Stefania Daghino

Bioweathering of chrysotile by fungi isolated in ophiolitic sites

Asbestos minerals are commonly found in serpentine rocks and because of the hazard to human health, research has recently focused on possible detoxification strategies. Some fungal species that inhabit serpentine sites (two disused chrysotile asbestos mines in the Western Alps) have been isolated and characterized in order to obtain data on their biodiversity and bioweathering abilities on chrysotile fibres. The three dominant species (Verticillium leptobactrum, Paecilomyces lilacinus and Aspergillus fumigatus) have proved to be able to actively remove iron from chrysotile fibres, V. leptobactrum being the most efficient. A wide range of serpentinicolous fungi release siderophores, iron-chelating compounds, that could play a role in iron extraction from fibres. Iron removal had been correlated previously with a decrease in the toxic potential of fibres, and a biotechnological application of fungi can be envisaged for asbestos detoxification.

Fungal Diversity Is Not Determined by Mineral and Chemical Differences in Serpentine Substrates

The physico-chemical properties of serpentine soils lead to strong selection of plant species. Whereas many studies have described the serpentine flora, little information is available on the fungal communities dwelling in these sites. Asbestos minerals, often associated with serpentine rocks, can be weathered by serpentine-isolated fungi, suggesting an adaptation to this substrate. In this study, we have investigated whether serpentine substrates characterized by the presence of rocks with distinct mineral composition could select for different fungal communities. Both fungal isolation and 454 pyrosequencing of amplicons obtained from serpentine samples following direct DNA extraction revealed some fungal taxa shared by the four ophiolitic substrates, but also highlighted several substrate-specific taxa. Bootstrap analysis of 454 OTU abundances indicated weak clustering of fungal assemblages from the different substrates, which did not match substrate classification based on exchangeable macronutrients and metals. Intra-substrate variability, as assessed by DGGE profiles, was similar across the four serpentine substrates, and comparable to inter-substrate variability. These findings indicate the absence of a correlation between the substrate (mineral composition and available cations) and the diversity of the fungal community. Comparison of culture-based and culture-independent methods supports the higher taxonomic precision of the former, as complementation of the better performance of the latter.

Weathering of chrysotile asbestos by the serpentine rock-inhabiting fungus Verticillium leptobactrum

Verticillium leptobactrum, a rare fungal species, has repeatedly been isolated from serpentinic rocks in the Western Alps, thus suggesting that it adapts easily to this selective mineral substrate. The rRNA internal transcribed spacer region of several isolates has been sequenced to confirm their identity and taxonomic position within Verticillium, a recently revised polyphyletic entity. Isolates of V. leptobactrum have also been investigated to establish their ability to weather asbestos chrysotile, the most common mineral in the isolation sites. The results of solubilization assays on magnesium and silicon, as well as measurement of the Mg/Si ratio in the asbestos fibres after exposure to fungal mycelia, indicate a high bioweathering activity of V. leptobactrum towards chrysotile. Comparison with data on Fusarium oxysporum shows differences among species, with V. leptobactrum being more active than F. oxysporum in removing structural ions from chrysotile. Asbestos weathering by fungi could be envisaged as a bioremediation strategy for hazardous asbestos-rich soils (e.g. abandoned mines). Fungi that have adapted to live in serpentine sites could be good candidates for this purpose.

OmZnT1 and OmFET, two metal transporters from the metal-tolerant strain Zn of the ericoid mycorrhizal fungus Oidiodendron maius, confer zinc tolerance in yeast.

Two full-length cDNAs (OmZnT1 and OmFET) encoding membrane transporters were identified by yeast functional screening in the heavy metal tolerant ericoid mycorrhizal isolate Oidiodendron maius Zn. OmZnT1 belongs to the Zn-Type subfamily of the cation diffusion facilitators, whereas OmFET belongs to the family of iron permeases. Their properties were investigated in yeast by functional complementation of mutants affected in metal uptake and metal tolerance. Heterologous expression of OmZnT1 and OmFET in a Zn-sensitive yeast mutant restored the wild-type phenotype. Additionally, OmZnT1 expression also restored cobalt tolerance in a Co-sensitive mutant. A GFP fusion protein revealed that OmZnT1 was targeted to the endoplasmic reticulum membrane, a result consistent with a function for OmZnT1 in metal sequestration. Similarly to other iron permeases, OmFET-GFP was localized on the plasma membrane. OmFET restored the growth of uptake-defective strains for iron and zinc. Zinc-sensitive yeast mutants expressing OmFET specifically accumulated magnesium, as compared to cells transformed with the empty vector. We suggest that OmFET may counteract zinc toxicity by increasing entry of magnesium into the cell.

