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At least 307 records · Page 17
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

The leucine-rich repeats in allelic barley MLA immune receptors define specificity towards sequence-unrelated powdery mildew avirulence effectors with a predicted common RNase-like fold

Nucleotide-binding domain leucine-rich repeat-containing receptors (NLRs) in plants can detect avirulence (AVR) effectors of pathogenic microbes. The Mildew locus a (Mla) NLR gene has been shown to confer resistance against diverse fungal pathogens in cereal crops. In barley, Mla has undergone allelic diversification in the host population and confers isolate-specific immunity against the powdery mildew-causing fungal pathogen Blumeria graminis forma specialis hordei (Bgh). We previously isolated the Bgh effectors AVR A1 , AVR A7 , AVR A9 , AVR A13 , and allelic AVR A10 /AVR A22 , which are recognized by matching MLA1, MLA7, MLA9, MLA13, MLA10 and MLA22, respectively. Here, we extend our knowledge of the Bgh effector repertoire by isolating the AVR A6 effector, which belongs to the family of catalytically inactive RNase-Like Proteins expressed in Haustoria (RALPHs). Using structural prediction, we also identified RNase-like folds in AVR A1 , AVR A7 , AVR A10 /AVR A22 , and AVR A13 , suggesting that allelic MLA recognition specificities could detect structurally related avirulence effectors. To better understand the mechanism underlying the recognition of effectors by MLAs, we deployed chimeric MLA1 and MLA6, as well as chimeric MLA10 and MLA22 receptors in plant co-expression assays, which showed that the recognition specificity for AVR A1 and AVR A6 as well as allelic AVR A10 and AVR A22 is largely determined by the receptors’ C-terminal leucine-rich repeats (LRRs). The design of avirulence effector hybrids allowed us to identify four specific AVR A10 and five specific AVR A22 aa residues that are necessary to confer MLA10- and MLA22-specific recognition, respectively. This suggests that the MLA LRR mediates isolate-specific recognition of structurally related AVR A effectors. Thus, functional diversification of multi-allelic MLA receptors may be driven by a common structural effector scaffold, which could be facilitated by proliferation of the RALPH effector family in the pathogen genome.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Cbp1, a fungal virulence factor under positive selection, forms an effector complex that drives macrophage lysis

Intracellular pathogens secrete effectors to manipulate their host cells. Histoplasma capsulatum (Hc) is a fungal intracellular pathogen of humans that grows in a yeast form in the host. Hc yeasts are phagocytosed by macrophages, where fungal intracellular replication precedes macrophage lysis. The most abundant virulence factor secreted by Hc yeast cells is Calcium Binding Protein 1 (Cbp1), which is absolutely required for macrophage lysis. Here we take an evolutionary, structural, and cell biological approach to understand Cbp1 function. We find that Cbp1 is present only in the genomes of closely related dimorphic fungal species of the Ajellomycetaceae family that lead primarily intracellular lifestyles in their mammalian hosts (Histoplasma, Paracoccidioides, and Emergomyces), but not conserved in the extracellular fungal pathogen Blastomyces dermatitidis. We observe a high rate of fixation of non-synonymous substitutions in the Cbp1 coding sequences, indicating that Cbp1 is under positive selection. We determine the de novo structures of Hc H88 Cbp1 and the Paracoccidioides americana (Pb03) Cbp1, revealing a novel “binocular” fold consisting of a helical dimer arrangement wherein two helices from each monomer contribute to a four-helix bundle. In contrast to Pb03 Cbp1, we show that Emergomyces Cbp1 orthologs are unable to stimulate macrophage lysis when expressed in the Hc cbp1 mutant. Consistent with this result, we find that wild-type Emergomyces africanus yeast are able to grow within primary macrophages but are incapable of lysing them. Finally, we use subcellular fractionation of infected macrophages and indirect immunofluorescence to show that Cbp1 localizes to the macrophage cytosol during Hc infection, making this the first instance of a phagosomal human fungal pathogen directing an effector into the cytosol of the host cell. We additionally show that Cbp1 forms a complex with Yps-3, another known Hc virulence factor that accesses the cytosol. Taken together, these data imply that Cbp1 is a fungal virulence factor under positive selection that localizes to the cytosol to trigger host cell lysis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Exploiting bacterial effector proteins to uncover evolutionarily conserved antiviral host machinery

