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Comparison Of Plant‐Adapted Rhabdovirus Protein Localization And Interactions, Kathleen Marie Martin

University of Kentucky Doctoral Dissertations

Sonchus yellow net virus (SYNV), Potato yellow dwarf virus (PYDV) and Lettuce Necrotic yellows virus (LNYV) are members of the Rhabdoviridae family that infect plants. SYNV and PYDV are Nucleorhabdoviruses that replicate in the nuclei of infected cells and LNYV is a Cytorhabdovirus that replicates in the cytoplasm. LNYV and SYNV share a similar genome organization with a gene order of Nucleoprotein (N), Phosphoprotein (P), putative movement protein (Mv), Matrix protein (M), Glycoprotein (G) and Polymerase protein (L). PYDV contains an additional predicted gene between N and P, denoted as X, that has an unknown function. In order to gain …

T-Phylloplanin And Cis-Abienol, Two Natural Products From Tobacco Have Broad Spectrum, Anti-Fungal Activities, Brian Christopher King

University of Kentucky Doctoral Dissertations

Tobacco phylloplanins (T-phylloplanin) are a group of closely-related glycoproteins that are formed and disposed at the interface between the plant aerial surface (the phylloplane) and the atmosphere. They are synthesized in short procumbent trichomes and are secreted to aerial surfaces where they are thought to serve the plant as a first line of defense against fungal pathogens. Here it is shown using in vitro and in planta assays that tobacco and sunflower phylloplanins have broad-spectrum antifungal activities against spores - and also hyphae for two species - of several true fungi. Field tests show that T-phylloplanin reduces diseases caused by …

Intermediate Steps Of Loline Alkaloid Biosynthesis, Jerome Ralph Faulkner

University of Kentucky Doctoral Dissertations

Epichloë species and their anamorphs, Neotyphodium species, are fungal endophytes that inhabit cool-season grasses and often produce bioprotective alkaloids. These alkaloids include lolines, which are insecticidal and insect feeding deterrents. Lolines are exo-1-aminopyrrolizidines with an oxygen bridge between carbons 2 and 7, and are usually methylated and formylated or acetylated on the 1-amine. In previously published studies lolines were shown to be derived from the amino acids L-proline and L-homoserine. In addition the gene cluster involved in loline-alkaloid biosynthesis has also been characterized. In this dissertation a survey of plant-endophyte symbioses revealed a phenotype with only N-acetylnorloline. This …

Role Of Lipids In Tombusvirus Replication, Monika Sharma

University of Kentucky Doctoral Dissertations

Positive-strand RNA virus group are the most abundant among viruses affecting plants and animals. To successfully achieve replication, these viruses usurp or co-opt host proteins. To facilitate the discovery of host factors involved in Tomato bushy stunt virus (TBSV), yeast has been developed as a surrogate model host. Genome-wide approaches covering 95% of yeast genes, has revealed approximately hundred factors that could affect virus replication. Among the identified host factors, there are fourteen yeast genes, which affect/regulate lipid metabolism of the host.

One of the identified host gene is ERG25, which is an important factor for sterol biosynthesis pathway, affecting …

Characterization Of G10h Promoter And Isolation Of Wrky Transcription Factors Involved In Catharanthus Terpenoid Indole Alkaloid Biosynthesis Pathway, Nitima Suttpanta

University of Kentucky Doctoral Dissertations

Catharanthus roseus produces a large array of terpenoid indole alkaloids (TIAs) that are an important source of natural or semi-synthetic anticancer drugs. Biosynthesis of TIAs is tissue-specific and induced by certain phytohormones and fungal elicitors, indicating the involvement of a complex transcriptional control network. However, the transcriptional regulation of the TIA pathway is poorly understood. This study reports the isolation and characterization of the G10H promoter and two WRKY transcription factors regulating TIA biosynthesis.

Geraniol 10-hydroxylase (G10H) controls the first committed step in the biosynthesis of terpenoid indole alkaloids (TIA). The C. roseus G10H promoter sequence was isolated by a …

Using Manual Defoliation To Simulate Soybean Rust: Effect On Growth And Yield Formation, Abdullah Mohammad Aqeel

University of Kentucky Doctoral Dissertations

Field experiments were conducted in Kentucky and Louisiana in 2008 and 2009 (split-plot in a randomized complete block design with four replications) to investigate it is possible to simulate with manual defoliation the effect of soybean rust (SBR) (Phakopsora pachyrhizi Syd. and P. Syd) injury on a healthy soybean [Glycine max, (L.) Merr.] canopy, understand how defoliation affects the growth dynamics and canopy light interception, and if defoliation affectsleaf senescence and nitrogen remobilization during the seed-filling period. Two manual defoliation treatments based on changes in effective leaf area index (ELAI) (calculated as the reduction in leaf area …

