A diffusible signal from arbuscular mycorrhizal fungi elicits a transient cytosolic calcium elevation in host plant cells

The implication of calcium as intracellular messenger in the arbuscular mycorrhizal (AM) symbiosis has not yet been directly demonstrated, although often envisaged. We used soybean (Glycine max) cell cultures stably expressing the bioluminescent Ca(2+) indicator aequorin to detect intracellular Ca(2+) changes in response to the culture medium of spores of Gigaspora margarita germinating in the absence of the plant partner. Rapid and transient elevations in cytosolic free Ca(2+) were recorded, indicating that diffusible molecules released by the mycorrhizal fungus are perceived by host plant cells through a Ca(2+)-mediated signaling. Similar responses were also triggered by two Glomus isolates. The fungal molecules active in generating the Ca(2+) transient were constitutively released in the medium, and the induced Ca(2+) signature was not modified by the coculture of germinating spores with plant cells. Even ungerminated spores were able to generate the signaling molecules, as proven when the germination was blocked by a low temperature. The fungal molecules were found to be stable to heat treatment, of small molecular mass (<3 kD), and, on the basis of extraction with an organic solvent, partially lipophilic. Evidence for the specificity of such an early fungal signal to the AM symbiosis is suggested by the lack of a Ca(2+) response in cultured cells of the nonhost plant Arabidopsis (Arabidopsis thaliana) and by the up-regulation in soybean cells of genes related to Medicago truncatula DMI1, DMI2, and DMI3 and considered essential for the establishment of the AM symbiosis.     

A switch in Ca2+ spiking signature is concomitant with endosymbiotic microbe entry into cortical root cells of Medicago truncatula

Ca(2+) spiking is a central component of a common signaling pathway that is activated in the host epidermis during initial recognition of endosymbiotic microbes. However, it is not known to what extent Ca(2+) signaling also plays a role during subsequent root colonization involving apoplastic transcellular infection. Live-tissue imaging using calcium cameleon reporters expressed in Medicago truncatula roots has revealed that distinct Ca(2+) oscillatory profiles correlate with specific stages of transcellular cortical infection by both rhizobia and arbuscular mycorrhizal fungi. Outer cortical cells exhibit low-frequency Ca(2+) spiking during the extensive intracellular remodeling that precedes infection. This appears to be a prerequisite for the formation of either pre-infection threads or the pre-penetration apparatus, both of which are fully reversible processes. A transition from low- to high-frequency spiking is concomitant with the initial stages of apoplastic cell entry by both microbes. This high-frequency spiking is of limited duration in the case of rhizobial infection and is completely switched off by the time transcellular infection by both microsymbionts is completed. The Ca(2+) spiking profiles associated with both rhizobial and arbuscular mycorrhizal cell entry are remarkably similar in terms of periodicity, suggesting that microbe specificity is unlikely to be encoded by the Ca(2+) signature during this particular stage of host infection in the outer cortex. Together, these findings lead to the proposal that tightly regulated Ca(2+) -mediated signal transduction is a key player in reprogramming root cell development at the critical stage of commitment to endosymbiotic infection.     

Analysis of calcium spiking using a cameleon calcium sensor reveals that nodulation gene expression is regulated by calcium spike number and the developmental status of the cell

Rhizobium-made Nod factors induce rapid changes in both Ca(2+) and gene expression. Mutations and inhibitors that abolish Nod-factor-induced Ca(2+) spiking block gene induction, indicating a specific role for Ca(2+) spiking in signal transduction. We used transgenic Medicago truncatula expressing a "cameleon" Ca(2+) sensor to assess the relationship between Nod-factor-induced Ca(2+) spiking and the activation of downstream gene expression. In contrast to ENOD11 induction, Ca(2+) spiking is activated in all root-hair cells and in epidermal or pre-emergent root hairs cells in the root tip region. Furthermore, cortical cells immediately below the epidermal layer also show slow Ca(2+) spiking and these cells lack Nod-factor-induced ENOD11 expression. This indicates a specialization in nodulation gene induction downstream of Nod-factor perception and signal transduction. There was a gradient in the frequency of Ca(2+) spiking along the root, with younger root-hair cells having a longer period between spikes than older root hairs. Using a Ca(2+)-pump inhibitor to block Ca(2+) spiking at various times after addition of Nod factor, we conclude that about 36 consecutive Ca(2+) spikes are sufficient to induce ENOD11-GUS expression in root hairs. To determine if the length of time of Ca(2+) spiking or the number of Ca(2+) spikes is more critical for Nod-factor-induced ENOD11 expression, jasmonic acid (JA) was added to reduce the rate of Nod-factor-induced Ca(2+) spiking. This revealed that even when the period between Ca(2+) spikes was extended, an equivalent number of Ca(2+) spikes were required for the induction of ENOD11. However, this JA treatment did not affect the spatial patterning of ENOD11-GUS expression suggesting that although a minimal number of Ca(2+) spikes are required for Nod-factor-induced gene expression, other factors restrict the expression of ENOD11 to a subset of responding cells.     

