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All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Control of Carbon Allocation in Phenylpropanoid Metabolism

Understanding the molecular mechanisms by which carbon flux is partitioned in plants, and how flux through the phenylpropanoid pathway in particular is regulated and allocated to different branches of the pathway is essential for the eventual rational manipulation of phenylpropanoid flux for bioenergy applications. The phenylpropanoid pathway is responsible for the biosynthesis of a diverse array of metabolites essential for structural integrity, water transport, UV protection, and defense against herbivores and pathogens. The products of the phenylpropanoid pathway, all of which are derived from phenylalanine, range from complex, insoluble polymers such as lignin and suberin, to soluble flavonoids and hydroxycinnamate esters, to volatile compounds used to attract pollinators. By far the most abundant of these is the complex heteropolymer lignin, which accounts for a significant portion of the dry weight of not only woody plants, but also of crop residues. Decreasing or altering lignin structure to provide increased cell wall digestibility could greatly increase the cost effectiveness of converting cell wall polysaccharides to ethanol or second-generation biofuels. On the other hand, it is also possible that plants engineered to accumulate higher levels of lignin could provide improved feedstock for direct combustion and electricity generation as part of a diverse bioenergy portfolio. Consistent with the enormous commitment of energy and carbon it requires, the synthesis of lignin is under tight regulatory control. In order to influence transcription, gene-specific activators and repressors must interact with basal transcription factors and the rest of the core transcriptional machinery. In eukaryotes, this interaction is bridged by the large, multi-subunit complex known as Mediator. Despite the clear importance of Mediator throughout the eukaryotes, the complex has only recently been studied in plants. We have shown that a pair of Mediator subunits, MED5a and MED5b are required for normal regulation of phenylpropanoid pathway product accumulation. Plants lacking these proteins show increased accumulation of phenylpropanoids in multiple tissues indicating that normally they directly or indirectly repress expression of phenylpropanoid pathway genes. Furthermore, we have recently shown that MED5 plays a role in dwarfing in some low lignin plants and feedback between and within metabolic pathways of secondary metabolism in Arabidopsis. Taken together with the established roles of Mediator in transcriptional regulation, and of MED5a and MED5b in the repression of phenylpropanoid metabolism, these observations suggest that MED5a and MED5b are key components of an active, transcriptional process by which phenylpropanoid homeostasis is maintained in wild-type plants. Our research focused on expanding our understanding of the role of Mediator, as well as elucidating the mechanisms by which these proteins influence phenylpropanoid biosynthesis and how metabolite accumulation within the pathway and between metabolic pathways is essential for feedback regulatory control. This research will inform future bioengineering efforts aimed at altering carbon allocation and cell wall engineering by contributing to our understanding of the role of Mediator in regulating lignin biosynthesis, a pathway of plant metabolism that is important on a global scale.

09 BIOMASS FUELS↗
DOE OSTI2023

Divergent Evolution of Early Terrestrial Fungi Reveals the Evolution of Mucormycosis Pathogenicity Factors

Abstract Fungi have evolved over millions of years and their species diversity is predicted to be the second largest on the earth. Fungi have cross-kingdom interactions with many organisms that have mutually shaped their evolutionary trajectories. Zygomycete fungi hold a pivotal position in the fungal tree of life and provide important perspectives on the early evolution of fungi from aquatic to terrestrial environments. Phylogenomic analyses have found that zygomycete fungi diversified into two separate clades, the Mucoromycota which are frequently associated with plants and Zoopagomycota that are commonly animal-associated fungi. Genetic elements that contributed to the fitness and divergence of these lineages may have been shaped by the varied interactions these fungi have had with plants, animals, bacteria, and other microbes. To investigate this, we performed comparative genomic analyses of the two clades of zygomycetes in the context of Kingdom Fungi, benefiting from our generation of a new collection of zygomycete genomes, including nine produced for this study. We identified lineage-specific genomic content that may contribute to the disparate biology observed in these zygomycetes. Our findings include the discovery of undescribed diversity in CotH, a Mucormycosis pathogenicity factor, which was found in a broad set of zygomycetes. Reconciliation analysis identified multiple duplication events and an expansion of CotH copies throughout the Mucoromycotina, Mortierellomycotina, Neocallimastigomycota, and Basidiobolus lineages. A kingdom-level phylogenomic analysis also identified new evolutionary relationships within the subphyla of Mucoromycota and Zoopagomycota, including supporting the sister-clade relationship between Glomeromycotina and Mortierellomycotina and the placement of Basidiobolus as sister to other Zoopagomycota lineages.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Stromules, functional extensions of plastids within the plant cell

