We used high-performance liquid chromatography to measure host metabolites in roots, shoots, and hyphae, focusing on catalpol, aucubin, and verbascoside. Our most striking result was that the metabolite catalpol was consistently found in AM fungal hyphae in host plants exposed to fungivores. When fungivores were absent, catalpol was undetectable in hyphae. Our results highlight the potential for plant-mediated protection of the mycorrhizal hyphal network.

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.

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.