Browsing by Author "McDowell, John M."

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    Application of alignment-free bioinformatics methods to identify an oomycete protein with structural and functional similarity to the bacterial AvrE effector protein
    Deb, Devdutta; Mackey, David; Opiyo, Stephen O.; McDowell, John M. (PLOS, 2018-04-11)
    Diverse plant pathogens export effector proteins to reprogram host cells. One of the most challenging goals in the molecular plant-microbe field is to functionally characterize the complex repertoires of effectors secreted by these pathogens. For bacterial pathogens, the predominant class of effectors is delivered to host cells by Type III secretion. For oomycetes, the predominant class of effectors is defined by a signal peptide that mediates secretion from the oomycete and a conserved RxLR motif. Downy mildew pathogens and Phytophthora species maintain hundreds of candidate RxLR effector genes in their genomes. Although no primary sequence similarity is evident between bacterial Type III effectors (T3Es) and oomycete RXLR effectors, some bacterial and oomycete effectors have convergently evolved to target the same host proteins. Such effectors might have evolved domains that are functionally similar but sequence-unrelated. We reasoned that alignment-free bioinformatics approaches could be useful to identify structural similarities between bacterial and oomycete effectors. To test this approach, we used partial least squares regression, alignment-free bioinformatics methods to identify effector proteins from the genome of the oomycete Hyaloperonospora arabidopsidis that are similar to the well-studied AvrE1 effector from Pseudomonas syringae. This approach identified five RxLR proteins with putative structural similarity to AvrE1. We focused on one, HaRxL23, because it is an experimentally validated effector and it is conserved between distantly related oomycetes. Several experiments indicate that HaRxL23 is functionally similar to AvrE1, including the ability to partially rescue an AvrE1 loss-of-function mutant. This study provides an example of how an alignment-free bioinformatics approach can identify functionally similar effector proteins in the absence of primary sequence similarity. This approach could be useful to identify effectors that have convergently evolved regardless of whether the shared host target is known.
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    The Arabidopsis PHD-finger protein EDM2 has multiple roles in balancing NLR immune receptor gene expression
    Lai, Yan; Lu, Xueqing Maggie; Daron, Josquin; Pan, Songqin; Wang, Jianqiang; Wang, Wei; Tsuchiya, Tokuji; Holub, Eric; McDowell, John M.; Slotkin, R. Keith; Le Roch, Karine G.; Eulgem, Thomas (PLoS, 2020-09-01)
    Plant NLR-type receptors serve as sensitive triggers of host immunity. Their expression has to be well-balanced, due to their interference with various cellular processes and dose-dependency of their defense-inducing activity. A genetic “arms race” with fast-evolving pathogenic microbes requires plants to constantly innovate their NLR repertoires. We previously showed that insertion of the COPIA-R7 retrotransposon into RPP7 co-opted the epigenetic transposon silencing signal H3K9me2 to a new function promoting expression of this Arabidopsis thaliana NLR gene. Recruitment of the histone binding protein EDM2 to COPIA-R7-associated H3K9me2 is required for optimal expression of RPP7. By profiling of genome-wide effects of EDM2, we now uncovered additional examples illustrating effects of transposons on NLR gene expression, strongly suggesting that these mobile elements can play critical roles in the rapid evolution of plant NLR genes by providing the “raw material” for gene expression mechanisms. We further found EDM2 to have a global role in NLR expression control. Besides serving as a positive regulator of RPP7 and a small number of other NLR genes, EDM2 acts as a suppressor of a multitude of additional NLR genes. We speculate that the dual functionality of EDM2 in NLR expression control arose from the need to compensate for fitness penalties caused by high expression of some NLR genes by suppression of others. Moreover, we are providing new insights into functional relationships of EDM2 with its interaction partner, the RNA binding protein EDM3/AIPP1, and its target gene IBM1, encoding an H3K9-demethylase.
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    Biochemical Characterization of Arabidopsis Enzymes Involved in Inositol Pyrophosphate Biosynthesis
    Adepoju, Olusegun Adeboye (Virginia Tech, 2019-09-05)
    To compensate for the sessile nature of plants, thousands of years of evolution have led to the development of many sophisticated signaling pathways that help plants sense and respond appropriately to different environmental cues. One such signaling pathway is called inositol phosphate signaling. This research dissertation focuses on the inositol phosphate signaling pathway in plants, with emphasis on elucidating how a new class of signaling molecules collectively referred to inositol pyrophosphates are synthesized. Inositol pyrophosphates are an emerging class of "high-energy" intracellular signaling molecules containing one or two diphosphate groups attached to an inositol ring, with suggested roles in bioenergetic homeostasis and inorganic phosphate sensing. Information regarding the biosynthesis of this unique class of signaling molecules in plants is scarce, however the enzymes responsible for their biosynthesis in other eukaryotes have been well described. This work aims to characterize the biochemical activity of the kinase domain (KD) of the Arabidopsis plant diphosphoinositol pentakisphosphate kinase enzymes (AtVIP1 and AtVIP2), and elucidate the biosynthesis pathway of inositol pyrophosphates in plants. Our data indicate that AtVIP1-KD and AtVIP2-KD function primarily as diphosphoinositol pentakisphosphate 5 kinases that phosphorylate this substrate at the 1-position. We also discovered a previously unreported inositol hexakisphosphate kinase activity for the Arabidopsis inositol(1,3,4) triphosphate 5/6kinase enzymes, that can convert InsP6 to InsP7. Together, these enzymes can function in plants to produce inositol pyrophosphates, which have been implicated in signal transduction and phosphate sensing pathways. The significance and potential application of these findings in terms of reduced phytate content and phosphate pollution, improved plant fitness, and improved nutrient use efficiency are discussed. The future outlook of inositol phosphate signaling research is also discussed.
