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At least 181 records · Page 10
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Evolutionary transition to the ectomycorrhizal habit in the genomes of a hyperdiverse lineage of mushroom-forming fungi

The ectomycorrhizal (ECM) symbiosis has independently evolved from diverse types of saprotrophic ancestors. In this study, we seek to identify genomic signatures of the transition to the ECM habit within the hyperdiverse Russulaceae. We present comparative analyses of the genomic architecture and the total and secreted gene repertoires of 18 species across the order Russulales, of which 13 are newly sequenced, including a representative of a saprotrophic member of Russulaceae, Gloeopeniophorella convolvens. The genomes of ECM Russulaceae are characterized by a loss of genes for plant cell wall-degrading enzymes (PCWDEs), an expansion of genome size through increased transposable element (TE) content, a reduction in secondary metabolism clusters, and an association of small secreted proteins (SSPs) with TE ‘nests’, or dense aggregations of TEs. Here, some PCWDEs have been retained or even expanded, mostly in a species-specific manner. The genome of G. convolvens possesses some characteristics of ECM genomes (e.g. loss of some PCWDEs, TE expansion, reduction in secondary metabolism clusters). Functional specialization in ECM decomposition may drive diversification. Accelerated gene evolution predates the evolution of the ECM habit, indicating that changes in genome architecture and gene content may be necessary to prime the evolutionary switch.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Local carbon reserves are insufficient for phloem terpene induction during drought in Pinus edulis in response to bark beetle–associated fungi

Stomatal closure during drought inhibits carbon uptake and may reduce a tree's defensive capacity. Limited carbon availability during drought may increase a tree's mortality risk, particularly if drought constrains trees' capacity to rapidly produce defenses during biotic attack. We parameterized a new model of conifer defense using physiological data on carbon reserves and chemical defenses before and after a simulated bark beetle attack in mature Pinus edulis under experimental drought. Attack was simulated using inoculations with a consistent bluestain fungus (Ophiostoma sp.) of Ips confusus, the main bark beetle colonizing this tree, to induce a defensive response. Trees with more carbon reserves produced more defenses but measured phloem carbon reserves only accounted for c. 23% of the induced defensive response. Our model predicted universal mortality if local reserves alone supported defense production, suggesting substantial remobilization and transport of stored resin or carbon reserves to the inoculation site. Our results show that de novo terpene synthesis represents only a fraction of the total measured phloem terpenes in P. edulis following fungal inoculation. Without direct attribution of phloem terpene concentrations to available carbon, many studies may be overestimating the scale and importance of de novo terpene synthesis in a tree's induced defense response.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Coevolution of the Ess1-CTD axis in polar fungi suggests a role for phase separation in cold tolerance

Most of the world’s biodiversity lives in cold (-2° to 4°C) and hypersaline environments. To understand how cells adapt to such conditions, we isolated two key components of the transcription machinery from fungal species that live in extreme polar environments: the Ess1 prolyl isomerase and its target, the carboxy-terminal domain (CTD) of RNA polymerase II. Polar Ess1 enzymes are conserved and functional in the model yeast, Saccharomyces cerevisiae. By contrast, polar CTDs diverge from the consensus (YSPTSPS) 26 and are not fully functional in S. cerevisiae. These CTDs retain the critical Ess1 Ser-Pro target motifs, but substitutions at Y1, T4, and S7 profoundly affected their ability to undergo phase separation in vitro and localize in vivo. We propose that environmentally tuned phase separation by the CTD and other intrinsically disordered regions plays an adaptive role in cold tolerance by concentrating enzymes and substrates to overcome energetic barriers to metabolic activity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Enzyme Discovery in Anaerobic Fungi (Neocallimastigomycetes) Enables Lignocellulosic Biorefinery Innovation

Lignocellulosic biorefineries require innovative solutions to realize their full potential, and the discovery of novel lignocellulose-active enzymes could improve biorefinery deconstruction processes. Enzymatic deconstruction of plant cell walls is challenging, as noncarbohydrate linkages in hemicellulosic sidechains and lignin protect labile carbohydrates from hydrolysis.

Microbiology↗
DOE OSTI2022

Identification of microRNAs responsive to arbuscular mycorrhizal fungi in Panicum virgatum (switchgrass)

