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Browsing by Author "Nishimura, Marc, committee member"

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    Candidate gene identification for glyphosate resistance and rapid cell death in Ambrosia trifida
    (Colorado State University. Libraries, 2024) Sparks, Crystal Devona, author; Gaines, Todd, advisor; Dayan, Franck, committee member; Beffa, Roland, committee member; Nishimura, Marc, committee member; Westra, Phil, committee member
    Glyphosate is one of the most widely used herbicides worldwide due to favorable chemical characteristics and availability of compatible transgenic biotechnology in crops. Resistance to glyphosate has evolved in many weed species capable of significant yield reduction in top production systems globally. One such species is Ambrosia trifida (giant ragweed), a monoecious broadleaf with imperfect flowers native to North America where it is highly competitive in corn, soybean, and cotton production. Some glyphosate resistant populations of A. trifida also display a rapid response with cell death in the mature leaves within 24-48 hours after treatment with glyphosate. Transcriptomic analysis revealed differential expression of multiple gene families associated with known glyphosate resistance mechanisms such as ATP-binding cassette (ABC) transporters and aldo-keto reductases. Gene ontology analysis showed an enrichment of many genes related to phytohormone response to biotic and abiotic stress in the differentially expressed genes. This could be related to a novel glyphosate resistance mechanism or a signaling cascade involved in the rapid cell death response. The A. trifida genome contains two loci of the glyphosate target site gene 5-enolpyruvylshikimate-3-phosphate-synthase (EPSPS), with a previously reported Pro106Ser mutation in EPSPS2. This locus showed up-regulation by three hours after treatment. Trait mapping revealed three genomic regions associated with glyphosate resistance and a single interval associated with the rapid response. Along with phenotypic segregation ratios, this indicates that resistance and rapid response traits are genetically independent and multiple genes likely contribute to resistance.
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    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 member
    As 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.
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    Design and quantification of a tissue type specific genetic circuit in plants
    (Colorado State University. Libraries, 2020) Oehmke, Sara, author; Medford, June, advisor; Argueso, Cris, committee member; Nishimura, Marc, committee member
    Synthetic biologists aim to rationally design genetic circuits and utilize plant platforms to photosynthetically drive, self-sustainable circuits. Although plants are excellent platforms, issues and unpredictability arise from the innate complexity of multicellularity. The ability to quantitatively control gene expression within specific cell types can address some issues arising from multicellularity. In my research, I developed a genetic circuit with the ability to induce and quantitatively control expression in Arabidopsis thaliana root epidermal cells. The circuit design uses an externally applied ligand that activates a computationally designed transcriptional response driven by a tissue specific promoter to control output (GFP expression). In addition, I engineered a circuit that adds a positive feedback motif. To quantify the behaviors of these circuits I developed a MATLAB program to remove background signals from confocal microscopy images and quantify GFP signal in a high-throughput manner. The genetic circuit is highly specific for root epidermal cells, controllable with external ligand, and has increased sensitivity and memory with positive feedback. The concepts and components of these circuits can be implemented in future designs to engineer and produce plants with more predictable and diverse behaviors affording the operator greater control.
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    Directed expression of R2R3 MYB transcription factors for ectopic suberin deposition
    (Colorado State University. Libraries, 2022) McKay Whiteman, Angel, author; Medford, June, advisor; Nishimura, Marc, committee member; Peebles, Christie, committee member
    Plants are susceptible to many stresses, which can lead to reduced growth, decreased crop yield, or even plant death. Plants contain natural barriers to protect them from stresses, including the hydrophobic biopolymer suberin. Suberin protects plants against a variety of factors, including water loss, toxic ions, loss of essential nutrients, and entry of microorganisms. Plant roots contain a suberized barrier called the endodermis, which regulates the entry and exit of materials from the cortex to the vascular cylinder. While the endodermis protects the vascular cylinder, the cortex is left susceptible to stresses. Engineering a suberized barrier in the root epidermis could protect the cortex and provide an additional point of regulation. Multiple R2R3 type MYB transcription factors have been found to be involved in suberin biosynthesis. Expressing these transcription factors in the root epidermis could provide a suberin barrier for protection. I expressed five R2R3 MYB transcription factors in the root epidermis of Arabidopsis thaliana to determine whether their expression led to suberization of the root epidermis. Constitutive expression of one transcription factor, MYB84, led to increased epidermal suberin. Two homozygous lines were further analyzed and found to have decreased root growth. Gene expression results from one homozygous line suggest that MYB84 overexpression may lead to increased expression of suberin biosynthetic genes involved in synthesis of aliphatic suberin monomers and monomer transport. Further analysis of these transgenic plants could provide insight into the potential protective barrier the root epidermal suberin provides.