Cellular response of Fusarium oxysporum to crocidolite asbestos as revealed by a combined proteomic approach.

Cellular mechanisms of asbestos toxicity rely, at least in part, on the chemical composition of these minerals. Iron ions are directly involved in the accepted mechanism of fiber toxicity because they constitute active centers where release of free radicals and reactive oxygen species takes place. Although no current technology is available for the remediation of asbestos polluted sites, the soil fungus Fusarium oxysporum was found to be very effective in iron extraction from crocidolite asbestos in vitro, and to cause a significant reduction in asbestos surface reactivity and oxidative damage to naked DNA. As little information is available on the molecular mechanisms of the fungus-asbestos interactions, a combined proteomic approach that used 2-DE, shotgun and quantitative iTRAQ proteomics was used to investigate the fungal metabolic activities in the presence of crocidolite, an iron-rich type of asbestos. Although global proteomic analyses did not show significant changes in the protein expression pattern of F. oxysporum when exposed to asbestos fibers, some proteins specifically regulated by asbestos suggest up-regulation of metabolic pathways involved in protection from oxidative stress. When compared with the response to crocidolite observed by other authors in human lung epithelial cells, that unlike fungi can internalize the asbestos fibres, a significant difference was the regulation of the pentose phosphate pathway.

Soil metaproteomics reveals an inter-kingdom stress response to the presence of black truffles

For some truffle species of the Tuber genus, the symbiotic phase is often associated with the presence of an area of scant vegetation, commonly known as the brûlé, around the host tree. Previous metagenomics studies have identified the microorganisms present inside and outside the brûlé of a Tuber melanosporum truffle-ground, but the molecular mechanisms that operate in this ecological niche remain to be clarified. To elucidate the metabolic pathways present in the brûlé, we conducted a metaproteomics analysis on the soil of a characterized truffle-ground and cross-referenced the resulting proteins with a database we constructed, incorporating the metagenomics data for the organisms previously identified in this soil. The soil inside the brûlé contained a larger number of proteins and, surprisingly, more proteins from plants, compared with the soil outside the brûlé. In addition, Fisher’s Exact Tests detected more biological processes inside the brûlé; these processes were related to responses to multiple types of stress. Thus, although the brûlé has a reduced diversity of plant and microbial species, the organisms in the brûlé show strong metabolic activity. Also, the combination of metagenomics and metaproteomics provides a powerful tool to reveal soil functioning.

Model systems to unravel the molecular mechanisms of heavy metal tolerance in the ericoid mycorrhizal symbiosis

Ericoid mycorrhizal plants dominate in harsh environments where nutrient-poor, acidic soil conditions result in a higher availability of potentially toxic metals. Although metal-tolerant plant species and ecotypes are known in the Ericaceae, metal tolerance in these plants has been mainly attributed to their association with ericoid mycorrhizal fungi. The mechanisms underlying plant protection by the fungal symbiont are poorly understood, whereas some insights have been achieved regarding the molecular mechanisms of heavy metal tolerance in the fungal symbiont. This review will briefly introduce the general features of heavy metal tolerance in mycorrhizal fungi and will then focus on the use of “omics” approaches and heterologous expression in model organisms to reveal the molecular bases of fungal response to heavy metals. Functional complementation in Saccharomyces cerevisiae has allowed the identification of several ericoid mycorrhizal fungi genes (i.e., antioxidant enzymes, metal transporters, and DNA damage repair proteins) that may contribute to metal tolerance in a metal-tolerant ericoid Oidiodendron maius isolate. Although a powerful system, the use of the yeast complementation assay to study metal tolerance in mycorrhizal symbioses has limitations. Thus, O. maius has been developed as a model system to study heavy metal tolerance mechanisms in mycorrhizal fungi, thanks to its high metal tolerance, easy handling and in vitro mycorrhization, stable genetic transformation, genomics, transcriptomic and proteomic resources.

