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At least 37 records · Page 2

A genetically encoded biosensor reveals spatiotemporal variation in cellular phosphate content in Brachypodium distachyon mycorrhizal roots

Arbuscular mycorrhizal (AM) symbiosis is accompanied by alterations to root cell metabolism and physiology, and to the pathways of orthophosphate (Pi) entry into the root, which increase with Pi delivery to cortical cells via arbuscules. How AM symbiosis influences the Pi content and Pi response dynamics of cells in the root cortex and epidermis is unknown. Using fluorescence resonance energy transfer (FRET)-based Pi biosensors, here we mapped the relative cytosolic and plastidic Pi content of Brachypodium distachyon mycorrhizal root cells, analyzed responses to extracellular Pi and traced extraradical hyphae-mediated Pi transfer to colonized cells. Colonized cortical cells had a higher cytosolic Pi content relative to noncolonized cortical and epidermal cells, while plastidic Pi content was highest in cells at the infection front. Pi application to the entire mycorrhizal root resulted in transient changes in cytosolic Pi that differed in direction and magnitude depending on cell type and arbuscule status; cells with mature arbuscules showed a substantial transient increase in cytosolic Pi while those with collapsed arbuscules showed a decrease. Directed Pi application to extraradical hyphae resulted in measurable changes in cytosolic Pi of colonized cells 18 h after application. Our experiments reveal that cells within a mycorrhizal root vary in Pi content and Pi response dynamics.

32Pi tracing↗

Routes to roots: direct evidence of water transport by arbuscular mycorrhizal fungi to host plants

Summary Arbuscular mycorrhizal fungi (AMF) can help mitigate plant responses to water stress, but it is unclear whether AMF do so by indirect mechanisms, direct water transport to roots, or a combination of the two. Here, we investigated if and how the AMF Rhizophagus intraradices transported water to the host plant Avena barbata , wild oat. We used two‐compartment microcosms, isotopically labeled water, and a fluorescent dye to directly track and quantify water transport by AMF across an air gap to host plants. Plants grown with AMF that had access to a physically separated compartment containing 18 O‐labeled water transpired almost twice as much as plants with AMF excluded from that compartment. Using an isotopic mixing model, we estimated that water transported by AMF across the air gap accounted for 34.6% of the water transpired by host plants. In addition, a fluorescent dye indicated that hyphae were able to transport some water via an extracytoplasmic pathway. Our study provides direct evidence that AMF can act as extensions of the root system along the soil–plant–air continuum of water movement, with plant transpiration driving water flow along hyphae outside of the hyphal cell membrane.

59 BASIC BIOLOGICAL SCIENCES↗

Transparent soil microcosms for live-cell imaging and non-destructive stable isotope probing of soil microorganisms

Microscale processes are critically important to soil ecology and biogeochemistry yet are difficult to study due to soil’s opacity and complexity. To advance the study of soil processes, we constructed transparent soil microcosms that enable the visualization of microbes via fluorescence microscopy and the non-destructive measurement of microbial activity and carbon uptake in situ via Raman microspectroscopy. We assessed the polymer Nafion and the crystal cryolite as optically transparent soil substrates. We demonstrated that both substrates enable the growth, maintenance, and visualization of microbial cells in three dimensions over time, and are compatible with stable isotope probing using Raman. We applied this system to ascertain that after a dry-down/rewetting cycle, bacteria on and near dead fungal hyphae were more metabolically active than those far from hyphae. These data underscore the impact fungi have facilitating bacterial survival in fluctuating conditions and how these microcosms can yield insights into microscale microbial activities.

59 BASIC BIOLOGICAL SCIENCES↗

The cell morphological diversity of Saccharomycotina yeasts

The ~1 200 known species in subphylum Saccharomycotina are a highly diverse clade of unicellular fungi. During its lifecycle, a typical yeast exhibits multiple cell types with various morphologies; these morphologies vary across Saccharomycotina species. Here, we synthesize the evolutionary dimensions of variation in cellular morphology of yeasts across the subphylum, focusing on variation in cell shape, cell size, type of budding, and filament production. Examination of 332 representative species across the subphylum revealed that the most common budding cell shapes are ovoid, spherical, and ellipsoidal, and that their average length and width is 5.6 µm and 3.6 µm, respectively. 58.4% of yeast species examined can produce filamentous cells, and 87.3% of species reproduce asexually by multilateral budding, which does not require utilization of cell polarity for mitosis. Interestingly, ~1.8% of species examined have not been observed to produce budding cells, but rather only produce filaments of septate hyphae and/or pseudohyphae. 76.9% of yeast species examined have sexual cycle descriptions, with most producing one to four ascospores that are most commonly hat-shaped (37.4%). Systematic description of yeast cellular morphological diversity and reconstruction of its evolution promises to enrich our understanding of the evolutionary cell biology of this major fungal lineage.

