Browsing by Author "Reddy, Anireddy, committee member"

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Open Access
A comprehensive compendium of Arabidopsis RNA-seq data
(Colorado State University. Libraries, 2020) Halladay, Gareth A., author; Ben-Hur, Asa, advisor; Chitsaz, Hamidreza, committee member; Reddy, Anireddy, committee member
In the last fifteen years, the amount of publicly available genomic sequencing data has doubled every few months. Analyzing large collections of RNA-seq datasets can provide insights that are not available when analyzing data from single experiments. There are barriers towards such analyses: combining processed data is challenging because varying methods for processing data make it difficult to compare data across studies; combining data in raw form is challenging because of the resources needed to process the data. Multiple RNA-seq compendiums, which are curated sets of RNA-seq data that have been pre-processed in a uniform fashion, exist; however, there is no such resource in plants. We created a comprehensive compendium for Arabidopsis thaliana using a pipeline based on Snakemake. We downloaded over 80 Arabidopsis studies from the Sequence Read Archive. Through a strict set of criteria, we chose 35 studies containing a total of 700 biological replicates, with a focus on the response of different Arabidopsis tissues to a variety of stresses. In order to make the studies comparable, we hand-curated the metadata, pre-processed and analyzed each sample using our pipeline. We performed exploratory analysis on the samples in our compendium for quality control, and to identify biologically distinct subgroups, using PCA and t-SNE. We discuss the differences between these two methods and show that the data separates primarily by tissue type, and to a lesser extent, by the type of stress. We identified treatment conditions for each study and generated three lists: differentially expressed genes, differentially expressed introns, and genes that were differentially expressed under multiple conditions. We then visually analyzed these groups, looking for overarching patterns within the data, finding around a thousand genes that participate in stress response across tissues and stresses.
Open Access
An exploration of viral RNA-mediated strategies to stall and repress the cellular exoribonuclease XRN1
(Colorado State University. Libraries, 2018) Charley, Phillida A., author; Wilusz, Jeffrey, advisor; Zabel, Mark, committee member; Perera, Rushika, committee member; Reddy, Anireddy, committee member
The regulation of mRNA decay plays a vital role in determining both the level and quality control of cellular gene expression in eukaryotes. Since they are likely recognized as foreign/unwanted transcripts, viral RNAs must also successfully navigate around the cellular host RNA decay machinery to establish a productive infection. This bypass of the cellular RNA decay machinery can be accomplished in many ways, including the sequestering of regulatory proteins or inactivating enzymatic components. One attractive way for RNA viruses to undermine the cellular RNA decay machinery is to target the cellular exoribonuclease XRN1 since this enzyme plays a major role in mRNA decay, appears to coordinate transcription rates with RNA decay rates, and is localized to the cytoplasm and thus readily accessible to cytoplasmic RNA viruses. We have previously shown that many members of Flaviviridae (e.g. Dengue, West Nile, Hepatitis C and Bovine Viral Diarrhea viruses) use RNA structures in their 5' or 3' untranslated regions (UTRs) to stall and repress XRN1. This results in the stabilization of viral RNAs while also causing significant dysregulation of cellular RNA stability (and thus dysregulation of overall cellular gene expression). In this dissertation we first extend this observation to another member of the Flaviviridae, Zika virus, by demonstrating that structures in the 3' UTR of the viral genomic RNA can stall and repress XRN1. Significantly, we also demonstrate that the 3' UTR of the N mRNA of the ambisense segment of Rift Valley Fever virus, as well as two other phleboviruses of the Phenuiviridae, also can effectively stall and repress XRN1. This observation establishes XRN1 stalling in an additional family of RNA viruses, in this case in the order Bunyavirales. We have mapped the region responsible for XRN1 stalling to a G-rich core of ~50 nucleotides and provide evidence that the formation of a G-quadruplex is contributing to stalling of XRN1. In addition to phleboviruses, we also detected RNA regions that stall XRN1 in the non-coding regions of two other virus families. The 3' UTRs of all four ambisense transcripts of Junin virus, an arenavirus, stall and repress XRN1. This observation was extended to two additional arenaviruses, suggesting that XRN1 stalling may be a conserved property of the 3' UTRs in the Arenaviridae. Finally, we demonstrate that the non-coding RNA from beet necrotic yellow vein virus RNA segment 3 is produced by XRN1 stalling and requires a conserved sequence called the coremin motif. Collectively, these observations establish XRN1 stalling and repression as a major strategy used by many virus families to effectively interface with the cellular RNA decay machinery during infection. We performed two proof of principle studies to extend the significance of the observation of XRN1 stalling during RNA virus infections. First, since XRN1 stalling may be associated with successful viral gene expression as well as cytopathology, we explored whether we could identify a small molecule compound that could interfere with the knot-like three helix RNA junction structure that stalls XRN1 in the 3' UTR of flaviviruses. We tested several triptycene-based molecules, compounds that have been previously shown to intercalate into three helix junctions and identified four triptycene derivatives that interfere with XRN1 stalling. Lastly, we explored whether there might be a cellular exoribonuclease that could navigate through the well-characterized flavivirus structure that effectively stalls XRN1. Our efforts focused on the mammalian Dom3z/DXO enzyme which contains both 5' decapping and 5'-3' exoribonuclease activity. Interestingly, recombinant Dom3z/DXO enzyme did not stall on RNAs containing the 3' UTR of either Dengue virus or the Rift Valley Fever Virus N mRNA. This may suggest that there is a molecular arms race of sorts between the cell and the virus for supremacy of regulating the 5'-3' decay of RNA during infection.
