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Genes and genetic engineering for arbuscular mycorrhiza technology and applications in sustainable agriculture

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Arbuscular mycorrhizal (AM) fungi are obligate biotrophs, which need to colonize appropriate host roots to complete their life cycle. In their symbiotic status, the fungi explore the soil surrounding the plant and help it in the absorption of relatively immobile nutrients such as P and N. Genes involved in phosphate metabolism and ammonium transport were isolated from the AM fungus Glomus intraradices. This fungus was monoxenically cultured, using compartmented dishes, to avoid contamination from any other soil organism. Both ammonium transporter (GintAMT1) and alkaline phosphatase (GintALP1) genes were isolated from the extraradical mycelium of the fungus by a combination of RT-PCR using degenerated primers and RACE techniques. GintAMT1 codes for a 479 amino acid protein with high homology to ammonium transporters of other fungi. The predicted topology is similar to that of ammonium transporters in eukaryotes: 11 transmembrane domains with an N terminusOUT/C terminusIN orientation. Heterologous expression of GintAMT1 in a yeast mutant defective in the three plasma membrane NH4+ transporters complemented the defect of this strain to grow in the presence of less than 1mM ammonium. The phenotipically restaured mutant yeast was used to study the kinetics of ammonium uptake by GintAMT1 through (14C)-methylammonium uptake experiments. These studies indicated GintAMT1 is a high affinity ammonium transporter with an approximate Km of 30microM. Expression studies using real time RT-PCR indicate that GintAMT1 is induced by low ammonium concentrations and inhibited by high concentrations. GintALP1 codes for a 525amino acid protein with high homology to yeast alkaline phosphatase genes. Expression studies of the gene by Real Time RT-PCR showed that GintALP1 expression in the extraradical mycelium of G. intraradices is inhibited by pulses with 350microM P. More information on the Genomyca -project can be found at: http://www.dijon.inra.fr/genomyca/
Candidatus Glomeribacter gigasporarum is an endocellular beta-proteobacterium present in the arbuscular mycorrhizal (AM) fungus Gigaspora margarita. Pulse Field Gel Electropheresis (PFGE) analyses of bacteria purified from spores of G. margarita (see e-TIP 16870Isolation and purification of Candidatus Glomeribacter gigasporarum from Gigaspora margarita) allowed us to estimate the genome size of Candidatus Glomeribacter gigasporarum to approximately 1.4Mb with a ca 750kb chromosome and a 600-650kb plasmid. This is the smallest genome known for any beta-proteobacterium, suggesting that the genome has evolved by genome reduction from an ancestor beta-proteobacterial genome. Such small genome sizes are typically found in endocellular bacteria living permanently in their host (obligate symbionts). More information on the Genomyca -project can be found at: http://www.dijon.inra.fr/genomyca/
Different methods were used to monitor heavy metal toxicity to mycorrhizal fungi. The germination test was shown to be a sensitive indicator of heavy metal toxicity. The germination level of control Gi. rosea spores was high. On the highest Pb concentration germination was strongly decreased. Also the appearance of the hyphae can indicate the presence of heavy metals. The problem with this test lies in the vulnerability to parasites. Gigaspora spores were more susceptible to parasitic attack than G. mosseae. Spores colonized with parasites showed decreased viability and no germination of infected spores was observed. Parasites can also alter the uptake and distribution of heavy metals. Three plant species (Zea mays, Plantago lanceolata and Medicago sativa) were used to assess the tolerance of AMF to soil toxicity and to compare heavy metal uptake by nonmycorrhizal and mycorrhizal plants. Mycorrhizal parameters were high only in plants inoculated with G. mosseae. In the case of both Gigaspora species, the colonisation level was low, although the arbuscule richness was high. The alkaline phosphatase test showed the impact of soil toxicity on the vitality of the fungus. G. mosseae was demonstrated to be the most tolerant and Gi. margarita the most sensitive. Therefore, G. mosseae was most suitable as an indicator of the influence of soil toxicity on mycorrhizal colonisation level and morphological alterations. The morphology of mycorrhizal structures was significantly altered. Arbuscules were often strongly septated or their branching was reduced. The accumulation of lipid like-bodies was observed. Arbuscule degeneration stages were different from those usually observed. The strongest morphological changes of arbuscules were observed in roots of plants grown on the Chrzanów waste, whereas in plants cultivated on the Trzebionka waste the arbuscules were the most healthy-looking. The same was observed in Gi. rosea. The extraradical mycelium often formed internal wall thickening, which were usually stained by rhodizonate. G. mosseae reacts to industrial wastes by the formation of abundant crystals in vesicles and spores within plant roots. Most of the intraradical mycelium was filled with oil droplets at much higher abundance than in control roots. The SUDAN IV and Oil Red 0 