Browsing by Author "Jelesko, John G."
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- Accession-Level Differentiation of Urushiol Levels, and Identification of Cardanols in Nascent Emerged Poison Ivy SeedlingsLott, Aneirin A.; Baklajian, Emily R.; Dickinson, Christopher C.; Collakova, Eva; Jelesko, John G. (MDPI, 2019-11-20)Poison ivy (Toxicodendron radicans (L.) Kuntze) shows accession-level differentiation in a variety of morphometric traits, suggesting local adaptation. To investigate whether the presumed defense compound urushiol also demonstrates accession-level accumulation differences, in vitro nascent germinated poison ivy seedlings from geographically isolated populations were germinated in vitro and then assayed for known urushiol congener accumulation levels. Significant accession-level differences in the accumulation levels of total C15- and C17-, total C15-, total C17-, specific C15 congeners, and specific C17 congeners of urushiol were identified. In addition, hereto novel C15- and C17-urushiol isomers were identified as well. Cardanols are assumed to be the penultimate metabolites giving rise to urushiols, but this assumption was not previously empirically validated. C15-cardanol congeners and isomers corresponding to expected substrates needed to produce the observed C15-urushiol congeners and isomers were identified in the same poison ivy seedling extracts. Total C15-cardanol and C15-cardanol congeners also showed significant accession-level differences. Based on the observed C15-cardanol congeners in poison ivy, the penultimate step in urushiol biosynthesis was proposed to be a cardanol-specific hydroxylase activity.
- AFLP Marker Analysis Of Monoploid PotatoVarrieur, John Michael (Virginia Tech, 2002-05-17)Potato haploids have been recent components in protoplast fusion research, strategies to combine wild and cultivated potato germplasm and the generation of economically valuable mutant phenotypes. Additionally, most major genetic mapping and QTL analyses in potato have utilized haploid germplasm to simplify linkage-mapping computations. The accuracy of genetic assumptions concerning the randomness and genetic purity of haploid genomes may directly affect the statistical validity of many results in current potato research. In the present study, AFLP analysis was conducted on two sibling S. phureja "BARD 1-3" monoploid populations derived by androgenesis in anther culture, and gynogenesis through the use of a haploid-inducing pollinator, S. phureja "IVP 101." Little indication of somaclonal variation and haploid-inducer gene introgression was found in the monoploid band data suggesting genomic stability. Segregation of marker alleles that were heterozygous in the parent was distorted from the expected 1:1 ratio in both populations, ranging from 35% in the gynogenic monoploids (GM) to 46% in the androgenic monoploids (AM). Genetic diversity appeared more random among the monoploid populations after skewed marker data was removed from phylogenetic analyses. Bilateral and unilateral marker skewness in the monoploid populations may respectively indicate common and unique segregation distorting loci (SDL) present in the AM and GM genomes. Representatives of both SDL types were located on a partial linkage map created using androgenic monoploid data.
- Biochemical and Functional Characterization of Induced Terpene Formation in Arabidopsis RootsSohrabi, Reza (Virginia Tech, 2013-08-13)Plants have evolved a variety of constitutive and induced chemical defense mechanisms against biotic stress. Emission of volatile compounds from plants facilitates interactions with both beneficial and pathogenic organisms. However, knowledge of the chemical defense in roots is still limited. In this study, we have examined the root-specific biosynthesis and function of volatile terpenes in the model plant Arabidopsis. When infected with the root rot pathogen Pythium irregulare, Arabidopsis roots release the acyclic C11-homoterpene (E)-4,8-dimethylnona-1,3,7-triene (DMNT), which is a common constituent of volatile blends emitted from insect-damaged foliage. We have identified a single cytochrome P450 monooxygenase of the CYP705 family that catalyzes a root-specific oxidative degradation of the C30-triterpene precursor arabidiol thereby causing the release of DMNT and a C19-degradation product named arabidonol. We found that DMNT shows inhibitory effects on P. irregulare mycelium growth and oospore germination in vitro, and that DMNT biosynthetic mutant plants were more susceptible to P. irregulare infection. We provide evidence based on genome synteny and phylogenetic analysis that the arabidiol biosynthetic gene cluster containing the arabidiol synthase (ABDS) and CYP705A1 genes possibly emerged via local gene duplication followed by de novo neofunctionalization. Together, our studies demonstrate differences and plasticity in the metabolic organization and function of terpenes in roots in comparison to aboveground plant tissues. Additionally, we demonstrated that the arabidiol cleavage product, arabidonol, is further modified by yet unknown enzymatic reactions into three products, which are found in root exudates. We suggested a pathway for their biosynthesis based on precursor feeding experiments and NMR analysis. Although DMNT biosynthetic genes are clustered on chromosome 4 along with several potential modification genes, we did not find a possible role of these genes in the derivatization of arabidonol. Preliminary experimental results using genetic and biochemical approaches for identifying genes involved in the modification steps are also presented. In summary, this study demonstrates an alternative route for volatile terpene formation belowground different from aboveground plant tissues via triterpene degradation and provides evidence for an unexplored triterpene catabolism pathway in Arabidopsis.