Arboviruses are a diverse group of insect-transmitted pathogens that pose global public health challenges. Identifying evolutionarily conserved host factors that combat arbovirus replication in disparate eukaryotic hosts is important as they may tip the balance between productive and abortive viral replication, and thus determine virus host range. Here, we exploit naturally abortive arbovirus infections that we identified in lepidopteran cells and use bacterial effector proteins to uncover host factors restricting arbovirus replication. Bacterial effectors are proteins secreted by pathogenic bacteria into eukaryotic hosts cells that can inhibit antimicrobial defenses. Since bacteria and viruses can encounter common host defenses, we hypothesized that some bacterial effectors may inhibit host factors that restrict arbovirus replication in lepidopteran cells. Thus, we used bacterial effectors as molecular tools to identify host factors that restrict four distinct arboviruses in lepidopteran cells. By screening 210 effectors encoded by seven different bacterial pathogens, we identify several effectors that individually rescue the replication of all four arboviruses. We show that these effectors encode diverse enzymatic activities that are required to break arbovirus restriction. We further characterize Shigella flexneri-encoded IpaH4 as an E3 ubiquitin ligase that directly ubiquitinates two evolutionarily conserved proteins, SHOC2 and PSMC1, promoting their degradation in insect and human cells. We show that depletion of either SHOC2 or PSMC1 in insect or human cells promotes arbovirus replication, indicating that these are ancient virus restriction factors conserved across invertebrate and vertebrate hosts. Collectively, our study reveals a novel pathogen-guided approach to identify conserved antimicrobial machinery, new effector functions, and conserved roles for SHOC2 and PSMC1 in virus restriction.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

19-LW-045 Full Length Final Report. Molecular Mechanisms of Bacterial Pathogenesis: Waging the Arms Race with Superbugs

As the current global pandemic makes abundantly clear, we need a better understanding of infectious disease to safeguard human health, the economy and global security. Modern omics techniques hold the promise of providing a comprehensive understanding of the molecular mechanisms of life, including causes of pathogenesis from infectious disease at the molecular level, but we there is a serious gap in annotation of gene function. For as much as half of the genes and gene products encoded in genomes the molecular and/or cellular function is unknown or only partially understood. Recent innovations in fluorescence microscopy for live cell imaging and genetic engineering make it possible to determine the temporal correlation between molecular events, such as a gene being expressed due to host-pathogen interaction, and cellular events, such as bacterial invasion of immune cells. This is turn allows us to gain new insight as to the molecular and cellular role of individual genes and will enable the discovery and validation of new molecular mechanisms essential for infectious disease. Knowing the molecular mechanisms of disease processes will provide new therapeutic targets or novel countermeasure strategies. We aimed to develop a lattice light sheet fluorescence microscope as a unique resource at LLNL for long time course live cell imaging experiments; to develop the reagents and cell lines needed to monitor molecular events during the course pathogenic bacteria infecting mammalian immune cells; and to demonstrate that we could capture molecular events during an infection. We fully commissioned the LLNL lattice light sheet microscope and conducted initial proof of principle imaging experiments on mammalian immune cells and pathogenic bacteria. It is clear from the experience gained that long time course live cell imaging has tremendous potential to help elucidate molecular mechanisms of host-pathogen interactions and to help annotate gene function, which would establish a basis for new countermeasures. It is also clear that if live cell imaging is to realize its full potential new data processing and analysis tools will need to be developed to facilitate analysis of molecular events within cells; new sample chambers and stages could facilitate studies with a wider range of cell and tissue types; and alternative molecular tagging methods need to be explored to enable more facile engineering of cells labeled with molecular specificity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Enabling generalized phage therapy to treat secondary infections in COVID-19 (CRADA Final Report)