staining, applied on roots stained with aniline blue, gave a positive reaction for both lipid droplets and crystals. The analysis of the extramatrical mycelium of several AMF species cultivated on industrial wastes showed that G. mosseae is much more sensitive than other strains of the genus Glomus studied. In this case, the reduction of the extramatrical mycelium was very high. On this basis, we can conclude that morphological changes observed in mycorrhiza of G. mosseae are good indicators of soil pollution. This was supported by the experiments using substratum spiked with individual heavy metals. The strongest changes in arbuscule morphology were observed in the case of Zn. The arbuscules formed in roots of plants treated with Zn were often cauliflower-like, with poorly developed branches and an oily appearance. At the highest Zn concentrations the intraradical mycelium was poorly stained, but remnants of arbuscules were well recognizable. Arbuscules from Cd treated cultures were usually much smaller and not branched. Hyphal walls often formed darkly stained thickenings. No obvious modifications of arbuscule morphology were found in the case of Pb. Instead, dark precipitates were observed most commonly within the vacuole-like structures. With the increase of heavy metal concentration also the decrease of mycorrhizal colonisation level was noted. Few irregular vesicles were observed within roots of plants treated with the tested elements. The above described differences show that observations done on mycorrhizal structures can clearly indicate not only the presence of the disturbance, but can also suggest the nature of this stress factor. This would lead to the conclusion that G. mosseae mycorrhiza could be used for the evaluation of soil toxicity, if soil samples are taken from a given area, sterilized and inoculated with the fungus of the known behaviour. Observations of native fungi in the soil are not appropriate, as their reaction patterns are mostly unknown. The second possibility involves spores of Gi. rosea, which is an excellent model for studying the impact of heavy metals on spore germination, also under laboratory conditions. More information on the Genomyca -project can be found at: http://www.dijon.inra.fr/genomyca/
Three cDNA libraries with each more than 300 clones have been established from extraradical hyphae of the arbuscular mycorrhizal (AM) fungus Glomus intraradices. These hyphae were obtained from an experimental system, where hairy root cultures of Daucus carota have been inoculated with the AM fungus and transferred onto an agar plate with two compartments. These libraries are enriched for genes regulated by: - Heavy metals, - Phosphate, or - Nitrate/ammonium using the subtractive suppressive hybridisation method as a strategy. The inserts from clones of the libraries were amplified, analysed by gel electrophoresis and transferred onto nylon membranes. Hybridisation of these membranes with complex cDNA probes identified numerous genes with differential expression patterns. In parallel, clones were sequenced and sequences were annotated. Although several AM fungal genes have previously been isolated and characterised which are regulated by phosphate, nitrate or by heavy metals, this is the first time that a non-targeted approach has been used to identify genes from these microorganisms which could be involved in central aspects of this symbiosis. This is on the one hand the supply of the plant with nutrients under conditions of low soil fertility, on the other hand the support of plant growth on heavy metal-contaminated soils. These libraries represent therefore a useful source for genes which might play a role in AM functioning. This is not only important for scientific purposes, but can be also used in research concerning the application of AM fungi. In addition, the cDNA libraries will contribute to the world wide EST collection from Glomus intraradices, which has to be built up on the background of the announced genomic sequencing of this organism. More information on the Genomyca -project can be found at: http://www.dijon.inra.fr/genomyca/
Arbuscular mycorrhizal (AM) fungi are ubiquitous soil-borne microorganisms that establish a mutualistic symbiosis with most plant species. In AM symbiosis, the fungus supplies the plant with slow-diffusing nutrients, such as P, N, Zn and Cu, but they are also able to reduce metal translocation to the host plant when present at toxic levels in the soil. Consequently, AM fungi play an important role in protecting the host plant against heavy metal stress. Among the mechanisms of heavy metal tolerance, metallothioneins and metal transporters are of primary importance. Genes involved in metal homeostasis have been isolated from the AM fungus Glomus intraradices. Two cDNA fragments corresponding to a metallothionein (GintMT1) and Zn transporter (GintZnT1) were identified by exploring a G. intraradices EST library. The full-length sequences were obtained by RACE using gene-specific primers and cDNA from extraradical mycelium of the monoxenically grown fungus. GintMT1 encodes a 71amino acid polypeptide with 13 Cys residues and a predicted molecular mass of 7.2kDa. It is related to gene members of the metallothionein family, showing 55% and 48 % similarity to the MT genes of the AM fungi G. rosea and G. margarita, respectively. Functional analysis