Amanita muscaria is an ectomycorrhizal mushroom that commonly grows at metal-polluted sites. Sporocarps from the lead smelter-polluted area near Příbram (Central Bohemia, Czech Republic) showed elevated concentrations of Cd and Zn. Size exclusion chromatography of the cell extracts of the sporocarps from both polluted and unpolluted sites indicated that substantial part of intracellular Cd and Zn was sequestered in 6-kDa complexes, presumably with metallothionein(s) (MT). When the cultured mycelial isolates were compared, those from Příbram were more Cd-tolerant and accumulated slightly less Cd and Zn than those from the unpolluted site. The analysis of the available A.muscaria sequence data returned a 67-amino acid (AA) MT encoded by the AmMT1 gene. Weak Cd and Zn responsiveness of AmMT1 in the mycelia suggested its metal homeostasis function in A.muscaria, rather than a major role in detoxification. The AmMT1 belongs to a ubiquitous peptide group in the Agaricomycetes consisting of 60-70-AA MTs containing seven cysteinyl domains and a conserved histidyl, features observed also in a newly predicted, atypical 45-AA RaMT1 of the Zn-accumulator Russula bresadolae in which the C-terminal cysteinyl domains VI and VII are missing. Heterologous expression in metal-sensitive yeast mutants indicated that AmMT1 and RaMT1 encode functional peptides that can protect cells against Cd, Zn, and Cu toxicity. The metal protection phenotype observed in yeasts with mutant variants of AmMT1 and RaMT1 further indicated that the conserved histidyl seems to play a structural, not metal binding role, and the cysteinyls of the C-terminal domains VI and VII are important for Cu binding. The data provide an important insight into the metal handling of site-associated ectomycorrhizal species disturbed by excess metals and the properties of MTs common in Agaricomycetes.
Crop inoculation with Glomus cubense isolate (INCAM-4, DAOM-241198) promotes yield in banana, cassava, forages, and others. Yield improvements range from 20 to 80% depending on crops, nutrient supply, and edaphoclimatic conditions. However, it is difficult to connect yield effects with G. cubense abundance in roots due to the lack of an adequate methodology to trace this taxon in the field. It is necessary to establish an accurate evaluation framework of its contribution to root colonization separated from native arbuscular mycorrhizal fungi (AMF). A taxon-discriminating primer set was designed based on the ITS nrDNA marker and two molecular approaches were optimized and validated (endpoint PCR and quantitative real-time PCR) to trace and quantify the G. cubense isolate in root and soil samples under greenhouse and environmental conditions. The detection limit and specificity assays were performed by both approaches. Different 18 AMF taxa were used for endpoint PCR specificity assay, showing that primers specifically amplified the INCAM-4 isolate yielding a 370 bp-PCR product. In the greenhouse, Urochloa brizantha plants inoculated with three isolates (Rhizophagus irregularis, R. clarus, and G. cubense) and environmental root and soil samples were successfully traced and quantified by qPCR. The AMF root colonization reached 41-70% and the spore number 4-128 per g of soil. This study demonstrates for the first time the feasibility to trace and quantify the G. cubense isolate using a taxon-discriminating ITS marker in roots and soils. The validated approaches reveal their potential to be used for the quality control of other mycorrhizal inoculants and their relative quantification in agroecosystems.
Mycorrhizal fungi are mutualists that play crucial roles in nutrient acquisition in terrestrial ecosystems. Mycorrhizal symbioses arose repeatedly across multiple lineages of Mucoromycotina, Ascomycota, and Basidiomycota. Considerable variation exists in the capacity of mycorrhizal fungi to acquire carbon from soil organic matter. Here, we present a combined analysis of 135 fungal genomes from 73 saprotrophic, endophytic and pathogenic species, and 62 mycorrhizal species, including 29 new mycorrhizal genomes. This study samples ecologically dominant fungal guilds for which there were previously no symbiotic genomes available, including ectomycorrhizal Russulales, Thelephorales and Cantharellales. Our analyses show that transitions from saprotrophy to symbiosis involve (1) widespread losses of degrading enzymes acting on lignin and cellulose, (2) co-option of genes present in saprotrophic ancestors to fulfill new symbiotic functions, (3) diversification of novel, lineage-specific symbiosis-induced genes, (4) proliferation of transposable elements and (5) divergent genetic innovations underlying the convergent origins of the ectomycorrhizal guild.
- MeSH
- ekosystém MeSH
- fungální proteiny genetika MeSH
- fylogeneze MeSH
- fyziologie rostlin MeSH
- genom fungální * MeSH
- houby klasifikace genetika fyziologie MeSH
- molekulární evoluce MeSH
- mykorhiza klasifikace genetika fyziologie MeSH
- rostliny mikrobiologie MeSH
- symbióza * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Specific quantification of root-colonizing arbuscular mycorrhizal fungi (AMF) by quantitative real-time PCR is a high-throughput technique, most suitable for determining abundances of AMF species or isolates in previously characterized experimental systems. The principal steps are the choice and validation of an appropriate assay to specifically amplify a gene fragment of the target AMF, preparation of templates from root samples, and quantification of the fungal gene copy numbers in these templates. The use of a suitable assay is crucial for a correct data collection but also highly specific for each experimental system and is therefore covered by general recommendations. Subsequently, specific steps are described for the validation of the assay using a standard dilution series, the determination of appropriate dilutions of DNA extracts from roots, and the quantification of the gene copy numbers in samples including calculations.
