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1.
Mycorrhiza ; 31(6): 755-766, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34432129

RESUMO

Ectomycorrhizal fungi contribute to the nutrition of many woody plants, including those in the Pinaceae family. Loblolly pine (Pinus taeda L.), a native species of the Southeastern USA, can be colonized by multiple species of ectomycorrhizal fungi. The role of these symbionts in P. taeda potassium (K+) nutrition has not been previously investigated. Here, we assessed the contribution of four ectomycorrhizal fungi, Hebeloma cylindrosporum, Paxillus ammoniavirescens, Laccaria bicolor, and Suillus cothurnatus, in P. taeda K+ acquisition under different external K+ availabilities. Using a custom-made two-compartment system, P. taeda seedlings were inoculated with one of the four fungi, or kept non-colonized, and grown under K+-limited or -sufficient conditions for 8 weeks. Only the fungi had access to separate compartments in which rubidium, an analog tracer for K+, was supplied before harvest. Resulting effects of the fungi were recorded, including root colonization, biomass, and nutrient concentrations. We also analyzed the fungal performance in axenic conditions under varying supply of K+ and sodium. Our study revealed that these four ectomycorrhizal fungi are differentially affected by external K+ and sodium variations, that they are not able to provide similar benefits to the host P. taeda in our growing conditions, and that rubidium may be used with some limitations to estimate K+ transport from ectomycorrhizal fungi to colonized plants.


Assuntos
Micorrizas , Pinus , Basidiomycota , Hebeloma , Laccaria , Pinus taeda , Potássio , Plântula
2.
PLoS One ; 15(11): e0242739, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33216794

RESUMO

Potassium (K+) acquisition, translocation and cellular homeostasis are mediated by various membrane transport systems in all organisms. We identified and described an ion channel in the ectomycorrhizal fungus Hebeloma cylindrosporum (HcSKC) that harbors features of animal voltage-dependent Shaker-like K+ channels, and investigated its role in both free-living hyphae and symbiotic conditions. RNAi lines affected in the expression of HcSKC were produced and used for in vitro mycorrhizal assays with the maritime pine as host plant, under standard or low K+ conditions. The adaptation of H. cylindrosporum to the downregulation of HcSKC was analyzed by qRT-PCR analyses for other K+-related transport proteins: the transporters HcTrk1, HcTrk2, and HcHAK, and the ion channels HcTOK1, HcTOK2.1, and HcTOK2.2. Downregulated HcSKC transformants displayed greater K+ contents at standard K+ only. In such conditions, plants inoculated with these transgenic lines were impaired in K+ nutrition. Taken together, these results support the hypothesis that the reduced expression of HcSKC modifies the pool of fungal K+ available for the plant and/or affects its symbiotic transfer to the roots. Our study reveals that the maintenance of K+ transport in H. cylindrosporum, through the regulation of HcSKC expression, is required for the K+ nutrition of the host plant.


Assuntos
Proteínas Fúngicas/biossíntese , Regulação Fúngica da Expressão Gênica/fisiologia , Hebeloma/fisiologia , Micorrizas/fisiologia , Pinus , Superfamília Shaker de Canais de Potássio/biossíntese , Simbiose/fisiologia , Pinus/microbiologia , Pinus/fisiologia , Potássio/metabolismo
3.
Mycorrhiza ; 30(6): 735-747, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32820366

RESUMO

While plants mainly rely on the use of inorganic nitrogen sources like ammonium and nitrate, soil-borne microorganisms like the ectomycorrhizal fungus Hebeloma cylindrosporum can also take up soil organic N in the form of amino acids and peptides that they use as nitrogen and carbon sources. Following the previous identification and functional expression in yeast of two PTR-like peptide transporters, the present study details the functions and substrates of HcPTR2A and HcPTR2B by analysing their transport kinetics in Xenopus laevis oocytes. While both transporters mediated high-affinity di- and tripeptide transport, HcPTR2A also showed low-affinity transport of several amino acids-mostly hydrophobic ones with large side chains.