Restoration Of Tall Fescue Pastures To Native Warm Season Grasslands: Does A Fungal Endophyte Symbiosis Play A Role In Restoration Success?, Sarah Lynn Hall

University of Kentucky Doctoral Dissertations

Tall fescue, a cool-season grass native to Europe, central Asia, and northern Africa, has been widely distributed throughout the U.S. for use as turf and forage. Following its widespread planting, its ability to associate with a toxic fungal endophyte, Neotyphodium coenophialum, was discovered. Research has linked this fescue-endophyte association with increased biotic and abiotic stress resistance in endophyte-infected (E+) versus endophyte-free (E-) plants, and these differences may affect the ability of land managers to eradicate tall fescue and restore native grasslands. I conducted three studies to examine whether E+ tall fescue plants respond differently to management than E- plants, …

Molecular, Genetic And Biochemical Characterization Of Resistance Protein-Mediated Signaling Against Turnip Crinkle Virus, Rae-Dong Jeong

University of Kentucky Doctoral Dissertations

Infection of the resistant Arabidopsis ecotype Di-17 with Turnip Crinkle Virus (TCV) elicits hypersensitive response (HR), accompanied by increased expression of defense genes. HR to TCV is conferred by HRT, which encodes a coiled-coil (CC)-nucleotide-binding site (NBS)-leucine-rich repeat (LRR) class of resistance (R) protein. In contrast to HR, resistance requires HRT and a recessive locus designated rrt. Unlike most CC-NBS-LRR R proteins, HRT-mediated resistance is dependent on EDS1 and independent of NDR1. Resistance is also dependent on salicylic acid (SA) pathway and light. A dark treatment, immediately following TCV inoculation, suppresses HR, resistance and activation of a majority of the …

Evolutionary Perspective Of Nicotine To Nornicotine Conversion, Its Regulation And Characterization Of Ein2 Mediated Ethylene Signaling In Tobacco, Manohar Chakrabarti

University of Kentucky Doctoral Dissertations

Nicotine, nornicotine, anabasine and anatabine are four major alkaloids in tobacco, of which nicotine is predominant. In many tobacco cultivars and also in other Nicotiana species, nicotine is converted to nornicotine, which in turn gives rise to potent carcinogen NNN. Nicotine to nornicotine conversion via nicotine-N-demethylation is mediated by the CYP82E family of P450 enzymes. Tobacco (Nicotiana tabacum) converts in senescing leaves, while its diploid progenitors N.tomentosiformis and N.sylvestris convert in both green and senescing and only in senescing leaves, respectively. Previously it has been shown that N.tomentosiformis has different active conversion loci in green and senescing leaves. …

Improvement Of New Oil Crops For Kentucky, Watchareewan Jamboonsri

University of Kentucky Doctoral Dissertations

Three oil crops, chia (Salvia hispanica L.), flax (Linum usitatissimum L.), and castor (Ricinus communis L.), were studied because of their nutritional and industrial values. Chia and flax are rich in an ω3 fatty acid, α-linolenic acid, and castor is a very high oil producer and high in a hydroxy fatty acid. Ethyl methanesulfonate (EMS) and gamma rays were employed to mutagenize chia seeds to produce early flowering mutants. The M₁ population was grown and induced to flower by short-day photoperiods. The M₂ population was planted in the field in Lexington, KY in 2008. Early …

Modeling Water Use In Nursery Crops, Amy Fulcher

University of Kentucky Doctoral Dissertations

Water use is an important topic in the global agriculture community and is a critical input in nursery crop production. Several plants in the genus Cornus are important nursery crops. Not only are they economically relevant, they are found in grafted and seedling forms and parents and their hybrid are readily available in the trade, facilitating an assessment of water requirements. Anecdotal information suggests that Cornus taxa have differing stress tolerance and water use requirements. Research was conducted to characterize and model water use among Cornus taxa. Scanning electron microscopy and anatomy‐based micromorphological studies as well as transpiration chamber‐based studies …

Phylloplanins: Novel Antifungal Proteins On Plant Leaf Surfaces, Ryan William Shepherd

University of Kentucky Doctoral Dissertations

Secreted surface proteins are an innate immune defense component employed by animals to inhibit invading microbes. Surface proteins have not been documented in plants, even though the aerial leaf surface, or phylloplane, is a major site of pathogen ingress. We have discovered novel proteins, termed phylloplanins, which accumulate on leaf surfaces of Nicotiana tabacum, and we have isolated the gene Phylloplanin that is unique in gene databases. Natural and E. coli-expressed phylloplanins inhibit spore germination and limit leaf infection by the oomycete pathogen Peronospora tabacina.

