Glomeromycota communities survive extreme levels of metal toxicity in an orphan mining site.

Abandoned tailing basins and waste heaps of orphan mining sites are of great concern since extreme metal contamination makes soil improper for any human activity and is a permanent threat for nearby surroundings. Although spontaneous revegetation can occur, the process is slow or unsuccessful and rhizostabilisation strategies to reduce dispersal of contaminated dust represent an option to rehabilitate such sites. This requires selection of plants tolerant to such conditions, and optimization of their fitness and growth. Arbuscular mycorrhizal fungi (AMF) can enhance metal tolerance in moderately polluted soils, but their ability to survive extreme levels of metal contamination has not been reported. This question was addressed in the tailing basin and nearby waste heaps of an orphan mining site in southern France, reaching in the tailing basin exceptionally high contents of zinc (ppm: 97,333 total) and lead (ppm: 31,333 total). In order to contribute to a better understanding of AMF ecology under severe abiotic stress and to identify AMF associated with plants growing under such conditions, that may be considered in future revegetation and rhizostabilisation of highly polluted areas, nine plant species were sampled at different growing seasons and AMF root colonization was determined. Glomeromycota diversity was monitored in mycorrhizal roots by sequencing of the ribosomal LSU. This first survey of AMF in such highly contaminated soils revealed the presence of several AMF ribotypes, belonging mainly to the Glomerales, with some examples from the Paraglomerales and Diversisporales. AMF diversity and root colonization in the tailing basin were lower than in the less-contaminated waste heaps. A Paraglomus species previously identified in a polish mining site was common in roots of different plants. Presence of active AMF in such an environment is an outstanding finding, which should be clearly considered for the design of efficient rhizostabilisation processes.

The transcriptome of the arbuscular mycorrhizal fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate symbiont.

• 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.

A beta-1,3 glucan sulfate induces resistance in grapevine against Plasmopara viticola through priming of defense responses, including HR-like cell death.

Sulfated laminarin (PS3) has been shown previously to be an elicitor of plant defense reactions in tobacco and Arabidopsis and to induce protection against tobacco mosaic virus. Here, we have demonstrated the efficiency of PS3 in protecting a susceptible grapevine cultivar (Vitis vinifera cv. Marselan) against downy mildew (Plasmopara viticola) under glasshouse conditions. This induced resistance was associated with potentiated H2O2 production at the infection sites, upregulation of defense-related genes, callose and phenol depositions, and hypersensitive response-like cell death. Interestingly, similar responses were observed following P. viticola inoculation in a tolerant grapevine hybrid cultivar (Solaris). A pharmacological approach led us to conclude that both callose synthesis and jasmonic acid pathway contribute to PS3-induced resistance.

Medicago truncatula gene responses specific to arbuscular mycorrhiza interactions with different species and genera of Glomeromycota.

Plant genes exhibiting common responses to different arbuscular mycorrhizal (AM) fungi and not induced under other biological conditions have been sought for to identify specific markers for monitoring the AM symbiosis. A subset of 14 candidate Medicago truncatula genes was identified as being potentially mycorrhiza responsive in previous cDNA microarray analyses and exclusive to cDNA libraries derived from mycorrhizal root tissues. Transcriptional activity of the selected plant genes was compared during root interactions with seven AM fungi belonging to different species of Glomus, Acaulospora, Gigaspora, or Scutellospora, and under widely different biological conditions (mycorrhiza, phosphate fertilization, pathogenic/beneficial microbe interactions, incompatible plant genotype). Ten of the M. truncatula genes were commonly induced by all the tested AM fungal species, and all were activated by at least two fungi. Most of the plant genes were transcribed uniquely in mycorrhizal roots, and several were already active at the appressorium stage of fungal development. Novel data provide evidence that common recognition responses to phylogenetically different Glomeromycota exist in plants during events that are unique to mycorrhiza interactions. They indicate that plants should possess a mycorrhiza-specific genetic program which is comodulated by a broad spectrum of AM fungi.

Expression profiling of up-regulated plant and fungal genes in early and late stages of Medicago truncatula-Glomus mosseae 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.

Common gene expression in Medicago truncatula roots in response to Pseudomonas fluorescens colonization, mycorrhiza development and nodulation.