59 BASIC BIOLOGICAL SCIENCES↗

Genome‐wide CRISPR‐Cas9 screen reveals a persistent null‐hyphal phenotype that maintains high carotenoid production in Yarrowia lipolytica

Abstract Yarrowia lipolytica is a metabolic engineering host of growing industrial interest due to its ability to metabolize hydrocarbons, fatty acids, glycerol, and other renewable carbon sources. This dimorphic yeast undergoes a stress‐induced transition to a multicellular hyphal state, which can negatively impact biosynthetic activity, reduce oxygen and nutrient mass transfer in cell cultures, and increase culture viscosity. Identifying mutations that prevent the formation of hyphae would help alleviate the bioprocess challenges that they create. To this end, we conducted a genome‐wide CRISPR screen to identify genetic knockouts that prevent the transition to hyphal morphology. The screen identified five mutants with a null‐hyphal phenotype— ΔRAS2 , ΔRHO5 , ΔSFL1 , ΔSNF2 , and ΔPAXIP1 . Of these hits, only ΔRAS2 suppressed hyphal formation in an engineered lycopene production strain over a multiday culture. The RAS2 knockout was also the only genetic disruption characterized that did not affect lycopene production, producing more than 5 mg L −1 OD −1 from a heterologous pathway with enhanced carbon flux through the mevalonate pathway. These data suggest that a ΔRAS2 mutant of Y. lipolytica could prove useful in engineering a metabolic engineering host of the production of carotenoids and other biochemicals.

59 BASIC BIOLOGICAL SCIENCES↗

Variation in hyphal production rather than turnover regulates standing fungal biomass in temperate hardwood forests

Abstract Soil fungi link above‐ and belowground carbon (C) fluxes through their interactions with plants and contribute to C and nutrient dynamics through the production, turnover, and activity of fungal hyphae. Despite their importance to ecosystem processes, estimates of hyphal production and turnover rates are relatively uncommon, especially in temperate hardwood forests. We sequentially harvested hyphal ingrowth bags to quantify the rates of Dikarya (Ascomycota and Basidiomycota) hyphal production and turnover in three hardwood forests in the Midwestern United States, where plots differed in their abundance of arbuscular (AM)‐ vs. ectomycorrhizal (ECM)‐associated trees. Hyphal production rates increased linearly with the percentage of ECM trees and annual production rates were 66% higher in ECM‐ than AM‐dominated plots. Hyphal turnover rates did not differ across the mycorrhizal gradient (plots varying in their abundance of AM vs. ECM trees), suggesting that the greater fungal biomass in ECM‐dominated plots relates to greater fungal production rather than slower fungal turnover. Differences in hyphal production across the gradient aligned with distinctly different fungal communities and activities. As ECM trees increased in dominance, fungi inside ingrowth bags produced more extracellular enzymes involved in degrading nitrogen (N)‐bearing relative to C‐bearing compounds, suggesting greater fungal (and possibly plant) N demand in ECM‐dominated soils. Collectively, our results demonstrate that shifts in temperate tree species composition that result in changes in the dominant type of mycorrhizal association may have strong impacts on Dikarya hyphal production, fungal community composition and extracellular enzyme activity, with important consequences for soil C and N cycling.