of the gene through complementation of a yeast mutant defective in the metallothionein locus CUP1 showed in vivo protection of GintMT1 against Cd and Cu. Expression analysis of the gene through real time RT-PCR revealed down regulation of GintMT1 in the extraradical mycelium by a long-time exposure of G. intraradices to Zn, Cd or Cu. Sudden exposure of the extraradical mycelium to heavy metals or paraquat (an oxidative agent) showed up-regulation by high Cu concentrations and paraquat, but down-regulation by Zn and Cd. These data suggest the involvement of GintMT1 in the protection against oxidative stress, as well as in heavy metal homeostasis. GintZnT1 encodes a 454 amino acid polypeptide with a predicted molecular weight of 54kDa. Based on its sequence and general structural features, GintZnT1 has been classified as a putative Zn transporter, included in the Cation Diffusion Facilitator (CDF) family of metal transporters. GintZnT1 shows the highest homology to the vacuolar transporters Zrc1 and Cot1 of Saccharomyces cerevisiae and Zhf of Schizosaccharomyces pombe. The predicted topology of the deduced amino acid sequence of GintZnT1 shows 6 transmembrane domains and two cytoplasmic motifs. All these regions are almost identical among GintZnT1 and the three previously characterized fungal CDF proteins. An effect of the GintZnT1 gene product on the cytoplasmic labile regulatory Zn pool of S. cerevisiae has been detected by using a Zn-regulated beta-galactosidase reporter gene, which indicates a role of GintZnT1 reducing cytoplasmic Zn availability. GintZnT1 expression in the extraradical mycelium of G. intraradices has been studied by using Real-Time RT-PCR. Up-regulation of GintZnT1 mRNA was detected upon short-time exposure of G. intraradices to Zn and when the mycelia were developed in 75 microM Zn supplemented plates. All these data suggest a role of GintZnT1 in Zn compartmentalization and in the protection of G. intraradices against Zn stress. More information on the Genomyca -project can be found at: http://www.dijon.inra.fr/genomyca/
To check whether the expression and activity of ToMO is achievable in a simple eukaryotic organism, as a preparative step for the transformation of arbuscular-mycorrhizal fungi with tou genes, the cloning of touABCDEF genes for the transformation of Saccharomyces cerevisiae has been performed. tou genes have been preliminary cloned from a vector carrying all the six structural genes into modified prokaryotic vectors. ToMO subunits have been expressed either as wild-type or as histidine-tagged proteins in Escherichia coli; policlonal antibodies against ToMO proteins have been produced by inoculation in rabbit, in order to be used for western blot analyses. tou genes have been subsequently cloned into centromeric yeast expression vectors, and two yeast strains have been transformed to achieve the expression of wild-type and recombinant ToMO proteins. By western blot we checked the expression of ToMO subunits; the expression of recombinant ToMOHisA,E,D,F was detected using commercial antibodies, while the polyclonal antibodies above mentioned have been used for the wild-type ToMO D,F proteins. While there is wealth of publications concerning the cloning and expression of genes belonging to complex organisms such as eukaryotes into simpler models, the converse is scarcely documented. The difficulties experienced during the experiments reflect the higher complexity of the eukaryotic system. In any case, to our knowledge, this is the first example of the cloning and expression of such a complex enzymatic system in eukaryotes. More information on the Genomyca -project can be found at: http://www.dijon.inra.fr/genomyca/
Three groups of Gi. rosea spores were provided by the consortium: non-bombarded (NB), bombarded with an empty vector (BP) and bombarded with the GmarMT1 gene (BG). The spores were placed on sterile filter disks on sterile sand moistened with different metal solutions: Cd (4,5, 45 and 450 µM), Zn (75, 750 and 7500 µM) and Cu (50, 500 and 5000 µM) and checked for germination. After 4 weeks approximately 30 spores were stained with INT to check their viability, divided into live (positive reaction) and dead (negative reaction) and counted. Generally, in the control group, the bombarded spores (BP and BG) showed decreased viability compared to non-bombarded spores, although this difference was not significant. The same tendency was noted in the Cd-treated groups (4.5 and 450 µM). In the case of Zn, the tendency was the opposite: BG spores showed the highest viability. In the Cu-treated group, the situation was similar at the low and medium Cu concentrations (the BG group spores). Spores at the lowest Cu and Zn concentrations died due to parasitic attack. At the highest Cu concentration the overall viability was very low and the spores turned dark. It was also observed that the dark spores incubated with high Cu concentrations (5000 µM) showed a thickening of the cell wall. Dithizone and rhodizonate treatment of unstained spores (highest concentrations) showed a positive reaction in the case of Cd and Zn-treated groups and a much weaker reaction in the Cu-treated group. These dyes are excellent indicators of the accumulation of heavy metals in tissues, especially when used for pilot screening of the material. Spores that formed at least 500 öm long hyphae