Open Access
Assessing and understanding the generation and function of RNA decay intermediates in non-insect borne flaviviruses
(Colorado State University. Libraries, 2019) Mundell, Cary T., author; Wilusz, Jeffrey, advisor; Geiss, Brian, committee member; Perera, Rushika, committee member; Reddy, Anireddy, committee member
Cellular gene expression is an intricate process regulated on many levels that allows the cell to react correctly to stimuli or to maintain homeostasis. RNA viruses must act to preferentially drive production of their own messenger RNAs (mRNAs) and proteins in order to successfully replicate and ensure continued infection. Due to the necessity for RNA viruses to remain in the cytoplasm, regulatory factors that affect host mRNAs likely also affect the transcripts of RNA viruses. RNA decay represents a major pathway of regulation for mRNAs. A multitude of RNA viruses possess unique mechanisms that act to prevent the decay of viral transcripts and allow for successful translation. Members of the viral family Flaviviridae are positive sense, single-stranded RNA viruses that do not possess a poly(A) tail. Therefore, it is highly likely that these transcripts would be marked as deadenylated and shuttled down one of the RNA decay pathways that exist in the cell. Interestingly, members of the genera Flavivirus of the family Flaviviridae possess a conserved structured 3' untranslated region (UTR) that acts to interfere with the decay processes of the major cytoplasmic cellular 5'-3' decay enzyme XRN1. In addition, members of the generas Hepacivirus, Hepatitis C Virus (HCV) and Pestivirus, Bovine Viral Diarrhea Virus (BVDV), possess XRN1 stalling elements within their 5' UTRs. These stalling sites block the action of the exonuclease and generate decay intermediates. The generation of these decay intermediates represses XRN1 activity in the infected cell. Herein we demonstrate a new method for studying RNA decay through the use of XRN1-resistant RNAs (xrRNAs). In this method we utilize the well characterized xrRNA of Dengue Virus Type 2 (DENV2) as a readout to study the decay rates of relatively large RNA constructs. We show that not only is utilizing an xrRNA an effective method for confirming XRN1-mediated decay, but that the accumulation of the readout xrRNA can be utilized to understand changes in the decay kinetics of RNA substrates. We further utilize this method to demonstrate a lack of XRN1 stalling elements within the poliovirus internal ribosomal entry site (IRES) element. We provide evidence that the stalling of XRN1 in the 5' UTR of BVDV is dependent on both the presence of the entire IRES structure and the presence of a stem loop 5' to the IRES element through the analysis of a series of truncations. Finally, we demonstrate one possible role for the HCV and BVDV decay intermediates as the truncated IRES element maintains translatability in an in vitro system. Collectively, these data better define the structural requirements for the novel XRN1 stalling elements located in the 5' UTR of non-insect borne members of the Flaviviridae as well as the potential function of the decay intermediates.
Open Access
Biology, comparative genomics and molecular diagnostics of Xanthomonas species infecting rice and corn
(Colorado State University. Libraries, 2017) Lang, Jillian M., author; Leach, Jan E., advisor; Bush, Daniel, committee member; Reddy, Anireddy, committee member; Verdier, Valérie, committee member
Emerging bacterial diseases on staple and economically important crops can pose critical threats to food security. Accurate identification of bacterial plant pathogens is the foundation of effective management for growers. This work advances the application of genomics to identify and characterize bacterial plant pathogens in the genus Xanthomonas that can cause destructive diseases on most agricultural crops, including rice and corn. In this thesis, taxonomy, host range, disease phenotypes and basic biology of the following pathogens were established: X. oryzae pv. oryzae, X. o. pv. oryzicola, X. o. pv. leersiae and X. vasicola pv. vasculorum. X. o. pv. oryzae and X. o. pv. oryzicola infect rice and cause bacterial blight and bacterial leaf streak, respectively. X. o. pv. leersiae infects cutgrass (Leersia sp.), weedy grasses that can serve as alternative hosts to X. oryzae and are endemic in all rice growing regions. X. vasicola pv. vasculorum was identified as the causal agent of bacterial leaf streak of corn, an emerging and now wide-spread disease in the United States, that was reported for the first time in 2017. This work established that X. vasicola pv. vasculorum can also infect sorghum and sugarcane and that the US strain is 99% similar to strains isolated over 20 years ago in S. Africa. To develop robust molecular diagnostic tools for these pathogens, unique features needed to be first identified. Using comparative genomics that included closely related bacteria and distant relatives, PCR-based diagnostic tools were developed, then validated using isolated cultures and field grown plant materials. Comparative genomics also contributed to elucidation of the taxonomy and phylogeny of X. o. pv. leersiae and X. v. pv. vasculorum. Characterization of X. o. pv. leersiae revealed adaptations to both the weedy grass hosts and rice. These features include virulence proteins that target homologous host genes (transcription activator like effectors, TALEs) to influence host gene expression. I conclude that X. oryzae is a complex that includes X. oryzae pv. oryzae, X. o. pv. oryzicola and X. o. pv. leersiae and that this complex can provide a unique window into pathogen evolution. By better understanding how pathogens adapt to their environments including new hosts, growers can manage surrounding ecosystems more effectively to minimize yield losses and therefore, contribute to food security.