59 BASIC BIOLOGICAL SCIENCES↗

Differences in soil organic matter between EcM- and AM-dominated forests depend on tree and fungal identity

As global change shifts the species composition of forests, we need to understand which species characteristics affect soil organic matter (SOM) cycling to predict future soil carbon (C) storage. Recently, whether a tree species forms a symbiosis with arbuscular (AM) versus ectomycorrhizal (EcM) fungi has been suggested as a strong predictor of soil C storage, but there is wide variability within EcM systems. In this study, we investigated how mycorrhizal associations and the species composition of canopy trees and mycorrhizal fungi related to the proportion of soil C and nitrogen (N) in mineral associations and soil C:N across four sites representing distinct climates and tree communities in the eastern US broadleaf forest biome. In two of our sites, we found the expected relationship of declining mineral-associated C and N and increasing soil C:N ratios as the basal area of EcM-associating trees increased. However, across all sites these soil properties strongly correlated with canopy tree and fungal species composition. Sites where the expected pattern with EcM basal area was observed were (1) dominated by trees with lower quality litter in the Pinaceae and Fagaceae families and (2) dominated by EcM fungi with medium-distance exploration type hyphae, melanized tissues, and the potential to produce peroxidases. Here, this observational study demonstrates that differences in SOM between AM and EcM systems are dependent on the taxa of trees and EcM fungi involved. Important information is lost when the rich mycorrhizal symbiosis is reduced to two categories.

mineral-associated organic matter↗

Exploring filamentous fungi depolymerization of corn stover in the context bioenergy queuing operations

Recalcitrance of lignocellulosic feedstocks to depolymerization is a significant barrier for bioenergy production approaches that require conversion of monomeric carbohydrates to renewable energy sources. This study assesses how low-cost modifications in the feedstock supply chain can be transformed into targeted pretreatments in the context of the entire bioenergy supply chain. The aim of this research is to overcome the physiochemical barriers in corn stover that necessitate increased severity in conversion in terms of chemical loading, temperature, and residence time. Corn stover samples were inoculated with a selective (Ceriporiopsis subvermispora) and non-selective (Phaenarochaete chrysosporium) lignin degrading filamentous fungal strains, then stored aerobically to determine the working envelope for fungal pretreatment to achieve lignin degradation. Dry matter loss and gross chemical makeup of corn stover varied by the length of treatment (2 and 4 weeks) and by the moisture content of the treated corn stover samples (40 and 60%, wet basis). Dry matter loss in P. chrysosporium inoculated biomass was elevated compared to the C. subvermispora inoculated biomass; however, treatment also induced additional chemical composition changes suggestive of depolymerization. Scanning electron microscope images reveal hyphae attached within cell lumen and suggest structural changes within P. chrysosporium treated corn stover after 60% moisture storage. These results highlight that fungal treatment approaches must balance loss of convertible material with the potential for reduction in recalcitrance. Techno-economic assessment (TEA) of fungal pretreatment in a short-term queuing system indicated the viability of this approach compared to conventional queuing operations. The total queuing system cost was estimated at $\$$1.65/tonne of biomass stored. After applying the credit of $\$$1.48/tonne from energy savings in the conversion phase using fungal pretreated biomass, the total system cost was $0.80 lower than traditional biomass queueing approach. While the TEA results suggested that treating biomass with C. subvermispora is the most economically viable storage method in the designed fungal-assisted queuing system, future research should focus on additional fungal depolymerization such as those observed in the P. chrysosporium inoculated biomass.

09 BIOMASS FUELS↗

Taxogenomic Analysis of a Novel Yeast Species, Lachancea rosae Sp. Nov. F.A., Isolated From the Wild Rose Rosa californica

A novel Saccharomycotina yeast strain, yHQL494, was isolated from the rose hip of the wild rose Rosa californica from Castle Crags State Park, California, USA. Phylogenetic analyses of both whole genome data and the sequences from the D1/D2 region of the large ribosomal subunit (LSU) rRNA gene placed strain yHQL494 within the genus Lachancea and grouped it into a clade with Lachancea lanzarotensis and Lachancea meyersii. Taxogenomic analyses were conducted on publicly available genome sequences to gain a deeper insight into the carbon and nitrogen gene-trait associations across the Lachancea clade. The results of these analyses were found to be consistent across Lachancea species. Growth assays and microscopic analyses were conducted to determine the physiological characteristics of strain yHQL494, including the presence of hyphae or pseudohyphae, ascospore formation, fermentation abilities, and assimilation of carbon and nitrogen compounds. Based on the phenotypic and genomic characteristics of the strain yHQL494 T (=NRRL Y-64858 T , =CBS 18,574 T ), we propose a new species, Lachancea rosae sp. nov. f.a.