were removed from the filters and attached to carbon holders. The mycelium and the attached spores, cut open to facilitate drying up, were air-dried in order to prevent element loss due to lyophilisation, coated with carbon and analysed with EDS coupled to SEM. No statistically important differences in Cd content in the mycelium and both spore layers were found in spores germinated in 4,5 µM Cd solution. The differences became clear only at the higher Cd levels. At the highest Cd concentration (450µM) the highest content of Cd was found within the mycelium. The high Cd level was accompanied by a high content of S. S can come from the sulphate salts of the metals used in the experiment. While the first two batches were analysed, in the 450µM Cd solution, only spores bombarded with the MT gene germinated. This suggested the successful improvement of resistance to Cd. The same tendency was noted for Cu. The high variation between individual spores can be explained by the effectiveness of transformation of about 50%. No differences were noted in the case of Zn. The third batch of spores appeared to be of the high vitality and the germination % was very high. Some of spores bombarded with the GmarMT1 gene clearly contained higher levels of Cd, although others accumulated much less that again could be caused either by the fact that not all spores were modified or by the fact that some hyphae were growing above the surface of the filter and had little contact with Cd. The clear increase of the mycelium growth rate was found in the case of the GmarMT1 gene bombarded spores that were exposed to lower Cd solutions within microtiter plates. More information on the Genomyca -project can be found at: http://www.dijon.inra.fr/genomyca/
Arbuscular mycorrhizal (AM) fungi cannot be grown alone in pure culture, which limits the source of available genomic DNA. Spores of AM fungi (produced in association with a host plant) house hundreds to thousands of nuclei, each of which generally contains 0.1 to 1pg DNA depending on the fungal species. Genomic libraries were constructed from DNA, which was extracted from a large number of spores of G. mosseae (50000), Gig. margarita (18000) and Gig. rosea (18000), isolated from pure pot cultures. Extracted DNA was purified on a CsCl gradient, restricted with BamHI or BglII, and ligated into a lambda-ZAPII or lambda-DASHII cloning vectors. l-ZAPII libraries (average insert size 1 to 4 kb) were obtained for all three fungi using BamHI, and absence of procaryotic DNA was confirmed in the libraries. The lambda-ZAPII libraries represent between 2 and 2.5 fold the fungal genome and they have proved useful for isolating repetitive DNA elements from each of the three fungi. Two genomic libraries of G. mosseae were constructed in the lambda-DASH-II vector without CsCl centrifugation of the sporal DNA (to limit DNA shearing) and after restriction with BamHI or BglII. Large subunit ribosomal DNA (25S rDNA) and several published protein encoding genes of known or unknown function were identified in the genomic library constructed from DNA restricted with BglII, and which is estimated to represent at least 6 times the fungal genome. This genomic library may be useful for the isolation of low copy genes. More information on the Genomyca -project can be found at: http://www.dijon.inra.fr/genomyca/
In the mutualistic AM symbiosis the plant supplies the fungus with carbon while the fungus assists the plant in its uptake of phosphate and other mineral nutrient from the soil. Genes involved in nitrogen and phosphate metabolism (amino acid permease and phosphate transporter) were investigated in the AM fungus Glomus mosseae. G. mosseae amino acid permease: A combination of PCR with degenerated oligonucleotides and 5’ and 3’ RACE-PCR assays led to the identification of a cDNA sequence (GmosAAP1) coding for a protein similar to previously described amino acid permeases. GmosAAP1 consists of 572 amino acids, a molecular mass of 61.7kDa and twelve putative transmembrane domains; this structure is in agreement with that of other fungal and yeast amino acid permeases. Gene expression analyses, performed by RT-PCR assays, demonstrated that GmosAAP1 was exclusively expressed in extraradical mycelium. Real-Time experiments indicated that GmosAAP1 expression was modulated by different nitrogen sources. G. mosseae phosphate transporter: By means of degenerate primers and RACE experiments we identified a 1120 bp cDNA sequence (GmosPT) showing the highest similarity with G. intraradices phosphate transporters (E value=e-177, identity 72%, similarity 85%). The sequence contains the complete 3’ end and corresponds to 80% of the expected full length protein (420 over 520 aminoacids). A phylogenetic analysis showed that GmosPT protein groups together with the other two phosphate transporters isolated from AM fungi, close to all of the fungal transporters and separated from the plant P transporter sequences. GmosPT expression was monitored by RT-PCR assays. GmosPT expression in extraradical mycelium responded to external concentration of phosphate: the transcript resulted more abundant in external hyphae exposed to 35 micromolar Na2HPO4 than in external hyphae treated with 3.5 millimolar Na2HPO4. More information on the Genomyca -project can be found at: http://www.dijon.inra.fr/genomyca/
