Actin versus tubulin configuration in arbuscule-containing cells from mycorrhizal tobacco roots

The involvement of the cytoskeleton in symbiotic interactions such as arbuscular mycorrhizas has received little attention. In this paper, we examine the organization of actin in tobacco mycorrhizal roots and compare actin and tubulin patterns within arbuscule-containing cells.
Our results show drastic reorganization of microfilaments and microtubules upon fungal infection and how those new cytoskeletal patterns relate to the host cytoplasm rearrangement and the intracellular fungal structures. Whereas in uninfected cells a network of cortical and perinuclear actin filaments was observed, in infected cells actin filaments closely follow the fungal branches and envelop the whole arbuscule in a dense coating network. Microtubules are less closely connected with the fungus surface. They run across the whole arbuscule mass, linking branches to each other and to the host cell cortex and nucleus.
These major differences between the two cytoskeletal components are used to advance some suggestions concerning their contribution to structural functions in the plant–fungus interactions during the mycorrhizal symbiosis.     

Building a mycorrhizal cell: How to reach compatibility between plants and arbuscular mycorrhizal fungi

Abstract

Arbuscular mycorrhizal fungi occur throughout the majority of ecosystems supporting host plant nutrition. Recent findings describe the accommodation of the fungus by the root cell as a crucial step for compatibility between the partners. We discuss here the novel aspects of cellular plant-fungus interactions, with a particular attention to the interface compartment, the unique apoplastic space hosting intracellular fungal structures. The main features of arbuscular mycorrhizal colonization are examined and recent information in the field of plant and fungal cell responses during the establishment of the symbiosis is discussed. Differences between the colonization of root epidermal and cortical tissues are discussed, highlighting the growing interest in the role of epidermal cells during the first and decisive steps of the symbiosis. New approaches such as root organ cultures, in vivo observations, GFP tagging and mutant plant analysis are commented on and information from these is compared with that gained from more traditional methods. In particular, the use of plant mutants is depicted as a powerful tool for dissecting and understanding the genetic and cellular aspects of plant/fungus compatibility. Finally, perspectives in this field are outlined through the application of these approaches to the currently unanswered questions.     

A morphological study of the mycorrhiza ofEntoloma clypeatum f.hybridum onRosa multiflora

Mycorrhizas ofEntoloma clypeatum f.hybridum onRosa multiflora in the field in Japan were studied by stereo, light and electron microscopy. In most mycorrhizas, the root cap, meristem, and apical region of the cortex disappeared, but in a few mycorrhizas, these tissues remained. Fungal hyphae of the mycorrhizas invaded root tissues and branched palmately. Hyphae in contact with cortical cells were larger than those far from the root cells and contained many mitochondria, cisternae of endoplasmic reticulum and transitional vesicles. Invading hyphae were undulate in the apical part of the mycorrhiza, and some of them lacked distinct organelles. Electron-dense granules accumulated in the root cells adjacent to the fungal hyphae. Both the remnants of the plant cells and the fungal hyphae were included in the amorphous materials on the tip of the stele. These observations suggest the destructive infection by fungal hyphae of the root cells and their collapse near the tip of the stele.     

Identification and expression analysis of two fungal cDNAs regulated by ectomycorrhiza and fruit body formation

Ectomycorrhiza formation is a complex developmental process that is still not well understood. To study this process, we identified genetic markers for mycorrhiza development by differential screening of a cDNA library obtained from fully developed Picea abies – Amanita muscaria mycorrhizas. Twenty-three cDNA clones were identified that showed significantly altered gene expression during the ectomycorrhizal interaction. A detailed analysis was performed for two fungal cDNA clones, SC13 and SC25, exhibiting the most pronounced differences. SC13 encodes a protein of 184 amino acid residues that shows no homology with any sequence in databases. It was highly expressed in non-mycorrhizal hyphae, whereas its expression was decreased at least 50-fold in mycorrhizas and fruit bodies. SC25 encodes a protein of 198 residues that shows weak sequence homology with extensin-like plant proteins. The expression of this gene was weak in non-mycorrhizal hyphae but approx. 30-fold higher in mycorrhizas and fruit bodies. Because the expression of both developmentally regulated fungal genes was identical for mycorrhizas and fruit bodies, a common regulation mechanism for both developmental processes is proposed.     