We report stromules are thin tubular extensions of the plastid compartment surrounded by the envelope membrane. A myriad of functions have been proposed for them, and they likely have multiple roles. Recent work has illuminated aspects of their formation, especially the important of microtubules in their movement and microfilaments in anchoring. A variety of biotic and abiotic stresses result in induction of stromule formation, and in recent years, stromule formation has been strongly implicated as part of the innate immune response. Both stromules and chloroplasts relocate to surround the nucleus when pathogens are sensed, possibly to supply signaling molecules such as reactive oxygen species. In addition to the nucleus, stromules have been observed in close proximity to other compartments such as mitochondria, endoplasmic reticulum, and the plasma membrane, potentially facilitating exchange of substrates and products to carry out important biosynthetic pathways. Much remains to be learned about the identity of proteins and other molecules released from chloroplasts and stromules and how they function in plant development and defense.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

A glycan receptor kinase facilitates intracellular accommodation of arbuscular mycorrhiza and symbiotic rhizobia in the legume Lotus japonicus

Receptors that distinguish the multitude of microbes surrounding plants in the environment enable dynamic responses to the biotic and abiotic conditions encountered. In this study, we identify and characterise a glycan receptor kinase, EPR3a, closely related to the exopolysaccharide receptor EPR3. Epr3a is up-regulated in roots colonised by arbuscular mycorrhizal (AM) fungi and is able to bind glucans with a branching pattern characteristic of surface-exposed fungal glucans. Expression studies with cellular resolution show localised activation of the Epr3a promoter in cortical root cells containing arbuscules. Fungal infection and intracellular arbuscule formation are reduced in epr3a mutants. In vitro , the EPR3a ectodomain binds cell wall glucans in affinity gel electrophoresis assays. In microscale thermophoresis (MST) assays, rhizobial exopolysaccharide binding is detected with affinities comparable to those observed for EPR3, and both EPR3a and EPR3 bind a well-defined β-1,3/β-1,6 decasaccharide derived from exopolysaccharides of endophytic and pathogenic fungi. Both EPR3a and EPR3 function in the intracellular accommodation of microbes. However, contrasting expression patterns and divergent ligand affinities result in distinct functions in AM colonisation and rhizobial infection in Lotus japonicus . The presence of Epr3a and Epr3 genes in both eudicot and monocot plant genomes suggest a conserved function of these receptor kinases in glycan perception.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

A multifunctional sesquiterpene synthase integrates with cytochrome P450s to reinforce the terpenoid defense network in maize

Terpenoids, the largest and most structurally diverse class of plant natural products, play essential roles in maize defense and ecological interactions. In this study, we identified and functionally characterized a sesquiterpenoid-based defense pathway in maize centered on α-santalenoic acid, a pathogen-inducible sesquiterpenoid antibiotic. Using a combination of metabolite-based genome-wide association studies (mGWAS), linkage mapping, and heterologous expression assays, we identified ZmTPS9 as a multiproduct terpene synthase that primarily produces α-santalene and β-bisabolene. Sequence analysis and site-directed mutagenesis revealed that threonine at position 413 is critical for enzyme activity, with its deletion resulting in a complete loss of enzyme activity. The sesquiterpene hydrocarbons produced by ZmTPS9 are further oxidized by three cytochrome P450 monooxygenases, ZmCYP71Z16, ZmCYP71Z18, and ZmCYP71Z19, to yield antimicrobial metabolites including α-santalenoic acid, zealexin D1 (ZD1), and zealexin D2 (ZD2). Together, these findings demonstrate a convergent biosynthetic strategy in maize, where multiproduct terpene synthases and promiscuous P450s collaboratively generate a flexible and robust terpenoid defense network.

a-santalenoic acid↗
DOE OSTI2023

A genome-informed higher rank classification of the biotechnologically important fungal subphylum Saccharomycotina