COVID-19 is a global health threat, with 32 millions infected and more than 500,000 deaths in the U.S. alone. A key aspect of COVID-19 deaths is bacterial secondary infections, with Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa identified as some of the major causative pathogens. Phage therapy, i.e., the administration of bacteriophages (viruses infecting these bacterial pathogens) to patients offers a complementary solution to antibiotics with multiple key advantages. Felix Biotechnology’s is developing a high-throughput platform to identify phages that not only kill pathogens, but also drive targeted trade-off of pathogenic phenotypes such as reduced virulence and antibiotic resistance, at a fraction of the time and R&D cost of any competitor. In this project, collaboration with scientists from the Lawrence Berkeley National Laboratory enabled Felix Biotechnology to establish robust analysis pipeline to investigate phage-host interactions in complex samples. These should enable a quick and robust identification of promising phages and key genetic determinants for therapy applications, paving the way towards a streamlined and safe use of phages in the clinic.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Defining Lipidomic Responses to Coronavirus Infection

Highly pathogenic human coronavirus infection can cause a severe atypical, rapid onset pneumonia with a mortality rate of up to 10% for severe acute respiratory syndrome coronavirus 1 (SARS-CoV 1), 35% for Middle East respiratory syndrome coronavirus (MERS-CoV), or 1% for severe acute respiratory syndrome coronavirus 2 (SARS-CoV 2 causative agent of COVID 19). While medical countermeasures successfully controlled the worldwide SARS-CoV epidemic, the MERS-CoV epidemic is still ongoing and continues to be a concern for travelers in the Middle East and the multi-year SARS-CoV 2 pandemic underscore the importance of defining biomarkers that are diagnostic and/or predictive of severe disease outcomes for respiratory viruses. Systems biology approaches provide global snapshots of infection induced changes in host cells/tissues and provide extremely rich datasets for understanding host pathogen interactions. Metabolites, especially lipids, are critical for viral replication but less is understood about infection induced changes to lipids due to limits in lipid species detection and identification. To characterize how individual lipid species and proteins with lipid associated functions contribute to highly pathogenic human coronavirus replication and disease severity, existing datasets were probed to determine cell type specific lipid responses to MERS-CoV infection and verification studies were performed to determine if modification of the host lipid signature (by inhibiting enzymatic functions that produce specific lipid species) can perturb CoV replication in human lung cells. MERS-CoV infects human lung epithelial, endothelial and fibroblast cells. All three cell types were infected with MERS-CoV and samples collected to analyze lipids, proteins, metabolites, and transcripts from 12 to 48 hours post infection. Time matched mock-infected cells were collected in parallel for each cell type. Following sample and statistical analysis, functional enrichment and bioinformatic analysis was performed to identify differentially expressed lipids and proteins. Two lipid species were found to be significantly upregulated following MERS-CoV infection, ceramides, and triacylglycerol both of which are indicative of cells undergoing apoptotic cell death. In contrast, sphingomyelins (lipid molecules that can serve as a precursor for one pathway for ceramide synthesis) had significantly decreased expression in MERS-CoV infected cells. An inhibitor of acid sphingomyelinase (that converts sphingomyelin to ceramides and phosphorylcholine) reduced MERS-CoV replication suggesting that production of ceramides is key for successful viral replication and transmission. Acyl-CoA-synthetase 3 (ACSL3), the only protein whose function is lipid associated and had increased differential expression in our dataset, regulates the synthesis of triacylglycerol (increased expression). Inhibitors that directly block ACSL3 (Triacsin C) but not steps later in the triacylglycerol synthesis pathway (Etomoxir) inhibit MERS-CoV replication suggesting that triacylglycerol production and/or ACSL3 activity is also key for viral replication. ACSL3 expression is also upregulated in lung cancer cells and is predicted to promote continued cell viability which would also enhance viral replication. The differentially expressed lipid and lipid-associated protein species identified in our studies suggest that MERS-CoV infection is activating cellular death pathways to limit the number of cells that become infected but also stimulating the production of lipid-associated enzymes that can prolong host cell viability and the amount of time progeny virions can be produced and released. As the inhibitors that worked against MERS-CoV infection were also efficacious against SARS-CoV 2 infection, countermeasures that target these host pathways may provide novel ways to block highly pathogenic human coronavirus infection and/or prevent severe disease outcomes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Using CRISPR/Cas13a technologies to detect miRNA [DOE SULI Internship Report]

