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Browsing by Author "Bombarely Gomez, Aureliano"

Now showing 1 - 9 of 9
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    Development of novel approaches to study Cuscuta campestris biology
    Bernal Galeano, Vivian Angelica (Virginia Tech, 2021-09-16)
    Cuscuta campestris is an obligate parasitic plant that lacks expanded leaves and roots and requires a host to complete its lifecycle. Parasite-host connections occur via an haustorium, a unique organ that acts as a bridge for the exchange of water, nutrients, macromolecules like mRNA, microRNA, and proteins, and microorganisms. Studies of Cuscuta spp. are challenging due to its dependence on the host and other host influences on the parasite. Recent research has shown intriguing aspects of Cuscuta biology like exchange genetic material with its hosts and loss of genes involved in processes such as high photosynthetic rates and defense. We developed new tools and methodologies that allow us to explore C. campestris biology in an unprecedent way. Foremost of these is an axenic method to grow C. campestris on an Artificial Host System (AHS). The AHS allows C. campestris to display its entire life cycle in vitro, including seed production. Using the AHS, we studied haustorial function, determining the role of nutrients and phytohormones on parasite haustorium development and growth, and found genes involved in haustorial function. The AHS allowed us to demonstrate the positive effect of light on C. campestris growth in the absence of a photosynthetic host and to investigate carotenoid- and ABA- related processes in the haustorial regions. We also wanted to understand how C. campestris defenses work independently from a plant host, so we studied the parasite responses to the bacterial epitope flg22 and the bacteria Peudomonas syringe. Our findings indicate that C. campestris is able to sense flg22, but its response differs from those observed in other non-parasite plants. Transcriptomic analysis revealed up-regulation of genes related to biotic and abiotic stresses, and downregulation of genes related to cuticle development. Our study contributes to understanding the C. campestris immune response in the absence of a host plant. Taken together, this research contributes novel methodologies that enable insights into C. campestris biology without the interference of a plant host on the parasite.
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    Development of tools to study the association of transposons to agronomic traits
    Yan, Haidong (Virginia Tech, 2020-05-21)
    Transposable elements (Transposons; TEs) constitute the majority of DNA in genomes and are a major source of genetic polymorphisms. TEs act as potential regulators of gene expression and lead to phenotypic plasticity in plants and animals. In crops, several TEs were identified to influence alleles associated with important agronomic traits, such as apical dominance in maize and seed number in rice. Crops may harbor more TE-mediated genetic regulations than expected in view of multifunctional TEs in genomes. However, tools that accurately annotate TEs and clarify their associations with agronomic traits are still lacking, which largely limits applications of TEs in crop breeding. Here we 1) evaluate performances of popular tools and strategies to identify TEs in genomes, 2) develop a tool 'DeepTE' to annotate TEs based on deep learning models, and 3) develop a tool 'TE-marker' to identify potential TE-regulated alleles associated with agronomic traits. As a result, we propose a series of recommendations and a guideline to develop a comprehensive library to precisely identify TEs in genomes. Secondly, 'DeepTE' classifies TEs into 15-24 super families according to sequences from plants, metazoans, and fungi. For unknown sequences, this tool can distinguish non-TEs and TEs in plant species. Finally, the 'TE-marker' tool builds a TE-based marker system that is able to cluster rice populations similar to a classical SNP marker approach. This system can also detect association peaks that are equivalent to the ones produced by SNP markers. 'TE-marker' is a novel complementary approach to the classical SNP markers that it assists in revealing population structures and in identifying alleles associated with agronomic traits.