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    Characterization of Effector Genes in Acidovorax citrulli the Causing Agent of Bacteria Fruit Blotch Disease of Cucurbits
    Traore, Sy M. (Virginia Tech, 2014-08-08)
    Bacterial fruit blotch (BFB) of cucurbits is caused by Acidovorax citrulli, a Gram-negative seedborne bacterium that can cause up to 100% fruit yield losses in the field. Currently, BFB is a major problem for the cucurbits industry worldwide. Thus far, attempts to identify resistance in cucurbit germplasm for controlling BFB have been unsuccessful. Despite the importance of the disease, little is known about the molecular mechanisms of A. citrulli pathogenicity, due to a lack of molecular tools for studying the A. citrulli/cucurbit interaction. The genomic sequence of A. citrulli strain AAC00-1 has been determined, and the components of type III secretion system have been identified. The goal of this research was to develop molecular tools for studying the BFB disease. Nineteen putative type III effector genes were cloned from two representative A. citrulli strains (AAC00-1 and M6). The distribution of 19 type III effectors among A. citrulli strains, collected worldwide, was studied. A novel Gateway-compatible binary vector was developed for transient expression of A. citrulli type III effectors genes in planta. A set of modified vectors for marker-exchange mutagenesis in A. citrulli were constructed. The model plant species Nicotiana benthamiana was found to be susceptible to A. citrulli, while Nicotiana tabacum was resistance to A. citrulli, so therefore could carry nonhost resistance genes. Two T3S effectors, Aave1548 and Aave2166, triggered water soaking-like cell death in N. benthamiana, but HR-like cell death in N. tabacum. Bacterial mutagenesis and in planta disease assay confirmed that both Aave1548 and Aave2166 have significant virulence contributions to A. citrulli in N. benthamiana plant and melon seeds. Aave2166 encodes a putative acetyltransferase that belongs to the YopJ super family, which is conserved in both animal and plant pathogenic bacteria. Wild type but not the putative catalytic mutant (C232A) of Aave2166 can trigger cell death phenotype in N. benthamiana and N. tabacum. N. benthamiana yeast two-hybrid cDNA library screening using Aave2166 identified six N. benthamiana proteins/peptides which specifically interacted with Aave2166. Further characterization of these Aave2166 interactors may allow us to understand the virulence mechanism provided by Aave2166. The identification of nonhost resistance genes that can recognize Aave2166 and other type III effectors may help to develop novel strategies to control BFB disease of cucurbit.
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    Characterization of Host Plant Defense Responses to Parasitization by Orobanche aegyptiaca
    Griffitts, Amanda Aline (Virginia Tech, 2001-05-07)
    Orobanche (spp.) are parasitic plants that attack the roots of many important crops. O. aegyptiaca penetrates the host root (aided by digestive enzymes) and forms connections to the host vascular tissue, from which it will withdraw all of its water and nutrient requirements. In order to control this weed, it is important to understand the relationship between the host and the parasite. To investigate how parasitism effects host defense pathways, we are studying the patterns of expression of host genes known to be involved in various aspects of plant defense responses. With respect to local defense responses, two genes of the isoprenoid pathway were studied, one of which is expressed in wounded tissue (hmg1), and another that is induced in response to wounding yet repressed in response to pathogen elicitors (squalene synthase). Genes analyzed that are associated with systemic defense include PR-1, PR-2, and PR-5, all of which are induced in response to pathogen attack as part of the systemic acquired resistance (SAR) response. Plant gene expression was studied using transgenic tomato plants containing hmg1-GUS fusions, and northern hybridization analysis of tobacco and Arabidopsis roots using gene-specific probes. Results indicated that expression of hmg1 is induced, PR-2 and squalene synthase are repressed, and PR-1a, PR-1, and PR-5 are not affected in tissue parasitized by O. aegyptiaca. Together, these results indicate a complex response to the parasite. Whereas hmg1 induction is consistent with O. aegyptiaca inflicting a simple wound-like injury, the repression of squalene synthase is consistent with plant recognition of a pathogen attack. In contrast, the failure of Orobanche to induce SAR- related PR-1 in tobacco and PR-1, PR-2, or PR-5 in Arabidopsis indicates an ability to avoid or perhaps inhibit some defense-related pathways. By comparing the regulation of these defense genes in response to O. aegyptiaca attack, we are able to gain a greater understanding of the host plant response to parasitization and explore potential gene candidates for future engineering strategies to create Orobanche resistant crops.