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 member
Plants 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.
Open Access
Characterizing the role of the Hec1 tail domain at the kinetochore-microtubule interface in human cells
(Colorado State University. Libraries, 2020) Wimbish, Robert T., author; DeLuca, Jennifer, advisor; Markus, Steven, committee member; Reddy, Anireddy, committee member; Ross, Eric, committee member
Chromosome segregation is powered by interactions between the mitotic spindle and kinetochores. Kinetochores – large, protein-rich machines built on the centromere of each sister chromatid – must bind to spindle microtubules and harness the forces from their dynamic instability to drive chromosome movement. This interaction must be robust enough to ensure chromosomes remain bound to the growing and shrinking microtubule polymers, yet must also be reversible: incorrectly oriented kinetochore-microtubule attachments can cause chromosome mis-segregation leading to aneuploidy, which can be catastrophic for the newly formed cell. Thus, cells must be able to actively regulate the strength with which kinetochores bind to spindle microtubules – such a regulatory scheme ensures that incorrect attachments can be released, and correct attachments can be preferentially stabilized. The direct linkage between kinetochores and microtubules is the highly conserved, kinetochore-anchored NDC80 complex. This complex is also an effector of attachment strength regulation; specifically, the N-terminal "tail" region of the NDC80 complex subunit Highly expressed in cancer 1 (Hec1) is a target for phosphorylation by the Aurora family of kinases, which ultimately weakens kinetochore-microtubule attachments. Here, we investigate the molecular basis for kinetochore-microtubule attachment regulation in human cells. We find that Hec1 tail phosphorylation regulates kinetochore-microtubule attachments independently of the spindle and kinetochore associated (Ska) complex, a critical factor for attachment stability, contrary to previous reports that the two pathways are functionally coupled. We additionally map the domains of the NDC80 complex required for its coordination with Ska complexes to strengthen attachments. We also find that the Hec1 tail domain is dispensable for the initial formation of kinetochore-microtubule attachments, but provide evidence it plays a role in force generation. We further interrogate this role and how phosphorylation of the tail regulates attachment formation and force generation, and find that the length requirements for these functions of the tail are different. Moreover, we demonstrate that the phospho-regulatory pathway for attachment regulation is deficient for short tails, suggesting a new model for the means by which attachments are regulated. Together these results provide novel insight into how attachments between chromosomes and the spindle are formed and regulated, and how errors in this process can lead to chromosome mis-segregation.
Open Access
Crop protection in industrial algae farming: detecting weedy algae and characterizing bacterial communities
(Colorado State University. Libraries, 2015) Fulbright, Scott Paul, author; Reardon, Kenneth F., advisor; Reddy, Anireddy, committee member; Laybourn, Paul, committee member; Wallenstein, Matthew, committee member; Tisserat, Ned, committee member
Microalgae are a promising source of feedstock for biofuel and bioproducts. Algae have higher rates of biomass production than terrestrial crops, and therefore can use less land for producing equivalent energy compared to other biofuels. Elite algae strains are chosen based on traits such as fast and robust growth, and rapid production of desired biochemical products, including fatty acids and other high-energy compounds. Monocultures of elite strains are grown in large algae production systems. A major challenge algae growers face is consistently growing robust cultures of elite algae. This is due to unwanted organisms invading cultures such as weedy algae that contain less desirable biochemical products, and bacteria that can detract from algae growth, thereby reducing overall system productivity. Historically, algae have not been grown at scales required for biofuels and bioproducts, and thus there is a lack of fundamental pest management knowledge and developed tools. In this work, we developed three polymerase chain reaction (PCR)-based tools for detecting and quantifying weedy and elite algae. We developed a simple and inexpensive CAPS (cleaved amplified polymorphic sequence) assay that can determine the presence of dominant algae species in cultures. Also, we developed and validated qPCR primers were able to detect one weedy algae cell in 108 cells in a culture. Compared to flow cytometry, the qPCR primers were 104 times more sensitive for detecting weedy algae. We validated tools by monitoring industrial algae systems, and exhibited their utility for assisting in culture management decisions. Bacteria are also prevalent in industrial algae cultures yet little is understood about their dynamics or role in the ecosystem of elite algae cultures. We sampled small, medium and large cultures from an industrial algae system growing elite algae Nannochloropsis salina, and sequenced the 16S rDNA gene and used QIIME bioinformatics program to analyze data. In this study, we characterized bacterial communities diversity, richness, and composition in industrial algae bioreactors during the scale-up process, through time and during various algae growth rates. We demonstrate that bacterial diversity richness increases as the size of the algae production system increases in the scale-up process. Therefore, larger cultures are comprised of more complex communities than smaller cultures, thus increasing the probability of detrimental algae-bacteria interactions. We identified a single core bacterium Saprospiraceae that was present in 100% of samples, and was on average the most abundant bacterium in all systems. Further, we identified a Deltaproteobacterium that was detected at abnormally high relative abundances in poorly growing algae cultures. Identifying pest bacteria that can detract from elite algae growth is an important step in developing crop protection strategies. We isolated bacteria from a poorly performing algae system and determined their influence on algae growth. We identified a single isolate, S7 as a growth inhibiting bacteria that was capable of completely inhibiting Nannochloropsis gaditana and N. salina growth. The bacterium was characterized as Bacillus pumilus. Additionally, we identified nutrients and cell concentrations required for inhibition of N. gaditana and N. salina. B. pumilus inhibition effect is species-specific as it did not inhibit weedy algae, Chlorella vulgaris and Tetraselmis striata. Due to this, B. pumilus is capable of manipulating algae population composition and reducing productivity. Contaminating organisms such as bacteria will often be prevalent in algae systems and understanding their influence on culture productivity is essential for successful large-scale cultivation of algae. In summary, we 1) developed molecular tools to monitor weedy algae that can be used by growers, 2) characterized bacterial communities in industrial algae system cultures, and 3) identified a novel pest for elite algae, N. gaditana and N. salina.