RiPEIP1, a gene from the arbuscular mycorrhizal fungus Rhizophagus irregularis, is preferentially expressed in planta and may be involved in root colonization

Transcriptomics and genomics data recently obtained from the arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis have offered new opportunities to decipher the contribution of the fungal partner to the establishment of the symbiotic association. The large number of genes which do not show similarity to known proteins witnesses the uniqueness of this group of plant-associated fungi. In this work, we characterize a gene that was called RiPEIP1 (Preferentially Expressed In Planta). Its expression is strongly induced in the intraradical phase, including arbuscules, and follows the expression profile of the Medicago truncatula phosphate transporter MtPT4, a molecular marker of a functional symbiosis. Indeed, mtpt4 mutant plants, which exhibit low mycorrhizal colonization and an accelerated arbuscule turnover, also show a reduced RiPEIP1 mRNA abundance. To further characterize RiPEIP1, in the absence of genetic transformation protocols for AM fungi, we took advantage of two different fungal heterologous systems. When expressed as a GFP fusion in yeast cells, RiPEIP1 localizes in the endomembrane system, in particular to the endoplasmic reticulum, which is consistent with the in silico prediction of four transmembrane domains. We then generated RiPEIP1-expressing strains of the fungus Oidiodendron maius, ericoid endomycorrhizal fungus for which transformation protocols are available. Roots of Vaccinium myrtillus colonized by RiPEIP1-expressing transgenic strains showed a higher mycorrhization level compared to roots colonized by the O. maius wild-type strain, suggesting that RiPEIP1 may regulate the root colonization process.

A PLAC8-containing protein from an endomycorrhizal fungus confers cadmium resistance to yeast cells by interacting with Mlh3p

Cadmium is a genotoxic pollutant known to target proteins that are involved in DNA repair and in antioxidant defence, altering their functions and ultimately causing mutagenic and carcinogenic effects. We have identified a PLAC8 domain-containing protein, named OmFCR, by a yeast functional screen aimed at identifying genes involved in cadmium resistance in the endomycorrhizal fungus Oidiodendron maius. OmFCR shows a remarkable specificity in mediating cadmium resistance. Both its function and its nuclear localization in yeast strictly depend on the interaction with Mlh3p, a subunit of the mismatch repair (MMR) system. Although proteins belonging to the PLAC8 family are widespread in eukaryotes, they are poorly characterized and their biological role still remains elusive. Our work represents the first report about the potential role of a PLAC8 protein in physically coupling DNA lesion recognition by the MMR system to appropriate effectors that affect cell cycle checkpoint pathways. On the basis of cell survival assays and yeast growth curves, we hypothesize that, upon cadmium exposure, OmFCR might promote a higher rate of cell division as compared to control cells.

OmGOGAT-disruption in the ericoid mycorrhizal fungus Oidiodendron maius induces reorganization of the N pathway and reduces tolerance to heavy-metals

Mycorrhizal fungi are key mediators of soil-to-plant movement of mineral nutrients, including essential and non-essential metals. In soil conditions that facilitate mobilization of metal ions, potentially toxic metals can interfere with nitrogen metabolism in both plants and microorganisms. Less is known about possible relationships between nitrogen metabolism and responses to heavy metals. Aim of this study was to investigate this aspect in the ericoid mycorrhizal fungus Oidiodendron maius strain Zn, a metal tolerant ascomycete. Growth of O. maius Zn on zinc and cadmium containing media was significantly affected by the nitrogen source. Screening of a library of O. maius Zn random genetic transformants for sensitivity to heavy metals (zinc and cadmium) and oxidative stress (menadione) yielded a mutant strain that carried a partial deletion of the glutamate synthase (NADH-GOGAT EC 1.4.1.14) gene and its adjacent gene, the APC15 subunit of the anaphase promoting complex. Comparison of WT and OmGOGAT-OmAPC15 mutant strains indicated an impaired N-metabolism and altered stress tolerance, and assays on the OmAPC15-recomplemented strains ascribed the observed phenotypes to the deletion in the OmGOGAT gene. OmGOGAT disruption modified the nitrogen pathway, with a strong reduction of the associated glutamine synthetase (GS, EC 6.3.1.2) activity and an up-regulation of the alternative NADP-glutamate dehydrogenase (NADP-GDH, EC 1.4.1.4) pathway for glutamate biosynthesis. Unless they were supplemented with glutamine, O. maius Zn transformants lacking OmGOGAT were very sensitive to zinc. These results highlight the importance of nitrogen metabolism not only for nitrogen assimilation and transformation, but also for stress tolerance. For mycorrhizal fungi, such as O. maius, this may bear consequences not only to the fungus, but also to the host plant.