The subphylum Saccharomycotina is a lineage in the fungal phylum Ascomycota that exhibits levels of genomic diversity similar to those of plants and animals. The Saccharomycotina consist of more than 1 200 known species currently divided into 16 families, one order, and one class. Species in this subphylum are ecologically and metabolically diverse and include important opportunistic human pathogens, as well as species important in biotechnological applications. Many traits of biotechnological interest are found in closely related species and often restricted to single phylogenetic clades. However, the biotechnological potential of most yeast species remains unexplored. Although the subphylum Saccharomycotina has much higher rates of genome sequence evolution than its sister subphylum, Pezizomycotina, it contains only one class compared to the 16 classes in Pezizomycotina. The third subphylum of Ascomycota, the Taphrinomycotina, consists of six classes and has approximately 10 times fewer species than the Saccharomycotina. These data indicate that the current classification of all these yeasts into a single class and a single order is an underappreciation of their diversity. Our previous genome-scale phylogenetic analyses showed that the Saccharomycotina contains 12 major and robustly supported phylogenetic clades; seven of these are current families (Lipomycetaceae, Trigonopsidaceae, Alloascoideaceae, Pichiaceae, Phaffomycetaceae, Saccharomycodaceae, and Saccharomycetaceae), one comprises two current families (Dipodascaceae and Trichomonascaceae), one represents the genus Sporopachydermia, and three represent lineages that differ in their translation of the CUG codon (CUG-Ala, CUG-Ser1, and CUG-Ser2). Using these analyses in combination with relative evolutionary divergence and genome content analyses, we propose an updated classification for the Saccharomycotina, including seven classes and 12 orders that can be diagnosed by genome content. This updated classification is consistent with the high levels of genomic diversity within this subphylum and is necessary to make the higher rank classification of the Saccharomycotina more comparable to that of other fungi, as well as to communicate efficiently on lineages that are not yet formally named.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Isolation of potential plant growth-promoting bacteria from nodules of legumes grown in arid Botswana soil

As the world population increases, improvements in crop growth and yield will be needed to meet rising food demands, especially in countries that have not developed agricultural practices optimized for their own soils and crops. In many African countries, farmers improve agricultural productivity by applying synthetic fertilizers and pesticides to crops, but their continued use over the years has had serious environmental consequences including air and water pollution as well as loss of soil fertility. To reduce the overuse of synthetic amendments, we are developing inocula for crops that are based on indigenous soil microbes, especially those that enhance plant growth and improve agricultural productivity in a sustainable manner. We first isolated environmental DNA from soil samples collected from an agricultural region to study the composition of the soil microbiomes and then used Vigna unguiculata (cowpea), an important legume crop in Botswana and other legumes as “trap” plants using the collected soil to induce nitrogen-fixing nodule formation. We have identified drought-tolerant bacteria from Botswana soils that stimulate plant growth; many are species of Bacillus and Paenibacillus . In contrast, the cowpea nodule microbiomes from plants grown in these soils house mainly rhizobia particularly Bradyrhizobium , but also Methylobacterium spp. Hence, the nodule microbiome is much more limited in non-rhizobial diversity compared to the soil microbiome, but also contains a number of potential pathogenic bacteria.

Kosty, Melissa↗
DOE OSTI2024

Diverse signatures of convergent evolution in cactus-associated yeasts

Many distantly related organisms have convergently evolved traits and lifestyles that enable them to live in similar ecological environments. However, the extent of phenotypic convergence evolving through the same or distinct genetic trajectories remains an open question. Here, we leverage a comprehensive dataset of genomic and phenotypic data from 1,049 yeast species in the subphylum Saccharomycotina (Kingdom Fungi, Phylum Ascomycota) to explore signatures of convergent evolution in cactophilic yeasts, ecological specialists associated with cacti. We inferred that the ecological association of yeasts with cacti arose independently approximately 17 times. Using a machine learning–based approach, we further found that cactophily can be predicted with 76% accuracy from both functional genomic and phenotypic data. The most informative feature for predicting cactophily was thermotolerance, which we found to be likely associated with altered evolutionary rates of genes impacting the cell envelope in several cactophilic lineages. We also identified horizontal gene transfer and duplication events of plant cell wall–degrading enzymes in distantly related cactophilic clades, suggesting that putatively adaptive traits evolved independently through disparate molecular mechanisms. Notably, we found that multiple cactophilic species and their close relatives have been reported as emerging human opportunistic pathogens, suggesting that the cactophilic lifestyle—and perhaps more generally lifestyles favoring thermotolerance—might preadapt yeasts to cause human disease. This work underscores the potential of a multifaceted approach involving high-throughput genomic and phenotypic data to shed light onto ecological adaptation and highlights how convergent evolution to wild environments could facilitate the transition to human pathogenicity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