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    Characterization of Transgenic Peanuts Expressing Oxalate Oxidase for Governmental Approval of Their Release for Control of Sclerotinia Blight
    Chriscoe, Shanna Marie (Virginia Tech, 2008-07-08)
    Sclerotinia minor Jagger is a fungal pathogen of cultivated peanut (Arachis hypogaea L.) that can cause crop losses in excess of 50%. Fungicides are not completely effective at controlling the disease and can cost up to $311 per hectare for three applications. The ability to produce oxalic acid is necessary for the pathogenicity of some Sclerotinia spp. With little to no naturally occurring resistance to Sclerotinia blight in Arachis spp., a biotechnological approach was used to confer resistance to the disease. Peanut plants were transformed with a gene from barley encoding oxalate oxidase, an enzyme that degrades oxalic acid. Transformed peanuts showed resistance to S. minor and increased yields under disease pressure compared to the parental lines. Before the resistant varieties can be marketed, they must be reviewed and approved by the governmental regulatory system. Responsibility for regulation of transgenic plants in the U.S. is shared among the U.S. Department of Agriculture (USDA) through the Animal and Plant Health Inspection Service (APHIS), the Food and Drug Administration (FDA), and the Environmental Protection Agency (EPA). These agencies require several different data sets including molecular characterization and field studies before each transformation event can be commercialized. This project was designed to characterize three different transformation events, N70, P39 and W171. Molecular characterization included determination of insertion number, copy number, intactness of the expression cassette and stable inheritance of the transgene. N70 was found to have two insertions and two copies while W171 had one insertion with one copy. The P39 event has two insertions and two or more copies. Each of the three events was stable over multiple generations. Phenotypic comparisons of each transgenic line to the parent cultivar were carried out in field studies. Characteristics such as oxalate oxidase expression, yield and quality, hay quality, disease occurrence, aflatoxin content and plant height were assessed. Transgenic peanuts showed few differences from the parent cultivar other than resistance to Sclerotinia blight and yield under disease pressure. Outcrossing studies were completed to determine the rate and distance of cross pollination. Outcrossing rates in N70, P39 and W171 were less than 2.5% and occurred up to 19 rows or 17.4 m from the nearest transgenic row. The molecular characterization and field performance of N70, P39 and W171 have been assembled into a document to petition APHIS for determination of non-regulated status.
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    Characterizing RNA translocation in the parasitic weed Cuscuta pentagona
    LeBlanc, Megan Leanne (Virginia Tech, 2013-06-03)
    The obligate stem parasite Cuscuta pentagona is able to take up host plant mRNA through a specialized organ known as the haustorium. Direct cell-to-cell symplastic connections between two different organisms are rare, and the translocation mechanisms and fate of these RNAs in the parasite is not understood. To characterize this phenomenon, mobile Arabidopsis and tomato mRNAs were identified from microarray and transcriptome sequencing projects and quantified in the host-parasite system. Mobile RNAs were quantified using real time (qRT)-PCR and were found to vary substantially in their rate of uptake and distribution in the parasite. Transcripts of tomato Gibberellic Acid Insensitive (SlGAI) and Cathepsin D Protease Inhibitor (SlPI) can be traced over 30-cm of parasite stem. SlPI was abundant in the C. pentagona stem, but the number of copies decreased substantially within the first eight hours post detachment. Additional studies of mobile RNAs from Arabidopsis, Translationally Controlled Tumor Protein (AtTCTP), Auxin Response Factor (AtARF) and a Salt-inducible Zinc Finger Protein (AtSZFP) supported the idea that mRNA molecules differ in their mechanisms of uptake and mobility between host and parasite. Known phloem-mobile RNAs (SlGAI and AtTCTP) have uptake patterns that differ from each other as well as from other RNAs that are not reported to be phloem mobile (SlPI and AtSZF1). The function of RNAs in plants extend beyond protein translation to include post transcriptional gene silencing or long distance signaling, and mobile RNA in C. pentagona systems offers novel insights into this aspect of plant biology. Studies of cell-to-cell trafficking of RNAs and other macromolecules would be facilitated by the ability to manipulate individual cells. To this end, work was initiated to explore alternative approaches to understanding single cell biology using laser-mediated approaches. Optoperforation, or the use of multiphoton processes to form quasi-free electron plasmas to initiate transient pore formation in plasma membranes, has been demonstrated, but not in cells of an intact plant. This work details a protocol for optoperforation of Arabidopsis epidermal cells to allow for uptake of external dye-labeled dextrans and retention for up to 72 hours, and has the potential for transformation and molecular tagging applications.
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    Cornichon Proteins: Unexpected Roles in Plant Pathogen Infection, ER Morphology Maintenance and Pollen Development
    Li, Jianhui (Virginia Tech, 2017-05-17)
    Cornichon (CNI) proteins are a conserved family of proteins among eukaryotes, from Erv14 in the yeast Saccharomyces cerevisiae to CNI homologs (CNIHs) in mammals and plants. Erv14 functions as a cargo receptor of coat protein complex II (COPII) for protein trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus, en route to their final destinations. By interacting with specific cargo proteins, CNI proteins regulate key steps of embryo polarity in Drosophila, budding in yeast, and synaptic transmission in the mammalian brain. However, we have very limited understanding of plant CNIHs. Positive-strand RNA viruses assemble their viral replication complexes (VRCs) at specific host organelle membranes. With a better understanding of host factors involved in targeting viral replication proteins to the preferred organelles, we expect to block trafficking of viral replication proteins and thus, viral infection, by manipulating the required host proteins. Brome mosaic virus (BMV) is a model of positive-strand RNA viruses and its replication can be recapitulated in yeast. Importantly, BMV replication protein 1a is the only required viral protein to form VRCs at the perinuclear ER membrane in yeast. I demonstrate that Erv14 and COPII coat proteins are required for targeting BMV 1a to the perinuclear ER in yeast, suggesting a novel function of COPII vesicles in protein trafficking to the perinuclear ER membrane and in the BMV VRC formation. As for cellular functions, I show that plant CNIHs complement the defective distribution of BMV 1a in yeast mutant lacking Erv14. Taking advantage of Arabidopsis thaliana knockout mutants and knockdown of gene expression in Nicotiana benthamina, I also discover that CNIHs unexpectedly play crucial roles in pollen development, infection of a bacterial pathogen, and maintenance of ER tubules. I further confirm that CNI proteins are also required for maintaining ER tubules in yeast, suggesting a novel and conserved role in shaping ER morphology. Therefore, these findings indicate the functional diversity and redundancy of CNI proteins in key cellular processes and suggest a novel strategy to control plant pathogenic viruses and bacteria by manipulating plant CNIHs.