- Biosynthesis of Steroidal Glycoakaloids in Solanum chacoense BitterMweetwa, Alice Mutiti (Virginia Tech, 2009-07-24)Steroidal glycoalkaloids (SGAs) are secondary metabolites produced by approximately 350 species in the Solanaceae family. SGAs are reported to be important for pest resistance and flavor enhancement at low concentrations but are toxic to humans and other mammals at high concentrations. Studies on sterol / SGA biosynthesis have implicated squalene synthase as a key regulatory enzyme because it catalyzes an irreversible step from the mevalonic acid pathway. However, the regulatory mechanisms of squalene synthase are not yet known. A study was conducted to elucidate the distribution pattern of SGAs and to clone the squalene synthase gene in order to determine a relationship between SGAs and gene expression levels. Solanum chacoense, a wild potato species was used as a model plant from which tissues were harvested at specified developmental stages and analyzed for SGA content. The results from the SGA analysis suggest a qualitative and quantitative tissue- and age-dependent accumulation of SGAs. Regenerative tissues such as, axiliary shoots, flowers and floral buds had the highest levels of 88, 49 and 63 µmole/g DW, respectively. The roots, stems and tubers showed the lowest amounts of SGAs of 1 to 8, 5 to 15 and 7 to 15 µmole/g DW, respectively. Stolons and tubers accumulated higher amounts of α-chaconine (59 to 67%) than α-solanine (61 to 64%) at all developmental stages analyzed. On the other hand, in young expanding, fully expanded, and old senescing leaves where leptine and leptinines tend to dominate, α-solanine and α-chaconine together accounted for only 8 to 15%, 7 to 15%, and 8 to 45%, respectively. Plant organs that showed the highest biosynthetic activity for SGA production also had high levels of transcripts coding for genes of isoprenoid biosynthesis. The results from the cloning and characterization of squalene synthase suggest that the cloned cDNA fragment is a putative S. chacoense squalene synthase gene with an open reading frame / predicted protein precursor of 411 amino acids. The cloned cDNA has high similarity (68-100%) to known plant squalene synthase genes and contains six deduced peptide domains observed in other species. The 3â untranslated regions of floral buds, young leaves (early vegetative stage), and fully expanded leaves (anthesis) were different in length with, 249, 335, and 202 nucleotides, respectively. The Southern blot analysis suggests a single copy gene although the existence of a gene family cannot be ruled out.
- Biotic and abiotic mechanisms shaping multi-species interactionsMaynard, Lauren Danielle (Virginia Tech, 2022-12-20)Interactions are important drivers of selection and community structure, which makes the study of multi-species interactions critical for understanding the ecology and evolution of organisms. This dissertation includes four data chapters that examine the biotic and abiotic mechanisms that shape multi-species interactions in both tropical and temperate ecosystems. The first three data chapters (Chapters 2–4) were completed within a Neotropical rainforest in Costa Rica and focus on one plant genus, Piper (Piperaceae). The final data chapter (Chapter 5) was conducted within a working landscape of soybean (Glycine max) fields in eastern Maryland, USA. In Chapter 2, I explore intra- and inter-specific dietary niche partitioning of Piper fruits among three frugivorous bats, illustrating the importance of fine-scale mechanisms that facilitate species coexistence and influence plant–animal interactions. In Chapter 3, I demonstrate how the chemical ecology of a Neotropical shrub, Piper sancti-felicis, shapes fruit interactions with antagonists (fruit fungi) and mutualists (frugivorous bats and birds), developing a foundation for understanding evolutionary ecology of plant chemical traits based on phytochemical investment patterns. In Chapter 4, I describe the direct and indirect impacts of elevated temperature and CO2 concentration on the plant traits and interactions in Piper generalense, improving our understanding of the effects of climate change on a Neotropical plant–herbivore system. In Chapter 5, I explore the biotic (herbivore-induced plant volatiles) and abiotic (fine-scale weather conditions) drivers affecting insectivorous bat foraging in soybean fields in eastern Maryland, providing a pathway to further investigate new strategies for integrated pest management. As a collective work, this dissertation disentangles the nuances of multi-species interactions, exploring foundational mechanisms underlying biodiversity maintenance as well as answering applied questions to address a changing climate and aid sustainable agriculture.
