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.     

Plant signals and fungal perception during arbuscular mycorrhiza establishment

Arbuscular mycorrhizal (AM) fungi are obligate symbionts that need their plant hosts to complete their life cycle. In the absence of the plant, germlings arrest growth after a few days and retract most of their cytoplasm back into the multinuclear spores. The spores can germinate again during more favorable conditions. How AM fungi recognize compatible host roots and activate their symbiotic program is not yet understood. However, research in this field in the last years has shed light into this topic. We, and others, have approached some of these aspects by studying changes in fungal gene expression observed at early stages of development, before and at the plant recognition stage in an attempt to identify genes and proteins featuring as key regulators in the switch between the asymbiotic and symbiotic style of life. The molecular bases of this recognition process are now starting to be understood and point to common signaling pathways shared with other microbe-plant associations and to arbuscular mycorrhiza specific signaling pathways.     

Identification of heavy metal-induced genes encoding glutathione S-transferases in the arbuscular mycorrhizal fungus Glomus intraradices

Arbuscular mycorrhizal fungi are able to alleviate the stress for plants caused by heavy metal contamination of soil. To analyze the molecular response of arbuscular mycorrhizal fungi to these pollutants, a subtractive cDNA library was constructed using RNA from Glomus intraradices extraradical hyphae of a root organ culture treated with a mixture of Cd, Zn, and Cu. Screening by reverse Northern blot analysis indicated that, among 308 clones, 17% correspond to genes up-regulated by heavy metals. Sequence analysis of part of the clones resulted, amongst others, in the identification of six genes putatively coding for glutathione S-transferases belonging to two different classes of these enzymes. Expression analyses indicated that the genes are differentially expressed during fungal development and that their RNA accumulation dramatically increases in extraradical hyphae grown in a heavy metal-containing solution.     

A Medicago truncatula mutant hyper-responsive to mycorrhiza and defective for nodulation

One key strategy for the identification of plant genes required for mycorrhizal development is the use of plant mutants affected in mycorrhizal colonisation. In this paper, we report a new Medicago truncatula mutant defective for nodulation but hypermycorrhizal for symbiosis development and response. This mutant, called B9, presents a poor shoot and, especially, root development with short laterals. Inoculation with Glomus intraradices results in significantly higher root colonisation of the mutant than the wild-type genotype A17 (+20% for total root length, +16% for arbuscule frequency in the colonised part of the root, +39% for arbuscule frequency in the total root system). Mycorrhizal effects on shoot and root biomass of B9 plants are about twofold greater than in the wild-type genotype. The B9 mutant of M. truncatula is characterised by considerably higher root concentrations of the phytoestrogen coumestrol and by the novel synthesis of the coumestrol conjugate malonyl glycoside, absent from roots of wild-type plants. In conclusion, this is the first time that a hypermycorrhizal plant mutant affected negatively for nodulation (Myc⁺⁺, Nod ^−/+ phenotype) is reported. This mutant represents a new tool for the study of plant genes differentially regulating mycorrhiza and nodulation symbioses, in particular, those related to autoregulation mechanisms.     

Effects of anthracene on development of an arbuscular mycorrhizal fungus and contribution of the symbiotic association to pollutant dissipation

The influence of anthracene, a low molecular weight polycyclic aromatic hydrocarbon (PAH), on chicory root colonization by Glomus intraradices and the effect of the root colonization on PAH degradation were investigated in vitro. The fungus presented a reduced development of extraradical mycelium and a decrease in sporulation, root colonization, and spore germination when exposed to anthracene. Mycorrhization improved the growth of the roots in the medium supplemented containing 140 mg l⁻¹ anthracene, suggesting a positive contribution of G. intraradices to the PAH tolerance of roots. Anthracene disappearance from the culture medium was quantified; results suggested that nonmycorrhizal chicory roots growing in vitro were able to contribute to anthracene dissipation, and in addition, that mycorrhization significantly enhanced anthracene dissipation. These monoxenic experiments demonstrated a positive contribution of the symbiotic association to anthracene dissipation in the absence of other microorganisms. In addition to anthracene dissipation, intracellular accumulation of anthracene was detected in lipid bodies of plant cells and fungal hyphae, indicating intracellular storage capacity of the pollutant by the roots and the mycorrhizal fungus.     