Root factors induce mitochondrial-related gene expression and fungal respiration during the developmental switch from asymbiosis to presymbiosis in the arbuscular mycorrhizal fungus Gigaspora rosea

During spore germination, arbuscular mycorrhizal (AM) fungi show limited hyphal development in the absence of a host plant (asymbiotic). In the presence of root exudates, they switch to a new developmental stage (presymbiotic) characterized by extensive hyphal branching. Presymbiotic branching of the AM fungus Gigaspora rosea was induced in liquid medium by a semipurified exudate fraction from carrot (Daucus carota) root organ cultures. Changes in RNA accumulation patterns were monitored by differential display analysis. Differentially appearing cDNA fragments were cloned and further analyzed. Five cDNA fragments could be identified that show induced RNA accumulation 1 h after the addition of root exudate. Sequence similarities of two fragments to mammalian Nco4 and mitochondrial rRNA genes suggested that root exudates could influence fungal respiratory activity. To support this hypothesis, additional putative mitochondrial related-genes were shown to be induced by root exudates. These genes were identified after subtractive hybridization and putatively encode a pyruvate carboxylase and a mitochondrial ADP/ATP translocase. The gene GrosPyc1 for the pyruvate carboxylase was studied in more detail by cloning a cDNA and by quantifying its RNA accumulation. The hypothesis that respiratory activity of AM fungi is stimulated by root exudates was confirmed by physiological and cytological analyses in G. rosea and Glomus intraradices. Oxygen consumption and reducing activity of both fungi was induced after 3 and 2 h of exposition with the root factor, respectively, and the first respiration activation was detected in G. intraradices after approximately 90 min. In addition, changes in mitochondrial morphology, orientation, and overall biomass were detected in G. rosea after 4 h. In summary, the root-exuded factor rapidly induces the expression of certain fungal genes and, in turn, fungal respiratory activity before intense branching. This defines the developmental switch from asymbiosis to presymbiosis, first by gene activation (0.5-1 h), subsequently on the physiological level (1.5-3 h), and finally as a morphological response (after 5 h).     

Increasing flavonoid concentrations in root exudates enhance associations between arbuscular mycorrhizal fungi and an invasive plant

Many invasive plants have enhanced mutualistic arbuscular mycorrhizal (AM) fungal associations, however, mechanisms underlying differences in AM fungal associations between introduced and native populations of invasive plants have not been explored. Here we test the hypothesis that variation in root exudate chemicals in invasive populations affects AM fungal colonization and then impacts plant performance. We examined flavonoids (quercetin and quercitrin) in root exudates of native and introduced populations of the invasive plant Triadica sebifera and tested their effects on AM fungi and plant performance. We found that plants from introduced populations had higher concentrations of quercetin in root exudates, greater AM fungal colonization and higher biomass. Applying root exudates more strongly increased AM fungal colonization of target plants and AM fungal spore germination when exudate donors were from introduced populations. The role of root exudate chemicals was further confirmed by decreased AM fungal colonization when activated charcoal was added into soil. Moreover, addition of quercetin into soil increased AM fungal colonization, indicating quercetin might be a key chemical signal stimulating AM fungal associations. Together these results suggest genetic differences in root exudate flavonoids play an important role in enhancing AM fungal associations and invasive plants’ performance, thus considering root exudate chemicals is critical to unveiling mechanisms governing shifting plant-soil microbe interactions during plant invasions.Subject terms: Population dynamics, Community ecology, Plant ecology     

