Suillus: an emerging model for the study of ectomycorrhizal ecology and evolution.

Research on mycorrhizal symbiosis has been slowed by a lack of established study systems. To address this challenge, we have been developing Suillus, a widespread ecologically and economically relevant fungal genus primarily associated with the plant family Pinaceae, into a model system for studying ectomycorrhizal (ECM) associations. Over the last decade, we have compiled extensive genomic resources, culture libraries, a phenotype database, and protocols for manipulating Suillus fungi with and without their tree partners. Our efforts have already resulted in a large number of publicly available genomes, transcriptomes, and respective annotations, as well as advances in our understanding of mycorrhizal partner specificity and host communication, fungal and plant nutrition, environmental adaptation, soil nutrient cycling, interspecific competition, and biological invasions. Here, we highlight the most significant recent findings enabled by Suillus, present a suite of protocols for working with the genus, and discuss how Suillus is emerging as an important model to elucidate the ecology and evolution of ECM interactions.

The elusive nature of the oligoclonal bands in multiple sclerosis.

Intrathecal immunoglobulin G (IgG) and oligoclonal bands (OCBs) detected in both the brain and cerebrospinal fluid (CSF) are seminal features of multiple sclerosis (MS). The presence of OCBs correlates with elevated disease burden and severity and supports the diagnosis of MS. Despite numerous investigations into the potential viral and autoantigen targets, the precise antigenic specificity of OCBs has remained elusive. We have little knowledge of the nature regarding these oligoclonal IgG bands. Here, we present compelling evidence highlighting the key findings that both OCBs and intrathecal IgG antibodies are under genetic control and that OCBs originate from clonal B-cells in both the periphery and CNS. We propose that MS OCBs are IgG immune complexes composed of IgG1 and IgG3 antibodies and that the pathological role of OCB stems from the IgG effector functions of these complexes, leading to demyelination and axonal injuries. We present additional evidence regarding the nature of MS OCBs: (1) disease-modifying therapies have been shown to affect CSF OCB; (2) OCBs have also been detected in several neuroinfectious diseases; (3) Epstein-Barr virus (EBV) has been particularly linked with MS pathogenesis, and its association with OCB is an important area of study. Although OCBs are closely associated with MS, more meticulously planned research is necessary to clarify the precise role of OCB in MS, both in terms of disease pathogenesis and diagnosis.

Herbarium specimens reveal that mycorrhizal type does not mediate declining temperate tree nitrogen status over a century of environmental change.

Rising atmospheric carbon dioxide concentrations (CO2 ) and atmospheric nitrogen (N) deposition have contrasting effects on ectomycorrhizal (EM) and arbuscular mycorrhizal (AM) symbioses, potentially mediating forest responses to environmental change. In this study, we evaluated the cumulative effects of historical environmental change on N concentrations and δ15 N values in AM plants, EM plants, EM fungi, and saprotrophic fungi using herbarium specimens collected in Minnesota, USA from 1871 to 2016. To better understand mycorrhizal mediation of foliar δ15 N, we also analyzed a subset of previously published foliar δ15 N values from across the United States to parse the effects of N deposition and CO2 rise. Over the last century in Minnesota, N concentrations declined among all groups except saprotrophic fungi. δ15 N also declined among all groups of plants and fungi; however, foliar δ15 N declined less in EM plants than in AM plants. In the analysis of previously published foliar δ15 N values, this slope difference between EM and AM plants was better explained by nitrogen deposition than by CO2 rise. Mycorrhizal type did not explain trajectories of plant N concentrations. Instead, plants and EM fungi exhibited similar declines in N concentrations, consistent with declining forest N status despite moderate levels of N deposition.

Defining a core microbial necrobiome associated with decomposing fungal necromass.

Despite growing interest in fungal necromass decomposition due to its importance in soil carbon retention, whether a consistent group of microorganisms is associated with decomposing necromass remains unresolved. Here, we synthesize knowledge on the composition of the bacterial and fungal communities present on decomposing fungal necromass from a variety of fungal species, geographic locations, habitats, and incubation times. We found that there is a core group of both bacterial and fungal genera (i.e. a core fungal necrobiome), although the specific size of the core depended on definition. Based on a metric that included both microbial frequency and abundance, we demonstrate that the core is taxonomically and functionally diverse, including bacterial copiotrophs and oligotrophs as well as fungal saprotrophs, ectomycorrhizal fungi, and both fungal and animal parasites. We also show that the composition of the core necrobiome is notably dynamic over time, with many core bacterial and fungal genera having specific associations with the early, middle, or late stages of necromass decomposition. While this study establishes the existence of a core fungal necrobiome, we advocate that profiling the composition of fungal necromass decomposer communities in tropical environments and other terrestrial biomes beyond forests is needed to fill key knowledge gaps regarding the global nature of the fungal necrobiome.