Assuntos
Hebeloma , Proteínas de Membrana Transportadoras , Micorrizas , Regulação Fúngica da Expressão Gênica , Hebeloma/genética , Proteínas de Membrana Transportadoras/genética
4.
Microbiologyopen ; 9(10): e1112, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32852125

RESUMO

l-amino acid oxidases (LAAOs) are flavoenzymes that catalyze the oxidative deamination of l-amino acids to the corresponding α-keto acids, ammonia, and hydrogen peroxide. Here, we show the overexpression, purification, and the characterization of LAAO4 from the fungus Hebeloma cylindrosporum in the yeast Pichia pastoris with a 9His-tag and compare this with the recently characterized 6His-hcLAAO4 expressed in E. coli. The expression of the enzyme with an ER-signal sequence in P. pastoris resulted in a glycosylated, secreted protein. The enzymatic activity without activation was higher after expression in P. pastoris compared to E. coli. Due to treatment with acidic pH, a striking increase of activity could be detected for both expression systems resulting in similar specific activities after acid activation. Regarding the substrate spectrum, temperature stability, Km, and vmax values, hcLAAO4 showed very few differences when produced in these two expression systems. A higher yield of hcLAAO4 could be obtained by fermentation.


Assuntos
Proteínas Fúngicas/genética , Hebeloma/enzimologia , L-Aminoácido Oxidase/genética , Estabilidade Enzimática , Fermentação , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Expressão Gênica , Hebeloma/química , Hebeloma/genética , Cinética , L-Aminoácido Oxidase/química , L-Aminoácido Oxidase/metabolismo , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
5.
J Biotechnol ; 323: 203-207, 2020 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-32653636

RESUMO

The biocatalytic oxidation of amino acids represents an attractive approach towards the synthesis of α-keto acids, which are interest for various industrial applications. As l-amino acids are readily available from fermentation processes, these natural amino acids can serve as substrates in combination with an l-amino acid oxidase. Besides an aqueous phase as reaction medium, a further advantage of such a process is the utilization of air as oxidation agent. In this study, we studied the organic-synthetic properties of a literature-known recombinant l-amino acid oxidase from the fungus Hebeloma cylindrosporum with respect to its suitability to catalyze the formation of α-keto acids exemplified for the synthesis of phenylpyruvic acid starting from l-phenylalanine as a substrate. In our study the enzyme displayed a reasonable operational stability in the reaction system and as well as promising applicability data with respect to substrate and product inhibition. In a biotransformation, 20 mM of substrate were converted after 4 h reaction. The formation of undesired by-products was suppressed using a commercially available catalase enzyme.


Assuntos
Hebeloma/metabolismo , L-Aminoácido Oxidase/metabolismo , Fenilalanina/metabolismo , Ácidos Fenilpirúvicos/metabolismo , Aminoácidos/metabolismo , Biocatálise , Biotransformação , Estabilidade Enzimática , L-Aminoácido Oxidase/genética , Especificidade por Substrato
6.
Mycologia ; 112(1): 172-184, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31900082

RESUMO

Hebeloma parvisporum is described as new and placed within H. sect. Porphyrospora. This mushroom is sold as an edible in markets of Laos under the local name "wai khom." Hebeloma sect. Porphyrospora is discussed and expanded to include the species formerly included in the genus Anamika and recently transferred to Hebeloma. Hebeloma sect. Porphyrospora currently comprises 16 species, 14 of which are known only from the western Pacific and Indian subcontinent. All species in this section share the character of having red-brown spores when fresh, atypical for other sections of Hebeloma, which causes the lamellae to be red-brown. However, this red-brown color fades when the material is dried. The close links, morphologically and molecularly, between H. parvisporum and other members of H. sect. Porphyrospora, particularly H. victoriense, are shown.


