Fungal Shaker-like channels beyond cellular K+ homeostasis: A role in ectomycorrhizal symbiosis between Hebeloma cylindrosporum and Pinus pinaster.

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.

Whole-plant frost hardiness of mycorrhizal (Hebeloma sp. or Suillus luteus) and non-mycorrhizal Scots pine seedlings.

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.

HcPT1.2 participates in Pi acquisition in Hebeloma cylindrosporum external hyphae of ectomycorrhizas under high and low phosphate conditions.

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.

HcTOK1 participates in the maintenance of K+ homeostasis in the ectomycorrhizal fungus Hebeloma cylindrosporum, which is essential for the symbiotic K+ nutrition of Pinus pinaster.

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.

The ectomycorrhizal basidiomycete Hebeloma cylindrosporum undergoes early waves of transcriptional reprogramming prior to symbiotic structures differentiation.

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.

The host plant Pinus pinaster exerts specific effects on phosphate efflux and polyphosphate metabolism of the ectomycorrhizal fungus Hebeloma cylindrosporum: a radiotracer, cytological staining and 31 P NMR spectroscopy study.

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.

A fungal conserved gene from the basidiomycete Hebeloma cylindrosporum is essential for efficient ectomycorrhiza formation.

We used Agrobacterium-mediated insertional mutagenesis to identify genes in the ectomycorrhizal fungus Hebeloma cylindrosporum that are essential for efficient mycorrhiza formation. One of the mutants presented a dramatically reduced ability to form ectomycorrhizas when grown in the presence of Pinus pinaster. It failed to form mycorrhizas in the presence of glucose at 0.5 g liter(-1), a condition favorable for mycorrhiza formation by the wild-type strain. However, it formed few mycorrhizas when glucose was replaced by fructose or when glucose concentration was increased to 1 g liter(-1). Scanning electron microscopy examination of these mycorrhizas revealed that this mutant was unable to differentiate true fungal sheath and Hartig net. Molecular analyses showed that the single-copy disrupting T-DNA was integrated 6,884 bp downstream from the start codon, of an open reading frame potentially encoding a 3,096-amino-acid-long protein. This gene, which we named HcMycE1, has orthologs in numerous fungi as well as different other eukaryotic microorganisms. RNAi inactivation of HcMycE1 in the wild-type strain also led to a mycorrhizal defect, demonstrating that the nonmycorrhizal phenotype of the mutant was due to mutagenic T-DNA integration in HcMycE1. In the wild-type strain colonizing P. pinaster roots, HcMycE1 was transiently upregulated before symbiotic structure differentiation. Together with the inability of the mutant to differentiate these structures, this suggests that HcMycE1 plays a crucial role upstream of the fungal sheath and Hartig net differentiation. This study provides the first characterization of a fungal mutant altered in mycorrhizal ability.

Strategies utilized by trophically diverse fungal species for Pinus sylvestris root colonization.

Physiological changes in host plants in response to the broad spectrum of fungal modes of infection are still not well understood. The current study was conducted to better understand the infection of in vitro cultures of Pinus sylvestris L. seedlings by three trophically diverse fungal species, Fusarium oxysporum E. F. Sm. & Swingle, Trichoderma harzianum Rifai and Hebeloma crustuliniforme (Bull.) Quél. Biochemical methods and microscopy were utilized to determine (i) which factors (apoplastic and cellular pH, reactive oxygen species, glutathione and cell death) play a role in the establishment of pathogenic, saprotrophic and mycorrhizal fungi, and (ii) whether cell death is a common response of conifer seedling tissues when they are exposed to trophically diverse fungi. Establishment of the pathogen, F. oxysporum, was observed more frequently in the meristematic region of root tips than in the elongation zone, which was in contrast to T. harzianum and H. crustuliniforme. Ectomycorrhizal (ECM) hyphae, however, were occasionally observed in the studied root zone and caused small changes in the studied factors. Colonization of the meristematic zone occurred due to host cell death. Independently of the zone, changes in cellular pH resulting in an acidic cytoplasm conditioned the establishment of F. oxysporum. Additionally, cell death was negatively correlated with hydrogen peroxide (H2O2) in roots challenged by a pathogenic fungus. Cell death was the only factor uniquely associated with the colonization of host roots by a saprotrophic fungus. The mechanism may differ, however, between the zones since apoplastic pH was negatively correlated with cell death in the elongation zone, whereas in the meristematic zone, none of the studied factors explained cell death. Colonization by the ECM fungus, H. crustuliniforme, was associated with a decreasing number of cells with acidic apoplast and by production of H2O2 in the elongation zone resulting in cell death. Saprotrophic and ECM fungi had a greater effect on cell acidification in the meristematic zone than the pathogenic fungus.

