Mortality from leukemia, cancer and heart disease among U.S. nuclear power plant workers, 1957–2011
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efam: an e xpanded, metaproteome-supported HMM profile database of viral protein fam ilies
Viruses infect, reprogram and kill microbes, leading to profound ecosystem consequences, from elemental cycling in oceans and soils to microbiome-modulated diseases in plants and animals. Although metagenomic datasets are increasingly available, identifying viruses in them is challenging due to poor representation and annotation of viral sequences in databases. Here, we establish efam, an expanded collection of Hidden Markov Model (HMM) profiles that represent viral protein families conservatively identified from the Global Ocean Virome 2.0 dataset. This resulted in 240 311 HMM profiles, each with at least 2 protein sequences, making efam >7-fold larger than the next largest, pan-ecosystem viral HMM profile database. Adjusting the criteria for viral contig confidence from ‘conservative’ to ‘eXtremely Conservative’ resulted in 37 841 HMM profiles in our efam-XC database. To assess the value of this resource, we integrated efam-XC into VirSorter viral discovery software to discover viruses from less-studied, ecologically distinct oxygen minimum zone (OMZ) marine habitats. This expanded database led to an increase in viruses recovered from every tested OMZ virome by ~24% on average (up to ~42%) and especially improved the recovery of often-missed shorter contigs (<5 kb). Additionally, to help elucidate lesser-known viral protein functions, we annotated the profiles using multiple databases from the DRAM pipeline and virion-associated metaproteomic data, which doubled the number of annotations obtainable by standard, single-database annotation approaches. Together, these marine resources (efam and efam-XC) are provided as searchable, compressed HMM databases that will be updated bi-annually to help maximize viral sequence discovery and study from any ecosystem.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Survey of Thirteen Novel Pseudomonas putida Bacteriophages
Bacteriophages have been widely investigated as a promising treatment of food, medical equipment, and humans colonized by antibiotic-resistant bacteria. Phages pose particular interest in combating those bacteria which form biofilms, such as the medically important human pathogen Pseudomonas aeruginosa and several plant pathogens, including P. syringae . In an undergraduate lab course, P. putida was used as the host to isolate novel anti-pseudomonal bacteriophages. Environmental samples of soil and water were collected, and purified phage isolates were obtained. After Illumina sequencing, genomes of these phages were assembled de novo and annotated. Assembled genomes were compared with known genomes in the literature and GenBank to identify taxonomic relations and to refine their functional annotations. The thirteen phages described are sipho-, myo-, and podoviruses in several families of Caudoviricetes , spanning several novel genera, with genomes ranging from 40,000 to 96,000 bp. One phage (DDSR119) is unique and is the first reported P. putida siphovirus. The remaining 12 can be clustered into four distinct groups. Six are highly related to each other and to previously described Autotranscriptaviridae phages: Waldo5, PlaquesPlease, and Laces98 all belong to the Waldovirus genus, whereas Stalingrad, Bosely, and Stamos belong to the Troedvirus genus. Zuri was previously classified as the founding member of a new genus Zurivirus within the family Schitoviridae . Ebordelon and Holyagarpour each represent different species within Zurivirus , whereas Meara is a more distantly related member of the Schitoviridae . Dolphis and Jeremy are similar enough to form a genus but have only a few distant relatives among sequenced phages and are notable for being temperate. We identified the lysis cassettes in all 13 phages, compared tail spike structures, and found auxiliary metabolic genes in several. Studies like these, which isolate and characterize infectious virions, enable the identification of novel proteins and molecular systems and also provide the raw materials for further study, evaluation, and manipulation of phage proteins and their hosts.
Pseudomonas putida↗
Inhibition of ethylene involved in resistance to E. turcicum in an exotic-derived double haploid maize population
Northern corn leaf blight (NCLB) is an economically important disease of maize. While the genetic architecture of NCLB has been well characterized, the pathogen is known to overcome currently deployed resistance genes, and the role of hormones in resistance to NCLB is an area of active research. The objectives of the study were (i) to identify significant markers associated with resistance to NCLB, (ii) to identify metabolic pathways associated with NCLB resistance, and (iii) to examine role of ethylene in resistance to NCLB. We screened 252 lines from the exotic-derived double haploid BGEM maize population for resistance to NCLB in both field and greenhouse environments. We used a genome wide association study (GWAS) and stepwise regression to identify four markers associated with resistance, followed by a pathway association study tool (PAST) to identify important metabolic pathways associated with disease severity and incubation period. The ethylene synthesis pathway was significant for disease severity and incubation period. We conducted a greenhouse assay in which we inhibited ethylene to examine the role of ethylene in resistance to NCLB. We observed a significant increase in incubation period and a significant decrease in disease severity between plants treated with the ethylene inhibitor and mock-treated plants. Our study confirms the potential of the BGEM population as a source of novel alleles for resistance. We also confirm the role of ethylene in resistance to NCLB and contribute to the growing body of literature on ethylene and disease resistance in monocots.