Photochemical processes, carbon assimilation and RNA accumulation of sucrose transporter genes in tomato arbuscular mycorrhiza

Arbuscular mycorrhizal fungi enhance CO₂ assimilation of their hosts which ensure the demand for carbohydrates of these obligate biotrophic microorganisms. Photosynthetic parameters were measured in tomato colonised or not by the arbuscular mycorrhizal fungus Glomus mosseae. In addition, carbohydrate contents and mRNA accumulation of three sucrose transporter genes were analysed. Mycorrhizal plants showed increased opening of stomata and assimilated significant more CO₂. A higher proportion of the absorbed light was used for photochemical processes, while non-photochemical quenching and the content of photoprotective pigments were lower. Analysis of sugar contents showed no significant differences in leaves but enhanced levels of sucrose and fructose in roots, while glucose amounts stayed constant. The three sucrose transporter encoding genes of tomato SlSUT1, SlSUT2 and SlSUT4 were up-regulated providing transport capacities to transfer sucrose into the roots. It is proposed that a significant proportion of sugars is used by the mycorrhizal fungus, because only amounts of fructose were increased, while levels of glucose, which is mainly transferred towards the fungus, were nearly constant.     

Alternative oxidase 1 (Aox1) gene expression in roots of Medicago truncatula is a genotype-specific component of salt stress tolerance

Alternative oxidase (AOX) is the central component of the non-phosphorylating alternative respiratory pathway in plants and may be important for mitochondrial function during environmental stresses. Recently it has been proposed that Aox can be used as a functional marker for breeding stress tolerant plant varieties. This requires characterization of Aox alleles in plants with different degree of tolerance in a certain stress, affecting plant phenotype in a recognizable way. In this study we examined Aox1 gene expression levels in Medicago truncatula genotypes differing in salt stress tolerance, in order to uncover any correlation between Aox expression and tolerance to salt stress. Results demonstrated a specific induction of Aox1 gene expression in roots of the tolerant genotype that presented the lowest modulation in phenotypic and biochemical stress indices such as morphologic changes, protein level, lipid peroxidation and ROS generation. Similarly, in a previous study we reported that induction of antioxidant gene expression in the tolerant genotype contributed to the support of the antioxidant cellular machinery and stress tolerance. Correlation between expression patterns of the two groups of genes was revealed mainly in 48 h treated roots. Taken together, results from both experiments suggest that M. truncatula tolerance to salt stress may in part due to an efficient control of oxidative balance thanks to (i) induction of antioxidant systems and (ii) involvement of the AOX pathway. This reinforces the conclusion that differences in antioxidant mechanisms can be essential for salt stress tolerance in M. truncatula and possibly the corresponding genes, especially Aox, could be utilized as functional marker.     

Aquaporin genes GintAQPF1 and GintAQPF2 from Glomus intraradices contribute to plant drought tolerance

Arbuscular mycorrhizal (AM) symbiosis, established between AM fungi (AMF) and roots of higher plants, occurs in most terrestrial ecosystems. It has been well demonstrated that AM symbiosis can improve plant performance under various environmental stresses, including drought stress. However, the molecular basis for the direct involvement of AMF in plant drought tolerance has not yet been established. Most recently, we cloned two functional aquaporin genes, GintAQPF1 and GintAQPF2, from AM fungus Glomus intraradices. By heterologous gene expression in yeast, aquaporin localization, activities and water permeability were examined. Gene expressions during symbiosis in expose to drought stress were also analyzed. Our data strongly supported potential water transport via AMF to host plants. As a complement, here we adopted the monoxenic culture system for AMF, in which carrot roots transformed by Ri-T DNA were cultured with Glomus intraradices in two-compartment Petri dishes, to verify the aquaporin gene functions in assisting AMF survival under polyethylene glycol (PEG) treatment. Our results showed that 25% PEG significantly upregulated the expression of two aquaporin genes, which was in line with the gene functions examined in yeast. We therefore concluded that the aquaporins function similarly in AMF as in yeast subjected to osmotic stress. The study provided further evidence to the direct involvement of AMF in improving plant water relations under drought stresses.     

Isolation and characterization of a novel plant promoter that directs strong constitutive expression of transgenes in plants

A novel, constitutively expressed gene, designated MtHP, was isolated from the model legume species Medicago truncatula. Sequence analysis indicates that MtHP most likely belongs to the PR10 multi-gene family. The MtHP promoter was fused to a -glucuronidase gene to characterize its expression in different plant species. Transient assay by microprojectile bombardment and hairy root transformation by Agrobacterium rhizogenes revealed GUS expression in leaf, stem, radicle and root in M. truncatula. Detailed analysis in transgenic Arabidopsis plants demonstrated that the promoter could direct transgene expression in different tissues and organs at various developmental stages; its expression pattern was similar to that of CaMV35S promoter, and the level of expression was higher than the reporter gene driven by CaMV35S promoter. Deletion analysis revealed that even a 107 bp fragment of the promoter could still lead to a moderate level of expression. The promoter was further characterized in white clover (Trifolium repens), a widely grown forage legume species. Strong constitutive expression was observed in transgenic white clover plants. Compared with CaMV35S promoter, the level of GUS activity in transgenic white clover was higher when the transgene was driven by MtHP promoter. Thus, the promoter provides a useful alternative to the CaMV35S promoter in plant transformation for high levels of constitutive expression.     