A shift from inorganic to organic nitrogen-dominance shapes soil microbiome composition and co-occurrence networks

Soil microbiomes are characterized by their composition and networks, which are linked to soil nitrogen (N) availability. In nature, inorganic N dominates at one end and organic N dominates at the other end along soil N gradients; however, little is known about how this shift influences soil microbiome composition and co-occurrence networks, as well as their controls. To this end, we conducted an experiment with the host plant Solidago canadensis , which was subject to three N regimes: inorganic N-dominated, co-dominated by inorganic and organic N (CIO), and organic N-dominated. Organic N dominance exhibited stronger effects on the composition and co-occurrence networks of soil microbiomes than inorganic N dominance. The predominant control was plant traits for bacterial and fungal richness, and soil pH for keystone species. Relative to the CIO regime, inorganic N dominance did not affect fungal richness and increased keystone species; organic N dominance decreased fungal richness and keystone species. Pathogenic fungi and arbuscular mycorrhizal fungi were suppressed by organic N dominance but not by inorganic N dominance. These findings suggest that the shift from soil inorganic N-dominance to soil organic N-dominance could strongly shape soil microbiome composition and co-occurrence networks by altering species diversity and topological properties.

Xin, Yue↗
DOE OSTI2025

The structural basis for the broad aldehyde specificity of the aminoaldehyde dehydrogenase PauC from the human pathogen Pseudomonas aeruginosa

Abstract Despite significant differences in size and formal charge, the aldehyde dehydrogenasePaPauC (PA5312) fromPseudomonas aeruginosaPAO1 efficiently catalyzes the NAD + ‐dependent oxidation of the aminoaldehydes formed in polyamines degradation. We report here thatPaPauC also oxidizes 4‐guanidinebutyraldehyde, formed in one arginine degradation pathway, trimethylaminobutyraldehyde, of unknown metabolic origin, and indole‐3‐acetaldehyde, a precursor of the plant growth‐promoting hormone indoleacetic acid.PaPauC has been proposed as a potential target for combatingP. aeruginosa. However, understanding its structure–function relationships, crucial for developing specific inhibitors, is lacking. Using X‐ray crystallography, we identified the structural characteristics that determinePaPauC broad aldehyde specificity: a spacious aldehyde‐entrance tunnel and six active‐site residues. Docking simulations, site‐directed mutagenesis, and kinetic analyses support the interactions of Lys479 with glutamylated aminoaldehydes; Phe169, Trp176, and Phe467 with amino and guanidinium groups through cation–π interactions and with the indole group via NH–π and CH–π interactions; Asp459 with amino and indole groups; and Thr303 with amide and guanidinium groups. Exploiting the distinctive structural features of thePaPauC active site could aid in developing specific inhibitors to combatP. aeruginosainfections in humans and animals, as well as in preventing its colonization of plants, which are abundantP. aeruginosareservoirs and, therefore, a significant source of human infections.

Biochemistry & Molecular Biology↗
DOE OSTI2023

Dynamic subcanopy leaf traits drive resistance of net primary production across a disturbance severity gradient