59 BASIC BIOLOGICAL SCIENCES↗
The future of fungi: threats and opportunities
The fungal kingdom represents an extraordinary diversity of organisms with profound impacts across animal, plant, and ecosystem health. Fungi simultaneously support life, by forming beneficial symbioses with plants and producing life-saving medicines, and bring death, by causing devastating diseases in humans, plants, and animals. With climate change, increased antimicrobial resistance, global trade, environmental degradation, and novel viruses altering the impact of fungi on health and disease, developing new approaches is now more crucial than ever to combat the threats posed by fungi and to harness their extraordinary potential for applications in human health, food supply, and environmental remediation. To address this aim, the Canadian Institute for Advanced Research (CIFAR) and the Burroughs Wellcome Fund convened a workshop to unite leading experts on fungal biology from academia and industry to strategize innovative solutions to global challenges and fungal threats. This report provides recommendations to accelerate fungal research and highlights the major research advances and ideas discussed at the meeting pertaining to 5 major topics: (1) Connections between fungi and climate change and ways to avert climate catastrophe; (2) Fungal threats to humans and ways to mitigate them; (3) Fungal threats to agriculture and food security and approaches to ensure a robust global food supply; (4) Fungal threats to animals and approaches to avoid species collapse and extinction; and (5) Opportunities presented by the fungal kingdom, including novel medicines and enzymes.
59 BASIC BIOLOGICAL SCIENCES↗
Genetic determinants of endophytism in the Arabidopsis root mycobiome
The roots of Arabidopsis thaliana host diverse fungal communities that affect plant health and disease states. Here, we sequence the genomes of 41 fungal isolates representative of the A. thaliana root mycobiota for comparative analysis with other 79 plant-associated fungi. Our analyses indicate that root mycobiota members evolved from ancestors with diverse lifestyles and retain large repertoires of plant cell wall-degrading enzymes (PCWDEs) and effector-like small secreted proteins. We identify a set of 84 gene families associated with endophytism, including genes encoding PCWDEs acting on xylan (family GH10) and cellulose (family AA9). Transcripts encoding these enzymes are also part of a conserved transcriptional program activated by phylogenetically-distant mycobiota members upon host contact. Recolonization experiments with individual fungi indicate that strains with detrimental effects in mono-association with the host colonize roots more aggressively than those with beneficial activities, and dominate in natural root samples. Furthermore, we show that the pectin-degrading enzyme family PL1_7 links aggressiveness of endophytic colonization to plant health.
59 BASIC BIOLOGICAL SCIENCES↗
Conserved genetic mechanisms for biotic stress in sorghum (Final report)
Developing durable disease resistance for biofuel crops is crucial, particularly as the range of biofuel crop production expands and pathogens of other plant species evolve to cause diseases of bioenergy feedstocks. Setosphaeria species are significant pathogens of the Andropogonae, and S. turcica can infect both maize and sorghum. Sorghum leaf blight (SLB), caused by S. turcica, is widespread and can decrease grain yields up to 50%, reduce forage quantity and quality, and predispose plants to other diseases, such as anthracnose. Our overall objective was to gain a systems-level understanding of the pathosystem by leveraging natural genetic variation, host specificity of the pathogen, and transcriptome analysis to improve biotic stress resistance in sorghum. We proposed three specific goals: 1) Identify sorghum alleles conferring resistance to S. turcica using natural genetic variation. 2) Characterize genes associated with biotic stress response in sorghum. 3) Identify fungal genes responsible for host specificity.
