Ph.D. Biotechnology
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Item Embargo The bipartite function of Clade I TGAs: involvement in basal immunity and systemic acquired resistance(Brock University) Barraco, Charles; Centre for BiotechnologyWhen pathogens attack, each plant cell must mount its defense response against the pathogen. Understanding the process of pathogenic infiltration, detection, signalling, and defense is required to produce more resistant crop species and understand the complexities involved. The transcription factor family TGAs contains two members, TGA1/4, which were found to be basal immunity and systemic acquired resistance in plants by large-scale transcriptomic analysis. TGA1/4 were found to negatively regulate WRKYs 15, 40, and 48 genes, which are negative regulators of basal immunity involved in the growth and immunity tradeoff controlled by the phytohormone Brassinoteroid. TGA1/4 was found to directly associate with each WRKY gene promoter and negatively regulate their expression. As a proxy to basal immunity, reactive oxygen species were quantified in COL-0 and tga1tga4 double mutant lines, showing a reduction in ROS expression in the mutant lines when treated with the brassinosteroid-pathway inhibitor, brassinazole. Additionally, TGA1/4 was found to form a repressosome with GRX480 and NPR1 to negatively regulate early-SAR inducible genes, like the SAR marker gene PR-1, by TGA1/4 and GRX480 directly interacting with PR-1 gene promoter. How TGA1/4 mechanistically regulates basal immunity, and SAR is not entirely known, in part due to the lack of structural information about TGA family members as a whole. In silico derived TGA1 bZip homodimer-DNA 3D model, developed through molecular dynamics simulations and alanine scanning, identified essential amino acids required for TGA1-DNA association and stabilization. Some residues outside the range of DNA interaction are critical in DNA stabilization, namely Y106, V107 and E111. Through EMSA experiments TGA1 WT and mutant-dsLS7 dissociation constant (KD) values were determined, showing TGA1-DNA K¬D to be about 22 nM, with a melting temperature of 66.5°C. Continued development of the TGA1-DNA model is required. However, the current model was successfully validated. Taken together, clade I TGAs possess bipartite functions within plant immunity, now being rooted in both the growth and immunity tradeoff and early-SAR induction.Item Open Access The Role of CRISPR-Mediated Phage Resistance in the Development of Phage-Based Biocontrol for Erwinia amylovora(Brock University) Parcey, Michael; Centre for BiotechnologyIn the post-antibiotic era, resistance in pathogenic bacteria is projected to significantly hinder crop production and become one of the leading causes of death. This has necessitated the development of therapies to address antibiotic resistant microbes and prolong the period for which antibiotics remain a viable treatment option. A prominent alternative technology that has recently re-emerged is the use of bacterial viruses known as phages. Phages selectively lyse their bacterial hosts during the replication process but must avoid phage resistance mechanisms to eliminate a bacterial population. In this dissertation, the impact of phage resistance on biocontrol efficacy is examined using the phytopathogen Erwinia amylovora. The primary source of acquired phage immunity in bacteria is the CRISPR-Cas system. However, the absence of methodologies to study Erwinia phages, and a lack of genomic data for E. amylovora, has previously hindered this avenue of research. Quantitative real time PCR assays were developed to simultaneously monitor both the E. amylovora and phage populations. The individual steps of the phage lytic cycle during infection were characterized by further modification of this methodology. Through this, phage candidates ΦEa46-1-A1 and ΦEa21-4, that previously demonstrated high biocontrol potential, were shown to produce a large number of progenies over a short period of time. A comparative genomic analysis using 127 sequenced isolates of E. amylovora was then completed. This study proposed three primary clades of E. amylovora which infect apples in North America. A novel bioinformatic pipeline was subsequently developed to analyse the CRISPR regions of E. amylovora and the activity of the CRISPR-Cas system was then confirmed. While each clade of E. amylovora exhibited a unique CRISPR