Phospholipase A2 up-regulation during mycorrhiza formation in Tuber borchii

TbSP1 is a secreted and surface-associated phospholipase A(2) previously found to be up-regulated in C- or N-deprived free-living mycelia from the ectomycorrhizal ascomycete Tuber borchii. As nutrient limitation is considered an important environmental factor favouring the transition to symbiotic status, TbSP1 was suggested to be involved in the formation of mycorrhizas. An in vitro symbiosis system between Cistus incanus and T. borchii was set up: TbSP1 mRNA levels in free-living mycelia and in mycorrhizas sampled in different districts of the plant-fungus interaction were examined. In the same samples, TbSP1 protein expression was analysed by immunoelectron microscopy. A substantially enhanced TbSP1 mRNA expression, compared with nutrient-limited but free-living mycelia, was detected in the presence of the plant and reached maximal levels in fully developed mycorrhizas. A similar expression trend was revealed by immunolocalization experiments. We have shown that TbSP1 appears to respond to two partially overlapping yet distinct stimuli: nutrient starvation and mycorrhiza formation.     

The auxin-inducible GH3 homologue Pp-GH3.16 is downregulated in Pinus pinaster root systems on ectomycorrhizal symbiosis establishment

In an attempt to determine whether auxin-regulated plant genes play a role in ectomycorrhizal symbiosis establishment, we screened a Pinus pinaster root cDNA library for auxin-upregulated genes. This allowed the identification of a cDNA, Pp-GH3.16, which encodes a polypeptide sharing extensive homologies with GH3 proteins of different plants. Pp-GH3.16 was specifically upregulated by auxins and was not affected by cytokinin, gibberellin, abscisic acid or ethylene, or by heat shock, water stress or anoxia. Pp-GH3.16 mRNAs were quantified in pine roots inoculated with two ectomycorrhizal fungi, Hebeloma cylindrosporum and Rhizopogon roseolus. Surprisingly, Pp-GH3.16 was downregulated following inoculation with both fungal species. The downregulation was most rapid on establishment of symbiosis with an indole-3-acetic acid (IAA)-overproducing mutant of H. cylindrosporum, which overproduced mycorrhizas characterized by a hypertrophic Hartig net. This indicates that, despite being auxin-inducible, Pp-GH3.16 can be downregulated on establishment of symbiosis with a fungus that releases auxin. By contrast, Pp-GH3.16 was not downregulated in pine root systems inoculated with a nonmycorrhizal mutant of H. cylindrosporum, suggesting that the downregulation we observed in mycorrhizal root systems was a component of the molecular cross-talk between symbiotic partners at the origin of differentiation of symbiotic structures.     

Fungal and plant gene expression in arbuscular mycorrhizal symbiosis

Arbuscular mycorrhizas (AMs) are a unique example of symbiosis between two eukaryotes, soil fungi and plants. This association induces important physiological changes in each partner that lead to reciprocal benefits, mainly in nutrient supply. The symbiosis results from modifications in plant and fungal cell organization caused by specific changes in gene expression. Recently, much effort has gone into studying these gene expression patterns to identify a wider spectrum of genes involved. We aim in this review to describe AM symbiosis in terms of current knowledge on plant and fungal gene expression profiles.An erratum to this article can be found at     

An intact microtubule cytoskeleton is not necessary for interfacial matrix formation in orchid protocorm mycorrhizas

 This paper reports the changes that occur in the microtubule cytoskeleton of cells of orchid protocorms during infection by a compatible mycorrhizal fungus. In cells of protocorms uninfected by a mycorrhizal fungus, microtubules occurred in regular arrays. In contrast, the cells of orchid protocorms with established mycorrhizas appeared to contain irregularly arranged microtubules. Double labelling with anti-β-tubulin and rhodamine-labelled wheat-germ agglutinin demonstrated that these irregularly arranged microtubules occurred only inside fungal hyphae and that microtubules were absent from host cells containing mycorrhizal fungi. Microtubule depolymerisation was shown to occur at the early stages of fungal infection. There was neither loss of nor obvious organisational change in microtubules in cells adjacent to others containing fungal hyphae. Electron microscopy confirmed the presence of an interfacial matrix between the host plasma membrane and the hyphal wall. The loss of microtubules from cells infected by mycorrhizal fungi suggests that an intact host microtubule cytoskeleton is not necessary for the formation of the interfacial matrix in mycorrhizas of orchid protocorms. Accepted: 9 November 1995     

