Browsing by Author "Leach, Jan E., advisor"
Now showing 1 - 10 of 10
Results Per Page
Sort Options
Item 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 memberEmerging 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.Item Open Access Defense response signaling for disease resistance in rice(Colorado State University. Libraries, 2011) Bruce, Myron Anthony, author; Leach, Jan E., advisor; Chisholm, Stephen, committee member; Ranu, Rajinder, committee member; Bush, Daniel R., committee memberPlant disease resistance is often associated with a type of programmed cell death (PCD) called the hypersensitive response (HR). Upon recognition of pathogen proteins or their activity, the affected cell and surrounding cells commit to the HR to limit pathogen spread throughout the plant. This mechanism of plant disease resistance renders a pathogen avirulent on the host plant. Lesion mimics (LM) are a class of mutant or transgenic plants that spontaneously show lesions resembling the HR in the absence of biotic stress. Based on the association of the LM phenotype to cell death and its similarity to disease symptoms and the HR, this phenotype is a useful tool to dissect and understand the plant defense response. To identify genes that when mutated result in the LM phenotype in rice, we used a microarray approach. By hybridizing labeled genomic DNA from an allelic series of deletion mutants to an oligonucleotide microarray, we identified candidate genes and genic regions that were deleted in a set of mutants. For one mutant, spl1, mutations in a cytochrome P450 gene were confirmed to confer the LM phenotype. A genome browser developed to handle these microarray data is a community resource that enables researchers to rapidly identify untagged deletion mutations in rice. Members of the 14-3-3 protein family were recently shown to be positive regulators of cell death and the HR in Arabidopsis. In contrast, the work herein shows that a rice 14-3-3 protein is a negative regulator of cell death and resistance. Transgenic plants carrying a construct that silences the rice 14-3-3 gene GF14e exhibit a LM phenotype and enhanced resistance to two distinct rice pathogens, Xanthomonas oryzae pv. oryzae and Rhizoctonia solani. These GF14e-silenced plants also showed enhanced expression of genes associated with salicylic acid (SA) mediated defense responses, including members of the peroxidase gene family. The GF14e-silenced plants did not show enhanced expression of marker genes associated with the ethylene response pathway, indicating that GF14e may negatively regulate SA mediated defense responses, but does not affect ethylene regulated responses. Silencing GF14e results in the up regulation of several defense responsive peroxidases. cDNA from GF14e silenced plants was used in quantitative PCR (qPCR) to assay expression of four peroxidase genes. Of these, three showed significant upregulation in 2 weeks after sowing (WAS) and 5 WAS GF14e-silenced plants. The promoters of the three upregulated genes (PO-C1, Pox8.1, Pox22.3) contain at least one W-box element. In contrast, the peroxidase (Pox5.1) that did not show upregulation and is not upregulated in R gene mediated responses had no W-box elements. W-box elements are binding sites for the WRKY class of transcription factors. This result, coupled with bioinformatic predictions of potential rice 14-3-3 clients, and the observation that some WRKY genes are upregulated in GF14e silenced lines indicates that GF14e may negatively regulate WRKY transcription factors related to cell death and defense responses. Based on the implication that one 14-3-3 protein negatively regulates defense responses related cell death, provides a framework to develop a model for how this protein might function in the plant disease resistance response.Item Open Access Exploiting rice diversity to uncover durable and broad-spectrum resistance(Colorado State University. Libraries, 2018) Bossa-Castro, Ana MarÃa, author; Leach, Jan E., advisor; Verdier, Valerie, committee member; Mosquera, Gloria, committee member; Argueso, Cristiana, committee member; Byrne, Patrick, committee memberRice is the staple food for human consumption and feeds over half the world's population. Major constraints towards a sustainable productivity of this cereal are losses caused by bacterial diseases, such as bacterial blight (BB) and bacterial leaf streak (BLS). Therefore, strategies aimed at increasing the global production of rice are essential. BB and BLS are caused by Xanthomonas oryzae pvs. oryzae (Xoo) and oryzicola (Xoc), respectively. This study aims to identify novel, broad-spectrum and durable sources of resistance to BB and BLS, and to pinpoint potential candidate genes for further characterization. We