09 BIOMASS FUELS↗
Conserved genetic mechanisms for biotic stress in sorghum (Final Report)
Developing durable disease resistance for biofuel crops is crucial, particularly as the range of biofuel crop production expands and pathogens of other plant species evolve to cause diseases of bioenergy feedstocks. Setosphaeria species are significant pathogens of the Andropogonae, and S. turcica can infect both maize and sorghum. Sorghum leaf blight (SLB), caused by S. turcica, is widespread and can decrease grain yields up to 50%, reduce forage quantity and quality, and predispose plants to other diseases, such as anthracnose. Our overall objective was to gain a systems-level understanding of the pathosystem by leveraging natural genetic variation, host specificity of the pathogen, and transcriptome analysis to improve biotic stress resistance in sorghum. We proposed three specific goals: 1) Identify sorghum alleles conferring resistance to S. turcica using natural genetic variation. 2) Characterize genes associated with biotic stress response in sorghum. 3) Identify fungal genes responsible for host specificity.
09 BIOMASS FUELS↗
The development of extracellular vesicle markers for the fungal phytopathogen Colletotrichum higginsianum
Fungal phytopathogens secrete extracellular vesicles (EVs) associated with enzymes and phytotoxic metabolites. While these vesicles are thought to promote infection, defining the true contents and functions of fungal EVs, as well as suitable protein markers, is an ongoing process. To expand our understanding of fungal EVs and their possible roles during infection, we purified EVs from the hemibiotrophic phytopathogen Colletotrichum higginsianum, the causative agent of anthracnose disease in multiple plant species, including Arabidopsis thaliana. EVs were purified in large numbers from the supernatant of protoplasts but not the supernatant of intact mycelial cultures. We purified two separate populations of EVs, each associated with over 700 detected proteins, including proteins involved in vesicle transport, cell wall biogenesis and the synthesis of secondary metabolites. We selected two SNARE proteins (Snc1 and Sso2) and one 14-3-3 protein (Bmh1) as potential EV markers and generated transgenic strains expressing fluorescent fusions. Each marker was confirmed to be protected inside EVs. Fluorescence microscopy was used to examine the localization of each marker during infection on Arabidopsis leaves. These findings further our understanding of EVs in fungal phytopathogens and will help build an experimental system to study EV interkingdom communication between plants and fungi.
14-3-3 proteins↗
Microbiome-enabled genomic selection improves prediction accuracy for nitrogen-related traits in maize
Root-associated microbiomes in the rhizosphere (rhizobiomes) are increasingly known to play an important role in nutrient acquisition, stress tolerance, and disease resistance of plants. However, it remains largely unclear to what extent these rhizobiomes contribute to trait variation for different genotypes and if their inclusion in the genomic selection protocol can enhance prediction accuracy. To address these questions, we developed a microbiome-enabled genomic selection method that incorporated host SNPs and amplicon sequence variants from plant rhizobiomes in a maize diversity panel under high and low nitrogen (N) field conditions. Our cross-validation results showed that the microbiome-enabled genomic selection model significantly outperformed the conventional genomic selection model for nearly all time-series traits related to plant growth and N responses, with an average relative improvement of 3.7%. The improvement was more pronounced under low N conditions (8.4–40.2% of relative improvement), consistent with the view that some beneficial microbes can enhance N nutrient uptake, particularly in low N fields. However, our study could not definitively rule out the possibility that the observed improvement is partially due to the amplicon sequence variants being influenced by microenvironments. Using a high-dimensional mediation analysis method, our study has also identified microbial mediators that establish a link between plant genotype and phenotype. Some of the detected mediator microbes were previously reported to promote plant growth. The enhanced prediction accuracy of the microbiome-enabled genomic selection models, demonstrated in a single environment, serves as a proof-of-concept for the potential application of microbiome-enabled plant breeding for sustainable agriculture.
60 APPLIED LIFE SCIENCES↗
SCCOUT: Surveying for Categorization and Control of Organism in Unwelcome Territory
Advancements in the fields of science, technology, and medicine have contributed largely to the growth of society over the last century. Thanks to these discoveries, all corners of the planet are connected both digitally and physically. Progress in the field of medicine has led to people living longer, fuller lives and a general increase in global population. Awareness of our world’s changing climate and the effects that has on local and global ecosystems has spurred great efforts to protect the environment. These discoveries along with many others have helped our world grow in a myriad of ways; however, certain efforts in agriculture, natural resource management, and ecosystem management have not scaled to accommodate this growth. Increasing prevalence and ease of dissemination of invasive plants and animals, diseases, and changing ecological conditions pose significant challenges to biodiversity, ecosystem health, and agricultural productivity. Moreover, as the world population grows, there are more people to feed and less viable farmland to produce food for them. Despite the increasing prevalence of these issues, methods for addressing and tracking them have stayed relatively the same for at least the last several decades.