arrays, none of the identified CRISPR spacers provided inherent protection against any biocontrol candidate. CRISPR-mediated phage resistance was confirmed in E. amylovora against biocontrol candidate ΦEa21-4 but only in isolates with primed CRISPR-Cas systems. Still, phage resistance to ΦEa21-4 was observed through an unknown resistance mechanism in wild-type isolates. Overall, this work demonstrates new techniques to improve trial outcome prediction and lays the foundation for further investigation into the phage resistance mechanisms of E. amylovora.Item Open Access (A). Regulation of B-/Z-DNA transition in modified oligonucleotides; (B). Synthesis of cationic BODIPY analogues for antimicrobial and anticancer applications(Brock University) Joshi, Dhruval Kumar; Centre for BiotechnologyA. In this work, LNA-dG was incorporated into d(CG)6 sequence in a site-specific manner through the phosphoramidite chemistry-based solid phase synthesis to investigate the impact of this modification on the B→Z-DNA transition. Circular dichroism study showed that the incorporation of a single LNA-dG unit into d(CG)6 at internal positions virtually suppressed Z-DNA formation at 4 M NaCl concentration, whereas the presence of single LNA-dG unit towards the terminal ends showed only partially inhibition of B→Z-DNA transition. To further understand the influence of chemical modification on B→Z-DNA transition, modification at C8-position on LNA-dG residue was explored. Towards this goal, compounds such as 8-bromo-5′-dimethoxytrityl-N-dimethylformamidine-(2′-O,4′-C-methylene)-guanosine-3′-O-(2-cyanoethyl)-N,N-diisopropy phosphoramidite and 8-bromo-2′-deoxy-5′-O-dimethoxy-N-[(dimethylamino)methylene]-2′-fluoroguanosine-3′-O-(2-cyanoethyl)-N,N-diisopropyl phosphoramidite were synthesized. B. In this study, N,N,N-trimethyl-2-(4,4-difluoro-2,6-diiodo-4-bora-3a,4a-diaza-s-indacen-8-yl) ethylammonium iodide 111 was successfully synthesized. Singlet oxygen generation experiments suggested that BODIPY 111 is ~2.5 times more efficient with respect to Rose Bengal (RB) in generating singlet oxygen. However, based on the cell-culture experiment, BODIPY 111 treated culture plates showed only slight reduction in CFUs compared to Rose Bengal and control plates. BODIPY compounds 107 (iodinated-fluoro-BODIPY), 130 (brominated-fluoro-BODIPY) and 110 (brominated-meso-dimethylamine-fluoro-BODIPY) showed excellent stability both in dark and in light conditions and were found to be very efficient in generating singlet oxygen when compared to RB.Item Open Access De novo sequencing, annotation, and characterization of the genome of Lavandula angustifolia (Lavender)(Brock University) Nattamai Malli Pooranachandhiran, Radesh; Centre for BiotechnologyLavender (Lavandula angustifolia) is a perennial plant native to the Mediterranean region, best known for its essential oil (EOs) that have numerous applications in the pharmaceutical, cosmetic and perfume industries. We performed sequencing of the L. angustifolia genome and report a detailed analysis of the assembled genome, focusing on genome size, ploidy, and repeat content. The lavender genome was estimated to be around 870 Mbp (1C=0.96 pg) using a quantitative PCR method. Genome size was further validated through analysis of raw genome sequences using Kmergenie, providing a conclusive end to the lavender genome size dispute. The repeat element composition of the genome was analyzed using de novo (RepeatModeler) and library-based methods (RepeatMasker) and was estimated to be around 45% of the full genome or ~57% of the non-gap genome sequences. Further characterization revealed Long Terminal Repeat (LTRs) retrotransposons as the major repeat type, which contribute to ~18% of the genome, followed by DNA transposons at ~8.5% of the genome. Interestingly, unlike most other plant genomes, the lavender genome has many more Copia than Gypsy elements, both showing a trend of recent increasing activity. Furthermore, these LTRs, especially Copia elements, have shown active participation in gene function including genes for essential oil production, with Copia elements contributing to ~30 % of the coding DNA sequence (CDS) regions, in addition to promoter, intron and untranslated (UTR) regions. The lavender genome also has an unusually high number of miniature inverted-repeat transposable elements (MITEs) compared to other model plant genomes, with the number being ~88,000, which is close to that (~90,000) of the much larger maize genome. Analysis also revealed the lavender genome with a high proportion at polyploidy level, which is strongly biased towards regions containing essential oil genes, with polyploidization events in the lavender genome occurred between 16 to 41 Mya. In conclusion, our results reveal the lavender genome to be highly duplicated and with past and ongoing active retrotransposition, making the genome optimized for EO production.Item Open Access Discovery of a novel cytochrome P450, (+)-vincadifformine 19-hydroxylase (V19H), distinguishes separate branch pathways forming aspidosperma-type monoterpenoid indole alkaloids in Catharanthus roseus roots(Brock University) Williams, Danielle; Centre for BiotechnologyInvestigation of Catharanthus roseus monoterpenoid indole alkaloid (MIA) biosynthesis and accumulation has been important in elucidating the formation of the antineoplastic drugs, vinblastine and vincristine. These pharmaceuticals are formed by the condensation of the MIAs catharanthine and vindoline, which accumulate in C. roseus leaves. While we had completed and expressed the seven-step pathway from the aspidosperma-type MIA (-)-tabersonine to vindoline in yeast, little was known about the reactions involved in the metabolism of aspidosperma-type MIAs in roots. C. roseus roots convert (-)-tabersonine to lochnericine, the precursor for a major root alkaloid hörhammericine, and the reasons for the production of different aspidosperma MIAs in above and below ground plant organs is unknown. The molecular and biochemical characterization of minovincinine-19-O-acetyltransferase (MAT), tabersonine-19-hydroxylase (T19H), tabersonine-6,7-epoxidase (TEX1/2), and tabersonine 19-O-acetyltransferase (TAT) suggests that biosynthesis of hörhammericine and its derivative, 19-O-acetyl-hörhammericine, involves an ordered series of reactions. Bioinformatic analysis led to the identification of a root specific homolog of tabersonine-3-oxygenase (T3O), a cytochrome P450 (P450) involved in the formation of tabersonine 2,3-epoxides, as part of the vindoline pathway in leaves. Characterization of the T3O-homolog revealed that it converts (+)-vincadifformine to its 19-hydroxyderivative, (+)-minovincinine, and it was named (+)-vincadifformine 19-hydroxylase (V19H). V19H did not accept (-)-tabersonine or tabersonine-derived (-)-vincadifformine. T19H, another root-specific P450, hydroxylates (-)-tabersonine and its derivatives, including (-)-vincadifformine, to their respective 19-hydroxyderivatives, but does not accept (+)-vincadifformine. TAT will only acetylate the (-)-tabersonine derivatives, whereas MAT only turns over the (+)-vincadifformine derivative to form (+)-echitovenine. This shows that two distinct aspidosperma pathways exist in C. roseus since endogenous vincadifformine must be the (+)-enantiomer instead of the tabersonine derived (-)-vincadifformine. Modelling studies revealed that V19H activity is competitively inhibited by (-)-vincadifformine, suggesting that the (-)-aspidosperma backbone could still be incorporated into the binding site, albeit without hydroxylation. Models of T3O and T19H were generated to compare their binding pockets with that of V19H, and there were four conserved residues in T3O and T19H that were missing in V19H. Using site-directed mutagenesis (SDM) of V19H at those four residues, the binding pocket became more T3O-like, and V19H gained T3O-like activity without the loss of V19H activity.Item Open Access Interactions and Population Dynamics Between Erwinia amylovora, Pantoea agglomerans, and their Bacteriophages for Effective Phage Therapy(Brock University) Gayder, Steven C; Centre for BiotechnologyFire blight is a globally devastating disease of apples, pears, and other rosaceous plants caused by the bacterial phytopathogen Erwinia amylovora. Our lab is developing a dual-action biological control product using the epiphytic bacterial antagonist Pantoea agglomerans and a cocktail of bacterial viruses called bacteriophages. P. agglomerans act as host cells for a cocktail of phages targeting E. amylovora in planta and also exclude the pathogen from the blossom pistil and hypanthia by natural antagonism and antibiotic production. The objective of this research is to understand the dynamics between the pathogen, the carrier, and their phages and use this information to develop an effective phage-carrier system for the control of