Assuntos
Hebeloma/classificação , Agaricales/classificação , Agaricales/citologia , Agaricales/genética , DNA Fúngico/genética , DNA Espaçador Ribossômico/genética , Genes Fúngicos/genética , Hebeloma/citologia , Hebeloma/genética , Laos , Filogenia , Pigmentação , Análise de Sequência de DNA , Esporos Fúngicos/classificação , Esporos Fúngicos/citologia , Esporos Fúngicos/genética
7.
Chemosphere ; 240: 124914, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31557642

RESUMO

Arsenic (As) contamination is one of the most daunting environmental problem bothering the whole world. Exploring a suitable bioremediation technique is an urgent need of the hour. The present study focusses on scrutinizing the ectomycorrhizal (ECM) fungus for its potential role in As detoxification and understanding the molecular mechanisms responsible for its tolerance. When exposed to increasing concentrations of external As, the ECM fungus H. cylindrosporum accumulated the metalloid intracellularly, inducing the glutathione biosynthesis pathway. The genes coding for GSH biosynthesis enzymes, γ-glutamylcysteine synthetase (Hcγ-GCS) and glutathione synthetase (HcGS) were highly regulated by As stress. Arsenic coordinately upregulated the expression of both Hcγ-GCS and HcGS genes, thus resulting in increased Hcγ-GCS and HcGS protein expressions and enzyme activities, with substantial increase in intracellular GSH. Functional complementation of the two genes (Hcγ-GCS and HcGS) in their respective yeast mutants (gsh1Δ and gsh2Δ) further validated the role of both enzymes in mitigating As toxicity. These findings clearly highlight the potential importance of GSH antioxidant defense system in regulating the As induced responses and its detoxification in ECM fungus H. cylindrosporum.


Assuntos
Arsênio/toxicidade , Glutationa/biossíntese , Hebeloma/efeitos dos fármacos , Micorrizas/efeitos dos fármacos , Poluentes do Solo/toxicidade , Antioxidantes/metabolismo , Arsênio/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Teste de Complementação Genética , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Glutationa/metabolismo , Glutationa Sintase/genética , Glutationa Sintase/metabolismo , Hebeloma/genética , Hebeloma/metabolismo , Inativação Metabólica , Mutação , Micorrizas/genética , Micorrizas/metabolismo , Saccharomyces cerevisiae/metabolismo , Poluentes do Solo/metabolismo
8.
Folia Microbiol (Praha) ; 64(6): 835-844, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31372834

RESUMO

Homeostatic mechanisms preventing the toxicity of heavy metal ions in cells involve, among others, compartmentalization and binding with peptidaceous ligands, particularly the cysteinyl-rich metallothioneins (MTs). We have previously shown that in natural conditions Zn-overaccumulating ectomycorrhizal (EM) fungus Russula bresadolae stores nearly 40% of Zn bound with cysteinyl- and hystidyl-containing RaZBP peptides, which resemble MTs, while the detoxification of Zn and Cd in EM Hebeloma mesophaeum relies upon compartmentalization in small vesicles and vacuoles, respectively. Here, we examined the performance of RaZBP1 gene expressed in H. mesophaeum mycelium with respect to handling of Zn and Cd. Expression of RaZBP1 impaired growth of the mycelium on low-Zn medium by 60%, the growth was partly ameliorated upon the addition of Zn and remained considerable up to 2 mmol/L Zn, while the growth of the wild-type and control mycelia transformed with empty T-DNA was severely reduced in the presence of 0.5 mmol/L Zn; furthermore, RaZBP1 slightly added to Cd tolerance in the range of Cd concentrations of 0.625 to 8 µmol/L. Staining of Zn- or Cd-exposed hyphal cells with Zn- or Cd-specific fluorescent tracers did not indicate that the expression of RaZBP1 would redirect the flow of the metals away from their innate sinks. Size exclusion chromatography of extracted metal species revealed that the complexes corresponding to Zn/Cd-RaZBP1 are present only in minute levels. Considering that RaZBP1 inhibited growth at low Zn, and despite the benefit that it provided to H. mesophaeum in the presence of high Zn and moderate Cd, these data indicate that the binding of excess Zn and Cd with RaZBP1 is not a trait that would be outright transmitted to H. mesophaeum.


