Browsing by Author "Leach, Jan, committee member"
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Item Open Access Analysis of genome-wide targets of Arabidopsis signal responsive 1 (AtSR1) transcription factor and its transcript stability in response to stress(Colorado State University. Libraries, 2017) Abdel-Hameed, Amira, author; Reddy, A. S. N., advisor; Bush, Daniel, committee member; Leach, Jan, committee member; Abdel-Ghany, Salah, committee memberAbiotic and biotic stresses cause significant yield losses in all crops. Acquisition of stress tolerance in plants requires rapid reprogramming of gene expression. SR1/CAMTA3, a member of signal responsive transcription factors (TFs), functions both as a positive and a negative regulator of biotic stress responses and as a positive regulator of cold stress-induced gene expression. Using high throughput RNA-seq, we identified ~3000 SR1-regulated genes. Promoters of about 60% of the differentially expressed genes have a known DNA binding site for SR1, suggesting that they are likely direct targets. Gene ontology analysis of SR1-regulated genes confirmed previously known functions of SR1 and uncovered a potential role for this TF in salt stress. Our results showed that SR1 mutant is more tolerant to salt stress than the wild type and complemented line. Improved tolerance of sr1 seedlings to salt is accompanied with the induction of salt-responsive genes. Furthermore, ChIP-PCR results showed that SR1 binds to promoters of several salt-responsive genes. These results suggest that SR1 acts as a negative regulator of salt tolerance by directly repressing the expression of salt-responsive genes. Overall, this study identified SR1-regulated genes globally and uncovered a previously uncharacterized role for SR1 in salt stress response. Soil salinity, one of the most prevalent environmental stresses, causes enormous losses in global crop yields every year. Therefore, it is imperative to generate salt tolerant cultivars. To achieve this goal, it is essential to understand the mechanisms by which plants respond to and cope with salt stress. Stress-induced reprogramming of gene expression at multiple levels contributes to the survival of plants under adverse environmental conditions. The control of mRNA stability is one of the post-transcriptional mechanisms that is highly regulated under stress conditions leading to changes in expression pattern of many genes. In this study, we show that salt stress increases the level of SR1 mRNA, by enhancing its stability. Multiple lines of evidence indicate that ROS generated by NADPH oxidase activity mediate salt-induced SR1 transcript stability. Furthermore, cycloheximide (CHX), a protein synthesis inhibitor, also increased SR1 mRNA stability, albeit to a higher level than in the presence of salt, suggesting a role for one or more labile proteins in SR1 mRNA turnover. Similar to salt, ROS generated by NADPH oxidase is also involved in CHX-induced SR1 mRNA accumulation. To gain further insights into mechanisms involved in saltand CHX-induced SR1 stability, the roles of different mRNA degradation pathways were examined in mutants that are impaired in either nonsense-mediated decay (NMD) or mRNA decapping pathways. These studies have revealed that neither the NMD pathway nor the decapping of SR1 mRNA is required for its decay. However, decapping activity is required for saltand CHXaccumulation of SR1 mRNA. To identify any specific regions within the open reading frame of the SR1 transcript (~3 kb) that are responsible for the salt-induced accumulation of SR1 mRNA, we generated transgenic lines expressing several truncated versions of the SR1 coding region in the sr1 mutant background. Then, we analyzed accumulation of each version in response to salt stress and CHX. Interestingly, we identified a 500 nts region in the 3' end of the SR1 coding sequence to be required for both saltand CHX-induced stability of SR1 mRNA. Potential mechanisms by which this region confers SR1 transcript stability in response to salt and CHX are discussed.Item Open Access Application of biotechnology in agriculture: a bioinformatic and market analysis of novel intervention methods(Colorado State University. Libraries, 2021) Frazier, Anthony Nathan, author; Yang, Hua, advisor; Metcalf, Jessica, advisor; Martin, Jennifer, committee member; Leach, Jan, committee memberTo view the abstract, please see the full text of the document.Item Embargo Characterization and insights into the molecular mechanism of cytokinin-induced priming of plant defenses(Colorado State University. Libraries, 2023) McIntyre, Kathryn, author; Argueso, Cristiana, advisor; Bush, Daniel, committee member; Leach, Jan, committee member; Stewart, Jane, committee member; Reddy, Anireddy, committee memberPlants have developed several mechanisms to cope with pathogenic challenges. One of these mechanisms, known as defense priming can be effective at reducing susceptibility to pathogens. Compared to unprimed plants, the immune response from primed plants, upon pathogen attack, is much stronger. This mechanism of induced disease resistance can be initiated by biological and chemical agents. The major benefit of priming is the induction of a high level of protection with considerably low fitness costs making it an attractive disease management strategy to preserve agricultural output. Recent research has demonstrated that the plant hormone cytokinin (CK) has a priming effect against biotrophic pathogens, a phenomenon referred to here as cytokinin-induced priming (CIP). This dissertation aims to gain further understanding of CIP against the hemibiotrophic bacterial pathogens Pseudomonas syringae pv. tomato (Pst) and Pseudomonas syringae pv. maculicola (Psm) in Arabidopsis thaliana (Arabidopsis) and Brassica napus, respectively as well as the necrotrophic fungal pathogen Botrytis cinerea in Arabidopsis. Chapter 2 focuses on characterizing CIP as a true priming agent by investigating the timeframe in which CIP is most effective at reducing susceptibility to Pst and Psm in both Arabidopsis and its closely related relative, B. napus and the impacts on plant growth due to CIP in these pathosystems. Moreover, we discovered that other known priming agents depend on endogenous CK signaling suggesting CK-mediated processes are involved in the priming of defense responses. The role of CK in primed defenses against B. cinerea is explored in chapter 3 where CIP is demonstrated to reduce necrotic lesion size caused by B. cinerea in a manner dependent on the JA-mediated defenses and partially on SA-mediated defenses. Transcriptome analysis revealed that during the priming stage, CK prepares the plants for pathogenic challenge through the accumulation of cellular components needed for translation and metabolites utilized for energy production and defense. Following B. cinerea inoculation, CIP suppresses defense while increasing photosynthetic-related processes. In the final chapter, molecular mechanisms are explored during CIP against Pst. Through transcriptome changes, priming by CK potentiates gene expression associated with systemic induction of defense, also known as systemic acquired resistance (SAR), following Pst challenge. Using this information, it is demonstrated that CK treatment can also induce SAR and that the known SAR inducer, L-pipecolic acid, is dependent