Open Access
Deep learning for bioinformatics sequences: RNA basecalling and protein interactions
(Colorado State University. Libraries, 2024) Neumann, Don, author; Ben-Hur, Asa, advisor; Beveridge, Ross, committee member; Blanchard, Nathaniel, committee member; Reddy, Anireddy, committee member
In the interdisciplinary field of bioinformatics, sequence data for biological problems comes in many different forms. This ranges from proteins, to RNA, to the ionic current for a strand of nucleotides from an Oxford Nanopore Technologies sequencing device. This data can be used to elucidate the fundamentals of biological processes on many levels, which can help humanity with everything from drug design to curing disease. All of our research focuses on biological problems encoded as sequences. The main focus of our research involves Oxford Nanopore Technology sequencing devices which are capable of directly sequencing long read RNA strands as is. We first concentrate on improving the basecalling accuracy for RNA, and have published a paper with a novel architecture achieving state-of-the-art performance. The basecalling architecture uses convolutional blocks, each with progressively larger kernel sizes which improves accuracy for the noisy nature of the data. We then describe ongoing research into the detection of post-transcriptional RNA modifications in nanopore sequencing data. Building on our basecalling research, we are able to discern modifications with read level resolution. Our work will facilitate research into the detection of N6-methyladeosine (m6A) while also furthering progress in the detection of other post-transcriptional modifications. Finally, we recount our recently accepted paper regarding protein-protein and host-pathogen interaction prediction. We performed experiments demonstrating faulty experimental design for interaction prediction which have plagued the field, giving the faulty impression the problem has been solved. We then provide reasoning and recommendations for future work.
Open Access
Determination of the functions of Rab32, Rab38, and their effector Myosin Vc in the biogenesis of melanosomes
(Colorado State University. Libraries, 2013) Bultema, Jarred, author; Di Pietro, Santiago, advisor; Ross, Eric, committee member; DeLuca, Jennifer, committee member; Chen, Chaoping, committee member; Reddy, Anireddy, committee member
In mammals, pigment produced within specialized cells is responsible for skin, hair, and eye coloration. Melanocytes are specialized cells that produce pigment within an organelle known as the melanosome. Melanosomes are a member of a specialized class of organelles, known as Lysosome-related organelles (LRO), which are responsible for a number of critical functions in mammals such as pigmentation, blood clotting, lung function, and immune function. LROs are related to the ubiquitous lysosome, and are formed using the same molecular mechanisms as lysosomes that rely upon the Adaptor Protein complexes -1 (AP-1) and -3 (AP-3), and the Biogenesis of Lysosome-related Organelles Complex (BLOC)-2 (BLOC-2). These protein complexes are critical for the trafficking of specialized cargoes to melanosomes required for proper melanin synthesis. But, these complexes are also used for the formation of lysosomes, and no mechanism is known to distinguish between trafficking to lysosomes and melanosomes. The melanosome serves as a model system to study the formation of LROs, and insights from the study of melanosomes help explain the biogenesis of other LROs. In this dissertation, I present the finding that Rab32 and Rab38 function as melanosome-specific trafficking factors that allow for the use of AP-3, AP-1, and BLOC-2 in melanosome biogenesis. Using biochemical approaches, I show that Rab32 and Rab38 bind directly to AP-3, AP-1, and BLOC-2 on membranes. In microscopy experiments, I demonstrate that Rab32 and Rab38 localize to early endosomal subdomains where AP-3, AP-1, and BLOC-2 function. Using a combination of biochemical and microscopic approaches, I show that Rab32 and Rab38 serve partially redundant functions in trafficking of specialized cargoes to melanosomes. I report the discovery that Myosin Vc, a class V myosin motor, interacts with Rab32 and Rab38 and serves novel functions in melanosomes trafficking. I show, using biochemical approaches, that Myosin Vc directly binds to several melanosomal Rab proteins and serves as an effector of these proteins in melanosome biogenesis. Using a combination of approaches, I demonstrate that depletion of Myosin Vc from melanocyte cells causes defects in the trafficking of cargoes to melanosomes, but also causes severe defects in the secretion of mature melanosomes. With biochemical and microscopic approaches, I compare the function and localization of Myosin Vc in melanocytes to related proteins Myosin Va and Myosin Vb, and provide evidence to suggest that all three of these proteins function in distinct steps of melanosome trafficking. My results answer outstanding questions about the use of ubiquitous trafficking machinery (AP-3, AP-1, and BLOC-2) in trafficking to a specialized organelle. I provide evidence to answer outstanding questions about the mechanism of action of Rab32 and Rab38 in melanosome trafficking through my studies with Myosin Vc. I also establish new areas of research in the comparison of Myosin Va, Myosin Vb, and Myosin Vc in melanosome trafficking. My results address numerous unknown areas in melanosome biogenesis, expand the knowledge of melanosome biogenesis, and provide numerous new avenues of research to explore to understand specialized trafficking to LROs.
