Master of Arts (MA), Bowling Green State University, 1950, Biological Sciences

Committee: R. Thelma D'Almaine (Advisor) Subjects: Biology

Doctor of Philosophy, The Ohio State University, 2021, Plant Pathology

Agrobacterium is a gram-negative soil bacterium and the causal agent of plant diseases such as crown gall and hairy root disease (HRD) in a variety of dicotyledonous plants. Plant pathogenic agrobacteria harbor a virulence plasmid (i.e., tumor-inducing or root-inducing) that contains a region of DNA that is transferred (also known as the T-DNA) from the bacteria into the cell of a host plant where it is integrated into the host cell's chromosomes. After T-DNA incorporation, the transformed plant cell(s) grow and divide due to genes encoded on the T-DNA that alter cellular and metabolic pathways that lead to tumor or transgenic root formation. Through rigorous research, the transformative power of agrobacteria was harnessed to become a useful tool for plant transformation, enabling the modification and/or insertion of useful traits into crop plants. My graduate work was focused on the biology and management of wild species of Agrobacterium rhizogenes, the causal agent of HRD on many vegetable crops including cucumber, tomato, eggplant, and pepper. Wild species of A. rhizogenes harbor a root-inducing (Ri) virulence plasmid that induces over proliferation of transgenic adventitious roots. These transgenic roots alter the plants' ability to maintain stable shoot/root relationships to favor root over-proliferation in lieu of shoot and/or fruit production. Today, HRD is a common problem in the hydroponic greenhouse industry where crops such as tomatoes, cucumber, eggplants, and peppers are grown. HRD has been present for many years in countries including the United Kingdom, France, Austria, Belgium, Denmark, Greece, the Netherlands, Poland, Switzerland, and Russia. Recently, the presence of the disease has been reported in Japan, South Korea, New Zealand, Canada, and now in hydroponic vegetable production systems in the USA. The first set of objectives for my research was to identify and characterize Pseudomonas strains that could be used as biocontrol agents for HRD (open full item for complete abstract)

Committee: Christopher Taylor (Advisor); Sally Miller (Committee Member); Michelle Jones (Committee Member); Ye Xia (Committee Member); Guo-Liang Wang (Committee Member) Subjects: Plant Pathology

Doctor of Philosophy (PhD), Ohio University, 2020, Chemistry and Biochemistry (Arts and Sciences)

Extensin peroxidases play a critical role in plant cell growth and are believed to play equally important roles in defense from pathogenesis and mechanical stress. By catalyzing the covalent polymerization of extensin proteins, they participate in the formation of the cell plate for cell division and help to reinforce the wall—preventing pathogen infection. Due to it's anionic character and catalytic processivity, TomEP is a particularly unique extensin peroxidase that requires much less time and enzyme than other extensin peroxidases to crosslink extensin substrate. Previous work identified the TomEP gene, and established methods to produce functional enzyme through heterologous expression in E. coli. This work aimed to expand upon these previous efforts by characterizing TomEP expression, TomEP function in vivo, and design a purification scheme to produce milligram-level quantities of pure enzyme for crystallization. An expression profile of TomEP was compiled using both qPCR analysis and promoter-GUS fusion experiments to provide data describing normal expression and response to wounding. Basal TomEP expression was demonstrated to be significantly higher in roots than in flowers, stems, or leaves. Through the same methods, wounding treatments were shown to increase TomEP expression in tomato roots from one to four hours, followed by attenuation for the following sixteen hours. The foundations of gain and loss-of-function experiments were pursued in an attempt to discern TomEP's influence on di-isodityrosine and pulcherosine content in tomato cell walls, using overexpression and CRISPR knock-out strategies. Overexpression lines of tomato and Arabidopsis were generated using Agrobacterium mediated methods, though these efforts failed to produce verifiable protein product, despite expression being observed on the RNA level. Transient expression in tobacco epidermal cells was successful however, allowing for in vivo analysis of TomEP activity, though no clea (open full item for complete abstract)

Committee: Michael Held II (Advisor); Marcia Kieliszewski (Committee Member); Showalter Allan (Committee Member); McMills Lauren (Committee Member) Subjects: Biochemistry; Botany; Plant Biology; Plant Sciences