on endogenous CK signaling. Due to the previously identified relationship between CK and source-sink relationships, amino acid transport was demonstrated to have a role in both CIP and CK-induced SAR. New agricultural practices that mitigate crop loss due to plant diseases are beneficial in terms of sustainability and economic costs. The use of CK as a priming agent offers an avenue for a new disease management strategy in that CIP protects plants against a broad range of pathogens with minimal effects on plant growth. The molecular mechanisms underlying CIP discovered here offers new insights into the relationship between plant metabolism and defense, where its exploitation could be used to create disease protection strategies.Item Open Access Characterization of a synthetic signal transduction system(Colorado State University. Libraries, 2012) Albrecht, Tessa, author; Medford, June, advisor; Bush, Daniel, committee member; Pilon-Smits, Elizabeth, committee member; Leach, Jan, committee memberThe Medford laboratory has developed a synthetic signal transduction system linking exogenous perception of a particular ligand to a transcriptional response. One application of this system is to produce plants that sense and respond to a specific ligand. The system was designed based on evolutionary conservation of histidine kinase signaling and uses bacterial components adapted to function in plants. The synthetic signaling system is responsive to extracellular ligand perception by a wild-type or modified ribose binding protein (RBP) scaffold. Upon ligand binding, RBP binds and activates a synthetic fusion histidine kinase made from the extracellular portion of the bacterial chemotactic receptor Trg and the cytoplasmic portion of the bacterial phosphate sensor PhoR. Activated Trg-PhoR transmits a phosphate signal to the bacterial response regulator PhoB. Upon phosphorylation PhoB translocates into the nucleus of a plant cell and activates transcription of the response gene(s). In addition to receiving a phosphate signal from Trg-PhoR, PhoB can be activated by exogenous cytokinin application suggesting that components of the cytokinin signaling pathway can interact with PhoB. Elimination or reduction of the interaction with cytokinin signaling components allows production of a more reliable signaling system. One goal of the following work was to reduce the interaction of PhoB with endogenous cytokinin signaling components. I attempted to identify a mutant form of PhoB that does not interact with cytokinin signaling components yet maintains function with the synthetic signaling system. I screened six different rationally selected PhoB mutants in plants for reduced response to exogenous cytokinin application. I concluded that a different approach will be needed to successfully reduce interaction with cytokinin signaling components. Another goal of this work was the identification cytokinin signaling components that interact with PhoB, possibly revealing a means to eliminate the interaction. I attempted to functionally express selected cytokinin signaling components in a bacterial testing system. After several failed cloning strategies, I conclude that the cytokinin sensor histidine kinase, AHK4, may be toxic and/or unstable in bacteria and expression of alternative genes will be needed to identify cytokinin signaling components that interact with PhoB. Additional work described here includes the independent testing of two computationally designed RBPs; one reported to bind the environmental pollutant methyl tert-butyl ether and the other reported to bind the explosive trinitrotoluene, for ligand dependent activation of the synthetic signaling system. These results show that the computationally designed RBPs do not function in a reliable manner and lead to the production of a detector plant using wild-type RBP to activate the synthetic signaling system that enables further analysis of the system components in plants.Item Open Access Combining quantitative genetics and genomics to identify polymorphisms associated with drought physiology in Arabidopsis and Brassica napus(Colorado State University. Libraries, 2014) Fletcher, Richard, author; McKay, John, advisor; Bauerle, William, committee member; Byrne, Patrick, committee member; Leach, Jan, committee memberTo view the abstract, please see the full text of the document.Item Open Access Constraints and capabilities of no-till dryland agroecosystems for bioenergy production(Colorado State University. Libraries, 2011) Lloyd, Grace Susanna, author; Hansen, Neil C., advisor; Brummer, Joe, committee member; Paustian, Keith, committee member; Leach, Jan, committee memberCrop residues are receiving attention as potential feedstocks for lignocellulosic biofuels. Sustainable residue harvest may be limited by soil erosion and the need to maintain soil organic carbon (SOC). Little attention has been given to the potential for residue harvest in the semi-arid Great Plains, largely due to assumptions of low production levels and the strong erosive forces of wind. Due to expanding interest in growing dedicated biofuel crops on marginal lands, these studies examined the capabilities and constraints of harvesting agricultural residues from dryland production systems in the semi-arid Great Plains. The first study examined long-term production levels of grain and stover for wheat (Triticum aestivum), corn (Zea mays), and grain sorghum (Sorghum bicolor) at three no-till dryland cropping sites in wheat-corn-fallow and wheat-sorghum-fallow rotations, and evaluated the impact of stover removal on wind and water erosion, soil organic carbon dynamics, and future productivity. The Revised Universal Soil Loss Equation and the Wind Erosion Equation were used to simulate water and wind erosion under various levels of residue removal. The DAYCENT model was used to estimate changes in soil organic carbon, grain yields, and soil fertility, if 50% of corn and wheat stover were harvested each crop year. Model validation was performed by comparing long-term production rates and measured changes in soil organic carbon to model simulated output. Total aboveground biomass production for corn and sorghum, averaged over site and soil type, was 5550 ± 2810 kg ha-1 yr-1, with average stover production of 2750 ± 1570 kg ha-1 yr-1 and 2800 ± 1570 kg ha-1 yr-1 for grain. The total aboveground annual biomass production of wheat across all sites averaged 5840 ± 2440 kg ha-1. Wheat annual stover yields were 3940 ±1880 kg ha-1 and grain yields averaged 1950 ± 820 kg ha-1. A 50% stover removal rate only slightly increased water erosion from 0.53 Mg ha-1 yr-1 (no removal) to a maximum of 1.4 Mg ha-1 yr-1. Wind erosion was a bigger risk, with rates surpassing the tolerable erosion levels after removing 10 - 30% of corn stover, depending on site and soil landscape position. However, at all three sites, up to 80% of wheat straw could be harvested without surpassing tolerable erosion rates. Soil organic carbon (SOC) declined 6-9% after 96 years of simulating 50% removal of corn and wheat stover. Under 0% removal, SOC levels appeared relatively stable, with maximum declines of 2.0%. As SOC levels are very low in these dryland systems, these declines represent a very small net loss of SOC when compared to losses observed in more humid regions. Under current wheat-corn-fallow