The Spectrum of Neurological Manifestations of Varicella-Zoster Virus Reactivation.

Varicella-Zoster virus (VZV) is a pathogenic human alpha herpes virus that causes varicella (chicken pox) as a primary infection and, following a variable period of latency in different ganglionic neurons, it reactivates to produce herpes zoster (shingles). The focus of this review is on the wide spectrum of the possible neurological manifestations of VZV reactivation. While the most frequent reactivation syndrome is herpes zoster, this may be followed by the serious and painful post-herpetic neuralgia (PHN) and by many other neurological conditions. Prominent among these conditions is a VZV vasculopathy, but the role of VZV in causing giant cell arteritis (GCA) is currently controversial. VZV reactivation can also cause segmental motor weakness, myelitis, cranial nerve syndromes, Guillain-Barre syndrome, meningoencephalitis, and zoster sine herpete, where a neurological syndrome occurs in the absence of the zoster rash. The field is complicated by the relatively few cases of neurological complications described and by the issue of causation when a neurological condition is not manifest at the same time as the zoster rash.

Climate change-induced stress disrupts ectomycorrhizal interaction networks at the boreal-temperate ecotone.

The interaction networks formed by ectomycorrhizal fungi (EMF) and their tree hosts, which are important to both forest recruitment and ecosystem carbon and nutrient retention, may be particularly susceptible to climate change at the boreal-temperate forest ecotone where environmental conditions are changing rapidly. Here, we quantified the compositional and functional trait responses of EMF communities and their interaction networks with two boreal (Pinus banksiana and Betula papyrifera) and two temperate (Pinus strobus and Quercus macrocarpa) hosts to a factorial combination of experimentally elevated temperatures and reduced rainfall in a long-term open-air field experiment. The study was conducted at the B4WarmED (Boreal Forest Warming at an Ecotone in Danger) experiment in Minnesota, USA, where infrared lamps and buried heating cables elevate temperatures (ambient, +3.1 °C) and rain-out shelters reduce growing season precipitation (ambient, ~30% reduction). EMF communities were characterized and interaction networks inferred from metabarcoding of fungal-colonized root tips. Warming and rainfall reduction significantly altered EMF community composition, leading to an increase in the relative abundance of EMF with contact-short distance exploration types. These compositional changes, which likely limited the capacity for mycelial connections between trees, corresponded with shifts from highly redundant EMF interaction networks under ambient conditions to less redundant (more specialized) networks. Further, the observed changes in EMF communities and interaction networks were correlated with changes in soil moisture and host photosynthesis. Collectively, these results indicate that the projected changes in climate will likely lead to significant shifts in the traits, structure, and integrity of EMF communities as well as their interaction networks in forest ecosystems at the boreal-temperate ecotone.

Melanization slows the rapid movement of fungal necromass carbon and nitrogen into both bacterial and fungal decomposer communities and soils.

