Identification of genes differentially expressed in extraradical mycelium and ectomycorrhizal roots during Paxillus involutus-Betula pendula ectomycorrhizal symbiosis

The development of ectomycorrhizal symbiosis leads to drastic changes in gene expression in both partners. However, little is known about the spatial regulation of symbiosis-regulated genes. Using cDNA array profiling, we compared the levels of expression of fungal genes corresponding to approximately 1,200 expressed sequenced tags in the ectomycorrhizal root tips (ECM) and the connected extraradical mycelium (EM) for the Paxillus involutus-Betula pendula ectomycorrhizal association grown on peat in a microcosm system. Sixty-five unique genes were found to be differentially expressed in these two fungal compartments. In ECM, a gene coding for a putative phosphatidylserine decarboxylase (Psd) was up-regulated by 24-fold, while genes coding for urea (Dur3) and spermine (Tpo3) transporters were up-regulated 4.1- and 6.2-fold in EM. Moreover, urea was the major nitrogen compound found in EM by gas chromatography-mass spectrometry analysis. These results suggest that (i) there is a spatial difference in the patterns of fungal gene expression between ECM and EM, (ii) urea and polyamine transporters could facilitate the translocation of nitrogen compounds within the EM network, and (iii) fungal Psd may contribute to membrane remodeling during ectomycorrhiza formation.     

四川都江堰亚热带针阔混交林中主要晚生型外生菌根真菌类群的空间分布

晚生型外生菌根真菌通常出现在森林演替的后期,是成熟林中的优势外生菌根真菌类群.对四川都江堰一片亚热带针阔混交林中的菌根真菌地上群落进行调查,并应用二元逻辑回归分析对晚生型外生菌根真菌的主要类群,即鹅膏菌科、牛肝菌科和红菇科,与周围(5 m×5 m样方)树种组成的关系进行研究.还应用次级变量分析方法对主要外生菌根真菌类群的空间格局进行了分析.结果表明,非外生菌根树种及某些外生菌根树种对特定类群菌根真菌子实体的出现有抑制作用,而不同类群外生菌根真菌在克隆生长上的差异并不是子实体空间分布的决定因素.我们认为,当研究自然林中外生菌根子实体的空间分布时,除了宿主植物的分布,也应考虑非宿主植物的分布以及菌根真菌相互作用的影响.     

四川都江堰亚热带针阔混交林中主要晚生型外生菌根真菌类群的空间分布

晚生型外生菌根真菌通常出现在森林演替的后期,是成熟林中的优势外生菌根真菌类群。对四川都江堰一片亚热带针阔混交林中的菌根真菌地上群落进行调查,并应用二元逻辑回归分析对晚生型外生菌根真菌的主要类群,即鹅膏菌科、牛肝菌科和红菇科,与周围(5m×5m样方)树种组成的关系进行研究。还应用次级变量分析方法对主要外生菌根真菌类群的空间格局进行了分析。结果表明,非外生菌根树种及某些外生菌根树种对特定类群菌根真菌子实体的出现有抑制作用,而不同类群外生菌根真菌在克隆生长上的差异并不是子实体空间分布的决定因素。我们认为,当研究自然林中外生菌根子实体的空间分布时,除了宿主植物的分布,也应考虑非宿主植物的分布以及菌根真菌相互作用的影响。     

Impact of an 8-Year-Old Transgenic Poplar Plantation on the Ectomycorrhizal Fungal Community