Open Access
Initiation and regulation of iron economy in Arabidopsis thaliana chloroplasts
(Colorado State University. Libraries, 2020) Kroh, Gretchen Elizabeth, author; Pilon, Marinus, advisor; Reddy, Anireddy, committee member; Bush, Daniel, committee member; Bedinger, Patricia, committee member; Argueso, Cristiana, committee member
Iron (Fe) is biologically important for all organisms because of its role as a protein cofactor which provides redox and catalytic functions. Fe cofactors come in 3 different forms (Fe-S clusters, heme, and non-heme Fe). Plants have a stronger requirement for Fe than non-photosynthetic organisms because the chloroplast has a high demand for Fe. Plants are commonly Fe deficient because soil Fe is typically found in the non-bioavailable, ferric (Fe3+) form, which limits plant growth in natural and agricultural settings. When grown on soils where Fe availability is low, plants can increase Fe uptake and use Fe more efficiently. The leaf response to Fe limitation in the model plant, Arabidopsis thaliana, is the topic of my dissertation. As a major contribution to a larger study, I first characterized the transcriptional response for specific leaf genes to Fe deficiency in the leaf and found that transcripts for abundant chloroplast Fe proteins were down-regulated, suggesting an Fe economy response. Specifically, photosynthetic electron transport and chloroplast Fe-S assembly were targeted for down-regulation. Fe deficiency affects photosynthesis and chloroplast Fe protein expression. I characterized a photosynthesis mutant and found that the regulation of Fe protein expression is maintained, suggesting that loss of electron transport does not trigger down-regulation of Fe protein expression. By using RNA-seq, I analyzed genome-wide transcriptomic changes to identify co-regulated transcripts early in the Fe economy response, including candidate transcription factors. The transcriptional responses in wild type Fe limited plants and a chloroplast Fe-S assembly mutant were independent of each other, suggesting that Fe-S assembly does not generate a signal to regulate chloroplast Fe proteins. The novel insights provided in this dissertation form a foundation for understanding how photosynthetic organisms cope with Fe limitation. From an applied perspective, the results of this dissertation open new avenues to minimize effects of Fe deficiency in agricultural settings.
Open Access
Molecular mechanisms of herbicide resistance in rice and kochia
(Colorado State University. Libraries, 2024) Gupta, Srishti, author; Dayan, Franck E., advisor; Gaines, Todd A., advisor; Reddy, Anireddy, committee member; Kumar, Vipan, committee member
Herbicide stress is an important challenge in agriculture and understanding how plants respond to herbicide exposure is crucial for developing effective weed management strategies. Transcription factors (TFs) play a pivotal role in regulating gene expression and mediating plant responses to various environmental stimuli, including herbicide stress. This dissertation aimed to elucidate the role of TFs in herbicide tolerance and sensitivity across plant species. A brief introduction was provided in Chapter 1. Subsequently, by analyzing transcriptomic data from different studies, we identified key TFs involved in herbicide responses. Our findings in Chapter 2 revealed distinct TF signatures, including bZIP, NAC, WRKY, and ERF, that were consistently upregulated in herbicide-tolerant plants. associated with herbicide tolerance or sensitivity, suggesting potential regulatory mechanisms in metabolic pathways and downstream signaling. These results underscore the importance of complex interplay between herbicide class, treatment duration, and plant species on TF expression patterns. In Chapter 3, we focused on herbicide resistance in rice, a critical staple crop. Transcriptomic analysis revealed upregulation of key detoxification genes, including glutathione S-transferase (GST) and cytochrome P450 (CYP450), in the NTSR mutant, suggesting their involvement in herbicide metabolism. Functional characterization confirmed increased glutathione S-transferase activity in the NTSR genotype. Additionally, computational studies identified a novel transcription factor, ZOS-1-16, with a potential role in regulating herbicide response. We investigated a novel non-target site resistance (NTSR) mechanism conferred by a mutation in the transcription factor ZOS-1-16. Our findings demonstrated that ZOS-1-16 upregulates genes like GSTs and CYPs involved in herbicide detoxification, leading to increased resistance to the herbicide quizalofop-p-ethyl (QPE). This study highlights the potential of targeting TFs for developing herbicide-resistant rice varieties. Finally, Chapter 4 explored glyphosate resistance in the invasive species Bassia scoparia (kochia). We investigated the inheritance of glyphosate resistance in kochia populations and found that it is primarily due to an increase in the copy number of the EPSPS (5‐enolpyruvyl‐3‐shikimate phosphate synthase) gene. Additionally, we estimated the outcrossing rate of kochia under field conditions and found a high level of outcrossing, which contributes to the rapid spread of glyphosate-resistant biotypes. Overall, this dissertation provides valuable insights into the role of TFs in herbicide responses and highlights the potential for developing novel strategies to enhance herbicide tolerance and manage herbicide-resistant weeds.