- Cell-type specificity and herbivore-induced responses of primary and terpene secondary metabolism in Arabidopsis rootsZhang, Jingyu (Virginia Tech, 2013-09-02)Plants employ diverse defense mechanisms to combat attack by harmful organisms. For instance, plants produce constitutive physical barriers or use chemical compounds such as specialized secondary metabolites to resist herbivore or pathogen invasion. Considering the cost-efficiency and energy balance between defense, growth and reproduction, defense reactions in plants have to be regulated temporally and spatially. As more cost-efficient strategies, plants may induce their defense response only in the presence of the attacker or restrict constitutive defenses to specific tissues or cells. In this study, we investigated aspects of the spatial regulation and induced changes of primary and secondary metabolism in roots of Arabidopsis thaliana. Roots represent important organs for anchoring plants in the soil and taking up water and nutrients. Hence, it is assumed that roots are as well protected as aerial tissues by different defense mechanisms. The first part of this work is focused on the cell-type-specific biosynthesis of volatile terpenes in Arabidopsis roots. Terpenes are the most abundant specialized metabolites in plants and play an important role in plant defense against pathogens or herbivores. Terpene biosynthetic enzyme activities are often coordinated in specific tissues and cellular compartments. Fine-scale transcriptome maps of Arabidopsis roots have shown that terpene biosynthesis is restricted to particular cell types. However, the reasons and significance of this cell-type specificity are not well understood. We hypothesized that the formation of terpene metabolites is not restricted to specific cells but can be supported by different cell types. We, therefore, probed the plasticity of the cell-specific formation of terpenes by swapping the expression of the terpene synthase (TPS) genes, TPS08, TPS13 and TPS25, between different root cell types in the respective mutant background. To investigate the ectopic expression of TPSs at different levels, quantitative real-time PCR (qRT-PCR), confocal microscopy, and gas chromatography-mass spectrometry (GC-MS) were performed. We found that terpene synthase TPS08, which produces the diterpene rhizathalene and is normally expressed in the root vascular tissue, is functionally active when expressed in the epidermis or cortex, although at substantially lower levels compared to the wild type. We did not find an obvious correlation between the volatile emission level and gene transcript level of TPS08, which may be attributed to a reduced activity of the expressed TPS08-yellow fluorescent protein (YFP) fusion protein. When expression of TPS13 (producing the sesquiterpene (Z)-"-bisabolene) was directed from the cortex to the epidermis or stele, TPS13 gene expression and (Z)-"-bisabolene formation was supported by these cell types although to varying levels in comparison to wild type. TPS13-YFP fluorescent signal driven by the epidermal WER and GL3 promoters was primarily detected at the root tip. Terpene production was also observed for the (E)-"-farnesene sesquiterpene synthase TPS25 when its expression was switched from the endodermis and non-hair producing epidermal cells to hair producing epidermal cells although only a weak fluorescent signal was detected from the expressed TPS25-mGFP protein. Together, the results provide preliminary evidence for a relaxed cell specificity of terpene biosynthesis in Arabidopsis roots and suggest that tissue-specific terpene metabolite patterns could change depending on different selective pressures in rhizosphere. In the second part of this study, we performed global gene transcript profiling and primary metabolite analysis of Arabidopsis roots upon feeding by the generalist root herbivore, Bradysia (fungus gnat). In a microarray analysis, we identified 451 of 22,810 genes that were up-regulated more than 2-fold. Gene ontology (GO) analysis showed that 26% of those genes with predicted or known functions play a role in primary or secondary metabolism, while 24% are involved in cell signaling or in responses to stimulating factors, such as jasmonic acid (JA), ethylene, wounding, and oxidative stress. At the metabolite level, we observed only marginal changes of amino acid, sugar and carboxylic acid relative levels over a time course of 4 days of Bradysia feeding. There was a trend for increased levels of amino acids and the relative levels of sucrose were increased significantly ("=0.05) at the fourth day of feeding. In conclusion, the study provided evidence for the induction of genes related to primary and secondary metabolism and stress responses in Arabidopsis roots, but showed only marginal changes at the primary metabolite level. In addition, the work indicated that the formation of terpene-specialized metabolites in Arabidopsis roots is not restricted to specific cells, but can be supported by different cell types.