Microbial necromass contributes significantly to both soil carbon (C) persistence and ecosystem nitrogen (N) availability, but quantitative estimates of C and N movement from necromass into soils and decomposer communities are lacking. Additionally, while melanin is known to slow fungal necromass decomposition, how it influences microbial C and N acquisition as well as elemental release into soils remains unclear. Here, we tracked decomposition of isotopically labeled low and high melanin fungal necromass and measured 13C and 15N accumulation in surrounding soils and microbial communities over 77 d in a temperate forest in Minnesota, USA. Mass loss was significantly higher from low melanin necromass, corresponding with greater 13C and 15N soil inputs. A taxonomically and functionally diverse array of bacteria and fungi was enriched in 13C and/or 15N at all sampling points, with enrichment being consistently higher on low melanin necromass and earlier in decomposition. Similar patterns of preferential C and N enrichment of many bacterial and fungal genera early in decomposition suggest that both microbial groups co-contribute to the rapid assimilation of resource-rich soil organic matter inputs. While overall richness of taxa enriched in C was higher than in N for both bacteria and fungi, there was a significant positive relationship between C and N in co-enriched taxa. Collectively, our results demonstrate that melanization acts as a key ecological trait mediating not only fungal necromass decomposition rate but also necromass C and N release and that both elements are rapidly co-utilized by diverse bacterial and fungal decomposers in natural settings. IMPORTANCE Recent studies indicate that microbial dead cells, particularly those of fungi, play an important role in long-term carbon persistence in soils. Despite this growing recognition, how the resources within dead fungal cells (also known as fungal necromass) move into decomposer communities and soils are poorly quantified, particularly in studies based in natural environments. In this study, we found that the contribution of fungal necromass to soil carbon and nitrogen availability was slowed by the amount of melanin present in fungal cell walls. Further, despite the overall rapid acquisition of carbon and nitrogen from necromass by a diverse range of both bacteria and fungi, melanization also slowed microbial uptake of both elements. Collectively, our results indicate that melanization acts as a key ecological trait mediating not only fungal necromass decomposition rate, but also necromass carbon and nitrogen release into soil as well as microbial resource acquisition.

Fungal necromass presents a high potential for Mercury immobilization in soil.

Past industrial activities have generated many contaminated lands from which Mercury (Hg) escapes, primarily by volatilization. Current phytomanagement techniques aim to limit Hg dispersion by increasing its stabilization in soil. Although soil fungi represent a source of Hg emission associated with biovolatilization mechanisms, there is limited knowledge about how dead fungal residues (i.e., fungal necromass) interact with soil Hg. This study determined the Hg biosorption potential of fungal necromass and the chemical drivers of passive Hg binding with dead mycelia. Fungal necromass was incubated under field conditions with contrasting chemical properties at a well-characterized Hg phytomanagement experimental site in France. After four months of incubation in soil, fungal residues passively accumulated substantial quantities of Hg in their recalcitrant fractions ranging from 400 to 4500 µg Hg/kg. In addition, infrared spectroscopy revealed that lipid compounds explained the amount of Hg biosorption to fungal necromass. Based on these findings, we propose that fungal necromass is likely an important factor in Hg immobilization in soil.

Knowns and unknowns of the soil fungal necrobiome.

Dead microbial cells, commonly referred to as necromass, are increasingly recognized as an important source of both persistent carbon as well as nutrient availability in soils. Studies of the microbial communities associated with decomposing fungal necromass have accumulated rapidly in recent years across a range of different terrestrial ecosystems. Here we identify the primary ecological patterns regarding the structure and dynamics of the fungal necrobiome as well as highlight new research frontiers that will likely be key to gaining a full understanding of fungal necrobiome composition and its associated role in soil biogeochemical cycling. Because many members of the fungal necrobiome are culturable, combining laboratory functional assays with field-based surveys and experiments will allow ongoing studies of the fungal necrobiome to move from largely descriptive to increasingly predictive.

Phenotype and Neuronal Cytotoxic Function of Glioblastoma Extracellular Vesicles.

Glioblastoma (GBM) is the most aggressive and lethal form of brain tumor. Extracellular vesicles (EVs) released by tumor cells play a critical role in cellular communication in the tumor microenvironment promoting tumor progression and invasion. We hypothesized that GBM EVs possess unique characteristics which exert effects on endogenous CNS cells including neurons, producing dose-dependent neuronal cytotoxicity. We purified EVs from the plasma of 20 GBM patients, 20 meningioma patients, and 21 healthy controls, and characterized EV phenotypes by electron microscopy, nanoparticle tracking analysis, protein concentration, and proteomics. We evaluated GBM EV functions by determining their cytotoxicity in primary neurons and the neuroblastoma cell line SH-SY5Y. In addition, we determined levels of IgG antibodies in the plasma in GBM (n = 82), MMA (n = 83), and controls (non-tumor CNS disorders and healthy donors, n = 50) with capture ELISA. We discovered that GBM plasma EVs are smaller in size and had no relationship between size and concentration. Importantly, GBM EVs purified from both plasma and tumor cell lines produced IgG-mediated, complement-dependent apoptosis and necrosis in primary human neurons, mouse brain slices, and neuroblastoma cells. The unique phenotype of GBM EVs may contribute to its neuronal cytotoxicity, providing insight into its role in tumor pathogenesis.