management, virtually all stover must remain in order to control wind erosion and maintain soil organic carbon. However, if dedicated non-grain bioenergy crops were grown on an annual basis, there could be 2500-2700 kg ha-1 of harvestable biomass yearly while still retaining enough residues to maintain SOC. Total biomass production of a dedicated non-grain energy crop could be higher than the biomass production of the grain crops examined, namely because energy is not diverted to grain, and non-grain crops are not as sensitive to the timing of water deficits. Replacement of the fallow period with a non-grain biomass crop could lower the amount of residue needed to control erosion. Elimination of the fallow period would likely reduce the amount of residue that must remain to maintain SOC, increasing the amount of biomass available for removal. The second study uses the DAYCENT model to simulate variable responses in fertility, yield, and soil organic carbon within two field landscapes in eastern Colorado. Grain yield, soil fertility, and soil carbon were all impacted by stover harvest, but the magnitude and direction of responses were dependent on soil type. Yield declines as great as 1615 and 1382 kg ha-1 were simulated for corn and wheat, respectively. Declines in annual mineralization rates as high as 13 g N m-2 yr-1 were observed with stover harvest, but simulated changes in mineralization rates were highly variable between soil types, with net mineralization rates increasing with stover removal in some years. The impact of stover harvest on soil organic carbon varied with soil type and landscape position. Results are used to highlight the variable impacts stover harvest could have within one field or management unit, and demonstrate the need for landscape scale predictive models to assess the impact of stover removal on soil fertility and SOM dynamics and transfer processes models. Simulations characterized by an average soil type are not sufficient to account for the complexity of soils or the interactions and feedbacks of sediment, nutrient, and water transport that occur within agricultural fields. In addition to predictive model support systems, management of soil-specific responses to stover harvest will likely require adoption of precision agriculture technologies and practices such as variable rate harvesting and fertilization.Item Open Access Cytokinin-mediated processes promote heat-induced disease susceptibility of plants to bacterial pathogens(Colorado State University. Libraries, 2021) Shigenaga, Alexandra Marie, author; Argueso, Cristiana, advisor; Bush, Dan, committee member; Leach, Jan, committee member; Heuberger, Adam, committee member; Nishimura, Marc, committee memberAs global human populations continue to grow and temperatures are expected to rise, the pressure to increase food productivity and develop more stress-resistant crop varieties intensifies. Increased temperatures, a consequence anticipated as a result of global climate change, is expected to have an overall negative impact on crop productivity and agricultural systems. When exposed to non-optimal, high temperature conditions plant defense responses to pathogen attack are attenuated, leading to a process referred to here as heat-induced disease susceptibility. The plant growth hormone cytokinin is known to regulate responses to both biotic and abiotic pressures, making it an ideal target to study heat-induced disease susceptibility. The overarching goal of this dissertation was to understand the role of cytokinin in heat-induced disease susceptibility, to identify novel strategies to combat this process and design new ways to teach future generations about the impact of climate change on agricultural systems and science policy. First, I identified that a plant lacking a functional cytokinin signaling pathway, ahk2,3 mutated on the cytokinin signaling receptors AHK2 and AHK3, was less susceptible at elevated temperatures to the bacterial pathogen, Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). My results show that ahk2,3 plants are less susceptible under high temperature conditions with Pst DC3000 populations proliferating at a lower rate compared to wild-type plants overtime, suggesting that heat-induced susceptibility is partially dependent on cytokininiii signaling. Our results show that differences in susceptibility under elevated temperatures of ahk2,3 and wild-type plants is not attributed to an increase in defense responses, but rather by a possible change in the availability of nutrients for Pst DC3000. Together the data reveals that under high temperature conditions cytokinin promotes late-physiological processes, centered around primary metabolism, that are contributing to increased pathogen proliferation. These results led to the identification of cytokinin-regulated genes that could be utilized for breeding efforts to obtain loss-of-heat induced disease susceptibility that could be translated to crop species. Second, I identified that another member of the Brassicaceae family, Brassica napus, also exhibited heat-induced disease susceptibility to the bacterial pathogen, P. syringae pv. maculicola (Psm ES4326). Gene expression analysis confirms that similar to Arabidopsis, B. napus plants increase cytokinin signaling in response to high temperature stress. To further address if cytokinin was important for heat-induced disease susceptibility of B. napus, I utilized a chemical approach. B. napus plants were sprayed with the cytokinin-signaling antagonist, PI-55, prior to inoculation and results show that a single application of PI-55 led to a loss of susceptibility under heat to Psm ES4326. Additionally, this application of PI-55 did not lead to any adverse vegetative growth parameters, suggesting a potential novel chemical approach to combat heat-induced disease susceptibility in Brassicaceae crops. Lastly, I constructed a new approach to teach future generations about the impact of climate change on plant diseases in agricultural systems. "Plant Diseases and Climate Change" is an active learning activity designed to give college students experience in synthesizing information and developing a solution, in the context of plant pathology. This exercise uses the issue of heat-induced susceptibility of rice in the Philippines to improve student understanding of the interactions between abiotic and biotic factors affecting global food security. By using an international agricultural pathosystem, I aim to inform students how environmental pressures can impact economically important plant systems, the role scientists and experts play in policy making to preserve food security, and the importance of agriculture on a global scale.Item Open Access EPSPS gene duplication in Palmer amaranth: relative fitness, inheritance, and duplication mechanism of the glyphosate resistance trait(Colorado State University. Libraries, 2015) Giacomini, Darci A., author; Westra, Philip, advisor; Ward, Sarah, advisor; Leach, Jan, committee member; Preston, Christopher, committee member; Bedinger, Patricia, committee memberGlyphosate resistant (GR) Palmer amaranth (Amaranthus palmeri S. Wats.) is a weedy plant species that has invaded agricultural fields in at least 25 states, raising the cost of weed control to more than 4x the original cost. In most areas, the resistance is conferred through a gene