E. amylovora. A novel DNA plasmid was created which allows simultaneous quantification of E. amylovora, P. agglomerans, and four Erwinia phage species using quantitative real-time PCR (qPCR). This plasmid standard-based qPCR was used to study the host range of 10 phages against a global collection of Erwinia strains (n=106) and potential carriers (n=30), and to study the population dynamics of Erwinia phages in different host and cocktail combinations in unprecedented detail. The host range on E. amylovora revealed global phage resistance is located largely in western North America, and this resistance is due in part to a greater effect of exopolysaccharide production by these hosts. Also three phages, ɸEa21-4, ɸEa46-1-A1, and φEa35-70, were chosen to investigate for potential biocontrol efficacy. Phage ɸEa35-70, while ineffective alone, synergized with both ɸEa21-4 and ɸEa46-1-A1 for enhanced reduction of E. amylovora growth over 24 h. The competition between ɸEa21-4 and ɸEa35-70 was exploited which maximized carrier survival and lead to the design of an effective phage-carrier combination which will be formulated and further investigated for the control of E. amylovora in planta. Finally, the quantitative host range data and host genomic sequences were used to identify 10 host genes that are potentially associated with phage infection using a novel, k-mer based, genome wide association study (GWAS). These genes are indicative of a potential phage receptor, two novel phage resistance mechanisms, and other metabolic and biological functions which may affect phage infection.Item Open Access A study on the dynamics of the symbiosis between Metarhizium and plants(Brock University) Barelli, Larissa Ruth; Centre for BiotechnologyMembers of the Metarhizium genus exist as both insect pathogens and plant endophytes. Agricultural formulations of these fungi are utilized for their biocontrol of insect pests and numerous additional benefits for crop plants (i.e., nutrient transfer, pathogen antagonism, increased biomass, etc.). In order to develop improvements to these formulations, it is vital to understand how specific factors, such as the nutrient availability and microbial community of the soil, as well as production of secondary metabolites, influence the interaction of Metarhizium with target crop species. Metarhizium is capable of translocating nitrogen obtained through insect parasitism to plant hosts in exchange for carbohydrates. Using wax moth larvae (Galleria mellonella) injected with 15N-ammonium sulfate, it was demonstrated that nutrient-rich soil effectively inhibited transfer of insect-derived 15N from Metarhizium into the leaves of haricot bean, Phaseolus vulgaris. Colonization was maintained under all conditions and was not correlated with nitrogen transfer. Nitrogen application reduced initial colonization, but it recovered at later timepoints. The persistence of Metarhizium within the rhizosphere is influenced by the microbial community but, reciprocally, the structure of the community may respond to Metarhizium application. Although soil amendment with M. robertsii did not affect overall diversity of the root microbiome of P. vulgaris, Illumina sequencing demonstrated significant effects on particular bacterial and fungal taxa. The relative abundance of several plant growth promoting microorganisms (e.g. Bradyrhizobium) increased after Metarhizium application. When challenged with the specific bean root rot pathogen Fusarium solani f. sp. phaseoli, both the microbiome and M. robertsii were able to suppress disease. The production of fungal secondary metabolites such as destruxins may dictate interactions with plant hosts. During co-culture with bean or corn, destruxin production varied by species of Metarhizium and plant. Similar to previous reports, M. robertsii and M. acridum generally produced relatively high and low levels of destruxins, respectively. However, numerous destruxins were synthesized by M. acridum during co-culture with corn. Unraveling the metabolomic profile of these fungal-plant interactions may provide insight into mechanisms behind maintaining symbioses and patterns of strain compatibility, as well as aid in strain selection for agriculture and discovery of novel bioactive metabolites.Item Open Access Considerations for the Development and Optimization of Wine made from Partially Dehydrated Grapes in Ontario, Canada(Brock University) Kelly, Jennifer; Centre