Assuntos
Cádmio/metabolismo , Proteínas Fúngicas/metabolismo , Hebeloma/metabolismo , Metalotioneína/metabolismo , Zinco/metabolismo , Basidiomycota/genética , Vesículas Citoplasmáticas/metabolismo , Proteínas Fúngicas/genética , Hebeloma/genética , Hebeloma/crescimento & desenvolvimento , Metalotioneína/genética , Micélio/genética , Micélio/crescimento & desenvolvimento , Micélio/metabolismo , Micorrizas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
9.
Chemosphere ; 224: 862-872, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30852466

RESUMO

Ectomycorrhizal (ECM) fungi can improve the growth of pine trees and enhance their tolerance to heavy metal stress, and may also be useful during the afforestation and phytoremediation of polluted regions with pine trees. Hebeloma vinosophyllum (Cr(VI)-sensitive strain) and Pisolithus sp1 ((Cr(VI)-tolerant strain) were selected through liquid culture experiment, and were used in symbiosis with Japanese black pine (Pinus thunbergii) in pot experiments, to determine their potential for improving phytoremediation of Cr(VI)-contaminated soils. Our results indicated that Pisolithus sp1 also had a significantly higher accumulation of Cr than H. vinosophyllum in mycelium under the same Cr(VI) treatments in liquid culture experiment. The tolerance index of Pisolithus sp1 ECM seedlings' shoots and roots to Cr(VI) were significantly higher than that of H. vinosophyllum ECM and non-ectomycorrhizal (NM) seedlings while the total accumulated Cr per seedling in Pisolithus sp1 ECM seedlings were 1.50-1.96 and 2.83-27.75 fold higher that of H. vinosophyllum ECM and NM seedlings, respectively, within 0-800 mg kg-1 Cr(VI) treatments in pot experiments. In addition, the significant differences ratios of photosynthetic rate, stomatal conductance, transpiration rate and intercellular CO2 concentration between Pisolithus sp1 ECM and NM seedlings were significantly higher than those between H. vinosophyllum ECM and NM seedlings under 400 and 800 mg kg-1 Cr(VI) treatments. Compared with the control (no plant), and planting NM or H. vinosophyllum ECM seedlings, the planting of Pisolithus sp1 ECM seedlings significantly reduced the percentage content of exchangeable Cr in the soil.


Assuntos
Biodegradação Ambiental , Cromo/metabolismo , Hebeloma/metabolismo , Micorrizas/metabolismo , Pinus/crescimento & desenvolvimento , Cromo/análise , Micélio/química , Raízes de Plantas/microbiologia , Plântula/crescimento & desenvolvimento , Solo/química
10.
Fungal Biol ; 123(3): 247-254, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30798880

RESUMO

Hebeloma mesophaeum is an ectomycorrhizal fungus frequently associated with metal disturbed environments. In this work, we examined Ag, Cd, and Zn tolerance of H. mesophaeum isolates from heavy metal-polluted (isolate Prib) and clean (isolate Rez) sites. Both mycelia showed essentially the same level of Ag and Zn tolerance, but Prib was more Cd tolerant. In short-term exposures, Prib accumulated slightly less Cd than Rez. Size exclusion chromatography of cell-free extracts and fluorescence microscopy of hyphae with a Cd-specific fluorescent tracer revealed that substantial proportion of Cd was contained in the vacuoles in both isolates. Considering that the proportion of Cd associated with fractions attributable to Cd complexes with cytosolic, metallothionein (MT) peptides was higher in Prib, we examined the copy number and basal levels of HmMTs genes in Rez and Prib. While no difference between the isolates was observed in the gene copy numbers and basal levels of HmMT1 transcripts, the basal transcription of HmMT3 was 3-fold higher in Prib. These observations suggest that MTs provide in Prib better protection against Cd. Furthermore, the higher Cd tolerance in Prib can be to some extent also supported by the efflux or reduced uptake of Cd in the hyphae.


Assuntos
Antifúngicos/metabolismo , Cádmio/toxicidade , Tolerância a Medicamentos , Poluentes Ambientais/metabolismo , Expressão Gênica , Hebeloma/efeitos dos fármacos , Metalotioneína/biossíntese , República Tcheca , Microbiologia Ambiental , Hebeloma/isolamento & purificação , Metalotioneína/genética , Metais Pesados/metabolismo , Micélio/efeitos dos fármacos , Prata/metabolismo , Zinco/metabolismo
11.
Appl Microbiol Biotechnol ; 103(5): 2229-2241, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30631897