The long-term impact of field-deployed genetically modified trees on soil mutualistic organisms is not well known. This study aimed at evaluating the impact of poplars transformed with a binary vector containing the selectable nptII marker and β-glucuronidase reporter genes on ectomycorrhizal (EM) fungi 8 years after field deployment. We generated 2,229 fungal internal transcribed spacer (ITS) PCR products from 1,150 EM root tips and 1,079 fungal soil clones obtained from the organic and mineral soil horizons within the rhizosphere of three control and three transformed poplars. Fifty EM fungal operational taxonomic units were identified from the 1,706 EM fungal ITS amplicons retrieved. Rarefaction curves from both the root tips and soil clones were close to saturation, indicating that most of the EM species present were recovered. Based on qualitative and/or quantitative α- and β-diversity measurements, statistical analyses did not reveal significant differences between EM fungal communities associated with transformed poplars and the untransformed controls. However, EM communities recovered from the root tips and soil cloning analyses differed significantly from each other. We found no evidence of difference in the EM fungal community structure linked to the long-term presence of the transgenic poplars studied, and we showed that coupling root tip analysis with a soil DNA cloning strategy is a complementary approach to better document EM fungal diversity.The poplar has become a model tree species in genetic engineering as it can easily be transformed and clonally propagated and has a small genome size (7, 77, 80). Tree growth, agronomic traits, and timber quality can be improved through genetic engineering (61), thereby avoiding the long reproductive cycles of conventional breeding (47, 59, 83). However, concerns have arisen about the potential impact of genetically modified (GM) trees on the environment (10). The potential environmental hazards linked to GM trees differ from those associated with transgenic crop plants at both spatial and temporal scales (84) because trees are long-lived perennials, unlike annual crop plants. They display several biotic interactions with soil microbial communities such as bacteria and fungi. Interactions between GM trees and these communities could result in exposure to the expression of new traits over several decades, a period longer than those for GM crop plants.Impact studies of GM plants on nontarget organisms usually focus on the potential risk linked to transgene expression (expected effects) that confers a genetic advantage to the transformed plant rather than on unforeseen (pleiotropic) effects from transgene insertion or the expression of other transgene components such as selection markers or reporter genes. The nptII gene, encoding neomycin phosphotransferase II (EC 2.7.1.95), and the GUS gene, encoding β-glucuronidase (GUS; EC 3.2.1.31), are frequently used for genetic selection of transformed cells and for monitoring transgene presence and expression during transgenic plant lifetime (76). The products of the nptII and GUS genes have been subjected to safety assessment studies and were shown to be nondeleterious to human and animal health (21, 23, 27, 51). Nevertheless, pleiotropic effects in crop plants transformed with the nptII and GUS genes have been observed (2, 15, 17, 43). Pleiotropic effects from GM trees coexpressing such selectable markers have also been recorded. For example, Pasonen et al. (56) showed a significant decrease in the number of root tips colonized by Paxillus involutus associated with a line of chitinase-transformed silver birch in vitro. Similar results have been observed in vivo with P. involutus associated with a line of lignin-modified silver birches (72).Many trees in temperate, boreal, tropical, and subtropical forests establish mutualistic interactions with ectomycorrhizal (EM) fungi (42, 66, 67, 68). EM fungi are a polyphyletic group comprising over 5,000 species (49) that play key roles in biogeochemical soil processes and plant health. They represent one-third of the total microbial biomass in the soil of boreal forests (32). Fine roots colonized by EM fungi, also called EM root tips or ectomycorrhizae, display a fungal mantle from which extends the extraradical mycelium to prospect the soil for nutrient uptake. These two anatomical parts can be sampled for EM fungus molecular identification, but some studies have highlighted dissimilarities between the EM fungal diversity recorded in root tip sampling and that recorded in extraradical mycelium sampling (26, 37, 39).Given the potential cumulative effects caused by the presence and stable constitutive expression of transgenes over years on GM tree fitness and on the environment, impact studies of GM trees require long-term field trials (5, 72, 84). In this study, we investigated the potential long-term impact on the EM fungal community of hybrid poplars transformed with the binary vector containing the selectable nptII marker and GUS reporter genes, field deployed for 8 years. This plantation was part of the first confined field trial of transgenic trees in Canada. Hybrid poplars constitutively expressed the nptII gene for kanamycin resistance driven by the NOS promoter (30). The activity of the NOS promoter has been shown to increase in the lower part of transgenic tobacco plants (4). Such a vertical gradient has also been observed in transgenic hybrid poplars, where the NOS promoter activity was 2.4-fold higher in roots than in leaves (87).As no direct negative impact of nptII or GUS gene expression on fungal organisms has been reported in the literature, we first tested the null hypothesis (H₀) that the EM fungal community recorded from transgenic poplars was similar to that from untransformed poplars. Second, since the EM fungal diversity picture can be influenced by the sampling method, we contrasted the EM fungal community recovered from root tips with that recorded in soil cloning analyses. Internal transcribed spacer (ITS) sequences from the nuclear rRNA were produced from both EM root tips and extraradical mycelia to compare the EM fungal communities associated with three control and three transgenic poplars. EM fungal communities were characterized by measuring the usual qualitative and quantitative EM species diversity within each community (α-diversity) and then estimating the nucleotide diversity between EM communities in relation to EM phylotype relative abundances (quantitative β-diversity).     