Frost hardiness of mycorrhizal (Hebeloma sp.) and non-mycorrhizal Scots pine roots.

The frost hardiness (FH) of mycorrhizal [ectomycorrhizal (ECM)] and non-mycorrhizal (NM) Scots pine (Pinus sylvestris) seedlings was studied to assess whether mycorrhizal symbiosis affected the roots' tolerance of below-zero temperatures. ECM (Hebeloma sp.) and NM seedlings were cultivated in a growth chamber for 18 weeks. After 13 weeks' growth in long-day and high-temperature (LDHT) conditions, a half of the ECM and NM seedlings were moved into a chamber with short-day and low-temperature (SDLT) conditions to cold acclimate. After exposures to a range of below-zero temperatures, the FH of the roots was assessed by means of the relative electrolyte leakage test. The FH was determined as the inflection point of the temperature-response curve. No significant difference was found between the FH of mycorrhizal and non-mycorrhizal roots in LDHT (-8.9 and -9.8 °C) or SDLT (-7.5 and -6.8 °C). The mycorrhizal treatment had no significant effect on the total dry mass, the allocation of dry mass among the roots and needles or nutrient accumulation. The mycorrhizal treatment with Hebeloma sp. did not affect the FH of Scots pine in this experimental setup. More information is needed on the extent to which mycorrhizas tolerate low temperatures, especially with different nutrient contents and different mycorrhiza fungi.

Suillus mycelia under elevated atmospheric CO2 support increased bacterial communities and scarce nifH gene activity in contrast to Hebeloma mycelia.

Bacterial communities associated with mycorrhizal roots are likely to respond to rising atmospheric CO(2) levels in terms of biomass, community composition and activity since they are supported by the carbon (C) flow outside the root tips, especially by exudation of low molecular weight organic compounds. We studied how general bacterial and diazotrophic communities associated with ectomycorrhizal (ECM) fungi respond to different belowground C supply conditions, mediated by elevated atmospheric CO(2) concentration under nitrogen (N) limited conditions. Microcosm systems were constructed using forest soil and Scots pine seedlings, which were either pre-inoculated with one of the ECM fungal species Hebeloma velutipes or Suillus variegatus, or non-inoculated. These fungal species differ in C allocation and exudation patterns. Seedlings were maintained under ambient (380 ppm) or elevated (700 ppm) CO(2) levels for 6 months. Quantitative polymerase chain reaction (PCR) showed a significant increase in 16S rRNA gene copy numbers for Suillus-inoculated microcosms under elevated CO(2) compared to ambient CO(2). The copy numbers of the nitrogenase reductase (nifH) gene were under the detection limit in all samples regardless the CO(2) treatments. Denaturing gradient gel electrophoresis analysis of PCR-amplified nifH genes revealed simple and consistent communities in all samples throughout the incubation period. A nested reverse transcription PCR approach revealed that expression of nifH genes were detected in some microcosms. Our findings suggest that the effect of mycorrhizal fungi on soil bacteria may vary depending on C supply and fungal species.

Impact of ectomycorrhizal colonization and rust infection on the secondary metabolism of poplar (Populus trichocarpa x deltoides).

Fungal colonization can significantly affect the secondary metabolism of the host plants. We tested the impact of a common below-ground symbiosis, i.e., ectomycorrhiza formation, on poplar leaf chemical components that are involved in the defence against a common disease, i.e., rust fungi, in N-deficient soil. A rust-susceptible poplar clone (Populus trichocarpa × deltoides 'Beaupré') was (a) non-associated with ectomycorrhizal fungus (EM) Hebeloma mesophaeum (Pers.) Quélet MÜN and non-infected with rust fungus Melampsora larici-populina Kleb. (isolate 98AG31), (b) associated with EM, (c) inoculated with rust fungus and (d) associated with EM and inoculated with rust fungus. Poplar leaves were analysed by photometric and mass spectrometric techniques (liquid chromatography-tandem mass spectrometry (LC-MS/MS), pyrolysis-field ionization mass spectrometry (Py-FIMS)). Both rust infection and mycorrhiza formation led to increased proportions of condensed tannins in relation to total phenolics (13% in the control, 18-19% in the fungal treatments). In contrast, salicylic acid concentration (6.8 µg g(-1) in the control) was higher only in the rust treatments (17.9 and 25.4 µg g(-1) with rust infection). The Py-FIMS analysis revealed that the rust-infected treatments were significantly separated from the non-rust-infected treatments on the basis of six flavonoids and one lipid. The relative abundance of these components, which have known functions in plant defence, was decreased after rust infection of non-mycorrhizal plants, but not in mycorrhizal plants. The results indicate that the ectomycorrhizal formation compensated the rust infection by a decrease in the flavonoid syntheses. The study provides new evidence for an interactive response of mycorrhizal colonization and infection with rust fungi in the metabolism of poplar.