Pharmacological evidence that multiple phospholipid signaling pathways link Rhizobium nodulation factor perception in Medicago truncatula root hairs to intracellular responses, including Ca2+ spiking and specific ENOD gene expression

Rhizobium nodulation (Nod) factors are specific lipochito-oligosaccharide signals essential for initiating in root hairs of the host legume developmental responses that are required for controlled entry of the microsymbiont. In this article, we focus on the Nod factor signal transduction pathway leading to specific and cell autonomous gene activation in Medicago truncatula cv Jemalong in a study making use of the Nod factor-inducible MtENOD11 gene. First, we show that pharmacological antagonists that interfere with intracellular ion channel and Ca2+ pump activities are efficient blockers of Nod factor-elicited pMtENOD11-beta-glucuronidase (GUS) expression in root hairs of transgenic M. truncatula. These results indicate that intracellular Ca2+ release and recycling activities, essential for Ca2+ spiking, are also required for specific gene activation. Second, pharmacological effectors that inhibit phospholipase D and phosphoinositide-dependent phospholipase C activities are also able to block pMtENOD11-GUS activation, thus underlining a central role for multiple phospholipid signaling pathways in Nod factor signal transduction. Finally, pMtENOD11-GUS was introduced into all three Nod-/Myc- dmi M. truncatula mutant backgrounds, and gene expression was evaluated in response to the mastoparan peptide agonist Mas7. We found that Mas7 elicits root hair MtENOD11 expression in dmi1 and dmi2 mutants, but not in the dmi3 mutant, suggesting that the agonist acts downstream of DMI1/DMI2 and upstream of DMI3. In light of these results and the recently discovered identities of the DMI gene products, we propose an integrated cellular model for Nod factor signaling in legume root hairs in which phospholipids play a key role in linking the Nod factor perception apparatus to downstream components such as Ca2+ spiking and ENOD gene expression.     

Fungal symbiosis in rice requires an ortholog of a legume common symbiosis gene encoding a Ca2+/calmodulin-dependent protein kinase

In natural ecosystems, many plants are able to establish mutually beneficial symbioses with microorganisms. Of critical importance to sustainable agriculture are the symbioses formed between more than 80% of terrestrial plants and arbuscular mycorrhizal (AM) fungi and between legumes and nitrogen-fixing rhizobial bacteria. Interestingly, the two symbioses share overlapping signaling pathways in legumes, suggesting that the evolutionarily recent root nodule symbiosis may have acquired functions from the ancient AM symbiosis. The Medicago truncatula DMI3 (DOESN'T MAKE INFECTIONS3) gene (MtDMI3) and its orthologs in legumes are required for both bacterial and fungal symbioses. MtDMI3 encodes a Ca(2+)/calmodulin-dependent protein kinase (CCaMK) essential for the transduction of the Ca(2+) signal induced by the perception of Nod factors. Putative orthologs of MtDMI3 are also present in non-legumes, but their function in AM symbiosis has not been demonstrated in any non-legume species. Here, we combine reverse genetic approaches and a cross-species complementation test to characterize the function of the rice (Oryza sativa) ortholog of MtDMI3, namely, OsDMI3, in AM symbiosis. We demonstrate that OsDMI3 is not only required for AM symbiosis in rice but also is able to complement a M. truncatula dmi3 mutant, indicating an equivalent role of MtDMI3 orthologs in non-legumes.     

Prepenetration apparatus assembly precedes and predicts the colonization patterns of arbuscular mycorrhizal fungi within the root cortex of both Medicago truncatula and Daucus carota