Micro ribonucleic acids (miRNA) give our immune systems the ability to recognize viruses and other pathogens by their complementary single-stranded RNA (ssRNA) produced in the reproduction of the pathogen in our cells. When miRNA of a specific sequence is detected in a cell sample, it can be assumed that the immune system is activated and attempting to track down the infection. This pathway can be utilized to diagnose infection from a pathogen before the individual even develops symptoms, aiding in early disease detection and proper treatment. One of the ways that we can detect miRNA is through an assay of clustered regularly interspaced short palindromic repeats or “CRISPR” and the bacterial protein Cas13a. This report details discoveries made while attempting to optimize this assay for miRNA detection. After looking at several different factors within the assay, it was determined that some factors, such as reporter type and metallic ion concentration, are more impactful on the overall assay sensitivity than other factors, such as the overall concentration of Cas13a, CRISPR RNA (crRNA), or ssRNA reporter. It was also discovered that different sequences with different lengths require renewed optimization efforts, as each target has a unique binding affinity determined by the sequence length and composition. This information is crucial in the development of point of care molecular detection devices as they become sensitive enough to identify pathogens before they spread.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

PNNL DataHub Project: Omics Lethal Human Viruses Project Profiling of the Host Response to West Nile Virus Infection, Processed Experimental Dataset Catalog

West Nile virus (WNV) is classified as a Category B priority pathogen (mosquito-borne Flavivirus) by the National Institute of Allergy and Infectious Diseases (NIAID), and are known to cause severe infections in humans where lethal host-associated mechanisms are not clearly defined. The NIAID Modeling Host Responses to Understand Severe Human Virus Infections Research Program project (2013 - 2018) aimed to develop an improved comprehensive understanding of the host response to a suite of viruses causing lethal infections leveraging a systems biology approach. The NIAID Modeling Host Responses to Understand Severe Human Virus Infections Research Program project (2013-2018) aimed to develop an improved comprehensive understanding of the host response to a suite of viruses causing lethal infections leveraging a systems biology approach. Herein, PNNL sub-projects provide a never before released comprehensive infectious disease collection of primary and secondary transformation multi-Omics data profiling a series of priority pathogen primary experimental studies for enhanced open-access to viral Omics datasets and project lifecycle metadata. Secondary host-pathogen viral dataset downloads contain one or more statistically processed (normalization data transformation) quantitative dataset collections resulting in qualitative expression analyses of primary host-pathogen experimental study designs. Leveraging unique high-resolution Omics capabilities for proteomics (P), metabolomics (M), lipidomics (L), and transcriptomics (T) dataset downloads each have a direct relationship to a primary sample submission corresponding a specific West Nile virus [NCBITAXON:11082] (WNV-NY99 382) experimental infection study. Host sample types include cerebellum ["CB", BTO:0000232], cortical neurons ["CN", BTO:0004102], cortex ["CT", BTO:0000233], dendritic cells ["DC", BTO:0002042], granule cell neurons ["GCN", BTO:0003393], lymph node ["LN", BTO:0000784], and serum ["SE", BTO:0001239] from mouse (Mus musculus) tissue collections.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

How Cooperative Engagement Programs Strengthen Sequencing Capabilities for Biosurveillance and Outbreak Response