RESUMO

L-Amino acid oxidases (LAAOs) are flavoproteins, which use oxygen to deaminate L-amino acids and produce the corresponding α-keto acids, ammonia, and hydrogen peroxide. Here we describe the heterologous expression of LAAO4 from the fungus Hebeloma cylindrosporum without signal sequence as fusion protein with a 6His tag in Escherichia coli and its purification. 6His-hcLAAO4 could be activated by exposure to acidic pH, the detergent sodium dodecyl sulfate, or freezing. The enzyme converted 14 proteinogenic L-amino acids with L-glutamine, L-leucine, L-methionine, L-phenylalanine, L-tyrosine, and L-lysine being the best substrates. Methyl esters of these L-amino acids were also accepted. Even ethyl esters were converted but with lower activity. Km values were below 1 mM and vmax values between 19 and 39 U mg-1 for the best substrates with the acid-activated enzyme. The information for an N-terminal aldehyde tag was added to the coding sequence. Co-expressed formylglycine-generating enzyme was used to convert a cysteine residue in the aldehyde tag to a Cα-formylglycine residue. The aldehyde tag did not change the properties of the enzyme. Purified Ald-6His-hcLAAO4 was covalently bound to a hexylamine resin via the Cα-formylglycine residue. The immobilized enzyme could be reused repeatedly to generate phenylpyruvate from L-phenylalanine with a total turnover number of 17,600 and was stable for over 40 days at 25 °C.


Assuntos
Enzimas Imobilizadas/metabolismo , Hebeloma/enzimologia , L-Aminoácido Oxidase/metabolismo , Fenilalanina/metabolismo , Ácidos Fenilpirúvicos/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , L-Aminoácido Oxidase/genética , Proteínas Recombinantes de Fusão/genética
12.
Biometals ; 32(1): 101-110, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30560539

RESUMO

Ectomycorrhizal fungi hold a potential role in bioremediation of heavy metal polluted areas because of its metal accumulation and detoxification property. We investigated the cadmium (Cd) induced bioaccumulation of glutathione (GSH) mediated by γ-glutamylcysteine synthetase (γ-GCS) in the ectomycorrhizal fungus Hebeloma cylindrosporum. In H. cylindrosporum, a demand driven synthesis of GSH has been observed in response to Cd. The expression and enzyme activity of H. cylindrosporum γ-GCS (Hcγ-GCS) increased as a function of external Cd stress resulting in increased GSH production. The function of Hcγ-GCS in providing heavy metal tolerance to H. cylindrosporum was justified by complementing the gene in gsh1Δ mutant of Saccharomyces cerevisiae. The metal sensitive mutant gsh1Δ successfully restored its metal tolerance ability when transformed with Hcγ-GCS gene. Sequence analysis of Hcγ-GCS showed homology with most of the reported γ-GCS proteins from basidiomycetes family. The active site of the Hcγ-GCS protein is composed of amino acids that were found to be conserved not only in fungi, but also in plants and mammals. From these results, it was concluded that Hcγ-GCS plays an important role in bioaccumulation of GSH, which is a core component in the mycorrhizal defense system under Cd stress for Cd homeostasis and detoxification.


Assuntos
Cádmio/farmacologia , Glutamato-Cisteína Ligase/metabolismo , Glutationa/metabolismo , Hebeloma/efeitos dos fármacos , Hebeloma/metabolismo , Glutamato-Cisteína Ligase/genética , Hebeloma/enzimologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
13.
Tree Physiol ; 39(4): 526-535, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30371901