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    Functional Characterization of Four Xanthomonas euvesicatoria Type III Effectors
    Wang, Zhibo (Virginia Tech, 2020-03-19)
    Pepper and tomato, as two common, popular, and important vegetables grown worldwide, provide human beings with high quality fruit of flavor and aroma, and a high concentration of vitamins and antioxidants. Pepper and tomato production is frequently affected by various pathogens, including nematodes, fungi, and bacteria. Among those phytopathogens, Xanthomonas euvesicatoria (Xe) causes a severe bacterial spot (BS) disease on pepper and tomato. The BS disease could cause a loss of approximately 10% of the total crop yield in the world. Breeding tomato and pepper cultivars with improved BS disease resistance is one of the most important breeding goals. A better understanding of the virulence mechanism of Xe could help breeders design new strategies for resistance breeding. In this dissertation, we characterized the virulence and avirulence functions of four Xe Type Three Secretion Effectors (T3Es): Xe-XopQ, Xe-XopX, Xe-XopN, and Xe-avrRxo1. Xe-XopQ is a Xe T3E that functions as a determinant of host specificity. Here, we further explored the virulent and avirulent functions of Xe-XopQ. We identified another T3E Xe-XopX that could interact with XopQ and subsequently elicit the hypersensitive response in N. benthamiana in the Agrobacterium-mediated transient assay and Xe-mediated disease assay. The interaction is confirmed by bimolecular fluorescence complementation, co-immunoprecipitation and split luciferase assay. Intriguingly, we also revealed that XopX also interacts with multiple Xe T3Es including AvrBS2, XopN, XopB, and XopD in the co-IP assay. The virulent and avirulent functions of XopQ and AvrBS2 are compromised in the absence of Xe-XopX. Since XopX is conserved in diverse Xanthomonas spp., we speculate that Xe-XopX may have a general role required for the pathogenesis of Xe. Xe-XopN has been reported to be a T3E with virulence function via targeting host defense-related proteins, including atypical receptor-like kinase named TARK1 and a 14-3-3 protein to suppress the PAMPs (pathogen-associated molecular patterns) triggered immunity upon Xe colonization of tomato. In this study, we revealed additional virulence mechanisms of Xe-XopN, where Xe-XopN, is required for triggering the water-soaking symptom on Nicotiana benthamiana and pepper plants infected with Xe. In addition, we identified that XopN interacts with a transcription factor, NbVOZ, and represses the expression of NPR1, a key component of the basal defense. Therefore, XopN has a role in maintaining a water-affluent environment for better replication of Xe, and it can also interact with NbVOZ1/2 to regulate plant immunity. AvrRxo1, a T3E of Xanthomonas oryzae pv. oryzicola (Xoc), was previously identified to function as a NAD kinase. Here, we characterized a Xe T3E, Xe avrRxo1, that is a functional homologue of AvrRxo1, which is required for the full virulence of Xe to colonize the pepper and N. benthamiana plants. Overexpression of AvrRxo1 in bacterial or plant cells is toxic. Our group previously demonstrated AvrRxo1-ORF2 functions as an antitoxin that binds to AvrRxo1 to suppress its toxicity. In this study, we identified Xe4429 as the homologue of AvrRxo1-ORF2, which could interact with Xe-avrRxo1 to suppress its toxicity. We also revealed that Xe4429 could bind to the promoter of Xe-avrRxo1 and suppress its transcription. Therefore, we found Xe4429 encodes protein functions as an antitoxin and a transcription repressor in Xe bacterial cells.
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    Genomic Reconstruction of the Domestication History of Sinningia speciosa (Lodd.) Hiern, and the Development of a Novel Genotyping Approach
    Hasing Rodriguez, Tomas Nestor (Virginia Tech, 2019-11-12)
    Most staple food crops were domesticated thousands of years ago through independent processes across different regions of the world. Studies of the history of such crops have been essential to our understanding of plant domestication as a process that started with the collection of wild material and continued with subsequent propagation, cultivation, and selection under human care. Domestication often involves a complex genetic structure with contributions from multiple founder populations, interspecific hybridization, chromosomal introgressions, and polyploidization events that occurred hundreds to thousands of years earlier. Such intricate origins complicate the systematic study of the sources of phenotypic variation. The analysis of recently domesticated, non-traditional, non-model species, such as Sinningia speciosa (Gesneriaceae), can expand the knowledge that we have on phenotypic variation under domestication, and help us to comprehend modern patterns of plant domestication and to broaden our understanding of the general trends. S. speciosa is commonly known