Arbuscular mycorrhizas (AM) are widespread, ancient endosymbiotic associations that contribute significantly to soil nutrient uptake in plants. We have previously shown that initial fungal penetration of the host root is mediated via a specialized cytoplasmic assembly called the prepenetration apparatus (PPA), which directs AM hyphae through the epidermis (Genre et al., 2005). In vivo confocal microscopy studies performed on Medicago truncatula and Daucus carota, host plants with different patterns of AM colonization, now reveal that subsequent intracellular growth across the root outer cortex is also PPA dependent. On the other hand, inner root cortical colonization leading to arbuscule development involves more varied and complex PPA-related mechanisms. In particular, a striking alignment of polarized PPAs can be observed in adjacent inner cortical cells of D. carota, correlating with the intracellular root colonization strategy of this plant. Ultrastructural analysis of these PPA-containing cells reveals intense membrane trafficking coupled with nuclear enlargement and remodeling, typical features of arbusculated cells. Taken together, these findings imply that prepenetration responses are both conserved and modulated throughout the AM symbiosis as a function of the different stages of fungal accommodation and the host-specific pattern of root colonization. We propose a model for intracellular AM fungal accommodation integrating peri-arbuscular interface formation and the regulation of functional arbuscule development.     

Germinating spore exudates from arbuscular mycorrhizal fungi: molecular and developmental responses in plants and their regulation by ethylene

Arbuscular mycorrhizal (AM) fungi stimulate root development and induce expression of mycorrhization-specific genes in both eudicots and monocots. Diffusible factors released by AM fungi have been shown to elicit similar responses in Medicago truncatula. Colonization of roots by AM fungi is inhibited by ethylene. We compared the effects of germinating spore exudates (GSE) from Glomus intraradices in monocots and in eudicots, their genetic control, and their regulation by ethylene. GSE modify root architecture and induce symbiotic gene expression in both monocots and eudicots. The genetic regulation of root architecture and gene expression was analyzed using M. truncatula and rice symbiotic mutants. These responses are dependent on the common symbiotic pathway as well as another uncharacterized pathway. Significant differences between monocots and eudicots were observed in the genetic control of plant responses to GSE. However, ethylene inhibits GSE-induced symbiotic gene expression and root development in both groups. Our results indicate that GSE signaling shares similarities and differences in monocots versus eudicots, that only a subset of AM signaling pathways has been co-opted in legumes for the establishment of root nodulation with rhizobia, and that regulation of these pathways by ethylene is a feature conserved across higher land plants.     

A dominant function of CCaMK in intracellular accommodation of bacterial and fungal endosymbionts

In legumes, Ca²⁺/calmodulin‐dependent protein kinase (CCaMK) is a component of the common symbiosis genes that are required for both root nodule (RN) and arbuscular mycorrhiza (AM) symbioses and is thought to be a decoder of Ca²⁺ spiking, one of the earliest cellular responses to microbial signals. A gain‐of‐function mutation of CCaMK has been shown to induce spontaneous nodulation without rhizobia, but the significance of CCaMK activation in bacterial and/or fungal infection processes is not fully understood. Here we show that a gain‐of‐function CCaMK^T265D suppresses loss‐of‐function mutations of common symbiosis genes required for the generation of Ca²⁺ spiking, not only for nodule organogenesis but also for successful infection of rhizobia and AM fungi, demonstrating that the common symbiosis genes upstream of Ca²⁺ spiking are required solely to activate CCaMK. In RN symbiosis, however, CCaMK^T265D induced nodule organogenesis, but not rhizobial infection, on Nod factor receptor (NFRs) mutants. We propose a model of symbiotic signaling in host legume plants, in which CCaMK plays a key role in the coordinated induction of infection thread formation and nodule organogenesis.     

Lipo-chitooligosaccharide signaling in endosymbiotic plant-microbe interactions

The arbuscular mycorrhizal (AM) and the rhizobia-legume (RL) root endosymbioses are established as a result of signal exchange in which there is mutual recognition of diffusible signals produced by plant and microbial partners. It was discovered 20 years ago that the key symbiotic signals produced by rhizobial bacteria are lipo-chitooligosaccharides (LCO), called Nod factors. These LCO are perceived via lysin-motif (LysM) receptors and activate a signaling pathway called the common symbiotic pathway (CSP), which controls both the RL and the AM symbioses. Recent work has established that an AM fungus, Glomus intraradices, also produces LCO that activate the CSP, leading to induction of gene expression and root branching in Medicago truncatula. These Myc-LCO also stimulate mycorrhization in diverse plants. In addition, work on the nonlegume Parasponia andersonii has shown that a LysM receptor is required for both successful mycorrhization and nodulation. Together these studies show that structurally related signals and the LysM receptor family are key components of both nodulation and mycorrhization. LysM receptors are also involved in the perception of chitooligosaccharides (CO), which are derived from fungal cell walls and elicit defense responses and resistance to pathogens in diverse plants. The discovery of Myc-LCO and a LysM receptor required for the AM symbiosis, therefore, not only raises questions of how legume plants discriminate fungal and bacterial endosymbionts but also, more generally, of how plants discriminate endosymbionts from pathogenic microorganisms using structurally related LCO and CO signals and of how these perception mechanisms have evolved.     