We investigated the site of phylloplanin biosynthesis using biochemical techniques. These techniques …

Soil Water And Crop Growth Processes In A Farmer's Field, Susmitha Surendran Nambuthiri

University of Kentucky Doctoral Dissertations

The study was aimed to provide information on local biomass development during crop growth using ground based optical sensors and to incorporate the local crop status to a crop growth simulation model to improve understanding on inherent variability of crop field. The experiment was conducted in a farmer’s field located near Princeton in Caldwell County, Western Kentucky. Data collection on soil, crop and weather variables was carried out in the farm from 2006 December to 2008 October. During this period corn (Zea mays L.) and winter wheat (Triticum sp) were grown in the field. A 450 m …

Grazing Evaluation Of A Novel Endophyte Tall Fescue Developed For The Upper Transition Zone, Jennifer Michelle Johnson

University of Kentucky Doctoral Dissertations

A wild-type endophyte (Neotyphodium coenophialum [(Morgan-Jones & Gams) Glen Bacon & Hanlin]) that infects tall fescue [Lolium arundinaceum (Schreb.) Darbysh. = Schedonorous arundinaceum (Schreb.) Dumort.] imparts tolerances to moisture, heat, and grazing stresses, but also produces ergot alkaloids that adversely affect performance and physiology of cattle. Novel endophytes, developed by AgResearch Ltd. NZ, can sustain fescue persistence and productivity, but do not produce toxic ergot alkaloids. University of Kentucky Plant Breeder, T. D. Phillips Ph.D, developed a tall fescue experimental population (KYFA9301) for the upper transition zone. A 2-yr grazing experiment was conducted with steers to evaluate steer …

Evaluating The Effects Of Organic And Conventional Inputs On Soil Chemical And Biological Properties In A Four-Year Vegetable Rotation And The Investigation Of Soil Microbial Properties On Plant Gene Expression, Audrey Law

University of Kentucky Doctoral Dissertations

The objective of this research was to determine the effects of conventional inputs on soil chemical and biological properties compared to organic systems in a four year vegetable rotation. Tillage and cover crops were the same in all treatments to avoid confounding factors often present in similar research. Additional experiments investigated plant gene expression in organic and conventional management systems and in soils with decreased microbial diversity. Experimental plots were prepared in the spring of 2004; four replications of three management treatments, organic, low-input and conventional, were arranged in a randomized complete block design. The rotation consisted of edamame soybean, …

Role Of P33 In Tombusvirus Replication, Jozsef Stork

University of Kentucky Doctoral Dissertations

Replication of the nonsegmented, plus-stranded RNA genome of Cucumber necrosis tombusvirus (CNV) requires two essential overlapping viral-coded replication proteins, the p33 replication co-factor and the p92 RNA-dependent RNA polymerase. In my thesis I describe (i) the effect of phosphorylation of p33, (ii) the RNA chaperone-like activity of p33, and (iii) the role of HSP70s a host proteins in the viral replication. To test the effect of phosphorylation on p33 function, I used in vitro phosphorylated p33. I found that phosphorylation inhibited the ability of p33 to bind to the viral RNA. Phosphorylation-mimicking mutations rendered p33 nonfunctional in plant protoplasts and …

Identification And Characterization Of Host Factors Involved In Tombusvirus Replication, Yi Jiang

University of Kentucky Doctoral Dissertations

Positive strand RNA viruses are intracellular parasites, and their genome replication and infection involves complex virus-host interactions. Therefore, identification of host factors and dissection of their functions during virus replication could facilitate our understanding of the mechanism of virus infection. Those host factors may also provide new targets for viral disease control. Tomato bushy stunt virus (TBSV) has recently become one of the model viruses to study positive strand RNA virus replication and hostvirus interactions. To identify host factors involved in TBSV replication we used yeast as a model host. Co-expression of the replication proteins and a replicon RNA (DI …

Towards Elimination And Genetic Manipulation Of Ergot Alkaloid Production In Fungal Endophytes, Simona Florea

University of Kentucky Doctoral Dissertations

Clavicipitaceous fungal endophytes provide several ecological benefits to their hosts. Besides improving host’s growth characteristics, Neotyphodium coenophialum, the endophyte of tall fescue (Lolium arundinaceum), produces ergot alkaloids that have been proposed to be involved in fescue toxicosis. One approach to address the toxicosis problem is to genetically manipulate and modify N. coenophialum by knocking out a pair of homologous genes, (dmaW1 and dmaW2), encoding dimethylallyltryptophan synthase, the enzyme for the first and determinant step in ergot-alkaloid biosynthesis. In this study, disruption of dmaW2 was attempted using several disruption methods. Out of 1522 transformants screened, three …