Ectomycorrhizal fungi: the symbiotic route to the root for phosphorus in forest soils

Many forest trees have evolved mutualistic symbioses with ectomycorrhizal (ECM) fungi that contribute to their phosphorus (P) nutrition. Forest productivity is frequently limited by P, a phenomenon that is likely to become more widespread under future conditions of elevated atmospheric CO₂ concentration [CO₂]. It is thus timely that this review considers current understanding of the key processes (absorption, translocation and transfer to the plant host) in ECM fungus-mediated P nutrition of forest trees. Solubilisation of inorganic P (P_i) and hydrolysis of organic P by ECM fungi in soil occurs largely at the growing mycelial front, where P_i absorption is facilitated by high affinity transporters. While large gaps remain in our understanding of the physiological and molecular mechanisms that underpin movement of P in ECM mycelia in soil and P transfer to the plant, host P demand seems likely to be a key driver of these processes. ECM fungi may make considerable contributions to meeting the likely increased P demand of trees under elevated [CO₂] via increased colonization levels, shifts in ECM fungal community structure and changed patterns of EMM production. Further research into the spatial scale of ECM-mediated P movements in soil, along with the interplay between ECM fungi and other soil microflora is advocated.     

Ectomycorrhizal Fungal Protein Degradation Ability Predicted by Soil Organic Nitrogen Availability

In temperate and boreal forest ecosystems, nitrogen (N) limitation of tree metabolism is alleviated by ectomycorrhizal (ECM) fungi. As forest soils age, the primary source of N in soil switches from inorganic (NH₄⁺ and NO₃⁻) to organic (mostly proteins). It has been hypothesized that ECM fungi adapt to the most common N source in their environment, which implies that fungi growing in older forests would have greater protein degradation abilities. Moreover, recent results for a model ECM fungal species suggest that organic N uptake requires a glucose supply. To test the generality of these hypotheses, we screened 55 strains of 13 Suillus species with different ecological preferences for their in vitro protein degradation abilities. Suillus species preferentially occurring in mature forests, where soil contains more organic matter, had significantly higher protease activity than those from young forests with low-organic-matter soils or species indifferent to forest age. Within species, the protease activities of ecotypes from soils with high or low soil organic N content did not differ significantly, suggesting resource partitioning between mineral and organic soil layers. The secreted protease mixtures were strongly dominated by aspartic peptidases. Glucose addition had variable effects on secreted protease activity; in some species, it triggered activity, but in others, activity was repressed at high concentrations. Collectively, our results indicate that protease activity, a key ectomycorrhizal functional trait, is positively related to environmental N source availability but is also influenced by additional factors, such as carbon availability.     

Regulation of genes involved in nitrogen utilization on different C/N ratios and nitrogen sources in the model ectomycorrhizal fungus Hebeloma cylindrosporum

Nitrogen (N) utilization by ectomycorrhizal fungi is an essential aspect of their ecosystem function. N deposition changes both the N pools and the carbon/nitrogen (C/N) ratio of the substrates where ectomycorrhizal fungi are found, and it is important to understand how these changes affect the N forms used by ectomycorrhizal fungi. To overcome the difficulties of studying ectomycorrhizal fungi in situ, we investigated all known N genes in the model fungus, Hebeloma cylindrosporum in a culture study. In addition to studying the regulation of all known N utilization genes, we aimed to understand whether there are gene clusters that undergo similar regulation. Lastly we studied how C/N ratio, N transporter type, and N source affected relative gene expression levels. We grew the D2 strain of H. cylindrosporum on a range of inorganic and organic N sources under low, medium, and high C/N ratios. We found three gene clusters that were regulated in a similar pattern. Lastly, we found C/N ratio, N source and N transporter type all affected gene expression levels. Relative expression levels were highest on the high C/N ratio, BSA and diLeucine N sources, and inorganic N transporters were always expressed at higher levels than organic N transporters. These results suggest that inorganic N sources may always the default preference for H. cylindrosporum, regardless of both the N sources and the C/N ratio of the substrate.     