Background: MicroRNAs (miRNAs) are important post-transcriptional regulators involved in the control of a range of processes, including symbiotic interactions in plants. MiRNA involvement in arbuscular mycorrhizae (AM) symbiosis has been mainly studied in model species, and our study is the first to analyze global miRNA expression in the roots of AM colonized switchgrass ( Panicum virgatum ), an emerging biofuel feedstock. AM symbiosis helps plants gain mineral nutrition from the soil and may enhance switchgrass biomass production on marginal lands. Our goals were to identify miRNAs and their corresponding target genes that are controlling AM symbiosis in switchgrass. Results: Through genome-wide analysis of next-generation miRNA sequencing reads generated from switchgrass roots, we identified 122 mature miRNAs, including 28 novel miRNAs. By comparing miRNA expression profiles of AM-inoculated and control switchgrass roots, we identified 15 AM-responsive miRNAs across lowland accession “Alamo”, upland accession “Dacotah”, and two upland/lowland F 1 hybrids. We used degradome sequencing to identify target genes of the AM-responsive miRNAs revealing targets of miRNAs residing on both K and N subgenomes. Notably, genes involved in copper ion binding were targeted by downregulated miRNAs, while upregulated miRNAs mainly targeted GRAS family transcription factors. Conclusion: Through miRNA analysis and degradome sequencing, we revealed that both upland and lowland switchgrass genotypes as well as upland-lowland hybrids respond to AM by altering miRNA expression. We demonstrated complex GRAS transcription factor regulation by the miR171 family, with some miR171 family members being AM responsive while others remained static. Copper miRNA downregulation was common amongst the genotypes tested and we identified superoxide dismutases and laccases as targets, suggesting that these Cu-miRNAs are likely involved in ROS detoxification and lignin deposition, respectively. Other prominent targets of the Cu miRNAs were blue copper proteins. Overall, the potential effect of AM colonization on lignin deposition pathways in this biofuel crop highlights the importance of considering AM and miRNA in future biofuel crop development strategies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Identification, characterization, and structure of a tRNA splicing enzyme RNA 5′-OH kinase from the pathogenic fungi Mucorales

Fungal Trl1 is an essential tRNA splicing enzyme composed of C-terminal cyclic phosphodiesterase and central polynucleotide kinase end-healing domains that convert the 2′,3′-cyclic-PO 4 and 5′-OH ends of tRNA exons into the 3′-OH,2′-PO 4 and 5′-PO 4 termini required for sealing by an N-terminal ATP-dependent ligase domain. Trifunctional Trl1 enzymes are present in most human fungal pathogens and are untapped targets for antifungal drug discovery. Mucorales species, deemed high-priority human pathogens by WHO, elaborate a noncanonical tRNA splicing apparatus in which a stand-alone monofunctional RNA ligase enzyme joins 3′-OH,2′-PO 4 and 5′-PO 4 termini. Here we identify a stand-aloneMucor circinelloidespolynucleotide kinase (MciKIN) and affirm its biological activity in tRNA splicing by genetic complementation in yeast. Recombinant MciKIN catalyzes magnesium-dependent phosphorylation of 5′-OH RNA and DNA ends in vitro. MciKIN displays a strong preference for GTP as the phosphate donor in the kinase reaction, a trait shared with the stand-alone RNA kinase homologs from Mucorales speciesRhizopus azygosporus(RazKIN) andLichtheimia corymbifera(LcoKIN) and with the kinase domains of fungal Trl1 enzymes. We report a 1.65 Å crystal structure of RazKIN in complex with GDP•Mg 2+ that illuminates the basis for guanosine nucleotide specificity.

Biochemistry & Molecular Biology↗
DOE OSTI2023

FUN-PROSE: A deep learning approach to predict condition-specific gene expression in fungi

mRNA levels of all genes in a genome is a critical piece of information defining the overall state of the cell in a given environmental condition. Being able to reconstruct such condition-specific expression in fungal genomes is particularly important to metabolically engineer these organisms to produce desired chemicals in industrially scalable conditions. Most previous deep learning approaches focused on predicting the average expression levels of a gene based on its promoter sequence, ignoring its variation across different conditions. Here we present FUN-PROSE—a deep learning model trained to predict differential expression of individual genes across various conditions using their promoter sequences and expression levels of all transcription factors. We train and test our model on three fungal species and get the correlation between predicted and observed condition-specific gene expression as high as 0.85. We then interpret our model to extract promoter sequence motifs responsible for variable expression of individual genes. We also carried out input feature importance analysis to connect individual transcription factors to their gene targets. A sizeable fraction of both sequence motifs and TF-gene interactions learned by our model agree with previously known biological information, while the rest corresponds to either novel biological facts or indirect correlations.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Imaging Intact Filamentous Fungi from Spore to Hyphal Tip on the Nanoscale

Previously, the Pacific Northwest National Laboratory was limited by a 3-order of magnitude gap in resolution for direct 2-D and 3-D imaging of chemical, biological, energy and materials science systems. Our imaging instrumentation could directly visualize 3-D ultrastructure without labeling at resolutions less than 10-nanometers or greater than 10-micrometers. To address this gap, we procured and commissioned a Sigray NanoFast Bio-Lambda laboratory-based x-ray nanotomography system. This equipment provides the capability to image through tens of micrometers of organic materials at down to 35-nanometer resolution to bridge the nanoscale to the mesoscale for biological systems, organic polymers, environmental and particulate samples.