Symbiotic associations between endocellular bacteria and eukaryotic cells are common among plants and animals. In fungi, the presence of endocellular bacteria has only been reported in some Glomeromycota species (arbuscular mycorrhizal fungi and Geosiphon pyriforme). In spite of the obligate endosymbiotic nature of the fungal host, the difficulty to access large fungal biomass and the relatively small endocellular bacterial density within the fungus, we have been able to isolate endocellular Candidatus G. gigasporarum bacteria from the arbuscular mycorrhizal fungus Gigaspora margarita. The isolation protocol is based on buffers containing sucrose 250mM and on centrifugations at different speeds. The crucial point was the use of a separation buffer containing Percoll, Ficoll and PEG that allowed satisfactory separation of bacteria from most cellular debris. The number of living isolated bacteria was estimated by using a Thoma cell and conventional fluorescence microscopy. Bacteria were stained with the Live/Dead BacLight bacterial viability kit (Molecular Probes). An average of ca 13 000 bacteria per fungal spore was obtained. A natural heterogeneity in the number of bacteria per fungal spore was observed ranging from 3 700 to 26 000. Electron microscopic observations of the isolated bacteria revealed that most bacteria are rod-shaped (0.8-1.2 by 1.5-2.0µm in size) with a laminated cell wall typical of Gram-negative bacteria and a cytoplasm rich in ribosomes. An electron transparent area, probably corresponding to the chromosome was often observed. Cell surface was particularly complex with a fibrillar coat but flagella or pili were not visible. To investigate the free living capacities of Candidatus G. gigasporarum, several media, suitable to sustain growth of a large spectrum of different microorganisms, supplemented with various vitamins or amino-acids, were tested. Candidatus G. gigasporarum growth was never observed in any of the tested media and chosen conditions. However, Candidatus G. gigasporarum is able to survive several weeks out of its fungal host. More information on the Genomyca -project can be found at: http://www.dijon.inra.fr/genomyca/.
Arbuscular mycorrhizal fungi are obligate endosymbionts that colonise the roots of almost 80% of land plants. We describe morphological and molecular data of a bacterial endosymbiont living in the cytoplasm of dormant or germinating spores and symbiotic mycelia of the fungal species Gigaspora margarita, Gi. decipiens, Scutellospora persica, and S. castanea. PCR-amplification of the near entire 16S ribosomal RNA gene, of a portion of the 23S rDNA and of 16S-23S rDNA spacer region of the Gi. margarita BEG 34 endosymbiont using universal bacterial primers, and subsequent sequence analysis (16S rDNA GenBank accession no. X89727, 23S rDNA accession no. AJ561042) demonstrated that this organism occupied a very distinct phylogenetic position within the beta subdivision of the Proteobacteria with the genera Burkholderia, Pandoraea, and Ralstonia as closest neighbours. The design of primers specific to the 16S rDNA (BLOf-BLOr) and the 23S rDNA (GlomGIGf-GlomGIGr) endosymbiotic bacteria of BEG 34 allowed amplification of spore DNA from endosymbionts of Gi. margarita, Gi. decipiens, S. persica, and S. castanea, but not from the Gi. gigantea endosymbiont (which was morphologically different) or from the cytoplasm of Gi. rosea (which did not contain endosymbiotic bacteria). These specific primers were successfully used as probe for the in situ hybridisation of endobacteria in Gi. margarita spores. The overall rod-shaped morphology of the Gi. margarita, Gi. decipiens, S. persica, and S. castanea endosymbionts was similar, and amplification and sequence analysis of the near complete 16S rDNA genes of several Gi. margarita, S. persica, and S. castanea endosymbionts revealed over 98% sequence similarity. These morphological and genomic characteristics were used to assign the endosymbionts of these three species (five isolates) of arbuscular mycorrhizal fungi as ‘Candidatus Glomeribacter gigasporarum’. More information on the Genomyca -project can be found at: http://www.dijon.inra.fr/genomyca/
The nuclei of most arbuscular mycorrhizal (AM) fungi (Glomeromycota) have previously been estimated to contain between 0.1 to 1 pg DNA per nucleus. This large genome size may be due to the presence of a high proportion of repeated DNA. Since repetitive genomic sequences have been used to promote integration of transgenes into genomes in other organisms, we isolated and identified repeated sequences of Gig. rosea, G. mosseae and Gig. margarita to select for transformation vector constructs. A total of 23 repetitive elements were obtained by screening l-ZAPII genomic libraries of the three fungi, obtained within the GENOMYCA project, with DNA from spores. Four were identified from Gig. rosea, 6 from G. mosseae, and 13 from Gig. margarita. All sequences are AT rich and most show no homology to database sequences. Two from Gig. margarita and one from Gig. rosea have significant similarities to retrotransposable-like elements, with protein sequences found in LTR (long terminal repeats) or non-LTR retrotransposons. This is the first evidence of retrotransposable-like elements in the genome of AM fungi. Their presence may account for the large genome size and the high genetic polymorphism that has been described