The threat of emerging and re-emerging infectious diseases continues to be a challenge to public and global health security. Cooperative biological engagement programs act to build partnerships and collaborations between scientists and health professionals to strengthen capabilities in biosurveillance. Biosurveillance is the systematic process of detecting, reporting, and responding to especially dangerous pathogens and pathogens of pandemic potential before they become outbreaks, epidemics, and pandemics. One important tool in biosurveillance is next generation sequencing. Expensive sequencing machines, reagents, and supplies make it difficult for countries to adopt this technology. Cooperative engagement programs help by providing funding for technical assistance to strengthen sequencing capabilities. Through workshops and training, countries are able to learn sequencing and bioinformatics, and implement these tools in their biosurveillance programs. Cooperative programs have an important role in building and sustaining collaborations among institutions and countries. One of the most important pieces in fostering these collaborations is trust. Trust provides the confidence that a successful collaboration will benefit all parties involved. With sequencing, this enables the sharing of pathogen samples and sequences. Obtaining global sequencing data helps to identify unknown etiological agents, track pathogen evolution and infer transmission networks throughout the duration of a pandemic. Having sequencing technology in place for biosurveillance generates the capacity to provide real-time data to understand and respond to pandemics. We highlight the need for these programs to continue to strengthen sequencing in biosurveillance. By working together to strengthen sequencing capabilities, trust can be formed, benefitting global health in the face of biological threats.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Anatomy of an agricultural antagonist: Feeding complex structure and function of three xylem sap‐feeding insects illuminated with synchrotron‐based 3D imaging

Abstract Many insects feed on xylem or phloem sap of vascular plants. Although physical damage to the plant is minimal, the process of insect feeding can transmit lethal viruses and bacterial pathogens. Disparities between insect‐mediated pathogen transmission efficiency have been identified among xylem sap‐feeding insects; however, the mechanistic drivers of these trends are unclear. Identifying and understanding the structural factors and associated integrated functional components that may ultimately determine these disparities are critical for managing plant diseases. Here, we applied synchrotron‐based X‐ray microcomputed tomography to digitally reconstruct the morphology of three xylem sap‐feeding insect vectors of plant pathogens: Graphocephala atropunctata (blue‐green sharpshooter; Hemiptera, Cicadellidae) and Homalodisca vitripennis (glassy‐winged sharpshooter; Hemiptera, Cicadellidae), and the spittlebug Philaenus spumarius (meadow spittlebug; Hemiptera, Aphrophoridae). The application of this technique revealed previously undescribed anatomical features of these organisms, such as key components of the salivary complex. The visualization of the 3D structure of the precibarial valve led to new insights into the mechanism of how this structure functions. Morphological disparities with functional implications between taxa were highlighted as well, including the morphology and volume of the cibarial dilator musculature responsible for extracting xylem sap, which has implications for force application capabilities. These morphological insights will be used to target analyses illuminating functional differences in feeding behavior.

3D imaging↗
DOE OSTI2023

Prevalence of Listeria monocytogenes , Salmonella spp., Shiga toxin-producing Escherichia coli , and Campylobacter spp. in raw milk in the United States between 2000 and 2019: A systematic review and meta-analysis

Raw (unpasteurized) milk is available for sale and direct human consumption within some states in the United States (US); it cannot be sold or distributed in interstate commerce. Raw milk may contain pathogenic microorganisms that, when consumed, may cause illness and sometimes may result in death. No comprehensive review for prevalence and levels of the major bacterial pathogens in raw milk in the US exists. The objective of the present research was to systematically review the scientific literature published from 2000 to 2019 to estimate the prevalence and levels of Listeria monocytogenes, Salmonella spp., Shiga toxin-producing Escherichia coli (STEC), and Campylobacter spp. in raw milk in the US. Peer-reviewed studies were retrieved systematically from PubMed®, Embase®, and Web of ScienceTM. The unique complete nonduplicate references were uploaded into the Health Assessment Work Collaborative (HAWC). Based on the selection criteria, twenty studies were included in the systematic review and meta-analysis. Comprehensive Meta-Analysis (CMA) was used for statistical analyses, specifically, random effects meta-analyses were used to synthesize raw bulk tank milk (BTM) and milk filters (MF) data. Data from studies using culture and non–culture-based detection methods were included. Forest plots generated in CMA (Biostat, Englewood, NJ) were used to visualize the results. The average prevalence (event rate) of L. monocytogenes, Salmonella spp., STEC, and Campylobacter spp. in raw BTM in the US was estimated at 4.3% (95% confidence intervals [CIs], 2.8–6.5%), 3.6% (95% CIs, 2.0–6.2%), 4.3% (95% CIs, 2.4–7.4%), and 6.0% (95% CIs, 3.2–10.9%), respectively. Estimated prevalence was generally larger in MF than in BTM. There was not enough data to perform a meta-analysis for the prevalence or levels of pathogens in raw milk from retail establishments or other milk categories.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