47 OTHER INSTRUMENTATION↗
DOE OSTI2026

Engineering Anti-Microbial Peptide Production in Fungi and Bioprocess Development (CRADA 502)

Crop Protection, specifically controlling agricultural diseases and pests, is essential for crop production at every agricultural scale. Biologics hold enormous potential in this equation to decrease use of more toxic pesticides. Currently, the widespread use of biologics in crop protection is severely hampered by the lack of available tools for their mass production. To ensure biologics are regulated under EPA Biopesticide regulations, the following must be true: The biopesticide is a naturally occurring substance or structurally similar and functionally identical to a naturally occurring substance with a history of exposure to humans and the environment demonstrating minimal toxicity. Since this pertains to the marketed product it is also essential that fermentation hosts have a history of exposure and safe use by humans This research focuses on developing a novel platform for the production and delivery of the cghSAMP peptide using the filamentous fungus Aspergillus niger. A. niger is a well-established industrial microorganism, widely recognized for its strong protein secretion capabilities, high-yield production of enzymes, and classification as a generally recognized as safe (GRAS) organism. By genetically modifying A. niger to express the cghSAMP peptide transgene, we aim to leverage the fungus's robust fermentation system to produce a high volume of the antimicrobial agent. This engineered system represents a potential breakthrough in sustainable disease management, offering an environmentally friendly method for localized application or large-scale production of a biopesticide designed to mitigate the effects of citrus greening disease. The main objectives of this project were: 1. Developing a high-yield and cost-efficient bioprocess to produce an AMP in a current ABF host or readily onboarded hosts. 2. Scale up the process in stirred tank reactors and produce at least 100 grams of AMP at 1- 5g/L

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Comparative Genomic Analysis of Ochratoxin A Biosynthetic Cluster in Producing Fungi: New Evidence of a Cyclase Gene Involvement

The widespread use of Next-Generation Sequencing has opened a new era in the study of biological systems by significantly increasing the catalog of fungal genomes sequences and identifying gene clusters for known secondary metabolites as well as novel cryptic ones. However, most of these clusters still need to be examined in detail to completely understand the pathway steps and the regulation of the biosynthesis of metabolites. Genome sequencing approach led to the identification of the biosynthetic genes cluster of ochratoxin A (OTA) in a number of producing fungal species. Ochratoxin A is a potent pentaketide nephrotoxin produced by Aspergillus and Penicillium species and found as widely contaminant in food, beverages and feed. The increasing availability of several new genome sequences of OTA producer species in JGI Mycocosm and/or GenBank databanks led us to analyze and update the gene cluster structure in 19 Aspergillus and 2 Penicillium OTA producing species, resulting in a well conserved organization of OTA core genes among the species. Furthermore, our comparative genome analyses evidenced the presence of an additional gene, previously undescribed, located between the polyketide and non-ribosomal synthase genes in the cluster of all the species analyzed. The presence of a SnoaL cyclase domain in the sequence of this gene supports its putative role in the polyketide cyclization reaction during the initial steps of the OTA biosynthesis pathway. The phylogenetic analysis showed a clustering of OTA SnoaL domains in accordance with the phylogeny of OTA producing species at species and section levels. The characterization of this new OTA gene, its putative role and its expression evidence in three important representative producing species, are reported here for the first time.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Unveiling the Arsenal of Apple Bitter Rot Fungi: Comparative Genomics Identifies Candidate Effectors, CAZymes, and Biosynthetic Gene Clusters in Colletotrichum Species

The bitter rot of apple is caused by Colletotrichum spp. and is a serious pre-harvest disease that can manifest in postharvest losses on harvested fruit. In this study, we obtained genome sequences from four different species, C. chrysophilum, C. noveboracense, C. nupharicola, and C. fioriniae, that infect apple and cause diseases on other fruits, vegetables, and flowers. Our genomic data were obtained from isolates/species that have not yet been sequenced and represent geographic-specific regions. Genome sequencing allowed for the construction of phylogenetic trees, which corroborated the overall concordance observed in prior MLST studies. Bioinformatic pipelines were used to discover CAZyme, effector, and secondary metabolic (SM) gene clusters in all nine Colletotrichum isolates. We found redundancy and a high level of similarity across species regarding CAZyme classes and predicted cytoplastic and apoplastic effectors. SM gene clusters displayed the most diversity in type and the most common cluster was one that encodes genes involved in the production of alternapyrone. Our study provides a solid platform to identify targets for functional studies that underpin pathogenicity, virulence, and/or quiescence that can be targeted for the development of new control strategies. With these new genomics resources, exploration via omics-based technologies using these isolates will help ascertain the biological underpinnings of their widespread success and observed geographic dominance in specific areas throughout the country.

59 BASIC BIOLOGICAL SCIENCES↗