Doctor of Philosophy, The Ohio State University, 1966, Graduate School

Committee: Not Provided (Other) Subjects: Biology

Doctor of Philosophy, Case Western Reserve University, 2015, Biology

The Linum Insertion Sequence (LIS-1) is a unique element appearing as a site-specific insertion event in flax responsive lines responding to certain growth conditions and can be transmitted to subsequent generations. LIS-1's origin is uncertain, but one theory is that LIS-1 is assembled from small sequences found scattered throughout the genome, and inducing (stress) conditions can result in these sequences rearranging and joining together to build the full LIS-1 sequence, which is inserted into the “target site.” This study's primary question was to determine whether LIS-1 was a unique event to the flax family or would it have similar behavior in other plant species. Therefore, this work focused to develop transgenic plants containing LIS-1 in their genome and to follow LIS-1 stability and inheritance in subsequent generations. The first part of the project focuses on Arabidopsis thaliana. The rationale was to determine whether LIS-1 is mobilized in other plants through the T1 and T2 generations. Different accessions of Arabidopsis thaliana were transformed with either LIS-1 or with its target site, using the well-established floral dipping method via Agrobacterium. In the second part of the project, the main question was how flax varieties deal with an intact “exogenous” LIS-1. A novel technique of transforming flax using Agrobacterium via floral dipping was developed to transform an “exogenous” LIS-1 to flax varieties. The fate was followed in the T1 and T2 generations under different growth conditions. Both projects show that “exogenous” LIS-1 is not found intact in any transformed plants. However, the results of LIS-1 transformation into flax were consistent with LIS-1 transformation into the Arabidopsis thaliana. In both plants, the regions flanking LIS-1 in the T-DNA construct were converted back to the flax's progenitor target site in all T1 generations, followed by either complete excision or stability of this form in the T2 generations. The results (open full item for complete abstract)

Committee: Christopher Cullis (Advisor); Stephen Haynesworth (Committee Member); Emmitt Jolly (Committee Member); Ahmad Khalil (Committee Member); David Burke (Committee Member); Ryan Martin (Committee Chair) Subjects: Biology; Genetics; Molecular Biology

Master of Science, The Ohio State University, 2014, Plant Pathology

Developing new tools that increase plant transformation efficiencies are needed for continued improvement of transgenic crop technology. Farmers are expected to plant an all-time record number of acres of soybeans in the United States in 2014. Approximately 90% of these acres will consist of genetically engineered (GE) soybeans. (www.ers.usda.gov). Most transgenic plants today are transformed using a lab modified soil-borne bacterium, Agrobacterium tumefaciens, which is known for its unique ability to transfer a piece of DNA (T-DNA) into the genome of its host plant. Removing the genes in the T-DNA region (also known as disarming) and using a vector containing gene sequences of particular interest, such as glyphosate resistance, field crops, i.e. Roundup Ready soybeans, have been developed and extensively deployed. Most Agrobacterium-mediated transformations to date have utilized a single strain of Agrobacterium called C58, which while good for transforming some crops, i.e. tobacco, can be inefficient for use in transforming recalcitrant crops, such as soybean. Currently there are many partially characterized wild-type strains of Agrobacterium spp. that have not been fully tested for use in plant transformations. In addition to these strains, soil samples from soybean fields in Ohio and nationwide, are being processed to isolate potential novel strains for further testing. From these strains a few will be selected for disarming with the intention of increasing plant transformation efficiencies.

Committee: Christopher Taylor (Advisor); Sally Miller (Committee Member); John Finer (Committee Member) Subjects: Agriculture; Plant Biology; Plant Pathology; Plant Sciences

Master of Science, The Ohio State University, 2014, Horticulture and Crop Science

Many laboratories routinely use Agrobacterium for the generation of transgenic plants; however, only a few disarmed bacterial strains are widely available and none of these strains were isolated and selected based on evaluation of transformation efficiency in target species. For this research, new Arobacterium strains were isolated from crown galls of various plants in Ohio and from rhizospheric soil throughout the Midwest US. These wild-type strains were isolated by plating gall and soil extracts on a semi-selective medium and screening the isolates for the presence of virG using PCR. The efficiency of plant transformation was evaluated by transforming hypocotyl and cotyledonary tissues of sunflower and soybean seedlings and in proliferative embryogenic tissue of soybean and then quantifying GFP expression. In sunflower, seedling cotyledonary tissue was not responsive to any of the strains tested, however hypocotyl tissues were very responsive with the highest transformation rates obtained with EHA105. With sunflower hypocotyl tissues transformed with disarmed strain EHA105, greater than 75% of the transformed cells were located in the vascular tissues. In soybean seedling tissues, tissue-specific transformation was not observed with any strain as transformed cells were evenly distributed throughout target tissue. With soybean hypocotyl, cotyledon, and embryogenic tissues, a single strain from a soybean field in North Dakota gave 5-10x higher transformation rates than EHA105, while a strain from Ohio soil gave 3-5x higher rates than EHA105.