screened an indica rice Multi-parent Advanced Generation Inter-Cross (MAGIC) population, a novel mapping resource that allows high resolution detection for quantitative trait loci (QTL). A total of 14 disease resistance QTL effective against multiple X. oryzae strains were mapped, 11 confer resistance to both pathovars, i.e. broad-spectrum resistance (BSR), and three are pathovar-specific. We also detected specific alleles conferring disease resistance and susceptibility to these bacterial pathogens. Then, we combined diverse approaches to identify promising candidate genes, putatively involved in PAMP-triggered immunity (PTI) and effector triggered immunity (ETI), by (1) evaluating the presence and polymorphisms in defense-responsive cis-regulatory modules (CRMs) in gene promoters, (2) predicting gene promoters targeted by multiple X. oryzae strains, and (3) assessing the presence of SNP markers associated with resistance to X. oryzae strains. We also analyzed a cluster of MATH-BTB genes in a rice BSR QTL on chromosome 4 for polymorphisms between resistant and susceptible MAGIC lines. As a parallel approach to identify sources of durable resistance, the indica MAGIC population was also screened with an Xoo strain containing Tal7b, a transcription activator-like (TAL) virulence effector that is common to many Xoo strains. We mapped disease resistance QTL unique to this specific virulence factor and hypothesize that the mechanism of resistance conferred by one QTL is through a loss of susceptibility. BSR QTL and QTL specifically effective against virulence enhancing TALs may offer increased durability in the field. Because MAGIC lines are derived from elite cultivars, the use of identified QTL will be facilitated for the development of improved varieties.Item Open Access Functional characterization of germin family genes contributing to broad-spectrum, quantitative disease resistance in rice(Colorado State University. Libraries, 2009) Davidson, Rebecca M., author; Leach, Jan E., advisorQuantitative trait loci (QTL) are predicted to confer broad-spectrum and durable disease resistance. Application of disease resistance QTL in crop improvement programs has been hindered because we lack an understanding of (1) the genes contributing to the QTL-governed phenotype and (2) why certain alleles are more effective than others in conferring resistance. In this study, QTL-associated genes in the germin protein family, germin-like proteins (GLP) and oxalate oxidases (OXO), were identified in the rice genome and their functions were tested. Paralogous multi-gene families underlie the physical QTL regions, with twelve OsGLP members on chromosome (chr) 8 and four OsOXO members on chr 3. Based on shared motifs in 5' regulatory regions and/or protein sequence similarities to cereal orthologues, rice OsGLP genes belong to two germin subfamily groups (GER3 and GER4), and OsOXOs belong to the GER1 group. Conserved sequences for each gene family were used in RNA-interference gene silencing experiments. As more OsGLP genes were silenced, the more susceptible the plants were to two distinct fungal pathogens, Magnaporthe oryzae (Mo) and Rhizoctonia solani (Rs). Similarly, OsOXO-RNAi plants showed enhanced susceptibility to Mo, Rs and the broad host range pathogen, Sclerotinia sclerotiorum. OsGLP alleles were compared in resistant (+chr8 QTL) and susceptible (-chr8 QTL) parental rice lines. Cultivar-specific combinations of OsGLP genes were constitutively expressed and transiently induced by both wounding and Mo infection. In agreement with the silencing data, expression profiles suggest that GER4 subfamily members are involved in rice defense response. Transient induction occurred before fungal penetration of the plant cuticle, and differential expression between resistant and susceptible cultivars correlated with differential hydrogen peroxide accumulation after fungal infection and abiotic stresses. Gene silencing data confirms the roles of OsGLP and OsOXO as contributors to broad-spectrum, basal disease resistance in rice. Studies of allelic diversity among rice varieties suggest that regulation of OsGLPs may explain the effectiveness of resistant alleles compared to susceptible. Germin family proteins are encoded by developmentally regulated gene families in rice and across plant taxa. The germin subfamily members studied here have acquired functions in broad-spectrum defense responses and are important loci for crop improvement.Item Open Access How stress affects rice: a characterization of the rice transcriptome during single and simultaneous abiotic and biotic stresses(Colorado State University. Libraries, 2019) Cohen, Stephen Philip, author; Leach, Jan E., advisor; Argueso, Cristiana T., advisor; Snow, Christopher