Bajgain, Yakendra [Villanova Univ., PA (United Sta↗

Convergent reductive evolution and host adaptation in Mycoavidus bacterial endosymbionts of Mortierellaceae fungi

Intimate associations between fungi and intracellular bacterial endosymbionts are becoming increasingly well understood. Phylogenetic analyses demonstrate that bacterial endosymbionts of Mucoromycota fungi are related either to free-living Burkholderia or Mollicutes species. The so-called Burkholderia-related endosymbionts or BRE comprise Mycoavidus, Mycetohabitans and Candidatus Glomeribacter gigasporarum. These endosymbionts are marked by genome contraction thought to be associated with intracellular selection. However, the conclusions drawn thus far are based on a very small subset of endosymbiont genomes, and the mechanisms leading to genome streamlining are not well understood. The purpose of this study was to better understand how intracellular existence shapes Mycoavidus and BRE functionally at the genome level. To this end we generated and analyzed 14 novel draft genomes for Mycoavidus living within the hyphae of Mortierellomycotina fungi. We found that our novel Mycoavidus genomes were significantly reduced compared to free-living Burkholderiales relatives. Using a genome-scale phylogenetic approach including the novel and available existing genomes of Mycoavidus, we show that the genus is an assemblage composed of two independently derived lineages including three well supported clades of Mycoavidus. Using a comparative genomic approach, we shed light on the functional implications of genome reduction, documenting shared and unique gene loss patterns between the three Mycoavidus clades. We found that many endosymbiont isolates demonstrate patterns of vertical transmission and host-specificity, but others are present in phylogenetically disparate hosts. We discuss how reductive evolution and host specificity reflect convergent adaptation to the intrahyphal selective landscape, and commonalities of eukaryotic endosymbiont genome evolution.

59 BASIC BIOLOGICAL SCIENCES↗

Unlocking the magic in mycelium: Using synthetic biology to optimize filamentous fungi for biomanufacturing and sustainability

Filamentous fungi drive carbon and nutrient cycling across our global ecosystems, through its interactions with growing and decaying flora and their constituent microbiomes. The remarkable metabolic diversity, secretion ability, and fiber-like mycelial structure that have evolved in filamentous fungi have been increasingly exploited in commercial operations. The industrial potential of mycelial fermentation ranges from the discovery and bioproduction of enzymes and bioactive compounds, the decarbonization of food and material production, to environmental remediation and enhanced agricultural production. Despite its fundamental impact in ecology and biotechnology, molds and mushrooms have not, to-date, significantly intersected with synthetic biology in ways comparable to other industrial cell factories (e.g. Escherichia coli,Saccharomyces cerevisiae, and Komagataella phaffii). In this review, we summarize a suite of synthetic biology and computational tools for the mining, engineering and optimization of filamentous fungi as a bioproduction chassis. A combination of methods across genetic engineering, mutagenesis, experimental evolution, and computational modeling can be used to address strain development bottlenecks in established and emerging industries. These include slow mycelium growth rate, low production yields, non-optimal growth in alternative feedstocks, and difficulties in downstream purification. In the scope of biomanufacturing, we then detail previous efforts in improving key bottlenecks by targeting protein processing and secretion pathways, hyphae morphogenesis, and transcriptional control. Bringing synthetic biology practices into the hidden world of molds and mushrooms will serve to expand the limited panel of host organisms that allow for commercially-feasible and environmentally-sustainable bioproduction of enzymes, chemicals, therapeutics, foods, and materials of the future.

59 BASIC BIOLOGICAL SCIENCES↗

Disentangling the role of ectomycorrhizal fungi in plant nutrient acquisition along a Zn gradient using X-ray imaging

Zinc (Zn) is a plant essential micronutrient involved in a wide range of cellular processes. Ectomycorrhizal fungi (EMF) are known to play a critical role in regulating plant Zn status. However, how EMF control uptake and translocation of Zn and other nutrients in plant roots under different Zn conditions is not well known. Using X-ray fluorescence imaging, we found the EMF species Suillus luteus increased pine root Zn acquisition under low Zn concentrations and reduced its accumulation under higher Zn levels. By contrast, non-mycorrhizal pine roots exposed to high Zn indiscriminately take up and translocate Zn to root tissues, leading to Zn stress. Regardless of S. luteus inoculation, the absorption pattern of Ca and Cu was similar to Zn. Compared to Ca and Cu, effects of S. luteus on Fe acquisition were more marked, leading to a negative association between Zn addition and Fe concentration within EMF roots. Besides, higher nutrient accumulation in the fungal sheath, compared to hyphae inhabiting between intercellular space of cortex cells, implies the fungal sheath serves as a barrier to regulate nutrient transportation into fungal Hartig net. Furthermore, our results demonstrate the crucial roles EMF play in plant nutrient uptake and how fungal partners ameliorate soil chemical conditions either by increasing or decreasing element uptake.