Committee: John Finer (Advisor); Leah McHale (Committee Member); Finer Kim (Committee Member) Subjects: Plant Sciences

Master of Science in Biological Sciences, Youngstown State University, 0, Department of Biological Sciences and Chemistry

Penicillium marneffei is a notorious, medically pertinent fungal pathogen responsible for causing penicilliosis within immunocompromised individuals, particularly those with HIV/AIDS. Existing as a mold at 25°C and yeast at 37°C, it is the only Penicillium species known to undergo a temperature-dependent dimorphic switch. Recently, Agrobacterium tumefaciens-mediated transformation (ATMT) was used to introduce randomly integrated T-DNA fragments into the P. marneffei genome. This study sought to genotypically and phenotypically characterize mutant I189. An inverse PCR protocol was employed to recover the DNA sequences flanking the T-DNA insertion site. BLAST analysis revealed an interruption of a putative ubiquitin fusion degradation protein (UfdB) mRNA. Orthologous in the human pathogen Aspergillus fumigatus as well as model yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, this protein has been documented to tag abnormal stress-induced proteins for degradation. Current investigations of ufdB in P. marneffei mutant I189 show that fungal growth is significantly diminished in the presence of nutrient limitation although a full mRNA transcript is being produced. However, exposure to Congo Red, sodium dodecyl sulfate (SDS), and heat stress had no effect on cell viability.

Committee: Chester R. Cooper, Jr. PhD (Advisor); David K. Asch PhD (Committee Member); Jonathan J. Caguiat PhD (Committee Member); Gary R. Walker PhD (Committee Member) Subjects: Bioinformatics; Biology; Microbiology; Molecular Biology; Statistics

Master of Science in Biological Sciences, Youngstown State University, 2012, Department of Biological Sciences and Chemistry

The dimorphic fungus Penicillium marneffei is an etiological agent of penicillosis and the third leading cause of AIDS related death in Southeast Asia. This fungus is unique to Penicillium species in that it displays thermal dimorphism. Specifically, at 25°C P. marneffei grows as a filamentous mold, whereas at 37°C hyphae or conidia differentiate into fission yeast. One of the key factors to this organism's pathogenicity is the dimorphic switch that it performs in vivo to establish an infection. Mutagenesis with focus on morphology has been used to discover certain pathways linked to dimorphism in P. marneffei. The technique of Agrobacterium-mediated transformation was used to produce random T-DNA insertion mutants. From this prior work, two mutants with outward phenotypic defects were chosen to be characterized. Initially, the mutants were characterized phenotypically by slide culture techniques to note any differences in morphology as compared to the wild type at 25°C and 37°C. The disrupted genes of each mutant were identified by amplification of the flanking regions of the T-DNA insertion through inverse PCR and sequencing. These results indicated that mutant I209 possessed a defect in the septin gene, designated aspC, whereas the mutation in the second mutant, strain I219, was found to be in the gene encoding 3-methylcrotonyl CoA carboxylase (MccB). The expression of the interrupted gene in mutant I209 was verified using RT-PCR. Verification of a single T-DNA insertion was confirmed by Southern blot analysis.

Committee: Chester R. Cooper Jr. PhD (Advisor); David K. Asch PhD (Committee Member); Jonathan Caguiat PhD (Committee Member) Subjects: Biology

Master of Science, The Ohio State University, 2012, Microbiology

Histoplasma capsulatum is a pathogenic fungus endemic to the Ohio and Mississippi River valleys. Infection of mammals by Histoplasma causes respiratory histoplasmosis of varying severity which can lead to disseminated life-threatening disease. Upon inhalation into the mammalian lung, Histoplasma yeast are taken up by alveolar macrophages in which they replicate and ultimately lyse the host immune cells. Histoplasma's virulence in these phagocytes and the inability of the immune system to control the infection highlights the need to understand the mechanisms underlying the pathogenicity of this fungus. We sought to identify Histoplasma genes required for survival and growth in macrophages. We optimized procedures for Agrobacterium-mediated transformation of Histoplasma to facilitate insertional mutagenesis, and developed a simple and efficient screen for Histoplasma mutants unable to lyse host macrophages. We identified 14 mutants from 6500 that had decreased virulence in phagocytes. One of the loci identified is a heat shock protein 90 homolog (HSP82) which we show is instrumental for Histoplasma yeast to adapt to infection-associated stresses. A second identified locus, a riboflavin biosynthesis enzyme (RIB2), indicates that de novo vitamin biosynthesis is required for fungal proliferation within host cells. Murine infections with these mutants confirm each is necessary for full Histoplasma virulence in mammalian hosts. The attenuation of the rib2::T-DNA mutant suggests that the mammalian phagosome is a vitamin-limiting environment. Using folate biosynthesis inhibitors to arrest intracellular Histoplasma growth, we show that Histoplasma also requires de novo synthesis of folate intermediates. Together, these data demonstrate that de novo vitamin biosynthesis enables Histoplasma yeast to replicate in the nutrient-limiting macrophage phagosome and highlights vitamin biosynthetic pathways as potential therapeutic targets for treatment of histoplasmosis.

Committee: Chad Rappleye (Committee Chair); Birgit Alber (Committee Member); Charles Daniels (Committee Member); Daniel Wozniak (Committee Member) Subjects: Genetics; Microbiology; Molecular Biology; Parasitology