Identificazione, tramite approcci molecolari, di geni implicati nella tolleranza allo stress ossidativo nel fungo micorrizico Oidiodendron maius

Due to increasing anthropogenic activities, large areas are highly contaminated by heavy metals which are affecting biological systems. Oidiodendron maius strain Zn could be an interesting organism in a bioremediation program being both an ericoid mycorrhizal fungus and a heavy metal-tolerant strain. To understand the mechanisms underlying the oxidative stress tolerance of this strain, three different approaches were used. The first approach allowed us to obtain superoxide dismutase 1 (SOD1) null mutants. The most important technical advance in this work was the first successful disruption of a gene by homologous recombination in a mycorrhizal fungus. We demonstrate that the lack of OmSOD may cause an imbalance in the redox homeostasis and an alteration in the delicate dialogue between the fungus and its host plant. The second approach was based on a yeast functional complementation screening using an O. maius cDNA library. In this work we report the first transporters of an ericoid mycorrhizal fungus capable of conferring Zn tolerance to yeast transformants. Two full-length cDNAs were isolated and named OmCDF and OmFET. The heterologous expression of these two genes in various yeast mutants conferred resistance to zinc. Additionally, OmCDF expression also conferred Co tolerance. We provide evidence that OmCDF functions as a Zn transporter responsible for relocating cytoplasmic Zn into the endoplasmic reticulum, whereas expression of OmFET could counteract Zn toxicity by increasing Fe content of cells. The third approach consisted in the screening of a collection of O. maius random-mutants on Zn, Cd and menadione. We report the characterization of an O. maius-mutant that carries a mutation in the nmr gene. In this mutant, a decrease of glutamine and asparagine pools, and a reduction of the activity of glutamine synthase were recorded. Possible links between the oxidative stress tolerance and the nitrogen metabolism are discussed.En raison des activités anthropiques croissantes, de larges sites sont contaminés par les métaux lourds qui affectent les systèmes biologiques. La souche Oidiodendron maius Zn pourrait être un organisme intéressant dans un programme de bioremédiation étant à la fois un champignon mycorhizien éricoïde et une souche tolérante aux métaux lourds. Pour comprendre les mécanismes de la tolérance de cette souche, trois approches différentes ont été menées.La première approche a abouti à la génération de mutants du gène superoxyde dismutase 1 (OmSOD1). Il s'agit de la première délétion d'un gène par recombinaison homologue chez un champignon mycorhizien. Nous démontrons que l'absence d'OmSOD1 cause un déséquilibre dans l'homéostasie rédox et un changement dans le dialogue entre le champignon et sa plante hôte.La deuxième approche a été basée sur la complémentation fonctionnelle d'un mutant de levure en utilisant une banque d'ADNc d'O. maius. Nous décrivons les premiers transporteurs d'un champignon mycorhizien éricoïde capables de conférer la tolérance au Zn dans des levures. Deux gènes ont été isolés et nommés OmCDF et OmFET. L'expression hétérologue de ces deux gènes dans différents mutants de levure a permis de conférer la tolérance au Zn. De plus, OmCDF a également permis de conférer la tolérance au Co. Nos données suggérent que OmCDF est un transporteur de Zn responsable du transfert du Zn cytoplasmique vers le réticulum endoplasmique, tandis que l'expression d'OmFET pourrait neutraliser la toxicité engendrée par le Zn en augmentant le contenu du Fe dans la cellule. La troisième approche a concerné le criblage d'une collection de mutants aléatoires d'O. maius sur Zn, Cd et ménadione. Nous décrivons la caractérisation d'un mutant dans le gène nmr. Dans ce mutant, une diminution de la teneur en glutamine et asparagine, ainsi qu'une réduction de l'activité de la glutamine synthétase ont été enregistrées. Les liens possibles entre la tolérance au stress oxydatif et le métabolisme azoté sont discutés