• Beneficial rhizosphere microorganisms may share similar molecular steps during root colonization. To test this hypothesis, we compared Medicago truncatula Gaertn. gene expression in roots colonized, or not colonized, by Glomus mosseae BEG12, Pseudomonas fluorescens C7R12 or Sinorhizobium meliloti 2011. • Pseudomonas fluorescens C7R12 formed colonies on the surface of M. truncatula roots and colonized root tissues intercellularly and intracellularly in a way similar to that previously described for other plants. • Semiquantitative reverse transcriptase polymerase chain reaction of a set of 12 mycorrhiza upregulated M. truncatula genes revealed different expression profiles in roots 3 weeks after inoculation with P. fluorescens or S. meliloti. Pseudomonas fluorescens colonization activated seven of the plant genes while nodulated root systems showed increased expression in only three genes and five appeared to be downregulated. • This first report of similar gene induction by a fluorescent pseudomonad and a mycorrhizal fungus in roots supports the hypothesis that some plant cell programmes may be shared during root colonization by these beneficial microorganisms. Less similarity existed in expression of the gene set with nodulation by S. meliloti.

25S rDNA-based molecular monitoring of glomalean fungi in sewage sludge-treated field plots.

Recycling of sewage wastes in agriculture is likely to affect the biological activity of soils through contamination of ecosystems by pathogens and metallic or organic micropollutants. The impact of sewage sludge spreading under field conditions on arbuscular mycorrhiza (AM) formation by a community of glomalean fungi was evaluated using a nested polymerase chain reaction (PCR) and discriminating primers based on 25S rDNA polymorphisms to detect different fungal species within root systems. Medicago truncatula was grown in soil of field plots amended or not with a composted sewage sludge, spiked or not with organic or metallic micropollutants. Overall AM development in roots decreased with sewage sludge application, and the relative abundance of five AM fungal morphotypes in root fragments was modified by the input of composted sludges. Sewage sludge spiked or not with organic pollutants had a generally positive effect on the relative diversity of AM fungal populations in planta, whereas after spreading of the sludge spiked with metallic pollutants, no variation was observed in the abundance of different species.

Medicago truncatula ENOD11: a novel RPRP-encoding early nodulin gene expressed during mycorrhization in arbuscule-containing cells.

Leguminous plants establish endosymbiotic associations with both rhizobia (nitrogen fixation) and arbuscular mycorrhizal fungi (phosphate uptake). These associations involve controlled entry of the soil microsymbiont into the root and the coordinated differentiation of the respective partners to generate the appropriate exchange interfaces. As part of a study to evaluate analogies at the molecular level between these two plant-microbe interactions, we focused on genes from Medicago truncatula encoding putative cell wall repetitive proline-rich proteins (RPRPs) expressed during the early stages of root nodulation. Here we report that a novel RPRP-encoding gene, MtENOD11, is transcribed during preinfection and infection stages of nodulation in root and nodule tissues. By means of reverse transcription-polymerase chain reaction and a promoter-reporter gene strategy, we demonstrate that this gene is also expressed during root colonization by endomycorrhizal fungi in inner cortical cells containing recently formed arbuscules. In contrast, no activation of MtENOD11 is observed during root colonization by a nonsymbiotic, biotrophic Rhizoctonia fungal species. Analysis of transgenic Medicago spp. plants expressing pMtENOD11-gusA also revealed that this gene is transcribed in a variety of nonsymbiotic specialized cell types in the root, shoot, and developing seed, either sharing high secretion/metabolite exchange activity or subject to regulated modifications in cell shape. The potential role of early nodulins with atypical RPRP structures such as ENOD11 and ENOD12 in symbiotic and nonsymbiotic cellular contexts is discussed.

Differential activation of H+-ATPase genes by an arbuscular mycorrhizal fungus in root cells of transgenic tobacco.

In arbuscular mycorrhizas, H+-ATPase is active in the plant membrane around arbuscules but absent from plant mutants defective in arbuscule development (Gianinazzi-Pearson et al. 1995, Can J Bot 73: S526-S532). The proton-pumping H+-ATPase is encoded by a family of genes in plants. Immunocytochemical studies and promoter-gusA fusion assays were performed in transgenic tobacco (Nicotiana tabacum L.) to determine whether the periarbuscular enzyme activity results from de-novo activation of plant genes by an arbuscular mycorrhizal fungus. The H+-ATPase protein was localized in the plant membrane around arbuscule hyphae. The enzyme was absent from non-colonized cortical cells. Regulation of seven H+-ATPase genes (pma) was compared in non-mycorrhizal and mycorrhizal roots by histochemical detection of beta-glucuronidase (GUS) activity. Two genes (pma2, pma4) were induced in arbuscule-containing cells of mycorrhizal roots but not in non-mycorrhizal cortical tissues or senescent mycorrhiza. It is concluded that de-novo H+-ATPase activity in the periarbuscular membrane results from selective induction of two H+-ATPase genes, which can have diverse roles in plant-fungal interactions at the symbiotic interface.