An economic approach to evaluate the role of mycorrhizas in managed ecosystems

The benefits of management of mycorrhizas in agricultural and horticultural croppiing systems remains problematic except where the indigenous fungal population is low. Most experiments have focused on the introduction of exotic fungal isolates. Promotion of plant growth by mycorrhizas can be enhanced by increasing the effectiveness of the indigenous fungi as well as by introducing more effective species. Lack of reliable methods for identification of fungal species colonizing roots is a major limitation to characterizing the change in mycorrhizal populations. Assessment of the role of mycorrhizas in commerical food production systems must include an economic analysis. To do so requires an evaluation of the response to increasing the effectiveness of the mycorrhizal symbiosis relative to increasing yield with addition of phosphorus fertilizer. Thus field experiments should be designed to measure the response to phosphorus addition with the existing mycorrhizal population as well as with the more effectively managed population. In this paper we discuss changes that may be induced in mycorrhizal fungi by management to increase their effectiveness in promoting plant growth. We then suggest an economic analysis approach to assessing the potential benefits of this increase in effectiveness. We conclude with a discussion of research approaches needed to determine, in a more objective manner, the role of mycorrhizas in managed ecosystems.     

Fungal root colonization responses in natural grasslands after long-term exposure to elevated atmospheric CO2

Arbuscular mycorrhizae, ubiquitous mutualistic symbioses between plant roots and fungi in the order Glomales, are believed to be important controllers of plant responses to global change, in particular to elevated atmospheric CO₂. In order to test if any effects on the symbiosis can persist after long-term treatment, we examined root colonization by arbuscular mycorrhizal (AM) and other fungi of several plant species from two grassland communities after continuous exposure to elevated atmospheric CO₂ for six growing seasons in the field. For plant species from both a sandstone and a serpentine annual grassland there was evidence for changes in fungal root colonization, with changes occurring as a function of plant host species. We documented decreases in percentage nonmycorrhizal fungal root colonization in elevated CO₂ for several plant species. Total AM root colonization (%) only increased significantly for one out of the five plant species in each grassland. However, when dividing AM fungal hyphae into two groups of hyphae (fine endophyte and coarse endophyte), we could document significant responses of AM fungi that were hidden when only total percentage colonization was measured. We also documented changes in elevated CO₂ in the percentage of root colonized by both AM hyphal types simultaneously. Our results demonstrate that changes in fungal root colonization can occur after long-term CO₂ enrichment, and that the level of resolution of the study of AM fungal responses may have to be increased to uncover significant changes to the CO₂ treatment. This study is also one of the first to document compositional changes in the AM fungi colonizing roots of plants grown in elevated CO₂. Although it is difficult to relate the structural data directly to functional changes, possible implications of the observed changes for plant communities are discussed.     

Meeting a non-host: the behaviour of AM fungi

 Arbuscular mycorrhizal (AM) fungi are obligately biotrophic organisms that live symbiotically with the roots of most plants. The establishment of a functional symbiosis between AM fungi and host plants involves a sequence of recognition events leading to the morphological and physiological integration of the two symbionts. The developmental switches in the fungi are triggered by host signals which induce changes in gene expression and a process leading to unequivocal recognition between the two partners of the symbiosis. It has been calculated that about 80% of plant families from all phyla of land plants are hosts of AM fungi. The remaining plant species are either non-mycorrhizal or hosts of mycorrhizas other than the arbuscular type. Non-host plants have been used to obtain information on the factors regulating the development of a functional symbiosis. The aim of this present review is to highlight present-day knowledge of the fungal developmental switches involved in the process of host/non-host discrimination. The following stages of the life cycle of AM fungi are analysed in detail: spore germination, presymbiotic mycelial growth, differential branching pattern and chemotropism, appressorium formation, root colonization. Accepted: 17 June 1998     

Expression profiling of up-regulated plant and fungal genes in early and late stages of<Emphasis Type="Italic"> Medicago truncatula-Glomus mosseae</Emphasis> interactions

Suppression subtractive hybridization (SSH), expression profiling and EST sequencing identified 12 plant genes and six fungal genes that are expressed in the arbuscular mycorrhizal symbiosis between Medicago truncatula and Glomus mosseae. All the plant genes and three of the fungal genes were up-regulated in symbiotic tissues. Expression of 15 of the genes is described for the first time in mycorrhizal roots and two are novel sequences. Six M. truncatula genes were also activated during appressorium formation at the root surface, suggesting a role in this early stage of mycorrhiza establishment, whilst the other six plant genes were only induced in the late stages of mycorrhization and could be involved in the development or functioning of the symbiosis. Phosphate fertilization had no significant influence on expression of any of the plant genes. Expression profiling of G. mosseae genes indicated that two of them may be associated with appressorium development on roots and one with arbuscule formation or function. The other three fungal genes were expressed throughout the life-cycle of G. mosseae.     