Molecular approaches to study oxidative stress tolerance mechanisms in the ericoid mycorrhizal fungus Oidiodendron maius

En raison des activités anthropiques croissantes, de larges sites sont contaminés par les métaux lourds qui affectent les systèmes biologiques. La souche Oidiodendron maius Zn pourrait être un organisme intéressant dans un programme de bioremédiation étant à la fois un champignon mycorhizien éricoïde et une souche tolérante aux métaux lourds. Pour comprendre les mécanismes de la tolérance de cette souche, trois approches différentes ont été menées.La première approche a abouti à la génération de mutants du gène superoxyde dismutase 1 (OmSOD1). Il s'agit de la première délétion d'un gène par recombinaison homologue chez un champignon mycorhizien. Nous démontrons que l'absence d'OmSOD1 cause un déséquilibre dans l'homéostasie rédox et un changement dans le dialogue entre le champignon et sa plante hôte.La deuxième approche a été basée sur la complémentation fonctionnelle d'un mutant de levure en utilisant une banque d'ADNc d'O. maius. Nous décrivons les premiers transporteurs d'un champignon mycorhizien éricoïde capables de conférer la tolérance au Zn dans des levures. Deux gènes ont été isolés et nommés OmCDF et OmFET. L'expression hétérologue de ces deux gènes dans différents mutants de levure a permis de conférer la tolérance au Zn. De plus, OmCDF a également permis de conférer la tolérance au Co. Nos données suggérent que OmCDF est un transporteur de Zn responsable du transfert du Zn cytoplasmique vers le réticulum endoplasmique, tandis que l'expression d'OmFET pourrait neutraliser la toxicité engendrée par le Zn en augmentant le contenu du Fe dans la cellule. La troisième approche a concerné le criblage d'une collection de mutants aléatoires d'O. maius sur Zn, Cd et ménadione. Nous décrivons la caractérisation d'un mutant dans le gène nmr. Dans ce mutant, une diminution de la teneur en glutamine et asparagine, ainsi qu'une réduction de l'activité de la glutamine synthétase ont été enregistrées. Les liens possibles entre la tolérance au stress oxydatif et le métabolisme azoté sont discutés.Due to increasing anthropogenic activities, large areas are highly contaminated by heavy metals which are affecting biological systems. Oidiodendron maius strain Zn could be an interesting organism in a bioremediation program being both an ericoid mycorrhizal fungus and a heavy metal-tolerant strain. To understand the mechanisms underlying the oxidative stress tolerance of this strain, three different approaches were used. The first approach allowed us to obtain superoxide dismutase 1 (SOD1) null mutants. The most important technical advance in this work was the first successful disruption of a gene by homologous recombination in a mycorrhizal fungus. We demonstrate that the lack of OmSOD may cause an imbalance in the redox homeostasis and an alteration in the delicate dialogue between the fungus and its host plant. The second approach was based on a yeast functional complementation screening using an O. maius cDNA library. In this work we report the first transporters of an ericoid mycorrhizal fungus capable of conferring Zn tolerance to yeast transformants. Two full-length cDNAs were isolated and named OmCDF and OmFET. The heterologous expression of these two genes in various yeast mutants conferred resistance to zinc. Additionally, OmCDF expression also conferred Co tolerance. We provide evidence that OmCDF functions as a Zn transporter responsible for relocating cytoplasmic Zn into the endoplasmic reticulum, whereas expression of OmFET could counteract Zn toxicity by increasing Fe content of cells. The third approach consisted in the screening of a collection of O. maius random-mutants on Zn, Cd and menadione. We report the characterization of an O. maius-mutant that carries a mutation in the nmr gene. In this mutant, a decrease of glutamine and asparagine pools, and a reduction of the activity of glutamine synthase were recorded. Possible links between the oxidative stress tolerance and the nitrogen metabolism are discussed.NANCY1-Bib. numérique (543959902) / SudocSudocFranceF