Across the globe, the forest carbon sink is increasingly vulnerable to an expanding array of low- to moderate-severity disturbances. However, some forest ecosystems exhibit functional resistance (i.e., the capacity of ecosystems to continue functioning as usual) following disturbances such as extreme weather events and insect or fungal pathogen outbreaks. Unlike severe disturbances (e.g., stand-replacing wildfires), moderate severity disturbances do not always result in near-term declines in forest production because of the potential for compensatory growth, including enhanced subcanopy production. Community-wide shifts in subcanopy plant functional traits, prompted by disturbance-driven environmental change, may play a key mechanistic role in resisting declines in net primary production (NPP) up to thresholds of canopy loss. However, the temporal dynamics of these shifts, as well as the upper limits of disturbance for which subcanopy production can compensate, remain poorly characterized. In this study, we leverage a 4-year dataset from an experimental forest disturbance in northern Michigan to assess subcanopy community trait shifts as well as their utility in predicting ecosystem NPP resistance across a wide range of implemented disturbance severities. Through mechanical girdling of stems, we achieved a gradient of severity from 0% (i.e., control) to 45, 65, and 85% targeted gross canopy defoliation, replicated across four landscape ecosystems broadly representative of the Upper Great Lakes ecoregion. We found that three of four examined subcanopy community weighted mean (CWM) traits including leaf photosynthetic rate (p = 0.04), stomatal conductance (p = 0.07), and the red edge normalized difference vegetation index (p < 0.0001) shifted rapidly following disturbance but before widespread changes in subcanopy light environment triggered by canopy tree mortality. Surprisingly, stimulated subcanopy production fully compensated for upper canopy losses across our gradient of experimental severities, achieving complete resistance (i.e., no significant interannual differences from control) of whole ecosystem NPP even in the 85% disturbance treatment. Additionally, we identified a probable mechanistic switch from nutrient-driven to light-driven trait shifts as disturbance progressed. Our findings suggest that remotely sensed traits such as the red edge normalized difference vegetation index (reNDVI) could be particularly sensitive and robust predictors of production response to disturbance, even across compositionally diverse forests. The potential of leaf spectral indices to predict post-disturbance functional resistance is promising given the capabilities of airborne to satellite remote sensing. We conclude that dynamic functional trait shifts following disturbance can be used to predict production response across a wide range of disturbance severities.

resistance↗
DOE OSTI2023

Sequencing the Genomes of the First Terrestrial Fungal Lineages: What Have We Learned?

The first genome sequenced of a eukaryotic organism was for Saccharomyces cerevisiae, as reported in 1996, but it was more than 10 years before any of the zygomycete fungi, which are the early-diverging terrestrial fungi currently placed in the phyla Mucoromycota and Zoopagomycota, were sequenced. The genome for Rhizopus delemar was completed in 2008; currently, more than 1000 zygomycete genomes have been sequenced. Genomic data from these early-diverging terrestrial fungi revealed deep phylogenetic separation of the two major clades—primarily plant—associated saprotrophic and mycorrhizal Mucoromycota versus the primarily mycoparasitic or animal-associated parasites and commensals in the Zoopagomycota. Genomic studies provide many valuable insights into how these fungi evolved in response to the challenges of living on land, including adaptations to sensing light and gravity, development of hyphal growth, and co-existence with the first terrestrial plants. Genome sequence data have facilitated studies of genome architecture, including a history of genome duplications and horizontal gene transfer events, distribution and organization of mating type loci, rDNA genes and transposable elements, methylation processes, and genes useful for various industrial applications. Pathogenicity genes and specialized secondary metabolites have also been detected in soil saprobes and pathogenic fungi. Novel endosymbiotic bacteria and viruses have been discovered during several zygomycete genome projects. Overall, genomic information has helped to resolve a plethora of research questions, from the placement of zygomycetes on the evolutionary tree of life and in natural ecosystems, to the applied biotechnological and medical questions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Towards engineering ectomycorrhization into switchgrass bioenergy crops via a lectin receptor‐like kinase

Summary Soil‐borne microbes can establish compatible relationships with host plants, providing a large variety of nutritive and protective compounds in exchange for photosynthesized sugars. However, the molecular mechanisms mediating the establishment of these beneficial relationships remain unclear. Our previous genetic mapping and whole‐genome resequencing studies identified a gene deletion event of a Populus trichocarpa lectin receptor‐like kinase gene PtLecRLK1 in Populus deltoides that was associated with poor‐root colonization by the ectomycorrhizal fungus Laccaria bicolor . By introducing PtLecRLK1 into a perennial grass known to be a non‐host of L. bicolor , switchgrass ( Panicum virgatum L.), we found that L. bicolor colonizes ZmUbipro‐PtLecRLK1 transgenic switchgrass roots, which illustrates that the introduction of PtLecRLK1 has the potential to convert a non‐host to a host of L. bicolor . Furthermore, transcriptomic and proteomic analyses on inoculated‐transgenic switchgrass roots revealed genes/proteins overrepresented in the compatible interaction and underrepresented in the pathogenic defence pathway, consistent with the view that pathogenic defence response is down‐regulated during compatible interaction. Metabolomic profiling revealed that root colonization in the transgenic switchgrass was associated with an increase in N‐containing metabolites and a decrease in organic acids, sugars, and aromatic hydroxycinnamate conjugates, which are often seen in the early steps of establishing compatible interactions. These studies illustrate that PtLecRLK1 is able to render a plant susceptible to colonization by the ectomycorrhizal fungus L. bicolor and shed light on engineering mycorrhizal symbiosis into a non‐host to enhance plant productivity and fitness on marginal lands.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Genomic Analysis of Diverse Members of the Fungal Genus Monosporascus Reveals Novel Lineages, Unique Genome Content and a Potential Bacterial Associate