D., committee member; Antunes, Mauricio S., committee memberEnvironmental stresses, both abiotic and biotic, are large contributors to pre-harvest crop loss. Abiotic stresses, such as drought, salinity, non-optimal temperature and others, are non-living factors in the environment that have a negative effect on plants. Biotic stresses are biological factors that can harm plants, including pathogens, pests and competition from other plants. With climate change increasing the incidence of abiotic stresses and the constant pressures of pests and pathogens, it is critical to world agriculture that varieties of plants broadly tolerant to stresses are developed. For this, it is necessary to understand how plants respond to multiple simultaneous stresses. The goal of this work is to characterize the stress response of the global staple food plant rice. Here, I present the results of two comprehensive transcriptome studies. In the first, I characterize how the rice transcriptome changes in response to simultaneous heat stress and infection by the bacterial pathogen Xanthomonas oryzae (Xo). Xo includes the causal agent for the economically important bacterial blight disease of rice, Xo pathovar oryzae (Xoo). Bacterial blight is more severe during abiotic stresses such as high temperature and drought. Most rice resistance (R) genes that target Xoo lose function at high temperature; however, function of the R-gene Xa7 is enhanced when the host is subjected to abiotic stresses. Understanding why Xa7 is more effective during heat stress gives insight into host processes that are important during combined abiotic and biotic stresses. The major finding of this study was that the abscisic acid (ABA) pathway is a node of cross-talk in the interactions between heat stress and pathogen attack, during both susceptible and resistant interactions. In the second comprehensive study, I characterize how the rice transcriptome is universally regulated by all stresses. Understanding universalities in rice stress response transcriptomes provides insight into how plants endure a wide variety of stresses in the field. To explore the universal rice transcriptome response, I developed a custom workflow to analyze publicly available RNA-Seq data from rice stress response studies, including the abiotic stresses drought, salinity, heat and cold, and the biotic stresses bacterial leaf streak, bacterial blight, rice blast, and two viral diseases. From this study, I concluded that the rice stress response is a robust system with many overlapping features. This core response includes down-regulation of photosynthetic processes and up-regulation of downstream signaling of the hormones ABA, salicylic acid and jasmonic acid. Within this dissertation, I present networks of gene regulation in four major rice responses: (1) response to a susceptible interaction with Xo during high temperature, (2) response to a resistant interaction with Xo during high temperature, (3) core response to abiotic stresses and (4) core response to biotic stresses. Common among all of these pathways are the pathways upstream and downstream of the plant hormone ABA. ABA-related processes are universally up-regulated by abiotic and biotic stresses, and are only repressed during the enhanced Xa7 response at high temperature. Because ABA signaling is critical for stress response, we need a thorough understanding of how genes in the ABA response network interact to most efficiently improve rice to be tolerant to multiple and simultaneous stresses. The gene networks I have characterized can be integrated with genome and transcriptome data from stress-tolerant rice varieties. By having a complete understanding of the rice stress response, we can develop an informed approach for developing new varieties of rice that are resistant to stress.Item Open Access 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 memberDurable 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.Item Open Access The interaction of free-living amoeba with rice bacterial and fungal pathogens(Colorado State University. Libraries, 2018) Long, Jia Jun, author; Leach, Jan E., advisor; Jahn, Courtney E., advisor; Jackson, Mary, committee memberFree-living amoebae are ubiquitous microbes found in the soil and water across the globe. Amoebae live a predominantly heterotrophic lifestyle – preying on a variety of organisms including bacteria, fungi and even other protists. Although extensively studied, their potential as a biocontrol for agricultural pathogens is largely unexplored. As many pathogens occupy the same habitat as amoeba, we investigated their interactions as a first step to determine if amoeba are possible biocontrol agents. Our research focuses on two important pathogens of rice, the