Profiling of differentially expressed genes in ectomycorrhizal fungus Pisolithus tinctorius responding to mycorrhiza helper Brevibacillus reuszeri MPt17

A cDNA library of the ectomycorrhizal (ECM) fungus Pisolithus tinctorius Pt2 after interaction with the mycorrhiza helper bacterium (MHB) Brevibacillus reuszeri MPt17 was constructed by suppression subtraction hybridization. Total RNA from B. reuszeri MPt17 exudates treated Pi. tinctorius Pt2 was used as a “tester” and total RNA from nonbacterial treated Pi. tinctorius Pt2 was used as a “driver.” Among the differentially expressed sequences, a BLASTX in the NCBI non-redundant protein sequence database revealed that 75% of the non-redundant sequences (147 out of 196) were highly similar to known proteins (E-value < e^?5). Twelve sequences were annotated as mycelium development function combining with a potential functional categories using gene ontology. Quantitative real-time PCR analysis showed that all of the 3 symbiosis regulated acidic polypeptide genes were all up-regulated in the MPt17-treated Pt2. These results provide evidence that the MHB B. reuszeri MPt17 could significantly change the expression of symbiosis-related genes and genes in mycelium development in ECM fungus, and also support the hypothesis that the MHB functions as helper though promotion on fungal mycelium.     

Spatial patterns of gene expression in the extramatrical mycelium and mycorrhizal root tips formed by the ectomycorrhizal fungus Paxillus involutus in association with birch (Betula pendula) seedlings in soil microcosms

Functional compartmentation of the extramatrical mycelium of ectomycorrhizal (ECM) fungi is considered important for the operation of ECM associations, although the molecular basis is poorly characterized. Global gene expression profiles of mycelium colonizing an ammonium sulphate ((NH4)2SO4) nutrient patch, rhizomorphs and ECM root tips of the Betula pendula-Paxillus involutus association were compared by cDNA microarray analysis. The expression profiles of rhizomorphs and nutrient patch mycelium were similar to each other but distinctly different from that of mycorrhizal tips. Statistical analyses revealed 337 of 1075 fungal genes differentially regulated among these three tissues. Clusters of genes exhibiting distinct expression patterns within specific tissues were identified. Genes implicated in the glutamine synthetase/glutamate synthase (GS/GOGAT) and urea cycles, and the provision of carbon skeletons for ammonium assimilation via beta-oxidation and the glyoxylate cycle, were highly expressed in rhizomorph and nutrient patch mycelium. Genes implicated in vesicular transport, cytoskeleton organization and morphogenesis and protein degradation were also differentially expressed. Differential expression of genes among the extramatrical mycelium and mycorrhizal tips indicates functional specialization of tissues forming ECM associations.     

Ectomycorrhizal fungal response to warming is linked to poor host performance at the boreal‐temperate ecotone

Rising temperatures associated with climate change have been shown to negatively affect the photosynthetic rates of boreal forest tree saplings at their southern range limits. To quantify the responses of ectomycorrhizal (EM) fungal communities associated with poorly performing hosts, we sampled the roots of Betula papyrifera and Abies balsamea saplings growing in the B4Warmed (Boreal Forest Warming at an Ecotone in Danger) experiment. EM fungi on the root systems of both hosts were compared from ambient and +3.4 °C air and soil warmed plots at two sites in northern Minnesota. EM fungal communities were assessed with high‐throughput sequencing along with measures of plant photosynthesis, soil temperature, moisture, and nitrogen. Warming selectively altered EM fungal community composition at both the phylum and genus levels, but had no significant effect on EM fungal operational taxonomic unit (OTU) diversity. Notably, warming strongly favored EM Ascomycetes and EM fungi with short‐contact hyphal exploration types. Declining host photosynthetic rates were also significantly inversely correlated with EM Ascomycete and EM short‐contact exploration type abundance, which may reflect a shift to less carbon demanding fungi due to lower photosynthetic capacity. Given the variation in EM host responses to warming, both within and between ecosystems, better understanding the link between host performance and EM fungal community structure will to clarify how climate change effects cascade belowground.     