36 MATERIALS SCIENCE↗

Getting to the root of the problem: Soil carbon and microbial responses to root inputs within a buried paleosol along an eroding hillslope in southwestern Nebraska, USA

Large quantities of soil carbon (C) can persist within paleosols for millennia due to burial and subsequent isolation from plant-derived inputs, atmospheric conditions, and microbial activity at the modern surface. Erosion exposes buried soils to modern root-derived C influx via root exudation and root turnover, thus stimulating microbial activity leading to SOC decomposition and accumulation through organo-mineral stabilization of modern C. With this study we aim to quantify how modern root-derived C inputs impact paleosol C decomposition and stabilization across varying degrees of isolation from modern surface conditions in southwestern Nebraska, USA, where hillslope erosion is bringing a buried Late-Pleistocene-early Holocene paleosol (the “Brady Soil”) closer to the modern surface. We collected Brady Soil samples from 0.2m, 0.4m, and 1.2m below the modern surface and conducted two lab-based incubations. Soils were amended with either (1) a lab-synthesized mixture of low molecular weight compounds (12 atom% 13 C), or (2) 13 C enriched root residues (92 atom% 13 C), in 30-day and 240-day incubation experiments, respectively. Here we determined microbial responses to synthetic root exudates and residues by partitioning the 13 C label from Brady Soil C, including measurements of total, root, and primed C respiration, microbial biomass C (MBC), microbial C use efficiency (CUE). To assess the capacity of isolated paleosols to accrue modern plant C, we used Nano-scale Secondary Ion Mass Spectrometry imaging. We found that: (1) adding root-derived C inputs primed Brady Soil C across all depths, and was mediated by depth and composition of root additions; (2) root-derived C inputs stimulated microbial biomass C (MBC) growth similarly across depths, but the magnitude of CUE and MBC varied by chemistry of root-derived additions; (3) new particulate organic matter was incorporated into mineral-associated pools over time; (4) material from the added root residues was found in association with bacterial cells and fungal hyphae as well as with soil aggregate and mineral surfaces. Our study shows that paleosols defy expectations of C content and reactivity with depth, and changes in land cover and climate will expose buried paleosols to modern surface conditions, increasing respired C. This work highlights the importance of evaluating the role resurfacing buried soils through landscape change plays in C cycle feedbacks to the climate system.

54 ENVIRONMENTAL SCIENCES↗

Lipo-chitooligosaccharides as regulatory signals of fungal growth and development

Lipo-chitooligosaccharides (LCOs) are signaling molecules produced by rhizobial bacteria that trigger the nodulation process in legumes, and by some fungi that also establish symbiotic relationships with plants, notably the arbuscular and ecto mycorrhizal fungi. Here, we show that many other fungi also produce LCOs. We tested 59 species representing most fungal phyla, and found that 53 species produce LCOs that can be detected by functional assays and/or by mass spectroscopy. LCO treatment affects spore germination, branching of hyphae, pseudohyphal growth, and transcription in non-symbiotic fungi from the Ascomycete and Basidiomycete phyla. Our findings suggest that LCO production is common among fungi, and LCOs may function as signals regulating fungal growth and development.