Volatiles from the necrophagous fly Cochliomyia macellaria (Diptera: Calliphoridae) as indicators of Salmonella exposure

Blow flies (Diptera: Calliphoridae) are crucial in forensic investigations due to their association with both living and dead humans and other animals. Additionally, their interactions with various resources and potential as vectors of pathogens of humans and other animals, thus, make them potential tools for biosurveillance. This study investigated the potential of monitoring volatile organic compounds (VOCs) emitted by blow flies exposed to Salmonella as a method for pathogen surveillance. Adult blow flies ( Cochliomyia macellaria ) were exposed, or not, to Salmonella enterica . Following exposure, VOCs released by the blow flies were collected and analyzed using gas chromatography-mass spectrometry (GC-MS). Results indicate a treatment by time interaction (P < 0.01). Indicator species analysis identified a single compound significantly associated with S. enterica exposure (P = 0.02), Nonane, 2,2,4,4,6,8,8-heptamethyl, potentially indicating an immune system response. Given a compound indicating exposure was detected, future research should determine if more replicates could detect more differences after Salmonella ingestion. This research highlights the potential of blow flies as biosurveillance tools and the potential value of volatiles for assessing their exposure to pathogens.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Emerging wild virus of native grass bioenergy feedstock is well–established in the Midwestern USA and associated with premature stand senescence

The North American native prairie grass Panicum virgatum (switchgrass) is a primary bioenergy feedstock candidate. Its widespread distribution and genetic diversity enable the possibility of developing this perennial grass for high production in a variety of conditions, including on marginal lands. A critical concern in feedstock development and deployment is the risk of novel pathogen emergence. Here we investigate the landscape-scale prevalence and epidemiology of a little-studied North American virus first detected in switchgrass and other grasses in bioenergy trials in the US Midwest. Switchgrass mosaic virus (SwMV, Genus Marafivirus, Family Tymoviridae) is transmitted by leafhoppers and phylogenetically sister to Maize rayado fino virus, a significant pathogen of maize in parts of the Americas. Our goal was to determine whether SwMV is uniquely limited to specific bioenergy trials or well-established and circulating more broadly. We used molecular diagnostics to quantify naturally occurring SwMV infection in leafhoppers and switchgrass in naturalistic stands throughout a large Midwestern landscape, and quantified leafhopper abundances and stand performance. Our analysis revealed that this apparently wild virus is well-established and widespread. Infection was present at nearly all sites, across diverse landscape contexts, with prevalences ranging as high as 33%–60%. Infection appeared to accumulate and persist in stands over time. It was associated with increases in premature stand senescence but not with reductions in stand height. Although wild viruses are believed to evolve benign relationships with their natural hosts, these data suggest that SwMV has potential to impact yield components. Viruses are frequently overlooked in crop development efforts, but represent the majority of emerging plant pathogens. For SwMV, it is imperative to quantify its impact on host performance, to identify the extent of any host resistance, and to assess any risks of virus spillover to agricultural plantings of other Poaceae species, including maize and sorghum.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Eucalyptus grandis MYB‐Like and RAN‐Like Zinc Finger Proteins Display Dual Roles in Regulating Plant Immunity and Symbiosis Pathways