Molecular Identification of Ectomycorrhizal Mycelium in Soil Horizons

Molecular identification techniques based on total DNA extraction provide a unique tool for identification of mycelium in soil. Using molecular identification techniques, the ectomycorrhizal (EM) fungal community under coniferous vegetation was analyzed. Soil samples were taken at different depths from four horizons of a podzol profile. A basidiomycete-specific primer pair (ITS1F-ITS4B) was used to amplify fungal internal transcribed spacer (ITS) sequences from total DNA extracts of the soil horizons. Amplified basidiomycete DNA was cloned and sequenced, and a selection of the obtained clones was analyzed phylogenetically. Based on sequence similarity, the fungal clone sequences were sorted into 25 different fungal groups, or operational taxonomic units (OTUs). Out of 25 basidiomycete OTUs, 7 OTUs showed high nucleotide homology (≥99%) with known EM fungal sequences and 16 were found exclusively in the mineral soil. The taxonomic positions of six OTUs remained unclear. OTU sequences were compared to sequences from morphotyped EM root tips collected from the same sites. Of the 25 OTUs, 10 OTUs had ≥98% sequence similarity with these EM root tip sequences. The present study demonstrates the use of molecular techniques to identify EM hyphae in various soil types. This approach differs from the conventional method of EM root tip identification and provides a novel approach to examine EM fungal communities in soil.     

Ectomycorrhizal fungal community of naturally regenerated European larch (Larix decidua) seedlings

An investigation was undertaken to assess the community structure of ectomycorrhizal (ECM) fungi on naturally regenerating European larch (Larix decidua Mill.) seedlings grown under forest conditions. The sites examined were in two managed monoculture larch forests, differentiated by soil chemistry and mature tree density. Morphological and molecular analyses revealed a total of 22 fungal taxa. From detected ECM fungal taxa, 13 were noted at Site I and 13 at Site II. Only four taxa were found in both sites (Russula ochroleuca, Thelephora terrestris, Lactarius tabidus and Paxillus involutus). The most abundant species at Site I (lower mineral concentration, high tree density) was Hydnotrya tulasnei (25.7?%), followed by Pseudotomentella tristis, Tomentella sublilacina and Russula puelaris. At Site II (higher mineral concentration, low tree density) the dominant fungal symbiont of larch seedlings was clearly Wilcoxina mikolae, which accounted for 74?% of mycorrhizal tips. The less abundant species comprised T. terrestris, L. tabidus, Xerocomus pruinatus and R. ochroleuca. The analysis of similarity (ANOSIM) and non-metric multidimensional scaling (NMDS) ordination clearly separated the ECM fungal assemblages in the two sites tested. Because our study sites were differentiated by many factors, it is not easy to distinguish one factor in particular to explain the differences observed between the ECM communities at Sites I and II. The results obtained significantly increase our knowledge about the diversity of the ECM fungi hosted by L. decidua. The large number of ECM fungi detected was the first observation showing these fungi as symbiotic partners of European larch.     