RESUMO

Ectomycorrhizal trees are common in the cold regions of the world, yet the role of the mycorrhizal symbiosis in plant cold tolerance is poorly known. Moreover, the standard methods for testing plant frost hardiness may not be adequate for roots and mycorrhizas. The aims of this study were to compare the frost hardiness of mycorrhizal and non-mycorrhizal Scots pine (Pinus sylvestris L.) seedlings and to test the use of reverse-flow root hydraulic conductance (Kr) measurement for root frost hardiness determination. Mycorrhizal (Hebeloma sp. or Suillus luteus) and non-mycorrhizal seedlings were grown in controlled-environment chambers for 13 weeks. After this, half of the plants were allotted to a non-hardening treatment (long day and high temperature, same as during the preceding growing season) and the other half to a hardening (short day and low temperature) 'autumn' treatment for 4 weeks. The intact seedlings were exposed to whole-plant freezing tests and the needle frost hardiness was measured by relative electrolyte leakage (REL) method. The seedlings were grown for three more weeks for visual damage assessment and Kr measurements using a high-pressure flow meter (HPFM). Mycorrhizas did not affect the frost hardiness of seedlings in either hardening treatment. The effect of the hardening treatment on frost hardiness was shown by REL and visual assessment of the aboveground parts as well as Kr of roots. Non-mycorrhizal plants were larger than mycorrhizal ones while nitrogen and phosphorus contents (per unit dry mass) were similar in all mycorrhiza treatments. In plants with no frost exposure, the non-mycorrhizal treatment had higher Kr. There was no mycorrhizal effect on plant frost hardiness when nutritional effects were excluded. Further studies are needed on the role of mycorrhizas especially in the recovery of growth and nutrient uptake in cold soils in the spring. The HPFM is useful novel method for assessment of root damage.


Assuntos
Hebeloma/fisiologia , Micorrizas/fisiologia , Pinus sylvestris/fisiologia , Temperatura Baixa , Congelamento , Pinus sylvestris/microbiologia , Raízes de Plantas/microbiologia , Raízes de Plantas/fisiologia , Transpiração Vegetal , Estações do Ano , Plântula/microbiologia , Plântula/fisiologia , Simbiose , Árvores
14.
Plant Signal Behav ; 13(10): e1525997, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30289375

RESUMO

Ectomycorrhizal fungi improve tree phosphorus nutrition through transporters specifically localized at soil-hyphae and symbiotic interfaces. In the model symbiosis between the fungus Hebeloma cylindrosporum and the maritime pine (Pinus pinaster), several transporters possibly involved in phosphate fluxes were identified, including three H+:Pi transporters. Among these three, we recently unraveled the function of one of them, named HcPT2, in both pure culture and symbiotic interaction with P. pinaster. Here we investigated the transporter named HcPT1.2, by analyzing inorganic phosphate transport ability in a yeast complementation assay, assessing its expression in the fungus associated or not with the plant, and immunolocalizing the proteins in ectomycorrhizas. We also evaluated the effect of external Pi concentration on expression and localization of HcPT1.2. Our results revealed that HcPT1.2 is involved in Pi acquisition by H. cylindrosporum mycelium, irrespective of the external Pi concentrations.


Assuntos
Hebeloma/fisiologia , Hifas/fisiologia , Micorrizas/metabolismo , Fosfatos/metabolismo , Regulação Fúngica da Expressão Gênica , Fósforo/metabolismo , Pinus/metabolismo , Pinus/microbiologia , Simbiose/fisiologia
15.
Plant Signal Behav ; 13(6): e1480845, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29939816

RESUMO

Most land plants rely on root symbioses to complement or improve their mineral nutrition. Recent researches have put forward that mycorrhizal fungi efficiently absorb and transfer potassium (K+) from the soil to host plant roots, but the molecular mechanisms involved are not completely elucidated yet. We have recently revealed that K+ is likely released from the fungal Hartig net to the plant by TOK channels in the ectomycorrhizal model Hebeloma cylindrosporum - Pinus pinaster. H. cylindrosporum harbours three TOK members. Herein, we report that one of them, HcTOK1, has similar features than the yeast ScTOK1. Moreover, we propose a role for this channel in the transport of K+ from the medium to ectomycorrhizal roots under K+ starvation.


Assuntos
Regulação Fúngica da Expressão Gênica/fisiologia , Micorrizas/fisiologia , Pinus/metabolismo , Pinus/microbiologia , Potássio/metabolismo , Simbiose/fisiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Hebeloma/genética , Hebeloma/fisiologia , Homeostase , Pinus/genética , Simbiose/genética
16.
New Phytol ; 220(4): 1185-1199, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29944179