amplification mechanism in which the target gene of glyphosate, 5-enolpyruvylshikimate-3-phosphate (EPSPS) is duplicated in the genome 100+ times, resulting in an overproduction of the EPSPS protein. With so much EPSPS enzyme available in each cell, glyphosate only inhibits a fraction of the proteins, leaving the rest to function as normal and ensuring plant survival. Understanding how this increase in EPSPS gene copy number and EPSPS protein production impacts relative fitness of the resistant plants was one objective of this research. Through greenhouse studies comparing high EPSPS copy GR plants with single copy sensitive plants, no difference was observed for any of the fitness characteristics measured. Both biotypes yielded similar numbers of offspring with no significant differences in germination or growth rate, revealing a complete lack of a fitness cost associated with the resistance trait. The second objective of this research was to quantify the stability of this resistance trait via multigenerational inheritance studies and within-plant EPSPS copy number variance measurements in the absence of glyphosate selection. The inheritance work found a complex pattern of EPSPS copy number transmission through the generations, a result that could be explained at least partially by the mosaic of EPSPS gene copy numbers patterns observed in both male and female Palmer amaranth plants. Copy numbers were inherited in a non-Mendelian pattern with transgressive segregation of the trait seen in both directions (more and fewer EPSPS copies found in the offspring than expected). This retention of high EPSPS copy number in the absence of a glyphosate selection pressure and no evidence of a fitness cost associated with the resistance trait possibly indicates a long-term loss of glyphosate as a control option in fields infested with GR Palmer amaranth. The last objective of this project was to better understand the mechanism of EPSPS gene duplication through sequence assembly of the EPSPS amplicon and chromosomal localization of this duplicated region. The amplicon was extended out to a little over 110kb and was found to contain mostly repetitive sequence including long direct repeats, microsatellites, and multiple transposable elements. A fluorescent in situ hybridization (FISH) assay found a single chromosomal location for the EPSPS genes, suggesting a tandem gene arrangement. These results further suggest that EPSPS duplication is achieved in Palmer amaranth via unequal recombination of the repeats surrounding the gene during mitosis and/or meiosis.Item Open Access Gene expression regulation by a stress-responsive transcription factor in rice seedlings(Colorado State University. Libraries, 2019) Williams, Seré., author; Reddy, A. S. N., advisor; Leach, Jan, committee member; Bush, Daniel, committee memberStress physiology is an inherently complex field. As plants cannot leave their environment when it becomes unfavorable, they have developed multiple mechanisms to cope with stresses. Many of these are unique to plants compared to mobile organisms. Plant stress physiology is of interest not only for this reason, but because the human population relies on agriculture for food. Additionally, our ecosystem relies on plants as primary producers as an integral component of life on earth. Plant stress physiology at the molecular level involves a symphony of signaling cascades that reshape cell physiology and communicate the stress signal to the whole plant and even nearby organisms. Over the last thirty years, enormous progress has been made to identify key genes, hormones, and signaling pathways that are involved in plant stress responses. To this end, we have yet to understand a cohesive picture of how plants respond to a combination of stresses. Given the variety of biotic stresses from bacteria, fungi, viruses, nematodes, and herbivores and their interaction with abiotic stresses including environmental extremes and resource availability, continued efforts are needed to understand the molecular nuances of plant stress responses. Not only are stresses variable and unique, plants have evolved to thrive in specific habitats, thereby developing unique strategies to cope with local environments. For example, rice grows well in flooded soils which would induce a stress-response in typical, non-aquatic organisms. Therefore, stress response will need to be decoded at the level of the organism. The goal of this work is to better elucidate stress response in rice. Specifically, I have looked at the influence of a transcription factor, SIGNAL RESPONSIVE 1 (OsSR1), that is regulated by Ca2+/CaM and known to be a dynamic regulator in a myriad of stresses in Arabidopsis. I have generated complemented lines of Ossr1 mutant and OsSR1 overexpressor transgenic rice lines. When compared with WT and mutant lines, these lines showed a range of OsSR1 expression. These lines will be of great help in deciphering the action of this transcription factor. Homozygous SR1 complemented and overexpressor lines along with WT and Ossr1 mutant will be used in future studies to better understand the action of SR1 in stress response in rice. Additionally, I performed a factorial global gene expression analysis using RNA-seq with WT and Ossr1 lines at the seedling stage in control and drought conditions, which will serve as a breeding ground for hypothesis generation and testing in future studies. Significant differentially expressed (DE) genes show down-regulation of genes encoding serine threonine-protein kinase receptor (SRK)-receptors, kinases, TCP family transcription factor, cytokinin-modifying enzyme and up-regulation of aquaporin, sucrose synthase, G-protein-related, and ferredoxin-nitrate reductase in the mutant when compared to WT. In response to polyethylene glycol (PEG)-induced drought stress, the mutant up-regulated transcription factors (homeobox [HOX]- containing TFs, WRKY, and DIVARICATA), signaling proteins (protein phosphatases), late embryogenesis abundant protein 1 (LEA1), nodulin-related genes, and senescence-associated gene 21 (SAG21), while down-regulating a CaM-dependent protein kinase, efflux transporters, peroxidases, aquaporins, and disease-related genes including Pathogenesis-related protein PRB1-2, disease resistance protein RPS2, and NB-ARC domain-containing protein. Lastly, significant DE genes in the WT illuminate how this important crop plant responds when exposed to PEG-induced drought. Drought induced the expression of MAPKKKs, ethylene-responsive transcription factors (ERFs), HOX TFs, as well as zinc-finger proteins and protein phosphatase 2Cs. In drought, WT down-regulated glycol-lipid transfer proteins, aquaporins, and salt stress-induced proteins. Gene ontology (GO) analysis of significant DE genes showed enrichment of GO terms related to membranes, oxidative stress, response to stimulus, and transcription regulation in both the WT and mutant when exposed to PEG. Future work will analyze the promoters of candidate genes for the OsSR1 DNA-binding motif (CG-1) to identify direct targets of OsSR1. Rice is the model organism for monocots and provides 15% of the calories consumed by humans. This study and other studies based on this work will help in elucidating the functions of this stress-responsive transcription factor, OsSR1, in this important crop plant.Item Open Access Interplay