Two different but converging messenger pathways to intracellular Ca(2+) release: the roles of nicotinic acid adenine dinucleotide phosphate, cyclic ADP-ribose and inositol trisphosphate

Hormones and neurotransmitters mobilize Ca(2+) from the endoplasmic reticulum via inositol trisphosphate (IP(3)) receptors, but how a single target cell encodes different extracellular signals to generate specific cytosolic Ca(2+) responses is unknown. In pancreatic acinar cells, acetylcholine evokes local Ca(2+) spiking in the apical granular pole, whereas cholecystokinin elicits a mixture of local and global cytosolic Ca(2+) signals. We show that IP(3), cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate (NAADP) evoke cytosolic Ca(2+) spiking by activating common oscillator units composed of IP(3) and ryanodine receptors. Acetylcholine activation of these common oscillator units is triggered via IP(3) receptors, whereas cholecystokinin responses are triggered via a different but converging pathway with NAADP and cyclic ADP-ribose receptors. Cholecystokinin potentiates the response to acetylcholine, making it global rather than local, an effect mediated specifically by cyclic ADP-ribose receptors. In the apical pole there is a common early activation site for Ca(2+) release, indicating that the three types of Ca(2+) release channels are clustered together and that the appropriate receptors are selected at the earliest step of signal generation.     

Calcium oscillations and Nod signal transduction in Rhizobium-legume symbiosis

Rhizobium nodulation (Nod) factors are lipo-chitooligosaccharides that act as symbiotic signals, eliciting a number of key developmental responses in the roots of legume hosts. One of the earliest responses of root hairs to Nod factors is the induction of sharp oscillations of cytoplasmic calcium ion concentration ("calcium spiking"). This response was first characterised in Medicago sativa and Nod factors were found to be unable to induce calcium spiking in a nodulation-defective mutant of M. sativa. The fact that this mutant lacked any morphological response to Nod factors raised the question of whether calcium spiking could be part of a Nod factor-induced signal transduction pathway leading to nodulation. More recently, calcium spiking has been described in a model legume, Medicago truncatula, and in pea. When nodulation-defective mutants were tested for the induction of calcium spiking in response to Nod factors, three loci of pea and two of M. truncatula were found to be necessary for Nod factor-induced calcium spiking. These loci are also known to be necessary for Nod factor-induction of symbiotic responses such as root hair deformation, nodulin gene expression and cortical cell division. These results therefore constitute strong genetic evidence for the role of calcium spiking in Nod factor transduction. This system provides an opportunity to use genetics to study ligand-stimulated calcium spiking as a signal transduction event.     

Absence of carbon transfer between Medicago truncatula plants linked by a mycorrhizal network, demonstrated in an experimental microcosm

Carbon transfer between plants via a common extraradical network of arbuscular mycorrhizal (AM) fungal hyphae has been investigated abundantly, but the results remain equivocal. We studied the transfer of carbon through this fungal network, from a Medicago truncatula donor plant to a receiver (1) M. truncatula plant growing under decreased light conditions and (2) M. truncatula seedling. Autotrophic plants were grown in bicompartmented Petri plates, with their root systems physically separated, but linked by the extraradical network of Glomus intraradices. A control Myc-/Nod- M. truncatula plant was inserted in the same compartment as the receiver plant. Following labeling of the donor plant with 13CO2, 13C was recovered in the donor plant shoots and roots, in the extraradical mycelium and in the receiver plant roots. Fatty acid analysis of the receiver's roots further demonstrated 13C enrichment in the fungal-specific lipids, while almost no label was detected in the plant-specific compounds. We conclude that carbon was transferred from the donor to the receiver plant via the AM fungal network, but that the transferred carbon remained within the intraradical AM fungal structures of the receiver's root and was not transferred to the receiver's plant tissues.     