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    Crosstalk between the Circadian Clock and Innate Immunity in Arabidopsis
    Zhang, Chong; Xie, Qiguang; Anderson, Ryan G.; Ng, Gina; Seitz, Nicholas C.; Peterson, Thomas; McClung, C. Robertson; McDowell, John M.; Kong, Dongdong; Kwak, June M.; Lu, Hua (PLOS, 2013-06-06)
    The circadian clock integrates temporal information with environmental cues in regulating plant development and physiology. Recently, the circadian clock has been shown to affect plant responses to biotic cues. To further examine this role of the circadian clock, we tested disease resistance in mutants disrupted in CCA1 and LHY, which act synergistically to regulate clock activity. We found that cca1 and lhy mutants also synergistically affect basal and resistance gene-mediated defense against Pseudomonas syringae and Hyaloperonospora arabidopsidis. Disrupting the circadian clock caused by overexpression of CCA1 or LHY also resulted in severe susceptibility to P. syringae. We identified a downstream target of CCA1 and LHY, GRP7, a key constituent of a slave oscillator regulated by the circadian clock and previously shown to influence plant defense and stomatal activity. We show that the defense role of CCA1 and LHY against P. syringae is at least partially through circadian control of stomatal aperture but is independent of defense mediated by salicylic acid. Furthermore, we found defense activation by P. syringae infection and treatment with the elicitor flg22 can feedback-regulate clock activity. Together this data strongly supports a direct role of the circadian clock in defense control and reveal for the first time crosstalk between the circadian clock and plant innate immunity.
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    DD34E DNA Transposable Elements of Mosquitoes: Whole-Genome Survey, Evolution, and Transposition
    Coy, Monique Royer (Virginia Tech, 2007-06-13)
    Transposable elements (TEs) are mobile genetic elements capable of replicating and spreading within, and in some cases, between genomes. I describe a whole-genome analysis of DD34E TEs, which belong to the IS630-Tc1-mariner superfamily of DNA transposable elements, in the African malaria mosquito, Anopheles gambiae. Twenty-six new transposons as well as a new family, gambol, were identified. The gambol family shares the DD34E catalytic motif with Tc1-DD34E transposons, but is distinct from these elements in their phylogenetic relationships. Although gambol appears to be related to a few DD34E transposons from cyanobacteria and fungi, no gambol elements have been reported in any other insects or animals thus far. This discovery expands the already expansive diversity of the IS630-Tc1-mariner TEs, and raises interesting questions as to the origin of gambol elements and their apparent diversity in An. gambiae. Several DD34E transposons discovered in An. gambiae possess characteristics that are associated with recent transposition, such as high sequence identity between copies, and intact terminal-inverted repeats and open reading frames. One such element, AgTango, was also found in a distantly related mosquito species, Aedes aegypti, at high amino acid sequence identity (79.9%). It was discovered that Tango transposons have patchy distribution among twelve mosquito species surveyed using PCR as well as genomic searches, suggesting a possible case for horizontal transfer. Additionally, it was discovered that in some mosquito genomes, there are several Tango transposons. These observations suggest differential evolutionary scenarios and/or TE-host interaction of Tango elements between mosquito species. This strengthened the case that AgTango may be a functional transposase, and I sought to test its potential activity in a cell culture-based inter-plasmid transposition assay using the Herves plasmids as a positive control (Arensburger et al., 2005). AgTango constructs were successfully constructed; however, no transposition events were detected for Tango or Herves. Because the positive control failed to work, no assessment can be made concerning Tango's transposase. Possible causes and solutions for these results, alternative means to detect transposition, as well as future directions with Tango are discussed.