for this group of organisms. One sequence which was studied in detail (GmarRT1) is present in about 2000 copies which are dispersed in the genome of Gig. rosea, G. mosseae and Gig. margarita. Different sequences in this same retrotransposon family were obtained and restriction enzyme analyses showed them to be methylated. Methylation of retrotransposons is known in many organisms as a mechanism by which genomes inactivate retrotransposons, and a similar process is associated with the inactivation of transgenes. This, together with the observation that introduction of GmarRT1 into vector constructs did not improve transgene frequency in bombarded spores of Gig. rosea, suggests that the use of a retrotransposon sequence in transformation vectors is not an appropriate strategy for transgene integration in AM fungal genomes. More information on the Genomyca -project can be found at: http://www.dijon.inra.fr/genomyca/
Arbuscular mycorrhizal (AM) fungi living in symbiotic association with the roots of vascular plants have also been shown to host endocellular rod-shaped bacteria. Based on their ribosomal sequences these endobacteria have been recently identified as a new taxon, Candidatus Glomeribacter gigasporarum. In order to investigate the cytoplasmic stability of the endobacteria in their fungal host and their transmission during AM fungi reproduction (asexual), a system using transformed carrot roots and single spore inocula of Gigaspora margarita was used. Under these in vitro, sterile conditions, with no risk of horizontal contamination, the propagation of endobacteria could be monitored. Parallel multisporal inocula (50 spores per pot) were used in clover pot cultures. MORPHOLOGICAL ANALYSIS: Endobacterial cells were constantly observed in all sporal generations produced in both Petri dish and pot cultures by using the Bacteria Counting Kit. In monosporal-inoculated Petri dish cultures, endobacteria were observed throughout all the mycelial structures produced during root colonisation and spore production: i.e. germination hyphae, extraradical mycelium, auxiliary cells and new spores. TRANSMISSION OF THE ENDOBACTERIA: In order to verify the identity of endobacteria contained in all spore generations (SGs), PCR experiments with specific primers for 16S and 23S rDNA were performed on single SG0, SG1, SG2, SG3, SG4 spores, harvested from both pot cultures and Petri dishes. A fragment of the expected size (565bp) was obtained from spore generation SG0, SG1, SG2, SG3 and SG4 multiplied in Petri dish cultures, by using the specific primers GlomGIGf-GlomGIGr (23S rDNA) The direct sequencing of the PCR fragments confirmed the presence of Candidatus G. gigasporarum. As in vitro SG4 generation included spores where bacterial DNA could not be amplified (also with16S rDNA specific primer, BLOf-BLOr), the reliability of DNA extraction was checked. PCR amplifications of SG4 spores that appeared lacking endobacteria were performed with the eukaryotic primers NS1-NS2. They systematically gave positive response. Fragments of the expected size were constantly amplified, from all spore generations (SG0 to SG4) obtained from pot cultures. DNA amplification with the non specific 16S rDNA bacterial primers 27f and 1495r from the five spore generations (SG0, SG1, SG2, SG3 and SG4) consistently produced a single 1500pb fragment. Restriction analyses performed with a total of 5 restriction enzymes led to identical profiles across the SGs, confirming the presence of the same bacterial population along fungal generations. The size of the digested DNA fragments rightly matched the profiles expected after in silico analysis for the Candidatus G. gigasporum 16S rDNA (X89727). No difference between SG0 spores and the four in vitro produced spore generations were observed. The results show that, at least in the four spore generations here investigated, only one bacterial population is present, and it corresponds to Candidatus G. gigasporarum. BACTERIAL CELL QUANTIFICATION: The repeated observation of sporal cytoplasm from successive generations revealed that the spores produced by monosporal-inoculated Petri dish cultures apparently harboured a progressively smaller population of endobacteria (eventually leading to some bacteria-free SG4 spores), while no decrease was evident through the spore generations produced by the multisporal-inoculated pot cultures. In order to better investigate this aspect, a quantification method was set up, exploiting the capabilities of confocal microscopy. An automatic quantification of the bacterial cells (based on particle size and counting algorithms) was discarded, as a human control of overestimates (due to double counting) turned out to be necessary. In addition, the counting of bacteria in each optical section rather than in a projected image of the whole volume was introduced in order to allow the discrimination of each bacterium, since in many cases their high density resulted in confluent fluorescent areas in the projected image. The average numbers of bacteria present in 100µm-sided cubes of sporal cytoplasm was calculated for each spore. Though a wide range of variability was a general decrease occurred from SG0 to SG4 spores in monosporal-inoculated Petri dish cultures. In conclusion, a method of confocal microscopy for quantifying the density of endobacteria in sporal cytoplasm was designed and applied; endobacteria were constantly found in all the SGs, although their number decreased progressively from SG0 to SG4. In conclusion, our experiments demonstrates that a vertical transmission of endobacteria takes place through the fungal vegetative generations (sporulation) of an AM fungus, indicating that active bacterial proliferation occurs in the coenocytic mycelium of the fungus and that these bacteria are stable endocellular component of their AM fungal host. More information on the Genomyca -project can be found at: http://www.dijon.inra.fr/genomyca/