Plant roots live in constant contact with diverse microbes in the soil. Plant fitness, therefore, relies on signaling pathways that mount an effective immune response against pathogens while fostering mutualistic symbioses. Plant pathways, and specifically immune genes that may act as "switches," discriminating between pathogenic or mutualistic fungi, remain largely unknown. Using Eucalyptus grandis as a model system, we investigate alterations to the root transcriptomic landscape during pre-symbiosis with either the pathogen Armillaria luteobubalina or the mutualistic fungus Pisolithus microcarpus. Comparative analyses identified three strongly counter-regulated genes that may act as immune switches to accommodate or to repress fungal colonization. We characterized two of these, a MYB-like and RAN-like zinc finger protein, using a transgenic approach and demonstrated that they have bifunctional roles in the regulation of cell death and a hypersensitive-like response, depending on the lifestyle of the associated fungus. Using co-expression network analysis, we identified hypothetical pathways correlated to these genes. We functionally validated these predictions using plants with transgenic roots with increased or decreased transcription of these genes, thereby showing the power of co-expression networks as an a priori approach to identify key immune response pathways in plants. Overall, our results demonstrate that prior to physical contact with microbes, MYB-like and RAN-like zinc finger proteins are key regulators of plant immune signaling that respond to fungal signals and enable or repress symbiotic establishment.

mycorrhizal fungi↗
DOE OSTI2023

Comparative genomics of the Liberibacter genus reveals widespread diversity in genomic content and positive selection history

‘Candidatus Liberibacter’ is a group of bacterial species that are obligate intracellular plant pathogens and cause Huanglongbing disease of citrus trees and Zebra Chip in potatoes. Here, we examined the extent of intra- and interspecific genetic diversity across the genus using comparative genomics. Our approach examined a wide set of Liberibacter genome sequences including five pathogenic species and one species not known to cause disease. By performing comparative genomics analyses, we sought to understand the evolutionary history of this genus and to identify genes or genome regions that may affect pathogenicity. With a set of 52 genomes, we performed comparative genomics, measured genome rearrangement, and completed statistical tests of positive selection. We explored markers of genetic diversity across the genus, such as average nucleotide identity across the whole genome. These analyses revealed the highest intraspecific diversity amongst the ‘Ca. Liberibacter solanacearum’ species, which also has the largest plant host range. We identified sets of core and accessory genes across the genus and within each species and measured the ratio of nonsynonymous to synonymous mutations (dN/dS) across genes. We identified ten genes with evidence of a history of positive selection in the Liberibacter genus, including genes in the Tad complex, which have been previously implicated as being highly divergent in the ‘Ca. L. capsica’ species based on high values of dN.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Methods for safely sharing dual-use genetic data

Background: Some genetic data has dual-use potential. Sharing pathogen data has shown tremendous value. For example therapeutic development and lineage tracking during the COVID pandemic. This data sharing is complicated by the fact that these data have the potential to be used for harm. The genome sequence of a pathogen can be used to enable malicious genetic engineering approaches or to recreate the pathogen from synthetic DNA. Standard data security methods can be applied to genetic data, but when data is shared between institutions, ensuring appropriate security can be difficult. Sensitive data that is shared internationally among a wide array of institutions can be especially difficult to control. Methods for securely storing and sharing genetic data with potential for dual-use are needed to mitigate this potential harm.Results: Here we propose new methods that allow genetic data to be shared in a data format that prevents a nefarious actor from accessing sensitive aspects of the data. Our methods obfuscate raw sequence data by pooling reads from different samples. This approach can ensure that data is secure while stored and during electronic transfer. We demonstrate that by pooling raw sequence data from multiple samples of the same organism, the ability to fully reconstruct any individual sample is prevented. In the pooled data, most genomic information remains, but reads or mutations cannot be directly attributed to any individual sample. To further restrict access to information, regions of a genome can be removed from the reads.Conclusion: Our methods obscure genomic information within raw sequence reads. This method can allow genetic data to be stored and shared while preventing a nefarious actor from being able to perfectly reconstruct an organism. Broad-scale sequence information remains, while fine scale details about specific samples are difficult or impossible to reconstruct. Our software is available at https://github.com/Geneinfosec-Inc/ReadMixer.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Genetic mapping of sorghum resistance to an Illinois isolate of Colletotrichum sublineola