Transcriptional profiling of wine yeast in fermenting grape juice: regulatory effect of diammonium phosphate

The nitrogen composition of grape musts affects fermentation kinetics and production of aroma and spoilage compounds in wine. It is common practice in wineries to supplement grape musts with diammonium phosphate (DAP) to prevent nitrogen-related fermentation problems. Laboratory strains of Saccharomyces cerevisiae preferentially use rich nitrogen sources, such as ammonia, over poor nitrogen sources. We used global gene expression analysis to monitor the effect of DAP addition on gene expression patterns in wine yeast in fermenting Riesling grape must. The expression of 350 genes in the commercial wine yeast strain VIN13 was affected; 185 genes were down-regulated and 165 genes were up-regulated in response to DAP. Genes that were down-regulated encode small molecule transporters and nitrogen catabolic enzymes, including those linked to the production of urea, a precursor of ethyl carbamate in wine. Genes involved in amino acid metabolism, assimilation of sulfate, de novo purine biosynthesis, tetrahydrofolate one-carbon metabolism, and protein synthesis were up-regulated. The expression level of 86 orphan genes was also affected by DAP.     

Temporal Changes in the Ectomycorrhizal Community in Two Soil Horizons of a Temperate Oak Forest

The species structure of an ectomycorrhizal (ECM) community was assessed monthly for 15 months in the two horizons (A1 and A2) of an oak temperate forest in northeastern France. Ectomycorrhizal species were identified each month by internal transcribed spacer sequencing. Seventy-five fungal symbionts were identified. The community was dominated by Tomentellaceae, Russulaceae, Cortinariaceae, and Boletales. Four species are abundant in the study site: Lactarius quietus, Tomentella sublilacina, Cenococcum geophilum, and Russula sp1. The relative abundance of each species varied depending on the soil horizon and over time. Some species, such as L. quietus, were present in the A1 and A2 horizons. C. geophilum was located particularly in the A2 horizon, whereas T. sublilacina was more abundant in A1. Some species, such as Clavulina sp., were detected in winter, while T. sublilacina and L. quietus were present all year long. Our results support the hypothesis that a rapid turnover of species composition of the ECM community occurs over the course of a month. The spatial and temporal unequal distribution of ECM species could be explained by their ecological preferences, driven by such factors as root longevity, competition for resources, and resistance to environmental variability.     

Ectomycorrhizal colonization slows root decomposition: the post-mortem fungal legacy

The amount of carbon plants allocate to mycorrhizal symbionts exceeds that emitted by human activity annually. Senescent ectomycorrhizal roots represent a large input of carbon into soils, but their fate remains unknown. Here, we present the surprising result that, despite much higher nitrogen concentrations, roots colonized by ectomycorrhizal (EM) fungi lost only one-third as much carbon as non-mycorrhizal roots after 2 years of decomposition in a piñon pine ( Pinus edulis ) woodland. Experimentally excluding live mycorrhizal hyphae from litter, we found that live mycorrhizal hyphae may alter nitrogen dynamics, but the afterlife (litter-mediated) effects of EM fungi outweigh the influences of live fungi on root decomposition. Our findings indicate that a shift in plant allocation to mycorrhizal fungi could promote carbon accumulation in soil by this pathway. Furthermore, EM litters could directly contribute to the process of stable soil organic matter formation, a mechanism that has eluded soil scientists.     

Global patterns of gene regulation associated with the development of ectomycorrhiza between birch (Betula pendula Roth.) and Paxillus involutus (Batsch) Fr

The formation of ectomycorrhizal (ECM) root tissue is characterized by distinct morphological and developmental stages, such as preinfection and adhesion, mantle, and Hartig net formation. The global pattern of gene expression during these stages in the birch (Betula pendula)-Paxillus involutus ECM association was analyzed using cDNA microarrays. In comparison with nonsymbiotic conditions, 251 fungal (from a total of 1,075) and 138 plant (1,074 in total) genes were found to be differentially regulated during the ECM development. For instance, during mantle and Hartig net development, there were several plant genes upregulated that are normally involved in defense responses during pathogenic fungal challenges. These responses were, at later stages of ECM development, found to be repressed. Other birch genes that showed differential regulation involved several homologs that usually are implicated in water permeability (aquaporins) and water stress tolerance (dehydrins). Among fungal genes differentially upregulated during stages of mantle and Hartig net formation were homologs putatively involved in mitochondrial respiration. In fully developed ECM tissue, there was an upregulation of fungal genes related to protein synthesis and the cytoskeleton assembly machinery. This study highlights complex molecular interactions between two symbionts during the development of an ECM association.