Arbuscular Mycorrhizal Symbiosis Changes the Colonization Pattern of Acacia tortilis spp. Raddiana Rhizosphere by Two Strains of Rhizobia

The aim of the study was to assess the effect of the mycorrhizosphere of A. tortillis spp. raddiana mycorrhized with Glomus intraradices on the root nodulation by Sinorhizobium terangae (ORS 1009) and/or Mesorhizobium plurifarium (ORS 1096) in two different culture substrates (sandy soil and sand). The endomycorrhizal fungus only stimulated plant growth in the sandy soil. Moreover, arbuscular mycorrhizal infection enhanced the nodulation process in both culture substrates. Beside the stimulatory effects of the mycorrhizosphere on both rhizobia development, fungal symbiosis induces two different dynamics of each bacterial strains in the sand-grown plants. These results suggest specific relationships could occur during the development of the tripartite symbiosis, at physiological and molecular level. From a practical point of view, the role of arbuscular mycorrhizas in improving nodulation and N2 fixation is universally recognized. The fungal symbiosis could modify the development of bacterial inoculants along the root systems. This effect is of particular interest in the controlled inoculation of selected rhizobia.     

Apoplastic plant subtilases support arbuscular mycorrhiza development in Lotus japonicus

In the arbuscular mycorrhiza (AM) symbiosis, plant roots accommodate Glomeromycota fungi within an intracellular compartment, the arbuscule. At this symbiotic interface, fungal hyphae are surrounded by a plant membrane, which creates an apoplastic compartment, the periarbuscular space (PAS) between fungal and plant cell. Despite the importance of the PAS for symbiotic signal and metabolite exchange, only few of its components have been identified. Here we show that two apoplastic plant proteases of the subtilase family are required for AM development. SbtM1 is the founder member of a family of arbuscular mycorrhiza-induced subtilase genes that occur in at least two clusters in the genome of the legume Lotus japonicus . A detailed expression analysis by RT-PCR revealed that SbtM1 , SbtM3 , SbtM4 and the more distantly related SbtS are all rapidly induced during development of arbuscular mycorrhiza, but only SbtS and SbtM4 are also up-regulated during root nodule symbiosis. Promoter–reporter fusions indicated specific activation in cells that are adjacent to intra-radical fungal hyphae or in cells that harbour them. Venus fluorescent protein was observed in the apoplast and the PAS when expressed from a fusion construct with the SbtM1 signal peptide or the full-length subtilase. Suppression of SbtM1 or SbtM3 by RNAi caused a decrease in intra-radical hyphae and arbuscule colonization, but had no effect on nodule formation. Our data indicate a role for these subtilases during the fungal infection process in particular arbuscule development.     

The exudate from an arbuscular mycorrhizal fungus induces nitric oxide accumulation in Medicago truncatula roots

Nitric oxide (NO) is a signaling molecule involved in plant responses to abiotic and biotic stresses. While there is evidence for NO accumulation during legume nodulation, almost no information exists for arbuscular mycorrhizas (AM). Here, we investigated the occurrence of NO in the early stages of Medicago truncatula–Gigaspora margarita interaction, focusing on the plant response to fungal diffusible molecules. NO was visualized in root organ cultures and seedlings by confocal microscopy using the specific probe 4,5-diaminofluorescein diacetate. Five-minute treatment with the fungal exudate was sufficient to induce significant NO accumulation. The specificity of this response to AM fungi was confirmed by the lack of response in the AM nonhost Arabidopsis thaliana and by analyzing mutants impaired in mycorrhizal capacities. NO buildup resulted to be partially dependent on DMI1, DMI2, and DMI3 functions within the so-called common symbiotic signaling pathway which is shared between AM and nodulation. Significantly, NO accumulation was not induced by the application of purified Nod factor, while lipopolysaccharides from Escherichia coli, known to elicit defense-related NO production in plants, induced a significantly different response pattern. A slight upregulation of a nitrate reductase (NR) gene and the reduction of NO accumulation when the enzyme is inhibited by tungstate suggest NR as a possible source of NO. Genetic and cellular evidence, therefore, suggests that NO accumulation is a novel component in the signaling pathway that leads to AM symbiosis.     

全球变化下菌根真菌的作用及其作用机制

全球气候、环境、经济与社会的发展变化,对环境与资源造成严重挑战和新的发展机遇。菌根真菌是陆地生态系统中的重要生物组份,占据不可替代的重要地位,充当调控生态系统稳定和保持可持续发展的多重角色。分析了全球变化对菌根真菌的影响,探讨了全球变化下菌根真菌的地位、角色和作用,以及菌根真菌应对全球变化的可能作用机制,旨在为加强全面应对全球变化提供新的思路和途径。