A family of five genes from Glomus intraradices has been identified which are all inducible by heavy metals. This is up to now the largest gene family of a mycorrhizal fungus. Since such genes are generally involved ibn abiotic stress responses they could serve as a tool in order to build up a molecular biology based detection system for soils with aversive features. On the other hand the expression pattern of these genes could mirror the characteristics of mycorrhizal fungal isolates for providing tolerance against abiotic stress. More information on the Genomyca -project can be found at: http://www.dijon.inra.fr/genomyca/
Specific ecological conditions have been found to ensure large scale production of spores of Gigaspora species. This method is based on the conventional dual culture method on host-plants, which assures the genetic stability of the AMF isolate. It is now possible to supply laboratories with large quantities of spores of this genus to permit fondamental research on Gigaspora species. More information on the Genomyca -project can be found at: http://www.dijon.inra.fr/genomyca/
ARS sequences trigger the replication of the transformation vector, making unnecessary the integration of the plasmid in the genome. In order to isolate ARS from AM fungi we used the integrating vector YIp5. Since Yip5 lacks a yeast replication origin, it can transform S cerevisiae to uracyl prototrophy only via integration at the chromosomal ura3 locus. Such integration by homologous recombination in S cerevisiae is highly inefficient. This vector gives therefore very low transformation rate unless its replication is supported by an autonomously replicating sequence. Gigaspora rosea DNA was extracted from surface-sterilized spores and partially digested with Sau3A (2U) for 20 min. Random Sau3A fragments were cloned into the dephosphorilated BamHI site of YIp5. The ligation reaction was used to transform an ura3-1 S. cerevisiae strain (BMA64N) to uracyl prototrophy. Eight transformants were obtained and the recombinant plasmid from two out of these eight transformants successfully rescued in E. coli and the insert sequenced. The two ARS sequences, named ARSGR2 and ARSGR6, were 1436 and 1663 bp long, respectively. They revealed an extremely high AT content (72-75%). Three elements with 10/11 nucleotide homology (two in the forward direction, one in the reverse direction) to the 11-bp ARS consensus sequences (ACS) defined for yeast vector replication (5’-(A/T)TTTA(T/C)(A/G)TTT(A/T)-3’) were found in ARSGR2. Five elements with 10/11 nucleotide homology to the S. cerevisiae ARS consensus element were found in the ARSGR6 sequence. No G. rosea ARS element showed a perfect sequence match to the S cerevisiae ACS element. Overall, the results indicated that a non-strict match of the ACS element does not interfere with ARS function and contradict a previous study (Van Houten & Newlon, Mol Cell Biol 10, 3917, 1990) indicating that any one of numerous single point mutations in the ACS abolish ARS function. Possibly, the presence of multiple ARS elements in ARSGR2 and ARSGR6 compensate for the lack of a perfect sequence match of these elements to the S cerevisiae ACS element. More information on the Genomyca -project can be found at: http://www.dijon.inra.fr/genomyca/
A fluorescent in situ hybridisation (FISH) protocol was optimised to localise target DNA sequences in interphase nuclei of G. mosseae, Gig. margarita and Gig. rosea. Spores of the fungi were fixed in paraformaldehyde, crushed in NUC buffer, incubated in proteinase K and nuclei collected by filtering. The nuclei were then purified by centrifugation in a saccharose solution and resuspended in PBS buffer. FISH efficiency was confirmed by quantifying loci of tandem ribosomal DNA repeats in the nuclei using 18S or 25S digoxigenin-labelled probes and a three-step immunoreaction to amplify the fluorescence signal. Quantification of the rDNA loci performed on nuclei of Gig. margarita, Gig. rosea and G. mosseae subsequently provided a reference control for in situ hybridisation efficiency in experiments to localise other gene sequences. Eight high copy number repetitive sequences isolated from Gig. margarita and Gig. rosea were also observed in nuclei. Their localisation using FISH showed that they were dispersed at numerous loci in nuclei making quantification unfeasible. The metallothionein gene from Gig. margarita (GmarMT1) gene was detected only at one or two loci in nuclei of Gig. margarita and detection of this low copy number gene necessitated further amplification of signal sensitivity. A similar distribution of the gene was observed also in Gig. rosea nuclei, which confirmed gene expression studies of the presence of a homologous gene to GmarMT1 in this fungus. The protocol detected increased frequency of GmarMT1-homologous sequences in nuclei of spores bombarded with a GmarMT1 vector construct. Using the above protocol, extended DNA fibres were prepared from isolated nuclei of AM fungi for more detailed gene localisation by FISH. More information on the Genomyca -project can be found at: http://www.dijon.inra.fr/genomyca/