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

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The Hydrophobin-Like OmSSP1 May Be an Effector in the Ericoid Mycorrhizal Symbiosis

Mutualistic and pathogenic plant-colonizing fungi use effector molecules to manipulate the host cell metabolism to allow plant tissue invasion. Some small secreted proteins (SSPs) have been identified as fungal effectors in both ectomycorrhizal and arbuscular mycorrhizal fungi, but it is currently unknown whether SSPs also play a role as effectors in other mycorrhizal associations. Ericoid mycorrhiza is a specific endomycorrhizal type that involves symbiotic fungi mostly belonging to the Leotiomycetes (Ascomycetes) and plants in the family Ericaceae. Genomic and RNASeq data from the ericoid mycorrhizal fungus Oidiodendron maius led to the identification of several symbiosis-upregulated genes encoding putative SSPs. OmSSP1, the most highly symbiosis up-regulated SSP, was found to share some features with fungal hydrophobins, even though it lacks the Pfam hydrophobin domain. Sequence alignment with other hydrophobins and hydrophobin-like fungal proteins placed OmSSP1 within Class I hydrophobins. However, the predicted features of OmSSP1 may suggest a distinct type of hydrophobin-like proteins. The presence of a predicted signal peptide and a yeast-based signal sequence trap assay demonstrate that OmSSP1 is secreted. OmSSP1 null-mutants showed a reduced capacity to form ericoid mycorrhiza with Vaccinium myrtillus roots, suggesting a role as effectors in the ericoid mycorrhizal interaction

Table_4_The Hydrophobin-Like OmSSP1 May Be an Effector in the Ericoid Mycorrhizal Symbiosis.xlsx

<p>Mutualistic and pathogenic plant-colonizing fungi use effector molecules to manipulate the host cell metabolism to allow plant tissue invasion. Some small secreted proteins (SSPs) have been identified as fungal effectors in both ectomycorrhizal and arbuscular mycorrhizal fungi, but it is currently unknown whether SSPs also play a role as effectors in other mycorrhizal associations. Ericoid mycorrhiza is a specific endomycorrhizal type that involves symbiotic fungi mostly belonging to the Leotiomycetes (Ascomycetes) and plants in the family Ericaceae. Genomic and RNASeq data from the ericoid mycorrhizal fungus Oidiodendron maius led to the identification of several symbiosis-upregulated genes encoding putative SSPs. OmSSP1, the most highly symbiosis up-regulated SSP, was found to share some features with fungal hydrophobins, even though it lacks the Pfam hydrophobin domain. Sequence alignment with other hydrophobins and hydrophobin-like fungal proteins placed OmSSP1 within Class I hydrophobins. However, the predicted features of OmSSP1 may suggest a distinct type of hydrophobin-like proteins. The presence of a predicted signal peptide and a yeast-based signal sequence trap assay demonstrate that OmSSP1 is secreted. OmSSP1 null-mutants showed a reduced capacity to form ericoid mycorrhiza with Vaccinium myrtillus roots, suggesting a role as effectors in the ericoid mycorrhizal interaction.</p

Data_Sheet_1_The Hydrophobin-Like OmSSP1 May Be an Effector in the Ericoid Mycorrhizal Symbiosis.docx