Different patterns of regulation for the copper and cadmium metallothioneins of the ectomycorrhizal fungus Hebeloma cylindrosporum.

Metallothioneins (MTs) are small cysteine-rich peptides involved in metal homeostasis and detoxification. We have characterized two MT genes, HcMT1 and HcMT2, from the ectomycorrhizal fungus Hebeloma cylindrosporum in this study. Expression of HcMT1 and HcMT2 in H. cylindrosporum under metal stress conditions was studied by competitive reverse transcription-PCR analysis. The full-length cDNAs were used to perform functional complementation in mutant strains of Saccharomyces cerevisiae. As revealed by heterologous complementation assays in yeast, HcMT1 and HcMT2 each encode a functional polypeptide capable of conferring increased tolerance against Cd and Cu, respectively. The expression levels of HcMT1 were observed to be at their maximum at 24 h, and they increased as a function of Cu concentration. HcMT2 was also induced by Cu, but the expression levels were lower than those for HcMT1. The mRNA accumulation of HcMT1 was not influenced by Cd, whereas Cd induced the transcription of HcMT2. Zn, Pb, and Ni did not affect the transcription of HcMT1 or of HcMT2. Southern blot analysis revealed that both of these genes are present as a single copy in H. cylindrosporum. While the promoters of both HcMT1 and HcMT2 contained the standard stress response elements implicated in the metal response, the numbers and varieties of potential regulatory elements were different in these promoters. These results show that ectomycorrhizal fungi encode different MTs and that each of them has a particular pattern of expression, suggesting that they play critical specific roles in improving the survival and growth of ectomycorrhizal trees in ecosystems contaminated by heavy metals.

Hebeloma species associated with Cistus.

The genus Hebeloma has a number of species highly specific to Cistus and others that occur with several host genera. This paper discusses the species of Hebeloma that appear to be ectomycorrhizal with Cistus, judging from their occurrence when Cistus is the only available host. The previously unknown species H. plesiocistum spec. nov. is described. We also provide a key to the known Hebeloma associates of Cistus. Molecular analyses based on ITS sequence data further illustrate the distinctness of the newly described species and difficulties in the species delimitation with view to H. erumpens. Specific associations with Cistus may have evolved more than once within the genus Hebeloma.

Root hydraulic properties and growth of balsam poplar (Populus balsamifera) mycorrhizal with Hebeloma crustuliniforme and Wilcoxina mikolae var. mikolae.

The effects of an E-strain fungus (Wilcoxina mikolae var. mikolae) and an ectomycorrhizal fungus (Hebeloma crustuliniforme) on growth and water relations of balsam poplar were examined and compared in the present study. Balsam poplar roots inoculated with W. mikolae var. mikolae (Wm) exhibited structures consistent with ectendomycorrhizal (EEM) associations, including a mantle surrounding the outside of the root and an extensive Hartig net that was located between cortical cells and extended to the vascular cylinder. Roots colonized with H. crustuliniforme (Hc) developed a mantle layer, indicative of an ectomycorrhizal (ECM) association, around the outer part of the root, but no distinct Hartig net was present. Wm-colonized balsam poplar also showed increased shoot growth, stomatal conductance (g(s)), and root volumes compared with non-inoculated and Hc-inoculated plants. However, Hc-inoculated plants had higher root hydraulic conductivity (L(pr)) compared with non-inoculated plants and Wm-inoculated plants. These results suggest that L(pr) was not a growth-limiting factor in balsam poplar and that hyphal penetration of the root cortex in itself may have little influence on root hydraulic properties.