The Possible Role of Neural Cell Apoptosis in Multiple Sclerosis.

The etiology of multiple sclerosis (MS), a demyelinating disease affecting the central nervous system (CNS), remains obscure. Although apoptosis of oligodendrocytes and neurons has been observed in MS lesions, the contribution of this cell death process to disease pathogenesis remains controversial. It is usually considered that MS-associated demyelination and axonal degeneration result from neuroinflammation and an autoimmune process targeting myelin proteins. However, experimental data indicate that oligodendrocyte and/or neuronal cell death may indeed precede the development of inflammation and autoimmunity. These findings raise the question as to whether neural cell apoptosis is the key event initiating and/or driving the pathological cascade, leading to clinical functional deficits in MS. Similarly, regarding axonal damage, a key pathological feature of MS lesions, the roles of inflammation-independent and cell autonomous neuronal processes need to be further explored. While oligodendrocyte and neuronal loss in MS may not necessarily be mutually exclusive, particular attention should be given to the role of neuronal apoptosis in the development of axonal loss. If proven, MS could be viewed primarily as a neurodegenerative disease accompanied by a secondary neuroinflammatory and autoimmune process.

Variable Gene Expression in Human Ganglia Latently Infected with Varicella-Zoster Virus.

Varicella-Zoster virus (VZV) is a pathogenic human herpes virus that causes varicella ("chicken pox") as a primary infection, following which it becomes latent in neuronal cells in human peripheral ganglia. It may then reactivate to cause herpes zoster ("shingles"). Defining the pattern of VZV gene expression during latency is an important issue, and four highly expressed VZV genes were first identified by Randall Cohrs in 1996 using cDNA libraries. Further studies from both his and other laboratories, including our own, have suggested that viral gene expression may be more widespread than previously thought, but a confounding factor has always been the possibility of viral reactivation after death in tissues obtained even at 24 h post-mortem. Recent important studies, which Randall Cohrs contributed to, have clarified this issue by studying human trigeminal ganglia at 6 h after death using RNA-Seq methodology when a novel spliced latency-associated VZV transcript (VLT) was found to be mapped antisense to the viral transactivator gene 61. Viral gene expression could be induced by a VLT-ORF 63 fusion transcript when VZV reactivated from latency. Prior detection by several groups of ORF63 in post-mortem-acquired TG is very likely to reflect detection of the VLT-ORF63 fusion and not canonical ORF63. The contributions to the VZV latency field by Randall Cohrs have been numerous and highly significant.

Best practices in metabarcoding of fungi: From experimental design to results.

The development of high-throughput sequencing (HTS) technologies has greatly improved our capacity to identify fungi and unveil their ecological roles across a variety of ecosystems. Here we provide an overview of current best practices in metabarcoding analysis of fungal communities, from experimental design through molecular and computational analyses. By reanalysing published data sets, we demonstrate that operational taxonomic units (OTUs) outperform amplified sequence variants (ASVs) in recovering fungal diversity, a finding that is particularly evident for long markers. Additionally, analysis of the full-length ITS region allows more accurate taxonomic placement of fungi and other eukaryotes compared to the ITS2 subregion. Finally, we show that specific methods for compositional data analyses provide more reliable estimates of shifts in community structure. We conclude that metabarcoding analyses of fungi are especially promising for integrating fungi into the full microbiome and broader ecosystem functioning context, recovery of novel fungal lineages and ancient organisms as well as barcoding of old specimens including type material.

Hyphae move matter and microbes to mineral microsites: Integrating the hyphosphere into conceptual models of soil organic matter stabilization.

Associations between soil minerals and microbially derived organic matter (often referred to as mineral-associated organic matter or MAOM) form a large pool of slowly cycling carbon (C). The rhizosphere, soil immediately adjacent to roots, is thought to control the spatial extent of MAOM formation because it is the dominant entry point of new C inputs to soil. However, emphasis on the rhizosphere implicitly assumes that microbial redistribution of C into bulk (non-rhizosphere) soils is minimal. We question this assumption, arguing that because of extensive fungal exploration and rapid hyphal turnover, fungal redistribution of soil C from the rhizosphere to bulk soil minerals is common, and encourages MAOM formation. First, we summarize published estimates of fungal hyphal length density and turnover rates and demonstrate that fungal C inputs are high throughout the rhizosphere-bulk soil continuum. Second, because colonization of hyphal surfaces is a common dispersal mechanism for soil bacteria, we argue that hyphal exploration allows for the non-random colonization of mineral surfaces by hyphae-associated taxa. Third, these bacterial communities and their fungal hosts determine the chemical form of organic matter deposited on colonized mineral surfaces. Collectively, our analysis demonstrates that omission of the hyphosphere from conceptual models of soil C flow overlooks key mechanisms for MAOM formation in bulk soils. Moving forward, there is a clear need for spatially explicit, quantitative research characterizing the environmental drivers of hyphal exploration and hyphosphere community composition across systems, as these are important controls over the rate and organic chemistry of C deposited on minerals.