- MeSH
- DNA fungální genetika izolace a purifikace MeSH
- genová dávka genetika MeSH
- kořeny rostlin genetika mikrobiologie MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- mykorhiza genetika izolace a purifikace MeSH
- půda MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Research efforts directed to elucidation of mechanisms behind trading of resources between the partners in the arbuscular mycorrhizal (AM) symbiosis have seen a considerable progress in the recent years. Yet, despite of the recent developments, some key questions still remain unanswered. For example, it is well established that the strictly biotrophic AM fungus releases phosphorus to- and receives carbon molecules from the plant symbiont, but the particular genes, and their products, responsible for facilitating this exchange, are still not fully described, nor are the principles and pathways of their regulation. Here, we made a de novo quest for genes involved in carbon transfer from the plant to the fungus using genome-wide gene expression array targeting whole root and whole shoot gene expression profiles of mycorrhizal and non-mycorrhizal Medicago truncatula plants grown in a glasshouse. Using physiological intervention of heavy shading (90% incoming light removed) and the correlation of expression levels of MtPT4, the mycorrhiza-inducible phosphate transporter operating at the symbiotic interface between the root cortical cells and the AM fungus, and our candidate genes, we demonstrate that several novel genes may be involved in resource tradings in the AM symbiosis established by M. truncatula. These include glucose-6-phosphate/phosphate translocator, polyol/monosaccharide transporter, DUR3-like, nucleotide-diphospho-sugar transferase or a putative membrane transporter. Besides, we also examined the expression of other M. truncatula phosphate transporters (MtPT1-3, MtPT5-6) to gain further insights in the balance between the "direct" and the "mycorrhizal" phosphate uptake pathways upon colonization of roots by the AM fungus, as affected by short-term carbon/energy deprivation. In addition, the role of the novel candidate genes in plant cell metabolism is discussed based on available literature.
- MeSH
- fosfor metabolismus MeSH
- Medicago truncatula genetika metabolismus mikrobiologie MeSH
- metabolické sítě a dráhy MeSH
- mykorhiza genetika fyziologie MeSH
- regulace genové exprese u rostlin MeSH
- rostlinné proteiny genetika MeSH
- sekvenování exomu MeSH
- stanovení celkové genové exprese MeSH
- symbióza MeSH
- uhlík metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- Basidiomycota genetika MeSH
- cytoplazmatické vezikuly metabolismus MeSH
- fungální proteiny genetika metabolismus MeSH
- Hebeloma genetika růst a vývoj metabolismus MeSH
- kadmium metabolismus MeSH
- metalothionein genetika metabolismus MeSH
- mycelium genetika růst a vývoj metabolismus MeSH
- mykorhiza genetika MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- zinek metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
The nuclear ribosomal DNA (nuc-rDNA) is widely used for the identification and phylogenetic reconstruction of Agaricomycetes. However, nuc-rDNA-based phylogenies may sometimes be in conflict with phylogenetic relationships derived from protein coding genes. In this study, the taxonomic position of the basidiomycetous mycobiont that forms the recently discovered sheathed ericoid mycorrhiza was investigated, because its nuc-rDNA is highly dissimilar to any other available fungal sequences in terms of nucleotide composition and length, and its nuc-rDNA-based phylogeny is inconclusive and significantly disagrees with protein coding sequences and morphological data. In the present work, this mycobiont was identified as Kurtia argillacea (= Hyphoderma argillaceum) residing in the order Hymenochaetales (Basidiomycota). Bioinformatic screening of the Kurtia ribosomal DNA sequence indicates that it represents a gene with a non-standard substitution rate or nucleotide composition heterogeneity rather than a deep paralogue or a pseudogene. Such a phenomenon probably also occurs in other lineages of the Fungi and should be taken into consideration when nuc-rDNA (especially that with unusual nucleotide composition) is used as a sole marker for phylogenetic reconstructions. Kurtia argillacea so far represents the only confirmed non-sebacinoid ericoid mycorrhizal fungus in the Basidiomycota and its intriguing placement among mostly saprobic and parasitic Hymenochaetales begs further investigation of its eco-physiology.