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    Investigating the relationship between cover crop species diversity, composition and function of the soil microbiome
    (Colorado State University. Libraries, 2023) Seitz, Valerie, author; Prenni, Jessica, advisor; Wrighton, Kelly, committee member; Schipanski, Meagan, committee member; Nishimura, Marc, committee member
    Cropping diversification, such as cover cropping, can contribute to sustainable agriculture by enhancing soil health and promoting ecosystem services through interactions with the soil microbial community. One important mechanism through which cover crops impact soil health is via root exudation, the release of organic compounds from plant roots into the soil region surrounding the roots, the rhizosphere. Root exudation varies among cover crop species, growth stages, and edaphic and environmental conditions resulting in a myriad of effects on the rhizosphere. Plant-derived inputs, like root exudates, modulate the soil microbial community, influencing microbial biomass, community structure, and catalyzing biogeochemistry. As a result, cover crops are linked to microbial changes that impact soil nutrient cycling and organic matter decomposition leading to a legacy impact on primary crop yield and health. Understanding the intricate relationship between cover crop root exudation composition and the soil microbiome is crucial for optimizing cover crop selection, management practices, and harnessing cover crops for precision microbiome management in agroecosystems. My dissertation demonstrates that cover crop root exudation differs considerably across cover crop species, and cultivars within species, and reveals cover crop metabolic impacts on soil microbial composition and function, which play a large role in the generation and maintenance of healthy soils to support our agricultural needs.
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    Sweet surprise: the search for genes conferring beet curly top virus resistance
    (Colorado State University. Libraries, 2023) Withycombe, Jordan, author; Nachappa, Punya, advisor; Nalam, Vamsi, committee member; Nishimura, Marc, committee member; Dorn, Kevin, committee member
    Sugar beets (Beta vulgaris L.) are grown across the western United States and suffer economic loss annually to curly top disease. Curly top disease is caused by the beet curly top virus (BCTV) and is spread by the only known insect vector the beet leafhopper, Circulifer tenellus Baker (BLH). Current management strategies for BCTV include chemical control using neonicotinoid seed treatments and foliar insecticidal sprays, as well as the use of BCTV-resistant sugar beet varieties. However, the underlying genetic mechanism surrounding resistance in sugar beet is unknown. The overarching goal of this study was to identify the mechanism of resistance in sugar beet to BCTV and identify potential genes conferring resistance. The objectives for this study were: 1) classify the nature of BCTV resistance in a resistant (EL10) and susceptible (FC709-2) genotype of sugar beet using host suitability and host preference insect assays, as well as assess viral load within each genotype and 2) characterize the transcriptional response to BCTV infection using RNA-sequencing. To classify the nature of BCTV resistance in each genotype of sugar beet, host suitability and preference assays were conducted using virus infected and uninfected BLH. In host suitability assays, the percentage of surviving BLH adults and the number of nymphs produced when reared on a single plant of either genotype was determined over a 3-week period. There was no difference in adult survival, or the number of nymphs produced on either genotype for the virus infected or uninfected leafhoppers. Host preference assays were used to assess settling behavior of BLH over time when given a choice between the two genotypes. It was concluded that virus infected leafhoppers had a clear choice to settle on the susceptible genotype at all timepoints after 4 hours, while uninfected leafhoppers did not make as strong of a settling choice. Average viral load for each genotype across three timepoints was estimated using qPCR. The results showed that the average viral load increased in each genotype over time, yet there was no difference in the average viral load between the genotypes at any individual timepoint. The global transcriptional response to BCTV infection