Higher Levels of IgG3 Antibodies in Serum, But Not in CSF, Distinguish Multiple Sclerosis From Other Neurological Disorders.

Increased intrathecal IgG and oligoclonal bands (OCB) are seminal features of multiple sclerosis (MS). Although no such differences in MS blood total IgG antibodies have been reported, serum OCB are a common and persistent finding in MS and have a systemic source. Recent studies showed that IgG3+ B cells and higher levels of serum IgG3 are linked to the development of MS. Additionally, intrathecal IgG synthesis in MS is associated with IgG3 heavy chain gene single nucleotide polymorphisms, and there is a strong relationship between susceptibility to MS and an IgG3 restriction fragment length polymorphism. These studies support the role of IgG3 in disease pathogenesis. Using multiple immunoassays, we investigated levels of total IgG, IgG1, and IgG3 in sera and CSF of 102 MS patients (19 paired CSF and sera), 76 patients with other neurological disorders (9 paired CSF and sera), and 13 healthy controls. We show that higher levels of total IgG and IgG3 antibodies were detected in MS serum, but not in CSF, which distinguishes MS from other inflammatory and non-inflammatory neurological disorders, with Receiver Operating Characteristic (ROC) Curves 0.79 for both IgG3 & total IgG. Our data support the notion that IgG3 antibodies may be a potential candidate for MS blood biomarker development.

Aberrant Immunoglobulin G Glycosylation in Multiple Sclerosis.

A hallmark of the inflammatory response in multiple sclerosis (MS) is the presence of intrathecal Immunoglobulin G (IgG) antibodies and oligoclonal bands (OCBs). The biological activity of IgGs is modulated by changes in glycosylation. Using multiple immunoassays with common lectins for sialylation and galactosylation, we investigated levels of IgG glycosylation in 28 MS and 37 control sera as well as paired CSF and serum. We demonstrated the presence of significantly lower levels of IgG sialylation in MS CSF compared to paired serum. Further, we showed that in MS there was no correlation between sialylated IgG and total IgG antibodies, or between sialylated IgG in CSF and serum. ELISA with native IgG antibodies showed significantly higher levels of sialylated and galactosylated IgG in MS compared to other neurological disorders and normal healthy controls. We conclude that lower levels of sialylated intrathecal IgG and higher levels of serum IgG galactosylation in MS may play significant role in disease pathogenesis. The unique IgG glycosylation profiles suggest a complexed nature of the IgG antibodies which may influence its effector functions in MS.

Warming drives a 'hummockification' of microbial communities associated with decomposing mycorrhizal fungal necromass in peatlands.

Dead fungal mycelium (necromass) represents a critical component of soil carbon (C) and nutrient cycles. Assessing how the microbial communities associated with decomposing fungal necromass change as global temperatures rise will help in determining how these belowground organic matter inputs contribute to ecosystem responses. In this study, we characterized the structure of bacterial and fungal communities associated with multiple types of decaying mycorrhizal fungal necromass incubated within mesh bags across a 9°C whole ecosystem temperature enhancement in a boreal peatland. We found major taxonomic and functional shifts in the microbial communities present on decaying mycorrhizal fungal necromass in response to warming. These changes were most pronounced in hollow microsites, which showed convergence towards the necromass-associated microbial communities present in unwarmed hummocks. We also observed a high colonization of ericoid mycorrhizal fungal necromass by fungi from the same genera as the necromass. These results indicate that microbial communities associated with mycorrhizal fungal necromass decomposition are likely to change significantly with future climate warming, which may have strong impacts on soil biogeochemical cycles in peatlands. Additionally, the high enrichment of congeneric fungal decomposers on ericoid mycorrhizal necromass may help to explain the increase in ericoid shrub dominance in warming peatlands.