- MeSH
- Basidiomycota klasifikace genetika MeSH
- elongační faktor 1 genetika MeSH
- fylogeneze * MeSH
- GC bohatá sekvence MeSH
- mezerníky ribozomální DNA genetika MeSH
- mitochondriální DNA genetika MeSH
- mykorhiza klasifikace genetika MeSH
- ribozomální DNA genetika MeSH
- zastoupení bazí MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A search of R. atropurpurea transcriptome for sequences encoding the transporters of the Zrt-, Irt-like Protein (ZIP) family, which are in eukaryotes integral to Zn supply into cytoplasm, allowed the identification of RaZIP1 cDNA with a predicted product belonging to ZIP I subfamily; it was subjected to functional studies in mutant Saccharomyces cerevisiae strains. The expression of RaZIP1, but not RaZIP1H208A or RaZIP1H232A mutants lacking conserved-among-ZIPs transmembrane histidyls, complemented Zn uptake deficiency in zrt1Δzrt2Δ yeasts. RaZIP1 substantially increased cellular Zn uptake in this strain and added to Zn sensitivity in zrc1Δcot1Δ mutant. The Fe uptake deficiency in ftr1Δ strain was not rescued and Mn uptake was insufficient for toxicity in Mn-sensitive pmr1Δ yeasts. By contrast, RaZIP1 increased Cd sensitivity in yap1Δ strain and conferred Cd transport activity in yeasts, albeit with substantially lower efficiency compared to Zn transport. In metal uptake assays, the accumulation of Zn in zrt1Δzrt2Δ strain remained unaffected by Cd, Fe, and Mn present in 20-fold molar excess over Zn. Immunofluorescence microscopy detected functional hemagglutinin-tagged HA::RaZIP1 on the yeast cell protoplast periphery. Altogether, these data indicate that RaZIP1 is a high-affinity plasma membrane transporter specialized in Zn uptake, and improve the understanding of the cellular and molecular biology of Zn in R. atropurpurea that is known for its ability to accumulate remarkably high concentrations of Zn.
- MeSH
- Basidiomycota genetika růst a vývoj MeSH
- biologický transport účinky léků MeSH
- fungální proteiny genetika metabolismus MeSH
- mykorhiza chemie genetika MeSH
- proteiny přenášející kationty MeSH
- regulace genové exprese u rostlin MeSH
- rostlinné proteiny chemie genetika metabolismus MeSH
- sekvence aminokyselin genetika MeSH
- zinek chemie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Forest management practices often severely affect forest ecosystem functioning. Tree removal by clearcutting is one such practice, producing severe impacts due to the total reduction of primary productivity. Here, we assessed changes to fungal community structure and decomposition activity in the soil, roots and rhizosphere of a Picea abies stand for a 2-year period following clearcutting compared to data from before tree harvest. We found that the termination of photosynthate flow through tree roots into soil is associated with profound changes in soil, both in decomposition processes and fungal community composition. The rhizosphere, representing an active compartment of high enzyme activity and high fungal biomass in the living stand, ceases to exist and starts to resemble bulk soil. Decomposing roots appear to separate from bulk soil and develop into hotspots of decomposition and important fungal biomass pools. We found no support for the involvement of ectomycorrhizal fungi in the decomposition of roots, but we found some evidence that root endophytic fungi may have an important role in the early stages of this process. In soil, activity of extracellular enzymes also decreased in the long term following the end of rhizodeposition by tree roots.
- MeSH
- biomasa MeSH
- borovice mikrobiologie MeSH
- ekosystém MeSH
- houby klasifikace genetika růst a vývoj izolace a purifikace MeSH
- kořeny rostlin mikrobiologie MeSH
- mykobiom MeSH
- mykorhiza klasifikace genetika růst a vývoj izolace a purifikace MeSH
- půda chemie MeSH
- půdní mikrobiologie * MeSH
- rhizosféra MeSH
- stromy mikrobiologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Root colonization by arbuscular mycorrhizal fungi (AMF) can be quantified by different approaches. We compared two approaches that enable discrimination of specific AMF taxa and are therefore emerging as alternative to most commonly performed microscopic quantification of AMF in roots: quantitative real-time PCR (qPCR) using markers in nuclear ribosomal DNA (nrDNA) and mitochondrial ribosomal DNA (mtDNA). In a greenhouse experiment, Medicago truncatula was inoculated with four isolates belonging to different AMF species (Rhizophagus irregularis, Claroideoglomus claroideum, Gigaspora margarita and Funneliformis mosseae). The AMF were quantified in the root samples by qPCR targeted to both markers, microscopy and contents of AMF-specific phospholipid fatty acids (PLFA). Copy numbers of nrDNA and mtDNA were closely related within all isolates; however, the slopes and intercepts of the linear relationships significantly differed among the isolates. Across all isolates, a large proportion of variance in nrDNA copy numbers was explained by root colonization intensity or contents of AMF-specific PLFA, while variance in mtDNA copy numbers was mainly explained by differences among AMF isolates. We propose that the encountered inter-isolate differences in the ratios of mtDNA and nrDNA copy numbers reflect different physiological states of the isolates. Our results suggest that nrDNA is a more suitable marker region than mtDNA for the quantification of multiple AMF taxa as its copy numbers are better related to fungal biomass across taxa than are copy numbers of mtDNA.
- MeSH
- buněčné jádro genetika MeSH
- DNA fungální genetika MeSH
- Glomeromycota genetika MeSH
- kořeny rostlin mikrobiologie MeSH
- kvantitativní polymerázová řetězová reakce * MeSH
- Medicago truncatula mikrobiologie MeSH
- mitochondriální DNA genetika MeSH
- mykorhiza genetika MeSH
- Publikační typ
- časopisecké články MeSH