54 ENVIRONMENTAL SCIENCES↗
Conservation and Expansion of Transcriptional Factor Repertoire in the Fusarium oxysporum Species Complex
The Fusarium oxysporum species complex (FOSC) includes both plant and human pathogens that cause devastating plant vascular wilt diseases and threaten public health. Each F. oxysporum genome comprises core chromosomes (CCs) for housekeeping functions and accessory chromosomes (ACs) that contribute to host-specific adaptation. This study inspects global transcription factor profiles (TFomes) and their potential roles in coordinating CC and AC functions to accomplish host-specific interactions. Remarkably, we found a clear positive correlation between the sizes of TFomes and the proteomes of an organism. With the acquisition of ACs, the FOSC TFomes were larger than the other fungal genomes included in this study. Among a total of 48 classified TF families, 14 families involved in transcription/translation regulations and cell cycle controls were highly conserved. Among the 30 FOSC expanded families, Zn2-C6 and Znf_C2H2 were most significantly expanded to 671 and 167 genes per family including well-characterized homologs of Ftf1 (Zn2-C6) and PacC (Znf_C2H2) that are involved in host-specific interactions. Manual curation of characterized TFs increased the TFome repertoires by 3% including a disordered protein Ren1. RNA-Seq revealed a steady pattern of expression for conserved TF families and specific activation for AC TFs. Functional characterization of these TFs could enhance our understanding of transcriptional regulation involved in FOSC cross-kingdom interactions, disentangle species-specific adaptation, and identify targets to combat diverse diseases caused by this group of fungal pathogens.
59 BASIC BIOLOGICAL SCIENCES↗
Engineering pathogen resistance in plants
Provided herein are compounds and methods for increasing disease resistance and/or root length in plants.
Ronald, Paul↗
A Novel Signaling Pathway Connects Thiamine Biosynthesis, Bacterial Respiration, and Production of the Exopolysaccharide Amylovoran in Erwinia amylovora
Erwinia amylovora is a plant pathogen causing necrotrophic fire blight disease of apple, pear, and other rosaceous plants. This bacterium colonizes host vascular tissues via the production of exopolysaccharides (EPSs) including amylovoran. It is well-established that the nearly ubiquitous plasmid pEA29 of E. amylovora is an essential virulence factor, but the underlying mechanism remains uncharacterized. Here, we demonstrated that pEA29 was required for E. amylovora to produce amylovoran and to form a biofilm, and this regulation was dependent on the thiamine biosynthesis operon thiOSGF. We then conducted carbohydrate and genetic analyses demonstrating that the thiamine-mediated effect on amylovoran production was indirect, as cells lacking thiOSGF produced an EPS that did not contain glucuronic acid, one of the key components of amylovoran, whereas the transcriptional activity and RNA levels of the amylovoran biosynthesis genes were not altered. Alternatively, addition of exogenous thiamine restored amylovoran production in the pEA29-cured strain of E. amylovora and positively impacted amylovoran production in a dose-dependent manner. Individual deletion of several chromosomal thiamine biosynthesis genes also affected amylovoran production, implying that a complete thiamine biosynthesis pathway is required for the thiamine-mediated effect on amylovoran production in E. amylovora. Finally, we determined that an imbalanced tricarboxylic acid cycle negatively affected amylovoran production, which was restored by addition of exogenous thiamine or overexpression of the thiOSGF operon. In summary, our report revealed a novel signaling pathway that impacts E. amylovora virulence in which thiamine biosynthesis enhances bacterial respiration that provides energetic requirements for the biosynthesis of EPS amylovoran.
59 BASIC BIOLOGICAL SCIENCES↗
Dynamic Reconfiguration of Switchgrass Proteomes in Response to Rust ( Puccinia novopanici ) Infection
Switchgrass (Panicum virgatum L.) can be infected by the rust pathogen (Puccinia novopanici) and results in lowering biomass yields and quality. Label-free quantitative proteomics was conducted on leaf extracts harvested from non-infected and infected plants from a susceptible cultivar (Summer) at 7, 11, and 18 days after inoculation (DAI) to follow the progression of disease and evaluate any plant compensatory mechanisms to infection. Some pustules were evident at 7 DAI, and their numbers increased with time. However, fungal DNA loads did not appreciably change over the course of this experiment in the infected plants. In total, 3830 proteins were identified at 1% false discovery rate, with 3632 mapped to the switchgrass proteome and 198 proteins mapped to different Puccinia proteomes. Across all comparisons, 1825 differentially accumulated switchgrass proteins were identified and subjected to a STRING analysis using Arabidopsis (A. thaliana L.) orthologs to deduce switchgrass cellular pathways impacted by rust infection. Proteins associated with plastid functions and primary metabolism were diminished in infected Summer plants at all harvest dates, whereas proteins associated with immunity, chaperone functions, and phenylpropanoid biosynthesis were significantly enriched. At 18 DAI, 1105 and 151 proteins were significantly enriched or diminished, respectively. Many of the enriched proteins were associated with mitigation of cellular stress and defense.