RESUMO

Through a mutualistic relationship with woody plant roots, ectomycorrhizal fungi provide growth-limiting nutrients, including inorganic phosphate (Pi), to their host. Reciprocal trades occur at the Hartig net, which is the symbiotic interface of ectomycorrhizas where the two partners are symplasmically isolated. Fungal Pi must be exported to the symbiotic interface, but the proteins facilitating this transfer are unknown. In the present study, we combined transcriptomic, microscopy, whole plant physiology, X-ray fluorescence mapping, 32 P labeling and fungal genetic approaches to unravel the role of HcPT2, a fungal Pi transporter, during the Hebeloma cylindrosporum-Pinus pinaster ectomycorrhizal association. We localized HcPT2 in the extra-radical hyphae and the Hartig net and demonstrated its determinant role for both the establishment of ectomycorrhizas and Pi allocation towards P. pinaster. We showed that the host plant induces HcPT2 expression and that the artificial overexpression of HcPT2 is sufficient to significantly enhance Pi export towards the central cylinder. Together, our results reveal that HcPT2 plays an important role in ectomycorrhizal symbiosis, affecting both Pi influx in the mycelium and efflux towards roots under the control of P. pinaster.


Assuntos
Proteínas Fúngicas/metabolismo , Hebeloma/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Micorrizas/fisiologia , Simbiose , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Hebeloma/genética , Hebeloma/crescimento & desenvolvimento , Proteínas de Membrana Transportadoras/genética , Modelos Biológicos , Micélio/metabolismo , Fosfatos/metabolismo , Radioisótopos de Fósforo , Pinus/microbiologia , Regulação para Cima/genética
17.
Environ Microbiol ; 20(5): 1873-1887, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29614209

RESUMO

Ectomycorrhizal fungi play an essential role in the ecology of boreal and temperate forests through the improvement of tree mineral nutrition. Potassium (K+ ) is an essential nutrient for plants and is needed in high amounts. We recently demonstrated that the ectomycorrhizal fungus Hebeloma cylindrosporum improves the K+ nutrition of Pinus pinaster under shortage conditions. Part of the transport systems involved in K+ uptake by the fungus has been deciphered, while the molecular players responsible for the transfer of this cation towards the plant remain totally unknown. Analysis of the genome of H. cylindrosporum revealed the presence of three putative tandem-pore outward-rectifying K+ (TOK) channels that could contribute to this transfer. Here, we report the functional characterization of these three channels through two-electrode voltage-clamp experiments in oocytes and yeast complementation assays. The expression pattern and physiological role of these channels were analysed in symbiotic interaction with P. pinaster. Pine seedlings colonized by fungal transformants overexpressing two of them displayed a larger accumulation of K+ in shoots. This study revealed that TOK channels have distinctive properties and functions in axenic and symbiotic conditions and suggested that HcTOK2.2 is implicated in the symbiotic transfer of K+ from the fungus towards the plant.


Assuntos
Micorrizas/metabolismo , Pinus/microbiologia , Potássio/metabolismo , Transporte Biológico , Hebeloma/genética , Minerais/metabolismo , Canais de Potássio , Saccharomyces cerevisiae/metabolismo , Plântula , Simbiose/genética
18.
Environ Microbiol ; 19(3): 1338-1354, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28076886

RESUMO

To clarify the early molecular interaction between ectomycorrhizal partners, we performed a RNA-Seq study of transcriptome reprogramming of the basidiomycete Hebeloma cylindrosporum before symbiotic structure differentiation with Pinus pinaster. Mycorrhiza transcriptome was studied for comparison. By reference to asymbiotic mycelium, 47 and 46 genes were specifically upregulated over fivefold (p ≤ 0.05) upon rhizosphere colonization and root adhesion respectively. Other 45 were upregulated throughout the symbiotic interaction, from rhizosphere colonization to differentiated mycorrhizas, whereas 274 were specifically upregulated in mycorrhizas. Although exoproteome represents 5.6% of H. cylindrosporum proteome, 38.5% of the genes upregulated upon pre-infectious root colonization encoded extracellular proteins. The proportion decreased to 23.5% in mycorrhizas. At all studied time points, mycorrhiza-induced small secreted proteins (MiSSPs), representing potential effectors, were over-represented among upregulated genes. This was also the case for carbohydrate-active enzymes (CAZymes). Several CAZymes were upregulated at all studied stages of the interaction. Consistent with a role in fungal morphogenesis and symbiotic interface differentiation, CAZymes over-expressed before and upon root attachment targeted fungal and both fungal and plant polysaccharides respectively. Different hydrophobins were upregulated upon early root adhesion, in mycorrhizas or throughout interaction. The functional classification of genes upregulated only in mycorrhizas pointed to intense metabolic activity and nutritional exchanges.