<p>Mutualistic and pathogenic plant-colonizing fungi use effector molecules to manipulate the host cell metabolism to allow plant tissue invasion. Some small secreted proteins (SSPs) have been identified as fungal effectors in both ectomycorrhizal and arbuscular mycorrhizal fungi, but it is currently unknown whether SSPs also play a role as effectors in other mycorrhizal associations. Ericoid mycorrhiza is a specific endomycorrhizal type that involves symbiotic fungi mostly belonging to the Leotiomycetes (Ascomycetes) and plants in the family Ericaceae. Genomic and RNASeq data from the ericoid mycorrhizal fungus Oidiodendron maius led to the identification of several symbiosis-upregulated genes encoding putative SSPs. OmSSP1, the most highly symbiosis up-regulated SSP, was found to share some features with fungal hydrophobins, even though it lacks the Pfam hydrophobin domain. Sequence alignment with other hydrophobins and hydrophobin-like fungal proteins placed OmSSP1 within Class I hydrophobins. However, the predicted features of OmSSP1 may suggest a distinct type of hydrophobin-like proteins. The presence of a predicted signal peptide and a yeast-based signal sequence trap assay demonstrate that OmSSP1 is secreted. OmSSP1 null-mutants showed a reduced capacity to form ericoid mycorrhiza with Vaccinium myrtillus roots, suggesting a role as effectors in the ericoid mycorrhizal interaction.</p

Table_3_The Hydrophobin-Like OmSSP1 May Be an Effector in the Ericoid Mycorrhizal Symbiosis.XLSX

<p>Mutualistic and pathogenic plant-colonizing fungi use effector molecules to manipulate the host cell metabolism to allow plant tissue invasion. Some small secreted proteins (SSPs) have been identified as fungal effectors in both ectomycorrhizal and arbuscular mycorrhizal fungi, but it is currently unknown whether SSPs also play a role as effectors in other mycorrhizal associations. Ericoid mycorrhiza is a specific endomycorrhizal type that involves symbiotic fungi mostly belonging to the Leotiomycetes (Ascomycetes) and plants in the family Ericaceae. Genomic and RNASeq data from the ericoid mycorrhizal fungus Oidiodendron maius led to the identification of several symbiosis-upregulated genes encoding putative SSPs. OmSSP1, the most highly symbiosis up-regulated SSP, was found to share some features with fungal hydrophobins, even though it lacks the Pfam hydrophobin domain. Sequence alignment with other hydrophobins and hydrophobin-like fungal proteins placed OmSSP1 within Class I hydrophobins. However, the predicted features of OmSSP1 may suggest a distinct type of hydrophobin-like proteins. The presence of a predicted signal peptide and a yeast-based signal sequence trap assay demonstrate that OmSSP1 is secreted. OmSSP1 null-mutants showed a reduced capacity to form ericoid mycorrhiza with Vaccinium myrtillus roots, suggesting a role as effectors in the ericoid mycorrhizal interaction.</p

Comparative genomics and transcriptomics depict ericoid mycorrhizal fungi as versatile saprotrophs and plant mutualists

Some soil fungi in the Leotiomycetes form ericoid mycorrhizal (ERM) symbioses with Ericaceae. In the harsh habitats in which they occur, ERM plant survival relies on nutrient mobilization from soil organic matter (SOM) by their fungal partners. The characterization of the fungal genetic machinery underpinning both the symbiotic lifestyle and SOM degradation is needed to understand ERM symbiosis functioning and evolution, and its impact on soil carbon (C) turnover. We sequenced the genomes of the ERM fungi Meliniomyces bicolor, M. variabilis, Oidiodendron maius and Rhizoscyphus ericae, and compared their gene repertoires with those of fungi with different lifestyles (ecto- and orchid mycorrhiza, endophytes, saprotrophs, pathogens). We also identified fungal transcripts induced in symbiosis. The ERM fungal gene contents for polysaccharide-degrading enzymes, lipases, proteases and enzymes involved in secondary metabolism are closer to those of saprotrophs and pathogens than to those of ectomycorrhizal symbionts. The fungal genes most highly upregulated in symbiosis are those coding for fungal and plant cell wall-degrading enzymes (CWDEs), lipases, proteases, transporters and mycorrhiza-induced small secreted proteins (MiSSPs). The ERM fungal gene repertoire reveals a capacity for a dual saprotrophic and biotrophic lifestyle. This may reflect an incomplete transition from saprotrophy to the mycorrhizal habit, or a versatile life strategy similar to fungal endophytes