Open Access
Phenotypic changes and DNA methylation status in cryopreserved seeds of rye (Secale cereale L.)
(Colorado State University. Libraries, 2017) Lu, Jie, author; Byrne, Patrick, advisor; Greene, Stephanie, committee member; Reddy, Anireddy, committee member
Conserving genetic diversity is one of the major tasks for seed banks worldwide. At present, there are two long-term preservation methods in the USDA-ARS National Laboratory of Genetic Resources Preservation (NLGRP)：storage in a -18 °C vault (conventional storage) and storage in a liquid nitrogen vapor phase ranging from -135 oC to -180 °C (cryopreservation). Cryopreservation is considered to be the best method for long-term storage of vegetatively propagated plants and sometimes can be effective for recalcitrant seeds, but its value for orthodox seeds has not been thoroughly tested. Rye (Secale cereale L.) is a diploid allogamous plant species with 2n=14, a relatively high degree of gametophytic self-incompatibility and orthodox seeds. It has been an important crop in Europe and one of its major uses nowadays is in wheat breeding. In order to test the effects of long-term cryopreservation of orthodox seeds, 40 rye accessions (20 with spring habit and 20 with winter habit) stored for 25 years under both conventional storage and cryogenic conditions were evaluated. In our research, field and seedling evaluation and DNA methylation experiments were conducted. Winter rye seeds were planted in October 2014 and spring rye seeds were planted in April 2015 at CSU's Agricultural Research Education and Development Center (ARDEC) near Fort Collins, CO. Seedling evaluation was conducted at NLGRP in 2014-2015. A methylation sensitive amplified fragment length polymorphism (metAFLP) technique was used to evaluate DNA methylation of two accessions of the 40 total accessions used in the field experiment. This experiment was conducted in the summer of 2016. In the field evaluation, only spike length in the winter trial was significantly different (P=0.045) between storage treatments. Spikes of plants grown from conventionally stored seeds were slightly longer than those from cryopreserved seeds. Seedlings from cryopreserved samples had significantly higher normal germination percentage (P<0.0001) and lower abnormal germination percentage (P<0.01) than those stored under conventional conditions. In addition, root length in the winter trial and average root diameter in both trials showed significant differences (P<0.05) between the two storage treatments. Seedlings from cryopreserved seeds had longer roots and smaller root diameter than seedlings from conventionally stored seeds in the winter trial and seedlings from conventionally stored seeds had smaller root diameter than seedlings from cryopreserved seeds in the spring trial. No other significant differences between storage methods were detected. Our results indicated that cryopreservation has only minimal affects on phenotypic variation and may preserve seed for a longer period than conventional storage in orthodox cereal seeds. In the metAFLP experiment, only 5 of 311 loci in accession V/108 and 3 of 308 loci in accession Omka showed unadjusted methylation status differences between the two storage treatments at the P=0.05 significance level. However, after false discovery rate (FDR) adjustment, no differences in methylation were detected between storage treatments on an individual locus basis. To my knowledge, this study was the first evaluation of long-term cryopreservation versus conventional storage in orthodox seeds. The results indicated that cryopreserved seeds had increased viability; plants grown from cryopreserved seeds has only minimal phenotypic differences; and no epigenetic differences were detected compared to conventionally stored seeds. Therefore, based on the results of this study, cryopreservation is an appropriate method for long-term storage of rye seeds.
Open Access
Physiological and biochemical mechanisms behind the fast action of glufosinate
(Colorado State University. Libraries, 2019) Kagueyama Takano, Hudson, author; Dayan, Franck E., advisor; Westra, Philip, advisor; Reddy, Anireddy, committee member; Preston, Christopher, committee member; Gaines, Todd, committee member
Glufosinate is one of the few herbicides that are still effective for controlling herbicide resistant weeds, but its performance is often inconsistent and affected by environmental conditions. It inhibits glutamine synthetase (GS) by competing with glutamate for the active binding site. Unlike other amino acid biosynthesis inhibitors, glufosinate is a fast-acting herbicide and susceptible plants develop visual symptoms within a few hours after treatment. Inhibition of GS leads to ammonia accumulation and photosynthesis inhibition, which have traditionally been proposed as the causes of the rapid phytotoxicity. This dissertation presents a new understanding of the mechanism(s) of action of glufosinate and a biochemical approach to improve its herbicidal efficacy. Glufosinate uptake is inhibited by glutamine levels in the plant, and translocation is not affected by the rapid phytotoxicity. Glufosinate translocates primarily through the apoplast (xylem) rather than the symplast (phloem) probably due to its physicochemical properties and the absence of an effective membrane transporter. Glufosinate efficacy is proportional to the herbicide concentration in leaf tissues. Neither ammonia accumulation nor carbon assimilation inhibition are directly associated with the fast action of glufosinate. Instead, rapid phytotoxicity results from a massive light-dependent accumulation of reactive oxygen species (ROS). Inhibition of GS blocks the photorespiration pathway leading to a massive photooxidation damage. Under full sunlight, the excess of electrons is accepted by molecular oxygen leading to ROS generation. These free radicals cause lipid peroxidation, which ultimately leads to rapid cell death. The addition of protoporphyrinogen oxidase (PPO) inhibitors to glufosinate enhances ROS accumulation and herbicidal activity. This enhanced activity results from protoporphyrin formation at high levels due to a transient accumulation of glutamate, the precursor for chlorophyll biosynthesis. The herbicide combination also showed enhanced activity in the field and may help to overcome the lack of glufosinate efficacy under certain environmental conditions.