between selenium hyperaccumulator plants and their microbiome(Colorado State University. Libraries, 2016) Cochran, Alyssa T., author; Pilon-Smits, Elizabeth, advisor; Leach, Jan, committee member; von Fischer, Joseph, committee member; Stromberger, Mary, committee memberThe plant microbiome includes all microorganisms that occur on the plant root (rhizosphere) and shoot (phyllosphere) or inside plants (endosphere). Many of these microbes benefit their host by promoting growth, helping acquire nutrients or by alleviating biotic or abiotic stress. In addition to its intellectual merit, better knowledge of plant-microbiome interactions is important for agriculture and medicine. Microbiome studies are gaining popularity in multiple research areas, particularly due to advances in next generation sequencing, which has advantages over cultivable methods by revealing the complete microbial community. Still relatively little is known about the microbiomes of plants with extreme properties, including plants that hyperaccumulate (HA) toxic elements such as selenium (Se). Selenium HAs may contain up to 1.5% of their dry weight in Se, which can cause toxicity to herbivores and pathogens as well as neighboring plants. Many advances are yet to be made with regard to the interaction of Se and the plant microbiome: does plant Se affect microbial diversity and composition, and do plant-associated microbes affect plant Se accumulation? The first chapter of this thesis will discuss aspects of the plant microbiome as well as the discoveries to date with regard to plant-associated microbes and Se, mostly explored through culture-dependent methods. Selenium HA appear to harbor equally diverse endophytic microbial communities as non-hyperaccumulators. Thus, plant Se does not impair associations with microbes. A variety of microbes have been isolated from plants or soil in seleniferous areas, including some bacteria and fungi with extreme Se tolerance. Inoculation of plants with individual strains or consortia of microbes was able to promote plant growth, Se uptake and/or Se volatilization. Thus, microbes may facilitate their host’s fitness in seleniferous areas. Exploiting and optimizing plant-microbe associations may facilitate applications like phytoremediation (bio-based environmental cleanup) or biofortification (nutritionally fortified crops). Plant-derived microbial isolates may also be applicable without their plant host, e.g. for cleanup of wastewaters. Culture-dependent studies have dominated the plant-microbe interactions research in regards to hyperaccumulators thus far, painting an elaborate but incomplete picture. In the second chapter of this thesis, we use a mix of culture based and culture-independent methods to investigate the bacterial rhizobiome of selenium Se HAs. Using 16S rRNA Illumina sequencing, we show that the rhizobiomes of Se HAs are significantly different from non-accumulators from the same naturally seleniferous site, with a higher occurrence of Pedobacter and Deviosa surrounding HAs. In addition, we found that HAs harbor a higher species richness when compared to non-accumulators on the same seleniferous site. Thus, hyperaccumulation does not appear to negatively affect rhizobiome diversity, and may select for certain bacterial taxa in the rhizobiome. The bacterial isolates, independent from site or host plant species were in general extremely resistant to toxic concentrations of Se (up to 200mM selenate or selenite) and could reduce selenite to elemental Se. Thus, microbial Se resistance may be widespread and not be under selection by Se HAs. In future studies it will be interesting to further investigate the mechanisms by which Se HA species similarly shape their rhizobiome; this is perhaps due to Se-related root exudates. Future studies may also focus on elucidating the effects of microbes on plant Se accumulation and tolerance.Item Open Access Intraspecific variability of Geosmithia morbida the causal agent of thousand cankers disease, and effects of temperature, isolate and host family (Juglans nigra) on canker development(Colorado State University. Libraries, 2012) Freeland, Emily, author; Tisserat, Ned, advisor; Leach, Jan, committee member; Cranshaw, Whitney, committee member; Klett, James, committee memberSince the mid-1990's widespread mortality of Black Walnut (Juglans nigra) in the western United States has been noted. This mortality is the result of aggressive feeding of the Walnut twig beetle (Pityophthorus juglandis) and subsequent canker development caused by the newly named fungus Geosmithia morbida. Thousand Canker Disease (TCD) has been confirmed in Oregon, Washington, Idaho, Utah, California, Colorado, New Mexico, Arizona and recently in the native range of J. nigra, in Tennessee, Pennsylvania, and Virginia. Intraspecific variability of isolates was determined using rDNA ITS partial sequences and partial beta tubulin sequences. Nested clade phylogeographic analysis was used to look for correlations between haplotype trees and geography of isolates collected in screenings for the disease. Patterns of restricted dispersal by distance were found for both markers and high variability was found in isolates from single locations. Indicating that the populations causing disease throughout the western United States are not the result of recent point introductions. G. morbida isolate and J. nigra family had inconsistent effects on canker development, while temperature had a consistent effect. At higher temperatures (32°C) canker development was reduced compared to 25°C. Several genetically different G. morbida isolates were compared and repeated differences in pathogenicity were produced.Item Open Access Investigating resistance and the rapid response to glyphosate in giant ragweed (Ambrosia trifida L.)(Colorado State University. Libraries, 2016) Van Horn, Christopher R., author; Westra, Phil, advisor; Reddy, Anireddy, advisor; Argueso, Cris, committee member; Gaines, Todd, committee member; Leach, Jan, committee memberThe introduction of glyphosate-resistant crops along with widespread multiple in-season applications of glyphosate as part of weed management strategies that fail to address long-term weed control have provided the perfect scenario to foster the recent boom in glyphosate-resistant weeds. In order to implement best strategies to manage glyphosate-resistant weeds, it is important to understand the mechanism of resistance. Glyphosate targets and inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which prevents the synthesis of aromatic amino acids. We have investigated the mechanism of glyphosate resistance using several geographically diverse giant ragweed populations. From these populations we have characterized three phenotypic responses to glyphosate treatment: susceptible (GS), resistant slow response (GR slow), and resistant rapid response (GR rapid). Glyphosate resistance in giant ragweed (Ambrosia trifida L.) was first discovered in 2004 and the rapid response biotype was first identified in 2008. The molecular basis for resistance and the rapid response remains unknown. Our objective is to analyze glyphosate resistance and the rapid response in giant ragweed using physiological and molecular techniques. In whole-plant dose-response experiments conducted under greenhouse conditions, the glyphosate ED50 values (the effective