- Characterization of symbiotically important processes in Sinorhizobium melilotiZatakia, Hardik M. (Virginia Tech, 2015-09-15)Bacteria perform biological nitrogen fixation (BNF) which leads to conversion of N2 to ammonia. One of the best studied models of BNF is the symbiotic association of Sinorhizobium meliloti - Medicago sativa (alfalfa). Since alfalfa is a major source of animal feed and the fourth largest crop grown in the USA, enhanced understanding of this symbiosis can have implications for increasing crop yields, reducing environmental contamination and food costs. Studies discussed here focus on two symbiotically important bacterial traits, type IVb pili and chemotaxis. Chapter 2 characterizes S. meliloti type IVb pili encoded by flp-1 and establishes their role in nodulation. Bundle-forming pili were visualized in wild-type cells, while cells lacking pilA1, the pilin-encoding gene, showed an absence of pili. Competitive nodulation assays with alfalfa concluded that cells lacking pili had a significant nodulation defect. Regulation of pilA1 expression via a quorum sensing regulator, ExpR, was confirmed. Chapter 3 describes the role of the flp-2 cluster in establishing symbiosis. PilA2 is a pilin subunit encoded from flp-2. The pilA2 deletion strain was defective in nodulation by 31% as compared to the wild type. A non-significant change in nodulation was seen in pilA1pilA2 strain. Thus, both flp-1 and flp-2 have a significant role in establishing symbiosis. Chapter 4 focuses on the deviations of S. meliloti chemotaxis from the enterobacterial paradigm. Transcriptional fusions showed that S. meliloti chemoreceptors (MCPs) are class III genes and regulated by FlbT. Quantitative immunoblots determined the cellular amounts of chemoreceptors. Chemoreceptors were grouped in three classes; high, low, and extremely-low abundance, similar to the high and low abundance chemoreceptors of Escherichia coli. Importantly, the MCP:CheA ratio in an S. meliloti cell was observed to be 37:1, similar to that in Bacillus subtilis of 24:1, but quite different from that in E. coli of 3.4:1. In conclusion, our data indicates that soil bacteria may have optimized their chemotaxis system based on their milieu, which is different from enteric bacteria. These studies have enhanced our understanding of two symbiotically important processes in S. meliloti, and pave the way for future manipulations of the system to increase symbiosis and reduce our dependence on synthetic fertilizers.
- Characterization of the A/B regulon in tobacco (Nicotiana tabacum)Reed, Deborah G. (Virginia Tech, 2003-07-08)Plant alkaloids are secondary metabolites that may be synthesized in an inducible defense response to herbivory (Baldwin 1999). Genetic engineering of secondary metabolic pathways in plants to enhance or reduce metabolite production is limited by the current understanding of these pathways and their regulation in response to environmental conditions. This study was intended to provide new insights into the mechanism and regulation of alkaloid biosynthesis in N. tabacum by identifying genes that are coordinately regulated during conditions that induce alkaloid biosynthesis and by comparing their expression in regulatory mutant backgrounds that differ at two quantitative alkaloid loci, A and B. In order to identify novel genes that are differentially expressed during alkaloid biosynthesis, the transcriptional profiling procedure, fluorescent differential display (FDD), was used to screen total RNA isolated from Burley 21 (WT, AABB) and LA21 (low alkaloid regulatory mutant, aabb) tobacco root cultures that were induced for alkaloid synthesis. Four of thirteen cloned FDD fragments showed sequence homology to genes with defense-related functions. The differential expression of genes represented by selected FDD gene fragments was confirmed by comparing Northern blots of transcripts of those genes to known alkaloid biosynthetic genes, putrescine methyl transferase (PMT3), ornithine decarboxylase (ODC3), arginine decarboxylase (ADC1), and quinolinate phosphoribosyltransferase (QPRT). The role of the A and B loci in differential expression of genes represented by FDD clones and of known nicotine biosynthetic genes was examined using quantitative real time polymerase chain reaction (QRT-PCR) to measure transcript levels of these genes in four tobacco genotypes differing in alkaloid content, Burley 21(AABB), HI21 (AAbb), LI21(aaBB), and LA21 (aabb). Results of this study suggest that the A/B regulon is not limited to alkaloid biosynthetic genes, but includes multiple genes with defense-related functions. QRT-PCR analysis of nicotine biosynthetic genes and genes represented by confirmed differentially expressed FDD clones showed increased mRNA accumulation in response to alkaloid induction in all the tested genotypes, which suggests that the A and B mutations affect overall mRNA accumulation levels, rather than gene inducibility, per se. Baldwin, I.T. 1999. Inducible nicotine production in native Nicotiana as an example of adaptive phenotypic plasticity. Journal of Chem. Ecol. 25: 3-30.