59 BASIC BIOLOGICAL SCIENCES↗
Genome evolution and transcriptome plasticity is associated with adaptation to monocot and dicot plants in Colletotrichum fungi
Colletotrichum fungi infect a wide diversity of monocot and dicot hosts, causing diseases on almost all economically important plants worldwide. Colletotrichum is also a suitable model for studying gene family evolution on a fine scale to uncover events in the genome associated with biological changes. Here we present the genome sequences of 30 Colletotrichum species covering the diversity within the genus. Evolutionary analyses revealed that the Colletotrichum ancestor diverged in the late Cretaceous in parallel with the diversification of flowering plants. We provide evidence of independent host jumps from dicots to monocots during the evolution of Colletotrichum, coinciding with a progressive shrinking of the plant cell wall degradative arsenal and expansions in lineage-specific gene families. Comparative transcriptomics of 4 species adapted to different hosts revealed similarity in gene content but high diversity in the modulation of their transcription profiles on different plant substrates. Combining genomics and transcriptomics, we identified a set of core genes such as specific transcription factors, putatively involved in plant cell wall degradation. These results indicate that the ancestral Colletotrichum were associated with dicot plants and certain branches progressively adapted to different monocot hosts, reshaping the gene content and its regulation.
59 BASIC BIOLOGICAL SCIENCES↗
Single-Cell Metabolic Profiling: Metabolite Formulas from Isotopic Fine Structures in Heterogeneous Plant Cell Populations
Characterization of the metabolic heterogeneity in cell populations requires the analysis of single cells. Most current methods in single-cell analysis rely on cell manipulation, potentially altering the abundance of metabolites in individual cells. Small sample volume and the chemical diversity of metabolites are additional challenges in single-cell metabolomics. Here, we describe the combination of fiber-based laser ablation electrospray ionization (f-LAESI) with 21 tesla Fourier transform ion cyclotron resonance mass spectrometry (21TFTICR-MS) for in situ single-cell metabolic profiling in plant tissue. Single plant cells infected by bacteria were selected and sampled directly from the tissue without cell manipulation through mid-infrared ablation with a fine optical fiber tip for ionization by f-LAESI. Finally, ultrahigh performance 21T-FTICR-MS enabled the simultaneous capture of isotopic fine structures (IFSs) for 47 known and 11 unknown compounds, thus elucidating their elemental compositions from single cells, and providing information on metabolic heterogeneity in the cell population.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
A Chromosome-Scale Genome Assembly of the Flax Rust Fungus Reveals the Two Unusually Large Effector Proteins, AvrM3 and AvrN
Rust fungi comprise thousands of species, many of which cause disease on important crop plants. The flax rust fungus Melampsora lini has been a model species for the genetic dissection of plant immunity since the 1940s; however, the highly fragmented and incomplete reference genome has so far hindered progress in effector gene discovery. Here, we generated a fully phased, chromosome-scale assembly of the two nuclear genomes of M. lini strain CH5, resolving an additional 320 Mbp of the sequence. The 482-Mbp dikaryotic genome is at least 79% repetitive, with a large proportion (approximately 40%) of the genome comprising young, highly similar transposable elements. The assembly resolves the known effector gene loci, some of which carry complex duplications that were collapsed in the previous assembly. Using a genetic map followed by manual correction of gene models, we identified the AvrM3 and AvrN genes, which encode unusually large fungal effector proteins and trigger defense responses when co-expressed with the corresponding resistance genes. We located the genes linked to the tetrapolar mating system on chromosomes 4 and 9, but in contrast to the cereal rusts that have one pheromone receptor gene per haplotype, in flax rust, three pheromone receptor genes were found, with two of them closely linked on one haplotype. Taken together, we show that a high-quality assembly is crucial for resolving complex gene loci, and given the increasing number of fungal effectors of large size, the commonly applied criterion for effector candidates of being small proteins needs to be reconsidered.
Melampsora↗