dose that reduced shoot mass 50% relative to non-treated plants) of GR rapid biotypes were 4.2- and 2.3-fold greater than the ED50 values of GS biotypes and GR slow biotypes were 3.3- and 3.6-fold greater than the ED50 values of GS biotypes. The effective concentration that increased shikimate accumulation 50% relative to non-treated leaf tissue (EC50) was 4.1- and 3.9-fold higher (P < 0.05) for GR rapid and GR slow biotypes, respectively, than GS biotypes based on values averaged across accessions of the same biotype. However, at high glyphosate concentrations (1,000 to 2,000 μM), shikimate accumulation in the GR biotypes was similar to or greater than the GS biotypes. EPSPS sequence analysis showed no nucleotide mutation at a position where mutations are known to confer resistance in multiple species. Genomic copy number analysis found no evidence of target gene amplification. Whole plant physiology experiments suggest the rapid response is carbon dependent and both Phenylalanine and Tyrosine play a role in the stimulation of the rapid response. Excised leaf discs show H2O2 accumulation in mature leaf tissue of the GR rapid response biotype within 30 minutes after glyphosate application. Transcriptomic analysis of GR rapid and GS giant ragweed has identified significant differentially expressed genes that may be linked to glyphosate resistance and/or the rapid response. RNA-seq data was validated through qRT-PCR analysis. Six candidate genes were analyzed using qRT-PCR, two of which were selected for further expression analysis in an F2 population segregating for resistance. The final two candidate genes, osmotin 34 and S-adenosylmethionine synthetase 3, did not show a similar segregation pattern as seen in the F2 individuals. The large amount of RNA-seq data collected will continue to be used to determine key marker genes that could provide further insight into the glyphosate resistance mechanism present in this species. These initial results provide a framework for the future of giant ragweed glyphosate resistance research, which becomes increasingly important as the use of glyphosate-resistant crops develops world-wide. With this research, we can continue to work toward sustainable forms of herbicide weed management.Item Open Access Investigation of dietary rice bran for protection against Salmonella enterica Typhimurium infection in mice(Colorado State University. Libraries, 2014) Kumar, Ajay, author; Ryan, Elizabeth P., advisor; Dow, Steven, committee member; Leach, Jan, committee member; Schenkel, Alan, committee member; Weir, Tiffany, committee memberRice bran is a byproduct of rice milling for white rice. Rice bran is a rich source of nutrients such as vitamins, minerals, soluble and insoluble fibers, fatty acids, polyphenols and proteins. Research has shown the beneficial health effects of rice bran in hyperlipidemia, diabetes, immune modulation, allergies and cancer. This dissertation focuses on evaluation of rice bran for protection against Salmonella using a mouse model of oral infection. Salmonella is a food and water borne pathogen that affects a variety of hosts including plants, animals and humans. Salmonella infections are a major public health challenge around the globe. Currently, salmonellosis is treated using high doses of synthetic antimicrobials and the problem of drug resistance has increased. In this scenario, alternative and sustainable interventions are needed to control Salmonella infections. Several dietary agents have been studied for protective effects in Salmonella infection models. We tested the prophylactic effects of dietary rice bran in a Salmonella model of infection using female 129S6/SvEvTac mouse model with infection of Salmonella enterica Typhimurium 14028s strain. Feeding of 10% dietary rice bran for one week prior to infection significantly (p<0.05) reduced fecal excretion of Salmonella in orally infected mice. Salmonella-infected, rice bran fed mice also showed a significant decrease in systemic inflammatory cytokines such as TNF-α, IFN-γ and IL-12 as compared to control diet fed animals. The colonization resistance against enteric pathogens is highly influenced by composition of gut microflora. Supplementation of dietary rice bran increased the number of Lactobacillus spp. in feces of mice as compared to mice that were fed control diet. Research has shown that oral administration of some species of Lactobacillus reduces the colonization of Salmonella. We hypothesized that rice bran components also enhance mucosal protection by preventing Salmonella entry into the epithelial cells. Methanolic rice bran extracts were assessed in mouse small intestinal epithelial (MSIE) cells for blocking Salmonella entry and intracellular replication. Rice bran extract significantly reduced Salmonella entry and intracellular replication into MSIE cells. These results suggest the potential mechanisms for dietary rice bran induced improvement of colonization resistance against Salmonella. Given that rice crops have a large variation in genotype and phenotype such as in yield, disease and pest resistance, drought resistance, and nutrient quality, we hypothesized that variation in rice bran across cultivars induces differential protection against Salmonella infection due to differences in their phytochemical profile. A panel of six varieties namely IAC 600, Jasmine 85, IL 121-1-1, Wells, Red Wells and SHU 121 were tested in the in vitro and in vivo model of Salmonella infection. We found that rice bran extracts across varieties inhibited Salmonella entry into the MSIE and Caco-2 cells to different extents. IAC 600 fed animals significantly (p<0.05) reduced Salmonella fecal excretion as compared to the control diet fed animals. IAC 600 fed animals also reduced Salmonella fecal shedding significantly (p<0.05) as compared to SHU 121 diet fed animals at 2 and 6 days post Salmonella infection. Histopathological analysis revealed that IAC 600 diet fed animals had better ileal pathological scores as compared to SHU 121 and the control diet fed animals post Salmonella infection. SHU 121 and the control diet fed groups showed higher ulceration and inflammatory changes in ileum as compared to IAC 600 fed animals. Next we analyzed the fatty acid profile, mineral profile and total phenolic contents of rice bran. Stearic acid, lignoceric acid, boron and total phenol content were significantly correlated with Salmonella fecal shedding in mice across varieties. However, further studies are required to confirm the role of these nutrients from rice bran in protection against Salmonella. These results suggest that the variety of rice plays an important role in bran-induced protection against Salmonella infection and this difference in protection across the varieties could be attributed to a combination of bioactive components. Our studies suggest that dietary rice bran improves colonization resistance against Salmonella in mice. Rice bran could have important role in prevention of enteric infections in resource scarce populations and further human clinical studies are required. Rice bran may also be evaluated for supplementing diets of food animals to prevent Salmonella infections and therefore could have a potential role in food safety.Item Open Access Metabolomic profiles of Oryza sativa and influence of genetic diversity(Colorado State University. Libraries, 