- Characterizing the role in amino acid sensing and signaling of Amino Acid Permease 1 in ArabidopsisShelley, Brett A. (Virginia Tech, 2021-07-28)Amino acids are necessary for protein synthesis and specialized metabolism in plants. Yet very little is known about how plants sense and regulate when and where to allocate amino acids to meet the demand for nitrogen in growing tissues. In particular, while characterized in yeast and mammals, no amino acid sensor has been identified in plants. Amino Acid Permease 1 (AAP1) has been previously characterized and was shown to mediate amino acid uptake from the soil. aap1 knockout plants and several EMS mutants affected in AAP1 sequence display enhanced tolerance to toxic concentrations of amino acids. Yet, two of the corresponding variant proteins appear to be functional transporters, effectively dissociating amino acid transport and phenotype. To understand this apparent discrepancy, I precisely studied AAP1 localization of expression at the plant and cellular level, and in specific tissue types of the root where AAP1 function is required for the tolerance phenotype and the amino acid uptake activity. I showed that AAP1 protein is present in the endoplasmic reticulum of the cortex in wild type plants Yet, its ectopic expression in root tip and phloem increased amino acid uptake, while expression in cortex could not. This and other of my results do not support the current model of AAP1 functioning in amino acid uptake by the root. I propose that the main effect of mutations in AAP1 is a disturbance in amino acid metabolism, possibly triggered by altered amino acid sensing. In this new model, AAP1 would be necessary for sensing amino acid status of cortex cells, possibly in the endoplasmic reticulum, and adjust amino acid metabolic activity and uptake to current availability. In effect, disruption of the sensing function, either by complete loss of AAP1 function (knockout) or by uncoupling the transport and sensing function (EMS mutants), would lead to the various characteristics of the phenotype of the aap1 mutants I observed. My main hypothesis is that AAP1 is a transporter endowed with sensing function, i.e., an amino acid transceptor.
- Compositions and methods comprising colletotrichum for controlling plant species(United States Patent and Trademark Office, 2017-05-09)Provided are methods and compositions for controlling at least one plant. Methods and compositions can comprise at least one strain of Colletotrichum spp., such as Colletotrichum fioriniae TR-123, Colletotrichum fioriniae TR-126, and combinations thereof. Methods and compositions of the invention are useful, for example, for controlling Toxicodendron radicans and/or Schinus terebinthifolus.
- The Curious Poisoned Weed: Poison Ivy Ecology and PhysiologyDickinson, Christopher Cody (Virginia Tech, 2019-07-11)Poison ivy (Toxicodendron radicans (L.) Kuntze) is a native perennial liana widely recognized for the production of urushiol, and the associated contact dermatitis it causes in humans. Poison ivy is predicted to become both more prevalent and more noxious in response to projected patterns of global change. Moreover, poison ivy is an important food source for avian species, and urushiol has numerous applications as a high-value engineering material. Thus, this curious weed has many avenues for future concern, and promise. Here, I address gaps in knowledge about poison ivy ecology and physiology so that we may better understand its weediness and utilize its benefits. I address three core areas: poison ivy establishment patterns; biotic interactions with multiple taxa; and the development of molecular tools for use in poison ivy. I found that the early life stage of seedling emergence is a critical linchpin in poison ivy establishment due largely to herbivore pressure from large grazers. I also describe the multifaceted relationship between poison ivy and avian frugivores that not only disperse the drupes of poison ivy but also aid in reduction of fungal endophytic phytopathogens. A survey of poison ivy urushiols yielded that while variation in urushiol congeners was high across individuals, relative congener levels were stable within individuals over a two month period. Lastly I demonstrate best practices for introducing and transiently expressing recombinant DNA in poison ivy as a step towards future reverse genetic procedures.