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    Detection of Acidovorax citrulli, the Causal Agent of Bacterial Fruit Blotch Disease of Cucurbits, Prevention via Seed Treatments and Disease Resistance Genes
    Kiremit, Merve (Virginia Tech, 2021-04-02)
    Melon (Cucumis melo L.) and watermelon (Citrullus lanatus (Thunb.) Matsum and Nakai) belong to the family Cucurbitaceae. Bacterial fruit blotch (BFB) disease of cucurbits is an economically devastating plant disease that has caused an estimated loss of up to $450M on watermelon crops and $75M (worldwide) to the seed and transplant industries since 1996. Disease symptoms include water-soaked cotyledons, leaf necrosis, and internal fruit rot. Current commercial management strategies are very limited and include: seed production field sanitation, greenhouse transplant sanitation, copper-based bactericide sprays, crop rotation, disease-free healthy seeds, isolating diseased plants, and peroxyacetic acid seed treatments. The seedborne disease is usually spread by contaminated seeds, and there is a zero-tolerance policy in the seed industry for infected seeds. No nondestructive assays are commercially available to detect BFB in seeds. This research investigated several different aspects of BFB disease such as non-destructive seed detection, green tea seed treatment, candidate NB-LRR genes for disease resistance, and optimization of virus induced gene silencing for melon and watermelon crops. The potential application of attenuated total reflectance (ATR) Fourier transform infrared spectroscopy (ATR-FTIR) and high-resolution X-ray analysis methods for detection of BFB on seeds were evaluated. It was possible to detect BFB in seeds that were pistil inoculated via x-ray imaging and pericarp inoculated via ATR FT-IR. In vitro and in vivo experiments evaluated the potential of tea (Camellia sinensis) and tea polyphenols as seed treatments to sanitize seeds infected with A. citrulli. Green tea unlike black tea inhibited growth of A. citrulli because of polyphenols. Eighty one melon and forty four watermelon NB-LRR genes were reidentified, and genes that have potential resistance against A. citrulli on melon plants were screened based on host selectivity of the pathogen. Finally, the virus-induced, gene-silencing method was optimized for melon and watermelon for further analysis of potential disease resistance genes. BFB can be nondestructively identified in seeds and green tea may be an effective seed treatment with further development. Promising candidate R genes were identified that might confer stable resistance in the right genetic background.
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    Dissecting Transcriptional Regulation of Rpp8 in Arabidopsis thaliana
    Mohr, Toni Jolene (Virginia Tech, 2005-06-15)
    Plants have evolved physical barriers and inducible defense responses to combat microbial pathogens. Inducible responses are mediated by R proteins, which recognize invading pathogens. R proteins must be precisely regulated to provide effective resistance, without inhibiting normal plant growth. However, little is known about R gene regulation under defense-inducing conditions. The interaction between the oomycete Hyaloperonospora parasitica and the model plant Arabidopsis thaliana provides an excellent model system to explore R gene regulation. My research focuses on RPP8, a CC-NBS-LRR gene, which provides resistance to the H. parasitica isolate Emco5. Previous work in the McDowell lab suggested that RPP8 is upregulated during defense responses. My research shows that RPP8 alleles from the Columbia and Landsberg erecta ecotypes are upregulated by H. parasitica and the defense signaling molecule salicylic acid, suggesting a potential feedback loop. RPP8-Ler is also systemically upregulated after infection of the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Additionally, RPP8-Ler expression is increased during wounding and heat stress. I also examined the role of regulatory cis elements in the RPP8 promoter. Three W-boxes are essential for basal and inducible RPP8 expression, and are required for resistance to Emco5. The X-box, a unique cis element in the RPP8 promoter, is essential for strong basal expression and wound-induced upregulation, and affects spatial expression of RPP8-Ler. However, the X-box is not required for RPP8-Ler upregulation during pathogen or SA treatment. R genes may be induced as part of global defense responses, which could prime the host for more effective pathogen recognition.
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    Dissection of Innate Immunity in Tomato and Tolerance to Bacterial Wilt in Solanaceae species
    Naumenko, Anastasia Nikolayevna (Virginia Tech, 2013-04-05)
    Unlike mammals, plants do not have specific immune cells. However, plants can still recognize pathogens and defend themselves. They do that by recognizing microbial-associated molecular patterns (MAMPs) and secreted pathogen proteins, called effectors. MAMP-triggered immunity (MTI) relies on recognition of MAMPs by leucine-rich repeats (LRRs) pattern-recognition receptors (PRRs). The best-studied LRR PRR is Flagellin-Sensitive 2 (Fls2), the receptor of a 22-amino acid long epitope of bacterial flagellin, called flg22. In this project, alleles of FLS2 of different tomato cultivars were sequenced and compared to each other to get insight into natural selection acting on FLS2 and to identify residues important for ligand binding. This information may be used in the future to engineer Fls2 for improved ability to recognize flagellin. MTI can be suppressed by effectors secreted by bacteria into plant cells through the type III secretion system. On the other hand, plants are equipped with repertoires of resistance proteins, which can recognize some pathogen effectors. If a pathogen carries an effector that is recognized, effector-triggered immunity (ETI) is activated and the plant is resistant. Here, eggplant breeding lines were screened for their ability to activate ETI upon recognition of effectors of the soil borne pathogen Ralstonia solanacearum, a causative agent of bacterial wilt. Four effectors were found to trigger plant defenses in some of the lines. This is the first step in cloning the genes coding for the responsible resistance proteins. These genes may be used in the future for engineering tomato and potato for resistance to bacterial wilt.