The genus Monosporascus represents an enigmatic group of fungi important in agriculture and widely distributed in natural arid ecosystems. Of the nine described species, two (M. cannonballus and M. eutypoides) are important pathogens on the roots of members of Cucurbitaceae in agricultural settings. The remaining seven species are capable of colonizing roots from a diverse host range without causing obvious disease symptoms. Recent molecular and culture studies have shown that members of the genus are nearly ubiquitous as root endophytes in arid environments of the Southwestern United States. Isolates have been obtained from apparently healthy roots of grasses, shrubs and herbaceous plants located in central New Mexico and other regions of the Southwest. Phylogenetic and genomic analyses reveal substantial diversity in these isolates. The New Mexico isolates include close relatives of M. cannonballus and M. ibericus, as well as isolates that represent previously unrecognized lineages. To explore evolutionary relationships within the genus and gain insights into potential ecological functions, we sequenced and assembled the genomes of three M. cannonballus isolates, one M. ibericus isolate, and six diverse New Mexico isolates. The assembled genomes were significantly larger than what is typical for the Sordariomycetes despite having predicted gene numbers similar to other members of the class. Differences in predicted genome content and organization were observed between endophytic and pathogenic lineages of Monosporascus. Several Monosporascus isolates appear to form associations with members of the bacterial genus Ralstonia (Burkholdariaceae).

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Genetic diversity, population structure and anthracnose resistance response in a novel sweet sorghum diversity panel

Sweet sorghum is an attractive feedstock for the production of renewable chemicals and fuels due to the readily available fermentable sugars that can be extracted from the juice, and the additional stream of fermentable sugars that can be obtained from the cell wall polysaccharides in the bagasse. An important selection criterion for new sweet sorghum germplasm is resistance to anthracnose, a disease caused by the fungal pathogen Colletotrichum sublineolum. The identification of novel anthracnose-resistance sources present in sweet sorghum germplasm offers a fast track towards the development of new resistant sweet sorghum germplasm. We established a sweet sorghum diversity panel (SWDP) of 272 accessions from the USDA-ARS National Plant Germplasm (NPGS) collection that includes landraces from 22 countries and advanced breeding material, and that represents ~15% of the NPGS sweet sorghum collection. Genomic characterization of the SWDP identified 171,954 single nucleotide polymorphisms (SNPs) with an average of one SNP per 4,071 kb. Population structure analysis revealed that the SWDP could be stratified into four populations and one admixed group, and that this population structure could be aligned to sorghum’s racial classification. Results from a two-year replicated trial of the SWDP for anthracnose resistance response in Texas, Georgia, Florida, and Puerto Rico showed 27 accessions to be resistant across locations, while 145 accessions showed variable resistance response against local pathotypes. A genome-wide association study identified 16 novel genomic regions associated with anthracnose resistance. Four resistance loci on chromosomes 3, 6, 8 and 9 were identified against pathotypes from Puerto Rico, and two resistance loci on chromosomes 3 and 8 against pathotypes from Texas. In Georgia and Florida, three resistance loci were detected on chromosomes 4, 5, 6 and four on chromosomes 4, 5 (two loci) and 7, respectively. One resistance locus on chromosome 2 was effective against pathotypes from Texas and Puerto Rico and a genomic region of 41.6 kb at the tip of chromosome 8 was associated with resistance response observed in Georgia, Texas, and Puerto Rico. This publicly available SWDP and the extensive evaluation of anthracnose resistance represent a valuable genomic resource for the improvement of sorghum.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Genetics and Genomics of Pathogen Resistance in Switchgrass (Final Report)