Assuntos
Hebeloma/genética , Micorrizas/genética , Simbiose , Transcriptoma , Proteínas Fúngicas/genética , Hebeloma/crescimento & desenvolvimento , Hebeloma/isolamento & purificação , Hebeloma/fisiologia , Micélio/genética , Micélio/crescimento & desenvolvimento , Micélio/isolamento & purificação , Micorrizas/crescimento & desenvolvimento , Micorrizas/isolamento & purificação , Micorrizas/fisiologia , Pinus/microbiologia , Pinus/fisiologia , Raízes de Plantas/microbiologia , Raízes de Plantas/parasitologia , Proteoma/genética , Regulação para Cima
19.
Plant Cell Environ ; 40(2): 190-202, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27743400

RESUMO

Ectomycorrhizal (ECM) association can improve plant phosphorus (P) nutrition. Polyphosphates (polyP) synthesized in distant fungal cells after P uptake may contribute to P supply from the fungus to the host plant if they are hydrolyzed to phosphate in ECM roots then transferred to the host plant when required. In this study, we addressed this hypothesis for the ECM fungus Hebeloma cylindrosporum grown in vitro and incubated without plant or with host (Pinus pinaster) and non-host (Zea mays) plants, using an experimental system simulating the symbiotic interface. We used 32 P labelling to quantify P accumulation and P efflux and in vivo and in vitro nuclear magnetic resonance (NMR) spectroscopy and cytological staining to follow the fate of fungal polyP. Phosphate supply triggered a massive P accumulation as newly synthesized long-chain polyP in H. cylindrosporum if previously grown under P-deficient conditions. P efflux from H. cylindrosporum towards the roots was stimulated by both host and non-host plants. However, the host plant enhanced 32 P release compared with the non-host plant and specifically increased the proportion of short-chain polyP in the interacting mycelia. These results support the existence of specific host plant effects on fungal P metabolism able to provide P in the apoplast of ectomycorrhizal roots.


Assuntos
Hebeloma/fisiologia , Interações Hospedeiro-Patógeno , Espectroscopia de Ressonância Magnética , Micorrizas/fisiologia , Radioisótopos de Fósforo/metabolismo , Fósforo/metabolismo , Pinus/microbiologia , Polifosfatos/metabolismo , Hifas/metabolismo , Pinus/metabolismo , Zea mays/metabolismo
20.
Fungal Biol ; 120(1): 72-103, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26693686

RESUMO

Hebeloma section Denudata includes the majority of the taxa commonly referred to as the Hebeloma crustuliniforme complex. In a recent paper we described in detail H. subsection Denudata and fifteen European species recognised within this subsection, using morphological and molecular methods. In this paper we continue this work and describe in detail three additional subsections and several new species. Within H. subsection Hiemalia we recognise just one species, Hebeloma hiemale. Here we propose an epitype in order to unambiguously define this taxon. Nine species occurring in Europe are assigned to H. subsect. Clepsydroida, namely Hebeloma ammophilum, H. cavipes, H. fragilipes, H. ingratum, H. laetitiae, H. limbatum sp. nov., H. matritense sp. nov., H. pseudofragilipes sp. nov., and H. vaccinum. Finally, we introduce H. subsection Echinospora with three species: Hebeloma echinosporum sp. nov., H. populinum, and H. rostratum sp. nov. We provide descriptions of all three of these species in order to clarify the taxonomy of this section. We provide a key to H. sect. Denudata and the discussed subsections. For the majority of the taxa there is good overall consistency between morphological and phylogenetic delimitation and, where the information exists, thanks to Aanen and Kuyper's work, biological delimitation.


Assuntos
Hebeloma/classificação , Filogenia , Europa (Continente) , Hebeloma/genética , Hebeloma/crescimento & desenvolvimento , Hebeloma/isolamento & purificação , Dados de Sequência Molecular , Esporos Fúngicos/classificação , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/isolamento & purificação
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