Seven years of carbon dioxide enrichment, nitrogen fertilization and plant diversity influence arbuscular mycorrhizal fungi in a grassland ecosystem

? We tested the prediction that the abundance and diversity of arbuscular mycorrhizal (AM) fungi are influenced by resource availability and plant community composition by examining the joint effects of carbon dioxide (CO(2) ) enrichment, nitrogen (N) fertilization and plant diversity on AM fungi. ? We quantified AM fungal spores and extramatrical hyphae in 176 plots after 7 yr of treatment with all combinations of ambient or elevated CO(2) (368 or 560 ppm), with or without N fertilization (0 or 4 g Nm(-2) ), and one (monoculture) or 16 host plant species (polyculture) in the BioCON field experiment at Cedar Creek Ecosystem Science Reserve, Minnesota, USA. ? Extramatrical hyphal lengths were increased by CO(2) enrichment, whereas AM spore abundance decreased with N fertilization. Spore abundance, morphotype richness and extramatrical hyphal lengths were all greater in monoculture plots. A structural equation model showed AM fungal biovolume was most influenced by CO(2) enrichment, plant community composition and plant richness, whereas spore richness was most influenced by fungal biovolume, plant community composition and plant richness. ? Arbuscular mycorrhizal fungi responded to differences in host community and resource availability, suggesting that mycorrhizal functions, such as carbon sequestration and soil stability, will be affected by global change.     

A diffusible factor from arbuscular mycorrhizal fungi induces symbiosis-specific MtENOD11 expression in roots of Medicago truncatula

Using dual cultures of arbuscular mycorrhizal (AM) fungi and Medicago truncatula separated by a physical barrier, we demonstrate that hyphae from germinating spores produce a diffusible factor that is perceived by roots in the absence of direct physical contact. This AM factor elicits expression of the Nod factor-inducible gene MtENOD11, visualized using a pMtENOD11-gusA reporter. Transgene induction occurs primarily in the root cortex, with expression stretching from the zone of root hair emergence to the region of mature root hairs. All AM fungi tested (Gigaspora rosea, Gigaspora gigantea, Gigaspora margarita, and Glomus intraradices) elicit a similar response, whereas pathogenic fungi such as Phythophthora medicaginis, Phoma medicaginis var pinodella and Fusarium solani f.sp. phaseoli do not, suggesting that the observed root response is specific to AM fungi. Finally, pMtENOD11-gusA induction in response to the diffusible AM fungal factor is also observed with all three M. truncatula Nod(-)/Myc(-) mutants (dmi1, dmi2, and dmi3), whereas the same mutants are blocked in their response to Nod factor. This positive response of the Nod(-)/Myc(-) mutants to the diffusible AM fungal factor and the different cellular localization of pMtENOD11-gusA expression in response to Nod factor versus AM factor suggest that signal transduction occurs via different pathways and that expression of MtENOD11 is differently regulated by the two diffusible factors.     

Arbuscular mycorrhizal dynamics in a chronosequence of Caragana korshinskii plantations

Arbuscular mycorrhizal (AM) fungi in a chronosequence of 5–42-year-old Caragana korshinskii plantations in the semi-arid Loess Plateau region of northwestern China were investigated. AM fungi colonization, spore diversity and PCR-denatured gradient gel electrophoresis-based AM fungal SSU rRNA gene sequences were analyzed. AM fungi colonization [measured as the percent of root length (%RLC), vesicular (%VC) and arbuscular (%AC) colonization] and spore density were significantly correlated with sampling month, but not with plant age, except for %RLC. The percent of vesicular colonization was negatively correlated with soil total nitrogen and organic carbon, and spore density was negatively correlated with soil moisture and available phosphorus. Ten distinguishable AM fungal spore morphotypes, nine Glomus and one Scutellospora species, were found. Nine AM fungal Glomus phylotypes were identified by sequencing, but at each sampling time only four to six AM fungal phylotypes were detected. The AM fungal community was significantly seasonal, whereas the AM fungal species richness did not increase with plantation age. A significant change in AM fungal colonization and community composition over an annual cycle was observed in this study, and our results suggest that the changes of AM are the product of the interaction between host phenology, soil characteristics and habitat. Understanding these interactions is essential if habitat restoration is to be effective.