This project was funded by DOE under Grant no. DE-SC0016108. Originally approved for the 2016-2019 period, two no-cost extensions were solicited and approved, which prolonged the lifespan through July 2021. This final report informs on the results obtained so far from the research implemented. The research hinged on integrating genomics (genomic selection, RNAseq, virus-plant interactions) with classical genetics (conventional breeding) to incorporate durable resistance to fungal (rust) and viral (mosaic) diseases in switchgrass (Panicum virgatum) populations being bred for bioenergy. Higher biomass yield, higher quality (low lignin content), and durable disease resistance are key features to make lignocellulosic switchgrass feedstocks economically competitive and sustainable. Genomic selection is being applied on three generations of a switchgrass population derived from crossing two ecotypes (Kanlow as lowland female and Summer as upland male) with differential performance in terms of biomass yield and quality, disease resistance, and winter survivability. Target populations were screened for rust and mosaic in field and/or lab and phenotyped for biomass yield and quality traits. Genetic analyses were applied across generations to capture the joint inheritance of the targeted traits and predict breeding values for parents and progeny with greater accuracy. Parental and a panel of different switchgrass populations were genotyped with the DArTseq technology to develop SNP (0, 1, 2) and in-silico (presence/absence) DArT markers. Rust inoculations techniques were developed and applied successfully on switchgrass. The original populations (Kanlow and Summer) were sequenced with RNAseq to capture the gene expression profiles across sequential time-points and appraise the basis of greater resistance in the Kanlow vs the Summer ecotype. Constructs of PMV and sPMV mosaic virus were assembled and tested first on proso millet to find the best protocol to use later on switchgrass. Results from the preliminary analyses indicate that 1) ample additive genetic variation is available for selection and improving this inter-ecotypic population for yield, quality, and disease traits, 2) significant gains are to be expected with the genetic correlations being favorable between yield and lignin content and between yield and disease ratings, 3) substantial differences exist in the genetic regions controlling rust resistance in the two ecotypes, 4) co-infection with PMV isolates from Nebraska and its satellite from Kansas elicit severe mosaic symptoms, and 5) two different genetic systems are responsible for imparting resistance to rust and virus in switchgrass.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

An Improved Sampling and Baiting Method for Phytophthora tropicalis and P. heveae Detection in Macadamia integrifolia

Macadamia nuts are, economically, the second most important crop in the state of Hawai’i. A recent decline in yield and acreage has been attributed to insect damage and diseases such as Macadamia Quick Decline (MQD) caused by Phytophthora tropicalis and P. heveae. To develop an improved methodology for the diagnosis and treatment of MQD, investigations were undertaken to better understand the pathosystem of the disease. These investigations included sampling from multiple locations from sectioned trees utilizing two methods of tissue collection and isolations using two baiting techniques. The collection of tissue from the cambium and phloem of trees after scraping away the bark and in locations of recent or current sap exudation using a narrow diameter steel awl proved to be an efficient means for the molecular detection of the MQD pathogens from infected trees exhibiting MQD symptoms. In addition, a more efficient and cost-effective baiting method using apple puree was developed.

Plant Sciences↗
DOE OSTI2024

Seasonal Patterns in Mobile Colistin Resistance Gene Variants in Wastewater Bioaerosols and Liquid Sludge

To address the growing threat of global antimicrobial resistance, a one-health approach is needed to understand the complex socioecological cycling of antibiotic resistance genes. In this study, a metagenomics approach using DNA shotgun sequencing, metagenome assembly, and antibiotic resistance gene (ARG) annotation was used to examine seasonal patterns in the abundance of mobile colistin resistance (mcr) gene variants in bioaerosols and liquid sludge in three wastewater treatment plants (WWTPs). ARGs represented 0.2–0.8 and 0.1–0.2% of the bioaerosol and liquid sludge metagenomes, respectively, while mcr genes represented 0–0.3 and 0–0.5% of the identified ARGs in bioaerosol and liquid sludge metagenomes. Seven of the ten known mcr variants were detected in wastewater bioaerosol and liquid samples, with mcr-5 and mcr-8 being the most prevalent across all seasons and sites. Furthermore, additional functional and taxonomic annotation of mcr-containing metagenomic contigs showed that mcr genes were often located on contigs with other co-occurring ARGs and mobile genetic elements and may be harbored by opportunistic human pathogens and other bacterial taxa not previously associated with mcr genes. Atmospheric dispersion modeling showed that mcr-containing bioaerosols can be transported kilometers away from the WWTPs, resulting in the possible dissemination of these ARGs into surrounding environments and communities.

Antimicrobial agents↗