bacteria Xanthomonas oryzae and the fungus Rhizoctonia solani. Much of this thesis centers on the interaction between amoebae and X. oryzae, which is explored in depth and presented in the first chapter. Experimentation involved five common amoebal species and two highly virulent X. oryzae pathovars. Microscopy and vitality assays of amoebae-bacteria co-cultures first established that X. oryzae does not grow or dies in the presence of our amoebae. On the other hand, amoebae are not adversely affected, with most cells remaining alive in the metabolically active trophozoite form. Although the bacteria are harmed in this interaction, it is likely not through phagocytosis, the most common and well-studied tactic that amoeba use to feed. Observations made through confocal microscopy revealed that X. oryzae was rarely detected inside amoebae. Furthermore, lysis of amoebae after exposure to bacteria did not yield any viable bacteria, suggesting that either bacteria are rarely internalized and/or that X. oryzae does not survive in the amoeba cell. Conversely, amoebal trophozoites have no impact on the biofilms of X. oryzae either. These data indicate that amoeba do not directly or physically interact with X. oryzae. Instead, our amoeba-conditioned media assays reveal that amoeba alter the media and render it harmful to X. oryzae. The most likely scenario is that amoeba secrete a bactericidal agent into their surroundings. At this time, we have yet to isolate or identify the compound, but its presence may prove to be a boon with a variety of applications. The dynamics between amoeba and R. solani were not studied as extensively, but the basic interaction is presented in the appendices of this thesis. Again, five amoeba species were incubated with mature R. solani mycelia. First, co-cultures were observed with a compound microscope. Acanthamoeba and Dictyostelium did not have any effect on the fungi. Acanthamoeba species physically associated with the mycelia, but also rapidly encysted – suggesting some antiprotozoal activity from R. solani. D. discoideum had no interaction with the fungi: the trophozoites did not attach to the mycelia and neither cysts nor spore-forming bodies were seen. V. vermiformis was the only amoeba with some effect on the fungi. In co-cultures, fungal mycelium developed a shriveled and wrinkled morphology. V. vermiformis was attached to the fungi and most amoebae remained as viable trophozoites. V. vermiformis and its interaction with R. solani was further examined using scanning electron microscopy, which further corroborated the light microscope observations. While the reason/effect of the shriveling is unknown, it is a potential avenue for further experiments. Also in the appendices are references to two published papers I co-authored. For more information, the two papers can be found in their respective open-access journals.Item Open Access Understanding broad-spectrum disease resistance in rice: prompting a genome-wide uprising(Colorado State University. Libraries, 2017) Tonnessen, Bradley William, author; Leach, Jan E., advisor; Ben-Hur, Asa, committee member; Jahn, Courtney E., committee member; Argueso, Cristiana T., committee member; Bush, Daniel R., committee memberRice is the main staple food crop of the world, and thus, the detriments caused by rice diseases are a threat to international food security. The emergence of new virulent strains of pathogens can significantly reduce yields, and there are continual efforts to develop more resistant rice cultivars. Utilization of single R-genes is effective, but has proven inadequate due to rapid pathogen evolution. Thus, there is a need for breeding multigenic, broad-spectrum disease resistance in new varieties. This study aims to understand the aspects of basal resistance and its contribution to tolerance to multiple, diverse pathogens. Phenylalanine ammonia-lyase is a key enzyme in phenylpropanoid metabolism, which contributes to the basal defense response (DR). In this project, the DR gene, OsPAL4, which colocalizes with a disease resistance Quantitative Trait Loci (QTL), was shown to contribute to resistance to three important rice diseases, rice blast, bacterial blight, and sheath blight, in experiments using an ospal4 mutant. The functional element of resistance QTL haplotypes of DR genes such as OsPAL4 are largely unknown, and this work searched for sequence patterns in the promoters of DR genes to discern a regulatory mechanism specific to DR. Multiple cis-regulatory Modules (CRMs), or groups of DR-related sequence motifs were identified in promoters of DR genes. These CRMs harbor structural organizations of cis-elements known to be involved in the DR, and also motifs involved in a putative epigenetic regulatory