2011) Heuberger, Adam Lawrence, author; Brick, Mark, advisor; Leach, Jan, committee member; Ryan, Elizabeth, committee member; Byrne, Patrick, committee member; Thompson, Henry, committee memberFood crops with enhanced health characteristics are being developed in many breeding programs. Rice (Oryza sativa L.) is an ideal candidate to study traits related to health due to its importance as both a global staple food and a model system for cereal crops. Evaluating metabolite profiles can be a high-throughput method to identify variation in health properties of dietary components. Metabolomics is a useful tool to assess the influence of genetics on total metabolite variation in the cooked grain. Cooked rice metabolite profiles for 10 diverse varieties were determined using ultra performance liquid chromatography coupled to mass spectrometry (UPLC-MS) on aqueous-methanol extracts. A total of 3,097 molecular features were detected, and 25% of the features varied among the 10 varieties (ANOVA, p < 0.001). Both z-score and partial least squares-discriminant analysis (PLS-DA) showed variation consistent with subspecies-based varietal groupings, and indicated genetic control over the metabolite profiles. Variation in total phenolics and vitamin E was also consistent with varietal groupings. Genes in biochemical pathways for health-related metabolites were interrogated for allelic variation by single nucleotide polymorphisms (SNPs). SNP variation may serve as an important mechanism by which genes influence metabolic variation. The influence of genetic diversity on the metabolite profile of the rice grain was also assessed for two interacting effects: genotype-environment interactions (GEI) and genotype-fermentation interactions (GFI). GEI was assessed by growing two diverse rice varieties in the field and the greenhouse. Gas-chromatography-MS (GC-MS) was used to detect primary metabolites from aqueous-methanol extracts of cooked rice. Genotype, environmental, and GEI effects were observed for many metabolites, including the amino acid phenylalanine, a precursor for many secondary metabolites related to human health. Genes associated with phenylalanine synthesis were screened in rice gene expression databases, and variation within and among the genes suggests they are a potential source of genetic variation for phenylalanine synthesis. Both the metabolite and gene expression patterns indicate a potential interaction between phenylalanine and serine synthesis. The GC-MS data implies the GEI effects on primary metabolism may correspond to variation in secondary metabolites that are predicted to affect human health. Additionally, human health attributes of the grain may be dependent on fermentation of rice metabolites by gut microorganisms. GFI effects were assessed by fermenting three highly similar rice varieties with Saccharomyces boulardii, a probiotic yeast. Metabolites were extracted and detected by GC-MS. A PLS-DA model showed evidence of fermentation (F) effects, but not GFI. However, when extracts were assessed for the ability to inhibit viability of lymphoma cells, both F and GFI effects were apparent. It is therefore likely that GFI effects may exist among diverse rice varieties, and that interactions affect the bioactivity of rice metabolites. In summary, total metabolite variation is largely influenced by the rice genotype, including interactions with environment and fermentation. These data describe both heritable and non-heritable sources of variation. Thus, although genetic variation in rice is sufficient to establish metabolite profiles specific to human health characteristics, the heritability of a secondary metabolite-associated health trait is likely influenced by both environment and fermentation effects.Item Open Access Phenotypic plasticity in the response of sorghum to water stress and recovery indicates pre-flowering drought tolerance(Colorado State University. Libraries, 2018) Miller, Sarah Bethany, author; Jahn, Courtney, advisor; Argueso, Cris, committee member; Broeckling, Corey, committee member; Leach, Jan, committee memberTo view the abstract, please see the full text of the document.Item Open Access Plant-microbe interactions of selenium hyperaccumulators: effects on plant growth and selenium metabolism(Colorado State University. Libraries, 2012) Lindblom, Stormy Dawn, author; Pilon-Smits, Elizabeth, advisor; Leach, Jan, committee member; Wangeline, Ami, committee member; von Fischer, Joe, committee memberTo view the abstract, please see the full text of the document.Item Open Access RNA sequencing identifies genes putatively involved at the aphid-Buchnera symbiotic interface(Colorado State University. Libraries, 2020) Acharya, Shailesh Raj, author; Nalam, Vamsi J., advisor; Leach, Jan, committee member; Sloan, Daniel, committee memberIn aphids, the supply of essential amino acids depends on an ancient nutritional symbiotic association with the gamma-proteobacterium, Buchnera aphidicola. The endosymbiont converts abundant non-essential amino acids into essential amino acids that are supplied to the aphid. The long-term goal of the proposed work is to exploit the biochemical interdependence that exists between soybean aphid (Aphis glycines) and its primary endosymbiont to develop effective resistance in soybean (Glycine max). Little is known of the A. glycines and soybean amino acid transporters (AATs) that facilitate this exchange. The soybean aphid is the most important arthropod pest on soybean in North America and aphid outbreaks in major soybean growing regions of the country in the past has resulted in yield losses of up to 40%. In the current study, we used RNA-seq to identify amino acid transporters involved in the exchange of amino acids between the aphid and its endosymbiont. A total of 2121 genes were differentially expressed between the aphid and bacteriocytes with 516 genes showing up-regulation, while 1605 genes were down-regulated in the bacteriocytes. Analysis of GO terms revealed enrichment in membrane and transport associated processes. Our RNA-seq analysis of differentially expressed genes showed that one putative amino acid transporter: 72-RA, is up-regulated in the bacteriocytes. This work represents a first step towards understanding aphid dependency on its endosymbiotic bacteria and target them as a means of a novel aphid control strategy.Item Open Access The role of plants as an environmental reservoir of chronic wasting disease prions(Colorado State University. Libraries, 2016) Ortega, Aimee Elise, author; Zabel, Mark, advisor; Mathiason, Candace, committee member; Leach, Jan, committee member; Wilusz, Jeffrey, committee memberTransmissible Spongiform Encephalopathies (TSEs) are a group of diseases caused by an abnormal version, PrPRES, of the normal cellular host protein prion protein (Prnp) termed PrPC. Disease is fatal resulting in amyloid deposits and spongiform degeneration in the brain in most but not all cases. Clinical signs can include wasting, increases in salivation, and general motor impairment but many other clinical signs exist and can vary between TSEs. PrPRES is incredibly resistant to inactivation and can withstand radiation, formalin treatment, and autoclaving to name a few tried decontamination methods whereas PrPC is degraded normally. This difference in degradation allows for differentiation between