Arbuscular mycorrhiza development regulates the mRNA abundance of Mtaqp1 encoding a mercury-insensitive aquaporin of Medicago truncatula.

The genome of the model legume Medicago truncatula Gaertn. was screened for the presence of genes encoding tonoplast intrinsic proteins, and a gene family was identified. The cDNA fragments of two members of the multigene family were cloned from roots inoculated with an arbuscular mycorrhizal fungus. Transcript accumulation in roots could be detected for both cDNA fragments, but only one gene was induced in the symbiosis when compared to non-mycorrhizal control roots. A full-length cDNA clone was obtained from the arbuscular-mycorrhiza-regulated gene, and injection of in-vitro-transcribed RNA into Xenopus oocytes revealed that the encoded protein MtAQP1 specifically facilitates water transport. The possible role of MtAQP1 in buffering osmotic fluctations in the highly compartmented vacuole of arbuscule cells is discussed.

Effects on growth and comparison of root tissue colonization patterns of Eucalyptus viminalis by pathogenic and nonpathogenic strains of Fusarium oxysporum.

Soilborne pathogens, especially Fusarium oxysporum, are responsible for damping-off and root necrosis in Eucalyptus nurseries. New technologies are increasingly considering strategies for plant disease control other than chemical fungicides. Among these, natural fungal antagonists, which are colonizers of the root cortex, are potential biocontrol agents. An in vitro system was used: (1) to test the pathogenic effects of F. oxysporum strain Foeu1 which was recovered from a forest nursery soil; (2) to explore the potential of the nonpathogenic F. oxysporum strain Fo47, which is known for its efficiency in biological control, to suppress damping-off of Eucalyptus seedlings; (3) to compare the patterns of root colonization and host response to invasion by the two Fusarium strains inoculated separately in a time-course study. Root inoculation of E. viminalis with F. oxysporum strain Foeu1 caused damping-off in young seedlings in vitro, whilst disease symptoms were not visible in plants inoculated with F. oxysporum strain Fo47 or when both strains (Foeu1 + Fo47) were inoculated simultaneously. Each strain showed similarities in patterns of root tissue colonization, and in the processes of root penetration and initial colonization. Differential effects on root tissue were observed with fungal development within the cortex: ingress of strain Foeu1 was accompanied by severe host-cell alterations whilst no tissue damage occurred with development of strain Fo47.

Comparative differential RNA display analysis of arbuscular mycorrhiza in Pisum sativum wild type and a mutant defective in late stage development.

In order to analyse gene expression associated with the late stages of arbuscular mycorrhizal development between Pisum sativum and Glomus mosseae, comparative differential RNA display was carried out using wild-type P. sativum and a mutant, RisNod24, where the fungal partner is not able to form functional arbuscules. Comparison of RNA accumulation patterns between controls, G. mosseae-colonized mutant and wild-type roots resulted in the identification of four differentially occurring cDNA fragments. One of the corresponding genes was from the fungus and three of plant origin. One plant gene, Psam4 (P. sativum arbuscular mycorrhiza-regulated), was analysed in more detail. Sequencing of a cDNA clone showed that Psam4 encodes a proline-rich protein. Northern blot analysis and quantitative RT-PCR revealed a higher basal level of Psam4 RNA accumulation in the mutant compared to the wild type. In both pea genotypes, RNA accumulation was reduced after inoculation with mycorrhiza- or nodule-forming symbiotic microorganisms, but enhanced after infection with a root pathogenic fungus.

Characterization of root colonization profiles by a microcosm community of arbuscular mycorrhizal fungi using 25S rDNA-targeted nested PCR.

The aim of the present work was to study colonization patterns in roots by different arbuscular mycorrhizal fungi developing from a mixed community in soil. As different fungi cannot be distinguished with certainty in planta on the basis of fungal structures, taxon-discriminating molecular probes were developed. The 5' end of the large ribosomal subunit containing the variable domains D1 and D2 was amplified by PCR from Glomus mosseae (BEG12), G. intraradices (LPA8), Gigaspora rosea (BEG9) and Scutellospora castanea (BEG1) using newly designed eukaryote-specific primers. Sequences of the amplification products showed high interspecies variability and PCR taxon-discriminating primers were designed to distinguish between each of these four fungi. A nested PCR, using universal eukaryotic primers for the first amplification and taxon-discriminating primers for the second, was performed on individual trypan blue-stained mycorrhizal root fragments of onion and leek, and root colonization by four fungi inoculated together in a microcosm experiment was estimated. More than one fungus was detected in the majority of root fragments and all four fungi frequently co-existed within the same root fragment. Root colonization by G. mosseae and G. intraradices was similar from individual mixed inoculum, whilst the frequency of S. castanea and Gig. rosea increased in the presence of the two Glomus species, suggesting that synergistic interactions may exist between some arbuscular mycorrhizal fungi.