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    Dissection of Regulatory Networks Mediating Resistance and Susceptibility of Arabidopsis thaliana to the Downy Mildew Pathogen Hyaloperonospora parasitica
    Hoff, Troy Colston (Virginia Tech, 2008-12-19)
    Plants and pathogenic microorganisms are in constant conflict with each other. Understanding the molecular networks that trigger resistance, along with the molecular networks that pathogens might co-opt to infect susceptible plants, is important for developing the integrated, holistic perspective that is necessary for innovative development of engineered resistance to current and emerging pathogens. The first objective of the dissertation was to increase the understanding of mechanisms by which plants recognize pathogen attack and mount an appropriate defense response. These experiments focused on resistance triggered by the Arabidopsis thaliana R gene, RPP7, which encodes a coiled-coil nucleotide binding-leucine-rich repeat (CC-NB-LRR) protein that activates race-specific resistance to the downy mildew pathogen, Hyaloperonospora parasitica (Hpa). Previously-published genetic epistasis tests have established that RPP7 activates defense responses through a signaling mechanism that does not require accumulation of salicylic acid (SA), or components of the ethylene and jasmonate response pathways. Furthermore, RPP7 is not strongly compromised by mutations in genes associated with defense signal transduction (PAD4, NDR1, NPR1, RAR1). Double mutant combinations of these signal transduction components were analyzed to detect additive or functionally-redundant contributions to RPP7-dependent resistance. Most of the double mutants support an enhanced level of asexual sporulation compared to the single mutant parental lines. Time-course experiments with histochemical stains revealed that these double mutants delay, but do not suppress, the oxidative burst and the hypersensitive response. These results suggest that RPP7 activates multiple signaling pathways, each of which makes incremental contributions to the timing of defense activation. The second objective of the dissertation was to investigate the role that auxin plays in enabling virulent H. parasitica to colonize Arabidopsis. Transcript profiling revealed induction of auxin-associated genes in response to infection of Arabidopsis thaliana by virulent strains of the oömycete pathogen, H. parasitica. Experiments with the DR5
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    Effect of light and dark on the growth and development of downy mildew pathogen Hyaloperonospora arabidopsidis
    Telli, Osman; Jimenez-Quiros, Catherine; McDowell, John M.; Tor, Mahmut (2020-09)
    Disease development in plants requires a susceptible host, a virulent pathogen, and a favourable environment. Oomycete pathogens cause many important diseases and have evolved sophisticated molecular mechanisms to manipulate their hosts. Day length has been shown to impact plant-oomycete interactions but a need exists for a tractable reference system to understand the mechanistic interplay between light regulation, oomycete pathogen virulence, and plant host immunity. Here we present data demonstrating that light is a critical factor in the interaction between Arabidopsis thaliana and its naturally occurring downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa). We investigated the role of light on spore germination, mycelium development, sporulation, and oospore formation of Hpa, along with defence responses in the host. We observed abundant Hpa sporulation on compatible Arabidopsis under day lengths ranging from 10 to 14 hr. In contrast, exposure to constant light or constant dark suppressed sporulation. Exposure to constant dark suppressed spore germination, mycelial development, and oospore formation, whereas exposure to constant light stimulated these three stages of development. A biomarker of plant immune system activation was induced under both constant light and constant dark. Altogether, these findings demonstrate that Hpa has the molecular mechanisms to perceive and respond to light and that both the host and pathogen responses are influenced by the light regime. Therefore, this pathosystem can be used for investigations to understand the molecular mechanisms through which oomycete pathogens like Hpa perceive and integrate light signals, and how light influences pathogen virulence and host immunity during their interactions.
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    The Effects of Cell Culture Oxygen Levels on the Replicative Senescence Processes of Primary Human Fibroblasts
    Stab II, Bernd Robert (Virginia Tech, 2009-07-17)
    Serial passaging of primary human fibroblasts leads to the formation of non-dividing senescent cells by a process termed replicative senescence. This tissue culture-based methodology is currently used as a model system to determine the underlying mechanisms of in vivo cellular aging and tumor suppression. Senescence is regarded as an alternative pathway to apoptosis, where cells undergo multiple changes in metabolic and cellular signaling pathways in order to prevent proliferation but still maintain a metabolically-active cell. Whether or not this model accurately reflects in vivo processes is presently controversial; however, replicative senescence is currently the most applicable model through which one can investigate the underlying causes of human cellular aging in the context of controlled environmental stress over time. This work was directed at understanding the molecular processes involved in replicative senescence with specific emphasis on the role of the mitochondria. A series of experiments were performed to assess changes during the induction of replicative senescence under conditions of low (3%) and high (20%) oxygen levels. Measurements were made at the transcriptional, protein, and metabolite levels. Microscopy wasalso utilized to monitor changes in mitochondrial morphology and volume. While previous studies have evaluated specific pathways and/or products; this work combines a more complete metabolomic, genomic, proteomic, and morphological picture of cells undergoing senescence and oxidative stress. Considering the low cell population densities of primary adherent fibroblasts and the subsequent low concentrations of small polar metabolites involved in glycolysis and the TCA cycle, methodologies needed to be developed in order to optimize metabolite extraction and liquid chromatography-mass spectrometric analysis. Protein kinase and transcriptional microarrays were also performed in order to quantify the changes in activated/deactivated signaling cascades as well as gene expression and relate these findings to metabolomic data. Mitochondrial dynamics of cells at different age time points and under different oxygen conditions were also assessed including mitochondrial size, shape, membrane potential, and percent volume per cell volume using confocal microscopy. The results obtained not only confirm the major pathways involved in senescence (p53/p21, PTEN/p27, and RTK/Raf/MAPK) but also provide evidence at both the transcriptional and protein levels for additional senescence-associated pathways. The majority of the changes observed were related to pathways involved in cellular stress, cell cycle control, and the survival response. Metabolic data suggested a –pooling effect– of glycolysis and TCA precursor molecules due to attenuation in enzyme function; this theory was also supported by an observed up regulation of gene expression as a compensatory mechanism. Mitochondria exhibited changes in membrane potential as well as volume and percent volume per cell which suggested compensatory hypertrophy and/or attenuation of mitochondrial fission processes. When the aforementioned analyses are tied together, a “theoretical model of senescence” can be formulated and is characterized by increased metabolic protein and associated metabolite levels due to attenuation in their respective enzyme function, resulting in increases in expression of their associated genes as a compensatory mechanism.