the two protein forms as PrPRES is resistant to degradation by Proteinase K. In the early 1980s this abnormal protein was discovered to be the sole causative agent of the various TSEs which at the time was a novel finding and a novel method of disease transmission. It is thought that slightly misfolded forms of PrPC occur which can then misfold further eventually forming PrPRES. PrPRES then has the ability to act as a template for conversion, converting PrPC. Numerous TSEs exist that affect both humans and a variety of animals. One of the animal TSEs is Chronic Wasting Disease (CWD) which affects cervids such as elk, deer, and moose (Cervus candensis, Odocoileus hemionus, Alces alces) and has become endemic in both free-ranging and captive herds. The exact mechanisms behind spread of CWD are unknown but research has shown that environmental reservoirs play a role in transmission dynamics. We chose to explore whether PrPRES can be detected on or inside grasses and plants naturally exposed to prions in CWD endemic areas by use of Protein Misfolding Cyclic Amplification (PMCA). Here we present novel environmental evidence showing that PrPRES can be found on the surface of multiple plants from Rocky Mountain National Park and mice inoculated with these samples are showing clinical signs of disease.Item Open Access Towards the systematic identification of low-cost ecosystem-mediated carbon sequestration opportunities in bioenergy supply chains(Colorado State University. Libraries, 2015) Field, John L., author; Willson, Bryan, advisor; Paustian, Keith, advisor; Bradley, Thomas, committee member; Leach, Jan, committee member; Marchese, Anthony, committee memberBecause the dedicated production of terrestrial biomass feedstocks involves the fixation of atmospheric carbon, carefully managed biofuel and bioenergy supply chains are increasingly recognized as an opportunity for carbon sequestration in soils or geological reservoirs in addition to their climate change mitigation value via the displacement of fossil fuel use. Bioenergy involves the coupling of agricultural systems and industrial supply chains, and finding optimal system designs often requires navigating a fundamental tension between maximizing overall system productivity while simultaneously limiting the intensification of feedstock exploitation to sustainable levels. Bioenergy sustainability analyses are further complicated by strong spatial heterogeneities in feedstock production performance, fundamentally different emission mechanisms across the agricultural and industrial phases of the biofuel lifecycle, and the tendency to perform environmental assessments and economic analyses in isolation. Well-designed integrated assessments are necessary to identify the total amounts and time dynamics of sequestration possible in such systems, to put those results in context relative to other supply chain impacts, and to understand tradeoffs between various environmental impact criteria and production costs. This dissertation starts with a thorough review of the bioenergy lifecycle assessment (LCA) literature to identify outstanding climate impact accounting challenges and inform the integration of production cost estimates. Two integrated assessment case studies are then undertaken to identify low-cost opportunities for improving carbon sequestration at different points in the bioenergy supply chain. The first focuses on feedstock production, assessing the potential for increasing soil carbon sequestration in bioenergy landscapes based on the cultivation of perennial grasses. A spatially-explicit landscape analysis system is created around a newly-parameterized version of the DayCent biogeochemistry model, and switchgrass productivity and soil greenhouse gas balance are assessed across gradients of land quality and cultivation intensity in a real-world bioenergy landscape in western Kansas. Integrating these ecosystem simulation results with existing LCA, farm enterprise budget, and biomass transport models allows for the quantification of landscape level cost – mitigation tradeoffs under various system design strategies and policy constraints. The second case study focuses downstream in the supply chain, considering the use of low-value conversion co-products as soil amendments to improve agroecosystem sustainability. The biochar co-product from a hypothetical thermochemical conversion system in the Colorado Front Range is assessed using simplified models of biochar recalcitrance and agronomic benefits as a function of feedstock material and conversion method. Together, these case study results are illustrative of the potential costs of improving ecosystem-mediated carbon sequestration in bioenergy systems, and the ongoing work required for full global supply chain optimization.Item Open Access Transcriptome and secretome of two pythium species during infection and saprophytic growth(Colorado State University. Libraries, 2016) Ibarra Caballero, Jorge Rafael, author; Tisserat, Ned A., advisor; Leach, Jan, committee member; Hess, Ann, committee member; Jahn, Courtney, committee memberIn the first part of this dissertation, I describe how we obtained and analyzed the full complement of transcripts –the transcriptome- and the set of secreted proteins –the secretome- of Pythium irregulare and Pythium iwayamai isolates when they were infecting plant hosts and when they were growing saprophytically. Additionally, these two treatments were performed at two different temperatures (4˚ and 19˚C). The assembled transcriptomes were annotated, and a closer analysis of the expression profiles of transcripts coding for pathogenicity-related proteins is shown. Secreted proteins were semi-quantified and their likely functions were determined based on the annotation of the corresponding transcripts. In the second part of this document, I include four appendices. Each one is about a different project that I contributed to during my stay at the Department of Bioagricultural Sciences and Pest Management. An article has been publish for each project. For the study included in appendix 1, I used basic plant pathology and microbiology techniques to fulfill Koch’s postulates for a disease affecting Turkish filbert trees. I also used some molecular techniques to aid in the identification of the isolated bacteria. The work presented in appendix 2 was a follow-up of the previous one. We sequenced the genome of Xanthomonas arboricola pv. Corylina isolated from Turkish filbert leaves. Then, we iii assembled a draft genome of the bacterium. The assembled genome was annotated and the robustness of the assembly was verified. Appendix 3 includes the report about the genome assemblies of 3 different isolates of Lonsdalea quercina subsp. quercina. As in the previous work, the genomes were annotated and the robustness of the assemblies was verified. In appendix 4, the report of a study on the population structure of Geosmithia morbida is presented. This fungus and the walnut twig beetle Pityophthorus juglandis are associated with the Thousand Cankers disease of walnut and wingnut trees. There is big concern because the disease has moved from the western United States to the native range of a highly susceptible walnut species in the eastern USA. I contributed to this research in the analysis of the data, the statistical analyses and the writing of the paper.