- Discovery, Characterization, and Functional Analysis of micro RNAs in CulicidaeMead, Edward (Virginia Tech, 2009-05-13)MicroRNAs (miRNAs) are non-coding RNAs that often play a fundamental role in gene regulation. Currently, hundreds to over a thousand miRNAs are predicted to be present in many eukaryote species, with many to be discovered; the functions of most are unknown. While much attention has gone towards model organisms, a much greater depth of understanding remains to be gained for the miRNAs of many organisms directly important to humans. There are few verified miRNAs for any mosquito species, despite the role of mosquitoes in many of humanity’s worst diseases. Anopheles gambiae and Aedes aegypti, carriers of malaria and dengue, respectively, are responsible for over a million deaths a year. To date, there are sixty-six microRNAs in An. gambiae in miRBase, a central repository for miRNA sequences. Many of these are based on homology to primarily Drosophila miRNAs. While sequence conservation suggests an important function for these miRNAs, expression has not been experimentally verified for most mosquito miRNAs. Using small RNA cloning and northern blots, I discovered and analyzed 27 different microRNAs in aged female An. stephensi mosquitoes, the age group responsible for transmission of malarial parasites. Three of these miRNAs are only found in mosquitoes (miR-1889, -1890, and –1891). Cloning and northern analysis revealed an abundance of a miRNA that is linked to longevity in flies, miR-14, across different life stages of mosquitoes. It was also shown that miR-989 was expressed almost exclusively in the adult ovary and its expression fluctuated in response to bloodfeeding, suggesting a possible role in reproduction, an area of great importance to controlling mosquito populations. Building upon the above cloning experiment, a later high-throughput sequencing effort uncovered 98 miRNA precursors from Ae. aegypti. There are a total of 13 novel miRNAs that have not been found in other organisms by bioinformatic predictions or experiments. These “mosquito-specific” miRNAs may play a role in processes such as blood-feeding or vector-host interactions. A detailed examination of the expression of eight of these miRNAs was conducted in An. gambiae, An. stephensi, Ae. aegypti, and T. amboinensis to determine their expression profile, conservation, and provide hints to their function. My work revealed conserved and sometime stage-specific expression profiles of some of the mosquito-specific miRNAs. I also provided evidence for three lineage-specific miRNAs that may shed light on the divergence of different mosquito lineages. Extending the finding that miR-989 may be involved in mosquito reproduction, we conducted a detailed analysis of its evolution, expression, possible targets and regulation. miR-989 is conserved in holometabolous insects. miR-989 expression in female An. stephensi and Ae. aegypti dramatically rises following pupal emergence until strong signal is observed, until a blood meal is taken. Expression remains quite strong then begins a steep decline in expression at 32-40 hours post blood meal (PBM), and even by 96 hours PBM, remains weak. Bioinformatic predictions of miR-989 targets coupled with a PCR-based approach uncovered three potential target leads, though preliminary results were artifacts. Although the miR-989 post-emergence expression profile correlates with the expression of Juvenile Hormone, a key reproductive hormone in mosquitoes, no observable induction occurred when abdominal ligation samples were administered methoprene, a JH analog. However, methoprene impacted a number of other miRNAs, with up to a 3.87 fold induction (miR-1891), and a 3.15 fold suppression (miR-9a) of signal. Subsequent northern analysis provided visual confirmation of observable fold changes for miR-1891 and miR-9a, but not for miRNAs that showed changes below two fold. This analysis provides a foundation to study Juvenile Hormone regulation of miRNAs in mosquitoes. In summary, we have expanded the understanding of microRNAs in mosquitoes. An improved understanding of mosquito physiology can assist in efforts to control mosquito-borne infectious diseases.
- Dissecting Transcriptional Regulation of Rpp8 in Arabidopsis thalianaMohr, 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.
- Evolutionary history of the canary grasses (Phalaris, Poaceae)Voshell, Stephanie (Virginia Tech, 2014-06-12)Canary grasses (Phalaris, Poaceae) include 21 species widely distributed throughout temperate and subtropical regions of the world with centers of diversity in the Mediterranean Basin and western North America. The genus contains annual/perennial, endemic/cosmopolitan, wild, and invasive species with basic numbers of x=6 (diploid) and x=7 (diploid/tetraploid/hexaploid). The latter display vastly greater speciation and geographic distribution. These attributes make Phalaris an ideal platform to study species diversification, dispersal, historic hybridization, polyploidy events, and chromosome evolution in the grasses. This body of research presents the first molecular phylogenetic and phylogeographic reconstruction of the genus based on the nuclear ITS and plastid trnT-F DNA regions allowing species relationships and the importance of polyploidy in speciation to be assessed. Divergence dates for the genus were determined using Bayesian methods (BEAST, version 1.6.2) and historic patterns of dispersal were analyzed with RASP (version 2.1b). Self-incompatibility and the feasibility of hybridization between major groups within the genus were studied with a series of greenhouse experiments. Acetocarmine and fluorescent staining techniques were used to study the morphology of the chromosomes in a phylogenetic context and the nuclear DNA content (C values) was quantified using flow cytometry. Four major clades were revealed in the genus with cytological and geographic affinities leading to the establishment of two subgenera and four sections in the first comprehensive infrageneric treatment of Phalaris. Divergence dating revealed a Miocene emergence (20.6-8.4 MYA) for the genus which is concurrent with studies of other genera in the Aveneae tribe. The hypothesis stating that Phalaris originated in the Mediterranean Basin and dispersed to the New World via a western route leading to a secondary center of diversification in western North America was supported by phylogeographic and cytological analyses. An empirical study comparing the weight, length, and width of the florets by morphological type and cytotype revealed significant differences that support a dispersal advantage among the New World and Arundinacea species. The x=6 species displayed greater intraspecific C value variation, higher DNA content per haploid chromosome set, and a distinct karyotype compared with the x=7 species indicating a complex history of chromosome evolution.