59 BASIC BIOLOGICAL SCIENCES↗

Allosteric regulation of glycogen breakdown by the second messenger cyclic di-GMP

Streptomyces are our principal source of antibiotics, which they generate concomitant with a complex developmental transition from vegetative hyphae to spores. c-di-GMP acts as a linchpin in this transition by binding and regulating the key developmental regulators, BldD and WhiG. Here we show that c-di-GMP also binds the glycogen-debranching-enzyme, GlgX, uncovering a direct link between c-di-GMP and glycogen metabolism in bacteria. Further, we show c-di-GMP binding is required for GlgX activity. We describe structures of apo and c-di-GMP-bound GlgX and, strikingly, their comparison shows c-di-GMP induces long-range conformational changes, reorganizing the catalytic pocket to an active state. Glycogen is an important glucose storage compound that enables animals to cope with starvation and stress. Our in vivo studies reveal the important biological role of GlgX in Streptomyces glucose availability control. Overall, we identify a function of c-di-GMP in controlling energy storage metabolism in bacteria, which is widespread in Actinobacteria.

59 BASIC BIOLOGICAL SCIENCES↗

Calcium promotes persistent soil organic matter by altering microbial transformation of plant litter

Abstract Calcium (Ca) can contribute to soil organic carbon (SOC) persistence by mediating physico-chemical interactions between organic compounds and minerals. Yet, Ca is also crucial for microbial adhesion, potentially affecting colonization of plant and mineral surfaces. The importance of Ca as a mediator of microbe-mineral-organic matter interactions and resulting SOC transformation has been largely overlooked. We incubated 44 Ca labeled soils with 13 C 15 N labeled leaf litter to study how Ca affects microbial transformation of litter and formation of mineral associated organic matter. Here we show that Ca additions promote hyphae-forming bacteria, which often specialize in colonizing surfaces, and increase incorporation of litter into microbial biomass and carbon use efficiency by approximately 45% each. Ca additions reduce cumulative CO 2 production by 4%, while promoting associations between minerals and microbial byproducts of plant litter. These findings expand the role of Ca in SOC persistence from solely a driver of physico-chemical reactions to a mediator of coupled abiotic-biotic cycling of SOC.

54 ENVIRONMENTAL SCIENCES↗

Study of wrap mode impact on Pseudomonas aeruginosa motion in the chemotactic field of fungi

An agent-based discrete computational model biologically calibrated to Pseudomonas aeruginosa migration is used to explore the impacts of bacterial reversals and wrap mode on the efficiency of motion in different environments, both with and without chemotaxis. It is first shown that wrap mode increases the exploration of continuous multimodal chemotactic profiles such as those produced by biologically relevant fungal networks. For cells undergoing a run-reverse pattern, it is shown that the bacteria are likely to remain at the first local chemoattractant maximal production site on a hypha they find. However, with wrap mode, the bacteria can more easily escape these local sites to further explore their neighboring environment along the fungi, suggesting that wrap mode may be beneficial for migration along the fungi in liquid. In a different set of simulations of bacterial motion close to an isolated chemotactic source, wrap mode is shown to increase the ability of a bacterium to reorient toward the source while reducing the overall motion required for similar chemotactic efficiency as a run-reverse strategy, suggesting a potential metabolic benefit. In contrast, model simulations show that wrap mode can increase the rate of dispersal of P. aeruginosa in a nonchemotactic environment.

Hansen, Austin [University of California, Riversid↗

Computer vision models enable mixed linear modeling to predict arbuscular mycorrhizal fungal colonization using fungal morphology

Abstract The presence of Arbuscular Mycorrhizal Fungi (AMF) in vascular land plant roots is one of the most ancient of symbioses supporting nitrogen and phosphorus exchange for photosynthetically derived carbon. Here we provide a multi-scale modeling approach to predict AMF colonization of a worldwide crop from a Recombinant Inbred Line (RIL) population derived from Sorghum bicolor and S. propinquum . The high-throughput phenotyping methods of fungal structures here rely on a Mask Region-based Convolutional Neural Network (Mask R-CNN) in computer vision for pixel-wise fungal structure segmentations and mixed linear models to explore the relations of AMF colonization, root niche, and fungal structure allocation. Models proposed capture over 95% of the variation in AMF colonization as a function of root niche and relative abundance of fungal structures in each plant. Arbuscule allocation is a significant predictor of AMF colonization among sibling plants. Arbuscules and extraradical hyphae implicated in nutrient exchange predict highest AMF colonization in the top root section. Our work demonstrates that deep learning can be used by the community for the high-throughput phenotyping of AMF in plant roots. Mixed linear modeling provides a framework for testing hypotheses about AMF colonization phenotypes as a function of root niche and fungal structure allocations.

59 BASIC BIOLOGICAL SCIENCES↗