Identification of functional FKB protein in Echinococcus granulosus: Its involvement in the protoscolicidal action of rapamycin derivates and in calcium homeostasis

FK506 (tacrolimus) and polyketide macrolides such as rapamycin and its derivates bind to FK506-binding proteins (FKBPs). These proteins display a peptidyl-prolyl rotamase function that is believed to catalyze protein folding and they are well-validated anti-proliferative drug targets in certain pathogenic microorganisms, and their functions have been characterized in parasitic protozoa. However, much less is known in helminths and trials with rapalogs on cestoda have not yet been reported. Due to a growing need for new treatment options for human cystic echinococcosis, the in vitro efficacy of rapalogs in Echinococcus granulosus was investigated. We determined the effect of ramapycin, FK506 and everolimus against this cestode, demonstrating their protoscolicidal ability. Also, we observed synergic scolicidal actions during combined therapy with rapalogs plus cyclosporine A, proposing dual administration of drugs to improve pharmacological effects in vivo. We have identified an E. granulosus (Eg)-fkb1 gene that encodes Eg-FKBP, an archetypal protein of the FKBP family, which includes all residues implicated in the binding of pharmacological ligands, in the enzymatic activity and in interactions with possible target proteins. Levels of Eg-fkb1 mRNA are over-expressed by acid but not rapalog treatment. We also described the presence of receptor-operated calcium channels in the larval stage, suggesting that exogenous ligands may dissociate the interaction of Eg-FKBP from these intracellular channels, enhancing the activity of the Ca²⁺ release and interfering with their normal regulatory functions. As rapamycin sensitivity is the major criterion used to detect targets of rapamycin kinase, we identified and analyzed in silico critical residues of putative homologs in the Echinococcus genome. These preliminary results will allow us to continue subsequent studies that could reveal the precise intracellular functions of Eg-FKBP, providing greater knowledge for further identification of downstream target proteins, a promising target for chemotherapy of cystic echinococcosis.     

Differential effects of fenpropimorph and fenhexamid, two sterol biosynthesis inhibitor fungicides, on arbuscular mycorrhizal development and sterol metabolism in carrot roots

Sterols composition of transformed carrot roots incubated in presence of increasing concentrations of fenpropimorph (0.02; 0.2; 2 mg l⁻¹) and fenhexamid (0.02; 0.2; 2; 20 mg l⁻¹), colonized or not by Glomus intraradices was determined. In mycorrhizal roots treated with fenpropimorph, normal Δ⁵-sterols were replaced by unusual compounds such as 9β,19-cyclopropylsterols (24-methylpollinastanol), Δ^8,14-sterols (ergosta-8,14-dienol, stigmasta-8,14-dienol), Δ⁸-sterols (Δ⁸ sitosterol) and Δ⁷-sterols (ergosta-7,22-dienol). After application of fenpropimorph, a drastic reduction of the mycorrhizal root growth, root colonization and extraradical fungal development was observed. Application of fenhexamid did not modify sterol profiles and the total colonization of roots. But the arbuscule frequency of the fungal partner was significantly affected.Comparison of the effects caused by the tested fungicides indicates that the usual phytosterols may be involved in symbiosis development. Indeed, observed modifications of root sterols composition could explain the high fenpropimorph toxicity to the AM symbiosis. However, the absence of sterolic modifications in the roots treated with fenhexamid could account for its more limited impact on mycorrhization.     

Flavonoids,benzoic acids and cinnamic acids isolated from shoots and roots of Italian rye grass (Lolium multiflorum Lam.) with and without endophyte association and arbuscular mycorrhizal fungus

Phenolic compounds present in Lolium multiflorum Lam. were isolated and characterized. Significant differences in their distribution were found, in shoots and roots of plants with (Lm+) and without (Lm−) endophyte association, grown with (Mic+) and without (Mic−) mycorrhizal fungi Glomus intraradices, indicating a systemic effect of these microorganisms on the phenolics metabolism of L. multiflorum.