as the 'florist's gloxinia', and it has been cultivated for 200 years as an ornamental houseplant. In our genomic study of S. speciosa, we examined an extensive diversity panel consisting of 115 individuals that included different species in the genus, wild representatives, and cultivated accessions, as well as 150 individuals from an F2 segregating population. Our analyses revealed that all of the domesticated varieties are derived from a single founder population that originated in or near the city of Rio de Janeiro in Brazil. We identified two loci associated with domesticated traits (flower symmetry and color) and did not detect any major hybridization or polyploidization events that could have contributed to the rapid increase in phenotypic diversity. Our findings, in conjunction with other features such as a small, low-complexity genome, ease of cultivation, and rapid generation time, makes this species an attractive model for the study of genomic variation under domestication. Basic research on non-model organisms with low economic importance is uncommon but necessary to understand the world from a broader perspective. In such cases, reduced representation approaches like Genotyping-by-Sequencing (GBS) are efficient low-cost alternatives to whole genome resequencing. However, most of these technologies are subject to patent protection, licensing processes, and fees that constrain genomic research for small non-profit research organizations. We have designed a protocol to construct reduced representation libraries from genomic DNA. Our approach, called Targeted Amplification of Scattered Sites (TASS), deviates from the traditional digestion-ligation-amplification process that is the subject of intellectual property that protects most current methods. Instead, TASS relies on 1) targeting and duplicating scattered regions in the genome by annealing and expanding long tail primers with short annealing sites, and 2) amplifying these regions using primers that are complementary to the added overhangs. At the moment GBS is more consistent and delivers more variants than TASS. However, we have established a foundation on which further optimization can produce an accessible, easy to implement, high-throughput genotyping approach.
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    Identification, Characterization, and Use of Precipitation-borne and Plant-associated Bacteria
    Mechan Llontop, Marco Enrique (Virginia Tech, 2020-01-10)
    Bacteria are ubiquitously present in every ecosystem on earth. While bacterial communities that reside in specific habitats, called the microbiota, have characteristic compositions, their constituents are exchanged between habitats. To understand the assembly processes and function of a microbial community in an ecosystem, it is thus important to identify its putative sources and sinks. The sources and sinks of the plant leaf microbiome, also called the phyllosphere microbiome, are still under debate. Here, I hypothesized that precipitation is a so far neglected source of the phyllosphere microbiome. Using 16S rRNA amplicon and metagenomic sequencing, I identified the genera Massilia, Sphingomonas, Methylobacterium, Pseudomonas, Acidiphilium, and Pantoea as members of the core rain microbiome in Blacksburg, VA. Further, I used rainwater as a bacterial inoculum to treat tomato plants. I showed that rain-borne bacteria of the genera Chryseobacterium, Enterobacter, Pantoea, Paenibacillus, Duganella, Streptomyces, Massilia, Shinella, Janthinobacterium, Erwinia, and Hyphomicrobium were significantly more abundant in the tomato phyllosphere 7 days post-inoculation, suggesting that these rain-borne bacteria successfully colonized the tomato phyllosphere and had a direct impact on the composition of its microbiome. These results were confirmed by comparing the phyllosphere microbiota of tomato plants grown under greenhouse conditions, and thus never exposed to rain, compared to plants grown outside under environmental conditions, including precipitation. Since a large diversity of bacteria is associated with rain, I also hypothesized that rain-borne bacteria are well adapted to environmental stresses, similar to the stressors microbial biopesticides are exposed to in the field. I thus explored rain as a source of resilient biopesticides to control fire blight, caused by the bacterial pathogen Erwinia amylovora, on apple. In an in-vitro dual culture assay, I identified rain-borne isolates displaying broad-range inhibition against E. amylovora and several other plant pathogens. Two rain-borne isolates, identified as Pantoea agglomerans and P. ananatis, showed the strongest inhibition of E. amylovora. Further experiments showed that these two Pantoea isolates survive under environmental conditions and have a strong protective effect against E. amylovora. However, protection from disease in an orchard was inconsistent, suggesting that the timing of application and formulations must be