Cellular localization of a plant protein PSAM 1 in arbuscular mycorrhizas of Pisum sativum.

Psam 1 is a single-copy gene which is activated during early plant-fungal interaction in wild-type pea inoculated with Glomus mosseae and which codes for PSAM 1, a putative protein of 108 amino acids. A synthetic peptide was designed of 108 amino acids. A synthetic peptide was designed in an antigenic region of this protein to produce a polyclonal antibody against PSAM 1 and to investigate its cellular localization. Western blot analysis revealed that a polypeptide of about 14.5 kDa accumulated more in mycorrhizal than non-mycorrhizal pea roots. The PSAM 1 antigen was immunolocated in planta in arbuscule-containing cells of mycorrhizal roots and especially in the cytoplasm surrounding young arbuscules in cortical cells, which suggests that its accumulation is somehow related to the symbiotic state of these cells.

Base composition of DNA from glomalean fungi: high amounts of methylated cytosine.

Glomales (Zygomycetes) are obligate fungal symbionts of roots of land plants and form arbuscular mycorrhiza. Sporal DNA of 10 isolates belonging to nine species was purified and the base composition was determined by RP-HPLC. Base composition fell in a narrow range between 30 and 35% G + C. A high amount of methylated cytosine (mC) accounting for 2-4% of the total nucleotides was found in all taxa. The DNA melting profile was defined for Scutellospora castanea. It corresponded to 32% G + C, and the shape of the denaturation curve suggested a heterogeneity in the GC content within the fungal genome. Knowledge of GC contents and variations between taxa are essential for evaluating nuclear DNA content using fluorimetric methods, and high proportions of mC/C + mC in the genomes of glomalean fungi could reflect the existence of repeated DNA families. Results are discussed in relation to data for other fungi and eukaryotes.

Differential display analysis of RNA accumulation in arbuscular mycorrhiza of pea and isolation of a novel symbiosis-regulated plant gene.

Differential RNA display was used to analyze gene expression during the early steps of mycorrhiza development on Pisum sativum following inoculation with Glomus mosseae. Seven out of 118 differentially displayed cDNA fragments were subcloned and sequenced. One fragment corresponded to part of the fungal 25S ribosomal RNA gene and a second one showed similarity to a human Alu element. The others were derived from plant genes of unknown function. One of the fragments was used for the isolation of a full-length cDNA clone. It corresponded to a single-copy gene (psam1) which is induced during early symbiotic interactions, and codes for a putative transmembrane protein. Northern and RNA dot blot analyses revealed enhanced accumulation of psam1 RNA after inoculation with G. mosseae of wild-type pea and an isogenic mutant deficient for nodule development (Nod-, Myc+).

The Vicia faba leghemoglobin gene VfLb29 is induced in root nodules and in roots colonized by the arbuscular mycorrhizal fungus Glomus fasciculatum.

To investigate similarities between symbiotic interactions of broad bean (Vicia faba) with rhizobia and mycorrhizal fungi, plant gene expression induced by both microsymbionts was compared. We demonstrated the exclusive expression of 19 broad bean genes, including VfENOD2, VfENOD5, VfENOD12 and three different leghemoglobin genes, in root nodules. In contrast, the leghemoglobin gene VfLb29 was found to be induced not only in root nodules, but also in broad bean roots colonized by the mycorrhizal fungus Glomus fasciculatum. In uninfected roots, none of the 20 nodulin transcripts investigated was detectable. VfLb29 has an unusually low sequence homology with all other broad bean leghemoglobins as well as with leghemoglobins from other legumes. It can be regarded as a novel kind of leghemoglobin gene not described until now and the induction of which is common to symbiotic interactions of broad bean with both Rhizobium and a mycorrhizal fungus.

Characterization of a highly repeated DNA sequence (SC1) from the arbuscular mycorrhizal fungus Scutellospora castanea and its detection in planta.

A highly repeated DNA sequence from the genome of an arbuscular mycorrhizal fungus has been isolated and characterized. This 1,202-bp sequence (SC1) represents about 0.24% of the Scutellospora castanea genome, estimated to be 1 pg by flow cytometry. The sequence was shown to be a Scutellospora-specific probe in Southern blots and dot blot hybridizations. After complete sequencing of SC1, PCR primers were generated and used to amplify a 907-bp fragment from spores of S. castanea or from colonized Allium porrum roots. No amplification products were obtained with DNA from either spores or mycorrhizal root of other species of arbuscular mycorrhizal fungi. These primers were sufficiently specific for unequivocal detection of S. castanea in planta.