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    Elucidating essential roles of oomycee effector proteins in immune suppression and in targeting hormonal pathways in the host plant
    Deb, Devdutta (Virginia Tech, 2013-09-25)
    Effector proteins are exported to the interior of host cells by numerous plant pathogens. Effector proteins have been well characterized in bacteria. However, the mechanisms through which these effectors promote virulence are largely unknown. Bioinformatic analysis of genome sequences from oomycete pathogens Phytophthora sojae, P. ramorum, P. infestans and Hyaloperonospora arabidopsidis (Hpa) have led to the identification of a large number of candidate effector genes. These effector genes have characteristic motifs (signal peptide, RxLR and dEER) that target the effectors into plant cells. Although these effector genes are very diverse, certain genes are conserved between P. sojae and H. arabidopsidis, suggesting that they play important roles in pathogenicity. The goal of my first project was to characterize a pair of conserved effector candidates from Hpa and P. sojae. We hypothesized that these effectors have important conserved roles with regard to infection. We found that the Hpa effector was expressed early during the course of infection of Arabidopsis and triggered an ecotype-specific defense response in Arabidopsis, suggesting that it was recognized by host surveillance proteins. Both the effectors from Hpa and P. sojae respectively could suppress immunity triggered by pathogen associated molecular patterns (PTI) and by effectors (ETI) in planta. They also enhanced bacterial virulence in Arabidopsis when delivered by the Type III secretion system. Similar results were seen with experiments with transgenic Arabidopsis expressing the effectors. My second project showed that a different Hpa effector protein, HaRxL10, targets the Jasmonate-Zim Domain (JAZ) proteins that repressed responses to the phytohormone jasmonic acid (JA). This manipulation activates a regulatory cascade that reduces accumulation of a second phytohormone, salicylic acid (SA) and thereby attenuates immunity. This virulence mechanism is functionally equivalent to but mechanistically distinct from activation of JA-SA crosstalk by the bacterial JA mimic coronatine. These results reveal a new mechanism underpinning oomycete virulence and demonstrate that the JA-SA crosstalk is an Achilles\' heel that is manipulated by unrelated pathogens through distinct mechanisms.
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    Elucidating the function of inositol pyrophosphate signaling pathways in Arabidopsis thaliana
    Cridland, Caitlin A. (Virginia Tech, 2022-04-12)
    Phosphate (Pi) is an essential nutrient for plants, required for plant growth and seed viability. When Pi is limited, plants undergo dynamic morphological and metabolic changes to leverage available Pi, known as the Phosphate Starvation Response (PSR). The inositol phosphate (InsP) signaling pathway is a crucial element of the plant's ability to regulate the PSR and respond to changing energy conditions. InsPs are synthesized from the cyclic 6-carbon polyol scaffold, myo-inositol. Inositol hexakisphosphate (InsP6) is the most abundant InsP signaling molecule and can be phosphorylated by the multifunctional inositol tetrakisphosphate 1-kinase 1 (ITPK1) and diphosphoinositol pentakisphosphate (VIP) kinases, resulting in inositol pyrophosphates (PP-InsPs). PP-InsPs have high energy bonds and have been linked to Pi maintenance and energy homeostasis in yeast, plants, and mammals. However, the precise mechanism(s) by which PP-InsPs act within plant signaling pathways remains to be determined. Two approaches to understand the role of PP-InsPs in plants are described within this dissertation. The first approach analyzes genetic loss-of-function vip1/vip2 double mutants, and their responses to low Pi conditions. Specifically, vip1/vip2 double mutant gene expression and lipid remodeling patterns in response to low Pi were characterized. We found that vip1-2/vip2-2 had an impacted lipid remodeling response under low Pi conditions, whereas ipk1 had altered lipid composition under Pi-replete conditions. In a complementary approach, a gain-of-function in either the ITPK1 or the kinase domain of VIP (VIP2KD) were constructed in transgenic Arabidopsis thaliana plants. Both ITPK1 and VIP2KD transgenic plants contain elevated levels of the specific inositol pyrophosphate, InsP8. Elevated InsP8 in both types of plants results in changes in growth and senescence phenotypes, delayed time to flowering, Pi accumulation, and altered PSR gene expression. The data from both approaches suggest new roles for PP-InsPs in the regulation of the PSR and other signaling pathways in plants. To enhance my teaching and leadership skills, I participated in the Graduate Teaching Scholars (GTS) program. As a GTS, I worked with the Virginia Tech Research and Extension Experiential Learning (VT-REEL) program where I developed a structured mentorship program for undergraduate and graduate students and created a professional development workshop series. During the COVID-19 pandemic, I developed an online version of the VT-REEL program. Using inclusive pedagogy practices and surveys from the participants, we compiled the best practices for moving a summer undergraduate research program online. These practices come from surveyed participants in the 2020 and provides strategies that can be tailored to various online research experiences and be implemented in both online and in-person formats.