improved for field applications. Using a UV-mutagenesis screen and whole-genome sequencing, I found that a phenazine antibiotic produced by the P. agglomerans isolate was the likely active molecule that inhibited E. amylovora. Bacterial communities are constantly released as aerosols into the atmosphere from plant, soil, and aquatic sources. When in the atmosphere, bacteria may play crucial roles in geochemical processes, including the formation of precipitation. To understand the potential role of decaying vegetation as a source of atmospheric Ice Nucleation Particles (INPs), I analyzed a historic leaf litter sample collected in 1970 that had maintained Ice Nucleation Activity (INA) for 48 years. A culture-dependent analysis identified the bacterial species Pantoea ananatis and the fungal species Mortierella alpina to have INA and to be present in the leaf litter sample. Further, I determined that both P. ananatis and M. alpina produced heat-sensitive sub-micron INPs that may contribute to atmospheric INPs. The development of new sequencing technologies has facilitated our understanding of microbial community composition, assembly, and function. Most research in bacterial community composition is based on the sequencing of a single region of the 16S rRNA gene. Here, I tested the potential of culture-independent 16S rRNA sequencing of the phyllosphere microbiome for disease diagnosis. I compared the community composition of the microbiome of the aerial parts of cheddar pinks (Dianthus gratianopolitanus) that showed disease symptoms with the microbiome of healthy plants to identify the causative agent. However, I found that the pathogen is probably ubiquitous on cheddar pinks since it was present at similar abundance levels in symptomatic as well as healthy plants. Moreover, the low-resolution of 16S rRNA sequencing did not allow to identify the pathogen at the species or strain level. In summary, in this thesis, I found support for the hypothesis that rain is one of the sources of the phyllosphere microbiome, that rain is a promising source of biopesticides to control plant diseases in the field, that leaf litter is a source of atmospheric INPs, and that 16S rRNA sequencing is not well suited for pathogen identification in support of plant disease diagnosis. Finally, in additional research to which I contributed but that is not included in this thesis, I found that metagenomic sequencing can identify pathogens at the species and strain level and can overcome the limitations of 16S rRNA sequencing.
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    The Impact of Iron Deficiency on Plant-Oomycete Interactions
    Herlihy, John H. (Virginia Tech, 2020-04-08)
    Plants are sessile organisms adapted to cope with dynamic changes in their environment. Abiotic stresses, such as heat, drought, or nutrient deficiency must be overcome simultaneously with biotic threats such as pathogens and herbivores. Oomycete pathogens represent a significant threat to global food production and natural ecosystems. Novel modes of oomycete disease control could increase crop yield and reduce pesticide application. Overlaps between the plant response to iron deficiency and pathogens have been documented, but the impact of simultaneous imposition of both stresses on the plant have not been studied. Additionally, nothing is known about the impact of iron deficiency on oomycete infection, or mechanisms of oomycete iron uptake. We adapted a hydroponic system to simultaneously impose iron deficiency and monitor pathogen infection. The oomycete pathogens Hyaloperonospora arabidopsidis, and Phytophthora capsici grew less well on iron-deficient Arabidopsis thaliana, at least in part because of observed activation of immunity due to iron stress. We screened A. thaliana T-DNA insertion mutants defective in iron metabolism and transport and identified potential mechanisms of H. arabidopsidis iron acquisition. We conducted RNA sequencing to understand how A. thaliana responds to iron deficiency and root infection of P. capsici. 323 genes were differentially upregulated in iron-starved plants over three days, irrespective of pathogen infection, representing a core iron deficiency response. This group of core genes included the primary A. thaliana iron uptake pathway and genes for coumarin biosynthesis. Salicylic acid responsive genes were observed in both treatments consistent with this defense hormone's previously identified role in iron deficiency. Genes related to glucosinolate production – shown to be important in defense against P. capsici – were down regulated during infection, potentially due to the activity of virulence effectors. Our work demonstrates crosstalk between the iron deficiency response and plant immunity, and that iron acquisition remains important to the plant even after pathogen invasion. These new insights provide a first step in developing novel resistance strategies to control oomycetes in agronomically important crops.