Open Access
Phytoalexin deficient4 (PAD4): a plant defense regulatory gene with distinct alternative splicing patterns in tomato (Solanum lycopersicum) and soybean (Glycine max)
(Colorado State University. Libraries, 2023) Schmidt, Rebecca, author; Nalam, Vamsi, advisor; Argueso, Cristiana, committee member; Reddy, Anireddy, committee member
Alternative splicing is an important post-transcriptional regulatory mechanism that contributes to a plant's ability to perceive and respond to a variety of biotic and abiotic stressors. Alternative splicing has a documented role in plant immunity, as many R genes, which are important for plant defense against specialized pathogens, undergo alternative splicing in response to pathogen perception. Despite this, the role of alternative splicing in other components of plant defense responses is not well documented. As transcriptome data diversify to include more species and conditions, the extent of alternative splicing in plants has become apparent. PHYTOALEXIN DEFICIENT4 (PAD4), plays an integral role in plant defense signaling to biotic stressors, and in regulating responses to abiotic stresses. PAD4 undergoes alternative splicing in Soybean (Glycine max). Additionally, the expression pattern of Glycine max PAD4, GmPAD4, and its splice variant GmPAD4-AS1 are further characterized in early growth stages. We hypothesize PAD4 produces full-length and alternatively spliced transcripts in multiple species, and that PAD4 gene structure may influence the occurrence of alternatively spliced transcripts. Here we characterize alternative splicing of PAD4 in tomato (Solanum lycopersicum), identifying two splice variants. We also investigate the conservation of PAD4 intron-exon structure conservation across diverse species. PAD4 expression patterns are characterized using available expression data.
Open Access
Regional whole plant and molecular response of Kochia scoparia to glyphosate
(Colorado State University. Libraries, 2012) Wiersma, Andrew, author; Westra, Philip, advisor; Leach, Jan, advisor; Reddy, Anireddy, committee member; Holtzer, Thomas, committee member
Globally, glyphosate (Roundup®) resistant weeds pose a serious challenge to modern agricultural practices that utilize glyphosate for weed control, including Roundup Ready® cropping regimes. Locally, glyphosate resistant Kochia scoparia have been identified throughout the central Great Plains, and the infested range is expanding rapidly. Glyphosate and Roundup Ready® crops form the foundation of no-till technology, which has considerably reduced water use and soil loss in arid to semi-arid regions of North America. Unfortunately, the continued spread of glyphosate-resistant K. scoparia will jeopardize the utility of glyphosate and the sustainability of no-till agricultural practices. In an effort to suppress glyphosate-resistant K. scoparia, more needs to be known about 1) the spread of resistance, 2) the level of resistance, and 3) the mechanism responsible for glyphosate resistance in K. scoparia. Suspected glyphosate-resistant K. scoparia accessions were collected from Kansas, Colorado, North Dakota, South Dakota, and Alberta. Whole plant glyphosate dose response and shikimate assays were used to confirm resistance and assess the level of resistance. Then PCR, quantitative PCR, sequencing, and immunoblotting techniques were used to determine the mechanism responsible for glyphosate resistance. Sequence of the EPSPS binding site proline confirmed that amino acid substitution at that residue was not responsible for resistance in K. scoparia. However, quantitative PCR estimates of EPSPS copy number revealed increased copy number in all glyphosate-resistant individuals —ranging from 3 to 9 EPSPS copies relative to the reference ALS gene. Furthermore, increased EPSPS copy number was correlated to increased transcript and protein abundance. Based on these finding, I confirm resistance for all tested accessions throughout the North American central Great Plains, and conclude that increased glyphosate rates will have little effect in controlling glyphosate-resistant K. scoparia. Furthermore, I suggest that EPSPS gene amplification may be the mechanism responsible for glyphosate resistance in K. scoparia, and that lower level increases in EPSPS expression (as compared to A. palmeri) are sufficient for glyphosate resistance. Moreover, this research, again, demonstrates the adaptability of plants and foreshadows the need for diversifying weed management practices.