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    Elucidating three novel mechanisms of Pseudomonas syringae pathogenicity
    Clarke, Christopher R. (Virginia Tech, 2012-02-10)
    Pseudomonas syringae is an important bacterial plant pathogen that, as a species, is known to cause disease on hundreds of different plant species. However, any individual pathovar of P. syringae typically only causes disease on one or a few plant species, which constitute the host range of the pathovar. Plants are generally resistant to most pathogens primarily because the plant innate immune system is capable of recognizing conserved microbial-associated molecular patterns (MAMPs). Adapted pathovars of P. syringae secrete effector proteins through a Type Three Secretion System (T3SS) to suppress the immune response elicited by their MAMPs. However, secretion of effectors can also trigger a strong plant immune response if the plant harbors resistance proteins capable of recognizing the secreted effectors. Successful pathovars, therefore, must secrete a combination of effectors capable of suppressing MAMP/Pattern-Triggered Immunity (PTI) without eliciting Effector-Triggered Immunity. Here we identify several novel strategies employed by P. syringae to overcome the plant immune system and cause disease. First, we demonstrate that, in place of the canonical T3SS used by all known pathogens of P. syringae, several apparently nonpathogenic isolates of P. syringae employ a novel T3SS that is functional but not necessary for colonization of plants. Despite being closely related to pathogenic isolates of P. syringae, the isolates employing the noncanonical T3SS do not cause disease on any tested plants and instead appear to act more as commensal organisms. Second, we advance the understanding of PTI by identifying a second region of bacterial flagellin that triggers PTI in addition to the archetypical MAMP flg22, which is recognized by the archetypical plant receptor FLS2. This new elicitor, termed flgII-28, is also detected by FLS2 and appears to be under selection in very closely related lineages of P. syringae. Alleles of flagellin present in one recently expanded and agriculturally problematic lineage of P. syringae appear to trigger less PTI on their host plant, tomato, than the ancestral allele suggesting that avoidance of PTI through allelic diversity in MAMPs is an effective alternative strategy to suppression of PTI through delivery of effectors. Finally, we start to elucidate a role for chemotaxis (chemical-directed movement) in P. syringae pathogenicity. Not only is chemotaxis required for pathogenicity of P. syringae on plants, but it also appears to contribute to delimiting the host range of several P. syringae pathovars. These results highlight that additional aspects of P. syringae pathogenicity, such as chemotaxis, can directly contribute to defining the host range of individual P. syringae pathovars. The current paradigm of P. syringae pathogenicity posits that MAMPS and the repertoire of effector proteins are the primary determinant of the host range of any P. syringae pathovar; in contrast these results inspire a more nuanced view of pathogenicity that considers multiple aspects of the infection process.
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    Endogenous Stress Signaling within Human Multicellular Aggregates (Spheroids)
    Jack, Graham Dillon (Virginia Tech, 2006-07-10)
    A wide variety of adherent mammalian cells can be induced into a reversible state of metabolic arrest (quiescence) by conversion to non-adherent multicellular aggregates. These "spheroids" can be maintained at room temperature under oxygen- and nutrient-deprived conditions for extended periods of time (weeks) as well as converted back to viable proliferating monolayers. Herein it is shown that HEK293 spheroid arrest and recovery requires the co-activation of both NF-kB and JNK, and chemical inhibition of either NF-κB nuclear translocation or JNK phosphorylation leads to cell death. Cytokine profiling within the aggregates during the arrest and recovery process is suggestive that a cyclical cascade was in operation, leading to endogenous cytokine production of TNF-Alpha, IL-1Beta, and IL-8, thereby propagating the cellular stress signal within cells as well as throughout the aggregate. Cytokines exist in vivo as mixtures, yet tissue culture studies delineating how cells respond to these molecules are often performed using individual effectors added exogenously. Are the results obtained in these studies true representations of physiological responses? As HEK293 multicellular aggregates (spheroids) survive long term arrest by endogenous cytokine (TNF-α and IL-1β) and chemokine (IL-8) signaling, adherent monolayer cells were evaluated for their ability to provide a "spheroid signal response" when exposed to TNF-α, IL-1β and IL-8 individually, and in combination, at concentrations observed in the aggregates. The spheroid signal transduction response was only observed when all three cytokines were present, demonstrating that signal transduction cascade mechanisms are cytokine-profile dependent. To determine if similar processes were involved in the arrest and recovery of multicellular aggregates derived from other cell types, the responses of primary human foreskin fibroblasts (HFF-2) and a glioblastoma cell line (T98G) were characterized, utilizing the procedures developed in the HEK293 study. Both the T98G and HFF-2 cell lines entered and exited from the long term arrest utilizing an autocrine response. However, while the carcinoma cell line was dependent upon NF-κB for survival, its signaling partner was Gadd45α and signaling occurred through the p38 pathway. Primary fibroblast arrest and recovery proceeded through the p38 pathway as well, but was independent of NF-κB. Thus, three different cell types and transformation states (HEK293, HFF-2, and T98G) provided three different routes to survival, all with cyclical cytokine production and signaling. These routes cannot be measured or modulated effectively in adherent monolayers. Multicellular aggregates provide higher ordered systems that can be used to describe signaling pathways within a cell, highlighting the role of autocrine responses and the synergistic relationships between cytokines and neighboring cells.