mechanism. Polymorhpisms in CRMs are found in resistant relative to susceptible QTL haplotypes in DR gene promoters. These CRMs are sequence patterns found across DR gene promoters. Thus, we hypothesize that DR-associated CRM can be used as breeding markers to select loci on a genome scale that encode traits supporting broad spectrum basal resistance to important rice diseases.Item Open Access Understanding the roles of TAL effectors in Xanthomonas oryzae interactions with rice(Colorado State University. Libraries, 2013) Corral, Rene, author; Leach, Jan E., advisor; Verdier, Valérie, advisor; Hess, Ann, committee memberTranscription activator like (TAL) effector proteins are virulence factors that are secreted by Xanthomonas oryzae. Some TAL effectors contribute to virulence by activating transcription of plant host susceptibility genes thereby modulating the plant's physiology and creating a more pathogen favorable environment. Some TAL effectors activate transcription of disease resistance genes. Because most X. oryzae strains encode many (between 8 and 26) genes for TAL effectors, it is difficult to evaluate the function of individual TAL in the plant-pathogen interaction. In this study, we introduced the use of a TAL deficient strain of X. oryzae that allows study of individual TAL effectors. We demonstrated that the TAL deficient strain could deliver TAL to rice, and that subtle differences in TAL virulence functions could be measured in interactions with rice when delivered by this strain. Plants have evolved resistance genes that detect or recognize TAL effectors and activate resistance responses. Using the TAL deficient X. oryzae delivery system, we isolated a TAL that activates resistance in plants which are homozygous for the recessive resistance gene xa5. The TAL, Avrxa5P86, is predicted to target many transcription factors. Avrxa5P86 not only interacted with xa5 to confer resistance, but also exhibited a novel resistance interaction with Azucena and Nipponbare, both homozygous for the Xa5 allele. Our discovery of Avrxa5P86 is of presents a novel interaction that raises new evolutionary questions about TAL effectors and/or resistance genes in rice.Item Open Access Variation in cell wall composition and bioenergy potential of rice straw(Colorado State University. Libraries, 2014) Tanger, Paul, author; Leach, Jan E., advisor; Ben-Hur, Asa, committee member; Bush, Daniel R., committee member; McKay, John K., committee memberIn most grain crops the leaf and straw is often under-utilized. This biomass is largely plant cell wall, whose heterogeneous composition and recalcitrance limits end uses such as forage or bioenergy. I review the desirable traits for several bioenergy pathways from this biomass and identify traits in biomass that need to be optimized for enzymatic or thermochemical conversion of the biomass to energy. Sufficient variation exists across species and varieties for improving these traits through breeding. I assess variation in cellulose, lignin, hemicellulose, ash, total glucose, total xylose, mixed linkage glucan, saccarification yield and efficiency, hydroxyproline content and bulk density across two environments in the leaf and stem tissue of five rice varieties. Environment and tissue type are highly influential on the composition and yield phenotypes, and some traits perform better than others at predicting bioenergy yield in the field environment. Optimizing specific bioenergy-related phenotypes in isolation is not sufficient as overall crop health relies on many components. The plant cell wall serves an important function in crop health as a critical barrier against pests and diseases. I investigate the role of a family of putative broad spectrum defense response genes in rice, OsOXOs, that degrade oxalic acid: a pathogenicity factor. When expression of these genes is modified, I find a large impact on disease resistance to Sclerotinia sclerotiorum but little impact in the presence of Rhizoctonia solani. OsOXOs must play an important role in defense against S. sclerotiorum which relies on oxalic acid as a pathogenicity factor, because OsOXOs can degrade oxalic acid. R. solani utilizes a broader range of enzymes and compounds, limiting the effectiveness of OsOXOs against R. solani. With the bioenergy phenotyping methods optimized above, I assess saccharification yield of a rice mapping population, along with other agronomic traits including total biomass, flowering time, grain yield, and plant height. Transgressive segregation is apparent for all traits and quantitative trait loci (QTL) mapping approaches are presented. With the methods and populations evaluated here, we are closer to identifying the conditions and genes that can maximize biomass tailored for many purposes.