- An expanding role for purine uptake permease-like transporters in plant secondary metabolismJelesko, John G. (Frontiers, 2012)For the past decade, our understanding of the plant purine uptake permease (PUP) transporter family was primarily oriented on purine nucleobase substrates and their tissue-specific expression patterns in Arabidopsis. However, a tobacco PUP-like homolog demonstrating nicotine uptake permease activity was recently shown to affect both nicotine metabolism and root cell growth. These new findings expand the physiological role for PUP-like transporters to include plant secondary metabolism. Molecular evolution analyses of PUP-like transporters indicate they are distinct group within an ancient super family of drug and metabolite transporters (DMTs). The PUP-like family originated during terrestrial plant evolution sometime between the bryophytes and the lycophytes. A phylogenetic analysis indicates that the PUP-like transporters were likely derived from a pre-existing nucleotide-sugar transporter family within the DMT super family. Within the lycophyte Selaginella, there are three paralogous groups of PUP-like transporters. One of the three PUP-like paralogous groups showed an extensive pattern of gene duplication and diversification within the angiosperm lineage, whereas the more ancestral PUP-like paralogous groups did not. Biochemical characterization of four closely related PUP-like paralogs together with model-based phylogenetic analyses indicate both subfunctionalization and neofunctionalization during the molecular evolution of angiosperm PUP-like transporters. These findings suggest that members of the PUP-like family of DMT transporters are likely involved in diverse primary and secondary plant metabolic pathways.
- Expression of recombinant porcine preprorelaxin in Nicotiana tabacumBuswell, Walter Scott (Virginia Tech, 2006-05-10)Relaxin is a small peptide hormone that has demonstrated potential therapeutic actions for cardiovascular disease and fibrosis. Additionally, relaxin has demonstrated the ability to protect the heart from injuries caused by ischemia and reperfusion, promote the healing of ischemic ulcers, and counteract allergic responses. The objective of this research was to express fully processed porcine relaxin in transgenic tobacco plants, as an alternative to current methods of producing relaxin. Two recombinant relaxin genes were constructed that contained the patatin signal peptide cDNA fused in frame to prorelaxin cDNA, which was codon-optimized for expression in Nicotiana tabacum, under the control of either the "super" promoter or the dual enhanced cauliflower mosaic virus 35S promoter. Eighteen transgenic tobacco plants were generated that were transformed with the above recombinant genes. Preprorelaxin, mRNA was detected in 12 of the transgenic plants. Fully processed relaxin protein was not found in any tobacco plants that had demonstrated gene expression by northern blot analysis. Preprorelaxin was only identified in extracts from transgenic plants that contained the insoluble protein fraction, as determined by western blot analysis. Additionally, an increased yield of preprorelaxin was identified after incubation of tobacco leaves in an ubiquitin inhibitor.
- The Genetic Basis of Phytate, Oligosaccharide Content, and Emergence in SoybeanGlover, Natasha M. (Virginia Tech, 2011-06-27)Soybean [Glycine max (L.) Merr] is one of the U.S.'s most economically important crops due to the protein and oil content of seeds. The major storage form of phosphorus in soybean seeds is found in the form of phytate, but because of its negative nutritional and environmental impacts, seed phytate and raffinosaccharide content have been a recent focus of breeders and molecular geneticists. The soybean line CX1834 is a low phytate mutant known to have two low phytate QTLs on linkage groups (LGs) L and N. The first objective of this research was to determine the genetic basis of the low phytate trait in CX1834. By using the whole genome sequence, we identified two candidate multidrug resistance-associated (MRP) ABC transporter genes. Sequencing the genes from CX1834 and comparing them to the reference genome sequence revealed a single nucleotide polymorphism (SNP) in the MRP gene located on LG N (causing a stop codon), and a SNP mutation in the MRP gene located on LG L (causing an amino acid change from arginine to lysine). One major concern with low phytate soybeans is the low seedling emergence. The second objective was to undertake a population-wide study of emergence in the recombinant inbred population CX1834 x V99-3337, over two years and two locations. We found a positive correlation between phytate level and emergence, and that variation among year, location, genotypic class, year x genotypic class, and year x location interactions were significantly affecting emergence. V99-5089, in addition to being low phytate, has high sucrose and low raffinosaccharide content. This phenotype of V99-5089 has been previously determined to be due to a SNP mutation in its myo-inositol phosphate synthase (MIPS) gene located on LG B1. The third objective was to use the recombinant inbred population derived from CX1834 x V99-5089 to observe the combinations of all three mutations to see how the different alleles impact phytate and raffinosaccharide content. The individuals with all three mutations, as well as those with the two MRP mutations together had lower phytate than the other genotypic classes. However, these lines (all three mutations) had unexpectedly high stachyose.