Open Access
Regulation of dynein activity during spindle positioning in budding yeast
(Colorado State University. Libraries, 2020) Lammers, Lindsay, author; Markus, Steven, advisor; DeLuca, Jennifer, committee member; Tao, Tingting, committee member; Reddy, Anireddy, committee member
Cytoplasmic dynein is a minus-end directed, microtubule motor that is highly regulated to ensure it is targeted to the correct location at a specific time for its function in cells. This is particularly important for the process of spindle positioning during mitosis. Dynein is targeted to the cell cortex and activated to pull on astral microtubules attached to spindle poles to move the spindle into position at the site of cytokinesis. The position of the spindle dictates the plane of division and influences whether a cell divides asymmetrically or symmetrically- an important distinction during embryonic development and homeostasis. Using the model organism budding yeast, we confirmed that dynein is held in an inactive state before reaching its destination at the cell cortex by identifying a key factor in dynein activation- the cortical receptor Num1. We determined that the mechanism of activation involves enhancing dynein-dynactin interaction and releasing the recruitment factor, Pac1/Lis1. Additionally, I determined the role of another regulator in the dynein pathway, Ndl1/NudE. Ndl1/NudE aids the recruitment factor, Pac1/Lis1 in targeting dynein to astral microtubule plus ends that then deliver the motor to the cortex. Interestingly, it appears Ndl1/NudE may have another function that competes Pac1/Lis1 off dynein in a specific context. Next, I explored the two possible mechanisms of Num1-mediated dynein activation. First, I established an in vitro motility assay to observe how the regulators dynactin, Num1 and Pac1/Lis1 may coordinate to affect dynein activity. I determined the purification conditions for complete dynactin complexes as well as Num1 constructs to test whether Num1 acts as an adapter to activate the dynein-dynactin complex. Finally, I examined the second mechanism of Num1-mediated activation by initiating the release of Pac1/Lis1 from dynein complexes. I predicted that Num1 may influence the conformational changes of dynein during its mechanochemical cycle in conjunction with dynein engaging the microtubule that could induce Pac1/Lis1 release. To test this, I mutated dynein in a way that restricted conformation changes and observed how this affected Pac1/Lis1 interaction. The results show that Pac1/Lis1 binding is profoundly affected by dynein structure. Further, Num1 can still initiate Pac1/Lis1 release despite restriction in conformational changes, which suggests Num1 may initiate Pac1/Lis1 disassociation in another way. Together these data reveal important details of how regulatory proteins coordinate to spatially and temporally regulate dynein during spindle positioning.
Open Access
The impacts of high temperature on bacterial blight resistance genes in rice
(Colorado State University. Libraries, 2024) Shipp, Jennifer, author; Leach, Jan E., advisor; Argueso, Cristiana, committee member; Reddy, Anireddy, committee member
Rice is cultivated around the world and serves as a primary source of income and calories for many people. However, rice yield is threatened by the bacteria Xanthomonas oryzae pv. oryzae (Xoo), and outbreaks can be devastating to global communities. Xoo is the causal agent of bacterial blight (BB) in rice, and it proliferates in rice-growing climates. As climate change progresses, the trend of increasing BB severity may result in increased losses for growers. Disease severity, quantified through lesion lengths, increases at high temperature in rice. Previous studies indicated this pattern of increased disease phenotypes occurs even when a resistance (R) gene is present, except for one, Xa7. Our rationale for these experiments is to determine if the classification of an R gene can predict its performance against BB outbreaks. The classification of R genes in rice is a recent addition to the scope of our knowledge of plant pathology and has been the result of studies on nucleotide polymorphisms, genetic mapping, and fluorescent imaging of protein localization. Grouping the underlying mechanisms of action of individual R genes, such as the executor genes Xa7 and Xa10, allow for comparative studies to further elucidate details of their assigned classes. Not all R genes have been classified, but establishing a trend that some R genes maintain efficacy under higher temperatures would provide breeders with more tools to develop climate-friendly rice lines. This study indicates that R genes that remain effective at high temperature may be classified into the same category of executor R genes. More research is needed to determine if R gene classification predicts durability under heat stress. This study explores BB lesion lengths and Xoo colony counts at high and low temperatures. We find that at high temperature relative to low temperature, disease lesions were more severe in IR24, containing no active R gene, and in plants containing the R genes Xa21, xa5, and Xa3. Lesions were shorter in plants with Xa7 and Xa10. Additionally, under the same treatments, bacterial numbers increased to higher levels in IR24, Xa21, xa5, and Xa3. Numbers in Xa7 were reduced while numbers in Xa10 were low early in infection, but eventually increased beyond those measured at low temperature. Degree of lesion restriction did not always correspond to degree of restricted bacterial numbers, suggesting that severity of lesions may not always be a predictor of bacterial multiplication in the plant. Xa7 and Xa10 are classified as executor R genes. The mechanism of action in these genes may play a role in their durability at high temperatures. We hypothesize that the success of executor R genes may be a result of protein accumulation in the nucleus. This mechanism might be analogous to instances of temperature sensitive pathogen defense related protein accumulation, as seen in Arabidopsis. This mechanism may be induced or enhanced by the presence of reactive oxygen species (ROS) or other heat-stress related markers. More research is needed to explore the signaling between heat-stress pathways and R genes.