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    Integrating bioinformatic approaches to promote crop resilience
    Cui, Chenming (Virginia Tech, 2019-10-09)
    Even under the best management strategies contemporary crops face yield losses from diverse threats such as, pathogens, pests, and environmental stress. Adding to this management challenge is that under current global climate projections these impacts are predicted to become even greater. Natural genetic variation, long used by traditional plant breeders, holds great promise for adapting high performing agronomic lines to these stressors. Yet, efforts to bolster crop plant resilience using wild relatives have been hindered by time consuming efforts to develop genomic tools and/or identify the genetic basis for agronomic traits. Thus, increasing crop plant resilience requires developing and deploying approaches that leverage current high-throughput sequencing technologies to more rapidly and robustly develop genomic tools in these systems. Here we report the integration of bioinformatic and statistical tools to leverage high-throughput sequencing to 1) develop a machine learning approach to determine factors impacting transcriptome assembly and quantitatively evaluate transcriptome completeness, 2) dissect complex physiological pathway interactions in Solanum pimpinellifolium under combined stresses—using comparative transcriptomics, and 3) develop a genome assembly pipeline that can be deployed to rapidly assemble a more contiguous genome, unraveling previously hidden complexity, using Phytopthora capsici as a model. As a result, we have generated strategic guidelines for transcriptome assembly and developed an orthologue and reference free, machine learning based tool "WWMT" to quantitatively score transcriptome completeness from short read data. Secondly, we identified "hub genes" and describe genes involved with "cross-talk" between drought and herbivore stress response pathways. Finally, we demonstrate a protocol for combining long-read sequencing from the Oxford Nanopore Technologies MinION, and short-read data, to rapidly assembly a cost-effective, contiguous and relatively complete genome. Here we uncovered hidden variation in a well-known plant pathogen finding that the genome was 92% bigger than previous estimates with more than 39% of duplicated regions, supporting a hypothesized recent whole genome duplication in this clade. This community resource will support new functional and evolutionary studies in this economically important pathogen.
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    Potato genomics three ways: quantification of endoreduplication in tubers, a romp through the transposon terrain, and elucidation of flower color regulation
    Laimbeer, Francis Parker Effingham (Virginia Tech, 2018-08-02)
    Investigations of potato (Solanum tuberosum) have been hampered by its complicated genetics and high genetic load. This dissertation applies genome reduction techniques to investigate a broad swath of genomic and physiological phenomena. It begins with the presentation and evaluation of a protocol to characterize endoreduplication within potato tubers, demonstrating substantial variation between tissue types and among wild species which may facilitate research into the genesis and growth of these starchy underground stems. Next, we transitioned to explore the distribution and consequences of a specific class of transposable element, Miniature Inverted Transposable Elements (MITEs), showing that they comprise approximately 5% of the potato genome, occur more frequently in genes with stress-related functions, and may be associated with changes, especially decreases, in gene expression. We then combined homology and sparsity based approaches to predict recent MITE activity, identifying five families as especially active. Finally, we expose the gene underlying the potato flower color locus, a homolog of AN2, while showing the effects it exerts on the flavonoid biosynthesis and fruit ripening pathways. This region was shown to be particularly dynamic, replete with MITEs and structural variants which we hypothesize to be the ultimate cause of differences in AN2 expression within the germplasm we examined. While the separate topics of this dissertation are quite disparate, each addresses an important topic in potato genetics, the in-depth study of which is only possible through the utilization of genomic reduction approaches to acquire homozygous genotypes for study and currently available genomic resources.
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    SmartPlants and SmartFarms for Global Food, Feed, and Fiber Security
    Abaye, Azenegashe Ozzie; Balota, Maria; Bombarely Gomez, Aureliano; Clark, Susan F.; Evanylo, Gregory K.; Fox, Thomas R.; Grabau, Elizabeth; Haak, David C.; Holliday, Jason A.; Kroehler, Carolyn J.; Lowman, James Scott; McDowell, John M.; Raun, Patricia; Saghai-Maroof, Mohammad A.; Schmale, David G. III; Tholl, Dorothea; Thomason, Wade E.; Tokekar, Pratap; Veilleux, Richard E.; Vinatzer, Boris A.; Westwood, James; Wynne, Randolph H.; Zhao, Bingyu (Virginia Tech, 2017-05-15)
    Meeting the food, feed, and fiber needs of a growing world population represents one of the signature challenges of this century. The UN FAO estimates that food production alone must increase by 70 percent by 2050 to meet the needs of a projected nine billion people. Demand will also increase for livestock feed, biofuel feedstocks, fiber for paper products, and construction materials. Meeting these demands will require implementation of advanced technologies, sustainable management of natural resources, and coordination of political forces. We must think beyond the boundaries of traditional agriculture, to integrate breakthroughs in plant science, engineering, environmental sciences, and other disciplines...