- Glutamate receptor homologs in plants: functions and evolutionary originsPrice, Michelle B.; Jelesko, John G.; Okumoto, Sakiko (Frontiers, 2012)The plant glutamate-like receptor homologs (GLRs) are homologs of mammalian ionotropic glutamate receptors (iGluRs) which were discovered more than 10 years ago, and are hypothesized to be potential amino acid sensors in plants. Although initial progress on this gene family has been hampered by gene redundancy and technical issues such as gene toxicity; genetic, pharmacological, and electrophysiological approaches are starting to uncover the functions of this protein family. In parallel, there has been tremendous progress in elucidating the structure of animal glutamate receptors (iGluRs), which in turn will help understanding of the molecular mechanisms of plant GLR functions. In this review, we will summarize recent progress on the plant GLRs. Emerging evidence implicates plant GLRs in various biological processes in and beyond N sensing, and implies that there is some overlap in the signaling mechanisms of amino acids between plants and animals. Phylogenetic analysis using iGluRs from metazoans, plants, and bacteria showed that the plant GLRs are no more closely related to metazoan iGluRs as they are to bacterial iGluRs, indicating the separation of plant, other eukaryotic, and bacterial GLRs might have happened as early on as the last universal common ancestor. Structural similarities and differences with animal iGluRs, and the implication thereof, are also discussed.
- Habitat Suitability and Establishment Limitations of a Problematic LianaDickinson, Christopher C.; Jelesko, John G.; Barney, Jacob (MDPI, 2021-01-29)The US native liana, poison ivy (Toxicodendron radicans), responsible for contact dermatitis in humans, is a competitive weed with great potential for expansion in disturbed habitats. To facilitate a better understanding of this threat, we sought to evaluate habitat suitability, population demography, and biotic interactions of poison ivy, using a series of complementary field studies in the two habitats where it most commonly occurs—forest interiors and edges. Of the 2500 seeds planted across both habitats, poison ivy initially colonized forest interiors (32% emergence) at a higher rate than edge habitats (16.5% emergence). However, forest interior seedlings were less likely to survive (interior n = 3; edge n = 15), which might be attributed to herbivore pressure when the seedlings were smaller in the less competitive forest interior. Once established, the poison ivy seedlings appeared to be more tolerant of herbivory, except that of large grazers such as deer. The early life stage of seedling emergence, survival, and establishment are critical in poison ivy success, with biotic pressure, especially from plant competition and deer, limiting recruitment. A suitable habitat of this expanding native liana would increase with increasing forest fragmentation, but might be buffered by the expanding deer population.
- Identification and Characterization of Late Pathway Enzymes in Phytic Acid Biosynthesis in Glycine maxStiles, Amanda Rose (Virginia Tech, 2007-07-30)Phytic acid, also known as myo-inositol hexakisphosphate or Ins(1,2,3,4,5,6)P6, is the major storage form of phosphorus in plant seeds. Phytic acid is poorly digested by non-ruminant animals such as swine and poultry, and it chelates mineral cations including calcium, iron, zinc, and potassium, classifying it as an anti-nutrient. The excretion of unutilized phytic acid in manure translates to an excess amount of phosphorus runoff that can lead to eutrophication of lakes and ponds. Understanding the phytic acid biosynthetic pathway will allow for the development of low phytic acid (lpa) soybeans by the down-regulation of specific genes. The goal of this research was to elucidate the pathway(s) for phytic acid biosynthesis in soybean (Glycine max). We have isolated several myo-inositol phosphate kinase genes in soybean as possible candidates for steps in the biosynthetic pathway. We have characterized the genes for four myo-inositol(1,3,4)P3 5/6-kinases (GmItpk1-4), one myo-inositol(1,4,5)P3 6/3/5-kinase (GmIpk2), and one myo-inositol(1,3,4,5,6)P5 2-kinase (GmIpk1). We have examined expression in developing seeds and other tissues by Northern blot analysis and quantitative RT-PCR. We have expressed all six genes as tagged fusion proteins in E. coli, and verified enzyme activity on the proposed substrates. For each enzyme, we have conducted biochemical characterization to determine enzyme kinetics and substrate specificities. We have verified in vivo activity of GmIpk2 and GmIpk1 by complementing yeast mutants in the respective genes. Our studies indicate the likelihood that three of the genes may be involved in phytic acid biosynthesis: GmItpk3, GmIpk2 and GmIpk1. For future work, to more fully understand the contribution of each kinase gene to phytic acid biosynthesis, an RNA interference approach will be employed. The gene sequences identified in this study will be used to construct silencing vectors for use in future transformation of soybean embryogenic cultures to determine the effects of down-regulation on myo-inositol phosphate profiles.