over time for a resistant and susceptible genotype of sugar beet was conducted using RNA-sequencing technology. Mock-inoculated and BCTV-inoculated plants from each genotype were sampled on day 1, 7 or 14 post inoculation resulting in the preparation of 36 mRNA sequencing libraries. Comparison between mock-inoculated and BCTV-inoculated plants of each genotype and timepoint were conducted separately to generate six list of differentially expressed transcripts (DETs). Each transcript was annotated with a description and further classified for its role in the plant biological, cellular or molecular processes. The results showed that both genotypes of sugar beet had a dynamic response to BCTV infection over time, although there was minimal overlap between the responses to one another. EL10, the resistant genotype, had DETs associated with phytohormone production including jasmonic acid and abscisic acid, along with proteins linked to stress reduction and the downregulation of plant primary metabolic processes. In contrast FC709-2, the susceptible genotype, was found to produce opposing phytohormones like salicylic acid and auxins, as well as the production of volatile organic compounds and an increase of primary plant metabolic processes. These opposing responses shed light on the differences in the transcriptional response of a resistant and susceptible genotype of sugar beet. Understanding and classifying the mechanisms of resistance or susceptibility to BCTV infection in sugar beet is beneficial to researchers and plant breeders as it provides a basis for further exploration of the host plant-virus-vector interactions.
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    TALE-bound QTL: a computational investigation of bacterial effector association with resistance quantitative trait loci in Oryza sativa
    (Colorado State University. Libraries, 2022) Sharkey, Jacob Emmett, author; Leach, Jan E., advisor; Huerta, Alejandra I., committee member; Nishimura, Marc, committee member; Roberts, Robyn, committee member
    Durable resistance to Xanthomonas oryzae pathovars oryzae (Xoo) and oryzicola (Xoc), which cause bacterial blight and bacterial leaf streak, respectively, is highly sought after in rice (Oryza sativa) due to the pathogens ability to impact maximum attainable yields. Regions of the rice genome associated with quantitative resistance to multiple strains of Xoo and Xoc, known as quantitative trait loci (QTL), were previously identified using a multi-parent advanced generation intercross (MAGIC) rice population and a combination of genome wide association studies and interval mapping. These QTL have been associated with decreased lesion lengths by Xoc and Xoo on rice. What remains unknown is the molecular basis for the induction of genes under these QTL during pathogen infection. Considering our biological question "what is the molecular basis for regulation of resistance QTL associated with Xoo and Xoc?", we predicted that part of the answer could be found by investigating the bacteria's direct interaction with the O. sativa genome. Upon infection, Xoo and Xoc injects the host with DNA-binding TALE (transcription activator-like effector) proteins. These effectors, when bound to their target plant gene promoter, induce gene transcription. We hypothesize that differential interactions of TALE with promoters of rice genes under the QTL lead to the resistant/susceptible phenotypes exhibited across varieties. To test this, we designed a pipeline that predicts TALE-regulated candidate genes involved in quantitative resistance. This pipeline identifies genes that meet three criteria: (1) the presence of a binding site for an X. oryzae TALE in the gene's promoter, a strong correlation between binding site presence, and disease phenotypes and overlap of the gene with a resistance QTL. We used this pipeline with genomic and phenotypic data for the eight MAGIC founders to identify candidate genes involved in resistance against seven Xoo and Xoc strains. Candidate genes identified include ones encoding a patatin-like phospholipase and multiple NB-ARC containing proteins such as the Mla1 protein. Here, we exploit the abundant genomic data for the rice-X. oryzae systems and the ability to predict direct associations between bacterial proteins and plant genomes, to propose a method that could streamline the identification of genes involved in quantitative resistance to TALE- harboring Xanthomonas.