Comparative Biology Of Seed Dormancy-Break And Germination In Convolvulaceae (Asterids, Solanales), Kariyawasam Marthinna Gamage Gehan Jayasuriya

University of Kentucky Doctoral Dissertations

The biology of seed dormancy and germination of 46 species representing 11 of the 12 tribes in Convolvulaceae were compared in laboratory (mostly), field and greenhouse experiments. Seeds were tested for kind of dormancy and storage behavior; artificial or simulated natural treatments were applied to break physical dormancy (PY); the initial route of water entry (“water gap”) into seeds was identified; the morphoanatomy of the water gap was compared in seeds of 17 species; ontogenetical differences between water gap and seed coat away from the hilum were described in Ipomoea lacunosa seeds; cycling of sensitivity to dormancy break was elucidated …

Map-Based Cloning Of An Anthracnose Resistance Gene In Medicago Truncatula, Shengming Yang

University of Kentucky Doctoral Dissertations

Anthracnose, caused by the fungal pathogen Colletotrichum trifolii, is one of the most destructive diseases of alfalfa worldwide. Cloning and characterization of the host resistance (R) genes against the pathogen will improve our knowledge of molecular mechanisms underlying host resistance and facilitate the development of resistant alfalfa cultivars. However, the intractable genetic system of cultivated alfalfa, owing to its tetrasomic inheritance and outcrossing nature, limits the ability to carry out genetic analysis in alfalfa. Nonetheless, the model legume Medicago truncatula, a close relative of alfalfa, provides a surrogate for cloning the counterparts of many agronomically important genes in …

Roles Of Micrornas In Plant Abiotic Stress, Development And Viral Infection, Venugopal Mendu

University of Kentucky Doctoral Dissertations

Plant microRNAs play important roles in plant growth and development. Here we investigated the roles of miRNAs in the plant abiotic stress, development and viral infection. MicroRNA membrane array analysis using five different abiotic stress treatments resulted in the identification of 8 novel stress inducible miRNA-families. Functional studies on novel stress inducible miR168 revealed its functional relation with abiotic stress. Over expression of miR168 in Arabidopsis showed upregulation of four stress related miRNAs (miR163, miR167, miR398 and miR408). Analysis of 9 independent transgenic lines showed induction of miR398, an oxidative stress responsive miRNA with a corresponding down regulation of its …

Microbial Community Structure Dynamics In Ohio River Sediments During Reductive Dechlorination Of Pcbs, Andres Enrique Nunez

University of Kentucky Doctoral Dissertations

The entire stretch of the Ohio River is under fish consumption advisories due to contamination with polychlorinated biphenyls (PCBs). In this study, natural attenuation and biostimulation of PCBs and microbial communities responsible for PCB transformations were investigated in Ohio River sediments.

Natural attenuation of PCBs was negligible in sediments, which was likely attributed to low temperature conditions during most of the year, as well as low amounts of available nitrogen, phosphorus, and organic carbon. Moreover, surface sediments were relatively oxidized, as indicated by the prevalence of aerobic bacteria such as beta- Proteobacteria, alpha-Proteobacteria, Sphingobacteria, and Nitrospira in 16S rRNA sediment …

Localization Of Diplodia Pinea In Diseased And Latently-Infected Pinus Nigra, Jennifer Lee Flowers

University of Kentucky Doctoral Dissertations

Diplodia pinea causes Diplodia tip blight on more than 30 different pine species. During the past 10 years, Diplodia tip blight has emerged as a serious problem in landscape and Christmas tree farms in this region. Surveys of diseased and symptomless Austrian pines revealed that latent infections of symptomless shoots by D. pinea were common. Latent infections may account for the recently observed rapid decline of mildly diseased pines in our region. To investigate the colonization habits of D. pinea within its host, molecular cytology was attempted and traditional histology was performed on naturally infected, diseased and asymptomatic Austrian pine …

Loline Alkaloid Biosynthesis In Neotyphodium Uncinatum, A Fungal Endophyte Of Lolium Pratense, Jimmy Douglas Blankenship

University of Kentucky Doctoral Dissertations

Some endophytes in mutualistic associations with Festuca, Lolium and other grass species produce insecticidal loline alkaloids (1-aminopyrrolizidines; LA). These loline alkaloids have a saturated pyrrolizidine ring system (two-rings sharing a carbon and nitrogen atom), a 1-amine substituted with methyl, acetyl, or formyl groups, and an oxygen bridge between C-2 and C-7. The development of a reliable system of production of LA in cultures of the Lolium pratense (meadow fescue) endophyte, Neotyphodium uncinatum, facilitated work on the LA biosynthetic pathway. N. uncinatum produced norloline, loline, methylloline, N-acetylnorloline (NANL), N-formylloline (NFL), and N-acetylloline as detected in culture filtrates. The total production …