Two AM isolates of Glomus intraradices Schneck and Smith obtained from two heavy metal (HM) contaminated localities of anthropogenic origin in the Czech Republic were tested to optimise protocol for cultivation of AMF isolates from heavy metal contaminated soils: - G. intraradices PH5 from Pb-contaminated waste in the proximity of the Pøíbram lead smelter and - G. intraradices BEG140 from Mn-contaminated waste deposit Chvaletice. Both isolates were obtained via trap cultures and subsequent multispore cultures. After successful sporulation in multi-spore culture, two lineages of each isolate were established either in the original HM-contaminated substrate or in inert metal-free substrate (clinoptilolite/sand). G. intraradices PH5 and BEG140 have been cultured for 5 years in these two lineages. Culture conditions for maintaining HM tolerance of AM fungi lineages were determined. It was shown that the isolates of AM fungi originated from contaminated soils could potentially loose their tolerance to heavy metal when sub-cultured for a long term in inert media without contamination. This phenomena was highly significant for G. intraradices isolate BEG140 obtained from substrate contaminated with Mn, however, it was less pronounced for the isolate of the same AM fungal species from lead polluted substrate. In general, AM fungi were found to be more resistant to elevated concentration of cadmium than maize plants, but for Pb and Mn the results were opposite. Mycorrhizal inoculation exhibited potential to increase HM uptake to the roots and also to elevate P uptake into plant tissue. The isolate of G. intraradices from Pb contaminated soil has shown higher accumulation of heavy metals (including Pb) in the ERM as compared to reference isolate of G. intraradices from non-contaminated soil. In conclusion, the cultivation protocol for AM fungi isolates with heavy metal tolerance requires continuation of heavy metal stress, i.e. either cultivation of fungi in original soils or in inert media with simulated HM stress, to ascertain maintenance of heavy metal tolerance during sub-culturing of the isolate. Similar behaviour might be expected for fungal tolerance to other kinds of environmental stress what should be further tested (salinity, pollution with xenobiotics etc.). Therefore, these results are potentially usable for industrial producers of mycorrhizal inocula targeted for bioremediation. Desired maintenance of heavy metal stress tolerance requires suggested optimisation of cultivation protocol for these AM fungal strains originated in contaminated soils. It is therefore essential to cultivate mother cultures of AM strains from contaminated soils in their original soils to maintain stress (and in consequence to preserve the stress tolerance of the fungal isolate. Alternatively, the stress can be simulated in inert cultivation media e.g. by adding solution of relevant heavy metals at similar levels of available elements as in the original soil. This should ascertain maintenance of HM tolerance of AM strains isolated from contaminated soils or AM strains transformed by insertion of targeted methallothionein gene (later is being currently tested for transformed strain of Gigaspora rosea). More information on the Genomyca -project can be found at: http://www.dijon.inra.fr/genomyca/
A mesocosm system was designed and constructed to evaluate the ability of arbuscular-mycorrhizal (AM) fungi to degrade BTEX hydrocarbons. Preliminar mycorrhization was achieved by inoculating 1-week leek seedlings with Glomus mosseae, Gigaspora margarita and Gi. rosea spores. After one month plants were transferred in pots, using a vermiculite-based substrate supplemented with benzene, toluene, ethylbenzene and xylene adsorbed on active carbon. The cultures were then transferred under isolated glass cabinets, and grown for 17 days. The residual concentration of BTEXs in pot substrates and inside the system was quantified by gas-chromatography. A strong decrease of hydrocarbon concentration resulted in the mycorrhized samples, while in the substrate samples and in substrate of non-mycorrhized plant samples the amounts remained considerably higher. This observations suggest a clear connection between the arbuscular mycorrhiza and the decrease of hydrocarbon concentration. This result provides new information about the possible use of AM fungi for the reclamation of BTEX hydrocarbon-polluted sites. These fungi are highly competitive-organisms, and therefore their use in remediation techniques could be more advantageous in comparison with bacteria, which are subjected to predation by other microorganisms and whose growth is conditioned by nutrient availability. The identification and selection of the most effective species could be an interesting topic also for small and medium enterprises, for the production of inocula especially for the reclamation of soils. More information on the Genomyca -project can be found at: http://www.dijon.inra.fr/genomyca/

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