Anthracnose leaf blight (ALB) is an economically important disease of sorghum [Sorghum bicolor (L.) Moench] caused by the fungal pathogen Colletotrichum sublineola Henn. ex Sacc. & Trotter. Although qualitative and quantitative resistance have been identified for ALB, the usefulness of resistance loci differs depending on the pathogen pathotype. Identifying resistance effective against unique pathogen pathotypes is critical to managing ALB, as the disease is managed primarily through the deployment of host resistance. We isolated C. sublineola from ALB-infected leaves collected in Illinois and found that the strain was a novel pathotype, as it produced a unique combination of virulence against a set of differential lines. Using this isolate, we inoculated 579 temperate-adapted sorghum conversion lines in 2019 and 2020. We then conducted a genome-wide association study (GWAS) and a metabolic pathway analysis using the Pathway Associated Study Tool (PAST). We identified 47 significant markers distributed across all chromosomes except chromosome 8. We identified 32 candidate genes based on physical proximity with significant markers, some of which have a known role in host defense. We identified 47 pathways associated with ALB resistance, indicating a role for secondary metabolism in defense to ALB. Our results are important to improve the understanding of the genetic basis of ALB resistance in sorghum and highlight the importance of developing durable resistance to ALB.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Protection against Chemical Warfare Agents and Biological Threats Using Metal–Organic Frameworks as Active Layers

The SARS-CoV-2 pandemic outbreak and the unfortunate misuse of toxic chemical warfare agents (CWAs) highlight the importance of developing functional materials to protect against these chemical and pathogen threats. Metal–organic frameworks (MOFs), which comprise a tunable class of crystalline porous materials built from inorganic nodes and organic linkers, have emerged as a class of heterogeneous catalysts capable of rapid detoxification of multiple classes of these harmful chemical or biological hazards. In particular, zirconium-based MOFs (Zr-MOFs) feature Lewis acidic nodes that serve as active sites for a wide range of catalytic reactions, including the hydrolysis of organophosphorus nerve agents within seconds in basic aqueous solutions. In addition, postsynthetic modification of Zr-MOFs enables the release of active species capable of reacting with and deactivating harmful pathogens. Despite this impressive performance, utilizing Zr- MOFs in powder form is not practical for application in masks or protective uniforms. To address this challenge, our team sought to develop MOF/fiber composite systems that could be adapted for use under realistic operating conditions to protect civilians, military personnel, and first responders from harmful pathogens and chemical warfare agents. Over the last several years, our group has designed and fabricated reactive and biocidal MOF/fiber composites that effectively capture and deactivate these toxic species. In this Account, we describe the evolution of these porous and reactive MOF/fiber composites and focus on key design challenges and considerations. First, we devised a scalable method for the integration of Zr-MOFs onto textile substrates using aqueous precursor solutions and without using pretreated textiles, highlighting the potential scalability of this method. Moving beyond standard textiles, we also developed a microbial synthesis strategy to prepare hierarchically porous MOF/bacterial cellulose nanofiber composite sponges that can both capture and detoxify nerve agents when exposed to contaminated gas flows. The mass loading of the MOF in the nanofibrous composite sponge is up to 90%, affording higher work capacities compared to those of textile-fiber-based composites with relatively lower MOF loadings. Next, we demonstrated that heterogeneous polymeric bases are suitable replacements for volatile liquid bases typically used in solution-phase reactions, and we showed that these composite systems are capable of effectively hydrolyzing nerve agents in the solid state by using only water that is present as humidity. Moreover, incorporating a reactive dye precursor into the composite affords a dual function sensing and detoxifying material that changes color from white to orange upon reaction with the byproduct following nerve agent hydrolysis, demonstrating the versatility of this platform for use in decontamination applications. We then created chlorine-loaded MOF/fiber composites that act as biocidal and reactive textiles that are capable of not only detoxifying sulfur-mustard-based chemical warfare agents and simulants but also deactivating both bacteria and the SARS-CoV-2 virus within minutes of exposure. Lastly, we synthesized a mixed-metal Ti/Zr-MOF coating on cotton fibers to afford a photoactive biocidal cloth that shows fast and broad-spectrum biocidal performance against viruses and Gram-positive and Gram-negative bacteria under visible light irradiation. Given the tunable, multifunctional nature of these MOF/fiber composites, we believe that this Account will offer new insights for the rational design and preparation of functional MOF/fiber composites and pave the way toward the development of next-generation reactive and protective textiles.

36 MATERIALS SCIENCE↗