for BiotechnologyThe appassimento process for making wine can mitigate climatic challenges associated with cool climate winemaking, as fruit is dried post-harvest, reducing vintage-to-vintage variation due to varying fruit quality. Resultant wines fermented from dried grapes are high in ethanol and described as rich and intensely flavoured. One of the quality challenges facing wine made from partially dehydrated grapes is elevated levels of undesirable oxidation compounds, such as ethyl acetate, acetic acid and acetaldehyde. In this study we aim to characterize wines made from a local yeast isolate, Saccharomyces bayanus CN1, which demonstrates limited osmotolerance and may have application to this wine style, as it is a lower producer of such compounds. Wines made with the yeast of interest were compared to wines made with the accepted commercial yeast, Saccharomyces cerevisiae, EC1118. Fermentations were established over two vintages at one and three target starting sugar concentrations and a control, respectively. Wines were chemically (enzymatic) and sensorially analyzed. Wines (year two) were subject to volatile organic compound (VOC) and volatile fatty acid (VFA) measurements via Gas Chromatography-Mass Spectrometry. Another consideration for the development of this wine style is the inclusion of Botrytis cinerea, a pathogenic fungus that commonly develops during grape drying, and may impart favourable sensorial characteristics. Grapes were dried to 28.0°Brix and were fermented with EC1118 at 0 and 10% B. cinerea infection. A consumer preference test (n=153) that measured liking of wines (CN1 and 0% and10% B. cinerea infection) was conducted. Results indicate that CN1’s upper limit for fermentation to dryness is 27.5°Brix. All CN1 wines had significantly lower concentrations (p<0.05) of oxidation compounds than the commercial yeast, and oppositely, higher glycerol levels, along with comparable ethanol concentration to EC1118 wines. Significant differences in the concentrations of VOCs and VFAs, such as 2-phenylethanol and hexanoic acid were observed both within °Brix treatments and amongst yeast strains. Sensorially, the wines differed in intensity for a number of attributes. The consumer study revealed no preference between wines vinified with the different yeast strains. This work will contribute to the optimization of this wine style in cool climate winemaking regions and beyond.Item Open Access Resveratrol slows cell growth by targeting the Warburg effect and stimulating mitochondria metabolism(Brock University) Fonseca, Joao; Centre for BiotechnologyResveratrol (RES) is a plant-derived polyphenol that has been widely studied due to its health promoting effects, which are associated with RES ability to positively impact mitochondria function. Here, I investigated the interaction between RES’s effects on growth and metabolism in PC3 prostate cancer cells and demonstrated that RES-mediated growth inhibition is coincidental with an increase in mitochondrial network fusion, biogenesis and cellular respiration. This indicates that a metabolic reprogramming towards oxidative phosphorylation might be essential for RES antiproliferative effects. Indeed, when RES-induced metabolic reprogramming was prevented either by growing cells in galactose or stabilizing hypoxia inducible factor-1α (HIF-1α) expression, RES effects on growth and metabolism were attenuated or even abolished. Furthermore, consistent with RES ability to reduce HIF-1α levels, I observed that RES’s cell growth inhibitory effects were enhanced under hypoxia. This denotes the importance of conducting in vitro studies under conditions that better represent the physiological environment. However, , most in vitro studies are performed at supraphysiological levels of oxygen (O2) (18% O2; compared to the usual 1-5% O2 range observed in vivo) and glucose (25mM, which is close to five-times higher than normal plasma (glucose) in a healthy human). This artificial environment can affect a wide variety of cellular activities that may compromise in vitro studies’ reliability. It is therefore important to determine how cell culture conditions might affect RES in vitro effects. This was achieved by growing PC3 human prostate cancer and C2C12 mouse myoblasts cells under different culture conditions: physiological O2 (here considered 5% O2) and glucose levels (5mM); physiological O2 and high glucose (25mM); supraphysiological O2 (18% O2) and high glucose (25mM). Overall, RES effects on cell proliferation and mitochondrial network were less effective when cells were grown at 5% O2 and 5mM glucose (media condition that best resembles the physiological environment). In conclusion, these findings demonstrate the importance of oxygen and glucose levels as key determinants of RES in vitro antiproliferative effects, which may contribute to the discrepancy observed for resveratrol’s effects both in vivo and in vitro.Item Open Access Investigation of the effect of microwave irradiation (2.45 GHz) on biological systems at constant bulk temperature(Brock University) MAZINANI, SINA; Centre for BiotechnologyIn this thesis the effect of MW irradiation (2.45 GHz) at constant bulk temperature was investigated on several biological systems. Studies on enzymatic activity revealed that MW irradiation could enhance the activity of trypsin, however the enzymatic activity of α-amylase and alkaline phosphatase towards the hydrolysis of starch and 4-nitrophenyl phosphate was not affected. We found that the incorporation of a BODIPY fatty acid (4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid) into the cell membrane of PC-3 (human prostate cancer) cells was facilitated by microwave treatment at constant temperature. Also, microwave treatment while non-apoptotic, significantly increased the rate of reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) by PC-3 cells. Further studies revealed that MW irradiation (10 W, SAR: 700 mW/ml) could significantly increase the uptake of an anticancer drug (doxorubicin) by PC-3 and MCF-7 (human breast cancer) cells at constant bulk temperature. Studies on bacterial growth revealed that MW irradiation could significantly decrease the growth of Escherichia coli at constant bulk temperature and the impact appeared to be transient. A 2D-gel electrophoresis-based proteomic analysis revealed that the expression of a series of proteins likely involved in metabolism was affected by the exposure to the MW irradiation. Overall, the results demonstrated in this study provided additional evidence for the “microwave-specific effects” that are capable of altering the behaviour of biological systems in a way that is quite different from conventional heating (through conduction). Appropriate interpretations of these observations have to also consider the possibility of local heating and/or micro-hotspot formation during MW irradiation. In this respect, the “microwave-specific effects” may not be interpreted either entirely or partially as non-thermal in nature.Item Open Access Evaluation of genetic and strain specific factors on root colonization in endophytic insect pathogenic fungi (EIPF) Metarhizium and Beauveria, with special emphasis on hydrophobins(Brock University) Moonjely, Soumya; Centre for BiotechnologyThe Ascomycete genera, Metarhizium and Beauveria, are traditionally known as insect pathogenic fungi and are widely used as mycopesticides in agricultural settings. More recently, an additional role of these fungi in nature as root symbionts, with the ability to transfer nitrogen from dead insects to host plants, has been recognized. In this study, the genetic as well as strain-specific factors of Metarhizium and Beauveria were assessed during interaction with the plant and insect hosts. Hydrophobins are small proteins, unique to filamentous fungi, that provide hydrophobicity to aerial hyphae and conidia, and also supports fungal attachment to host surfaces. The role of two hydrophobins (hyd1 and hyd2) in insect pathogenicity were previously described in Beauveria, but little is known about their possible role in root colonization. Gene expression and plant root colonization assays revealed that the deletion of hyd1 or hyd2 subjected Beauveria to stress, which subsequently altered the expression of genes involved in signaling pathways, pigment production, specific adhesins, as well as fungal association with the root. The involvement of six Metarhizium genes on plant root colonization and insect pathogenesis were also investigated. Nitrogen transporter genes, Mep2, MepC and Urease, were selected due to sequence similarity with previously characterized plant-associating fungal ammonium transporters. Root colonization assays showed that the targeted deletion of MepC and Mep2 genes in M. robertsii enhanced the rhizoplane colonization on barley roots and insect-derived nitrogen transfer to plant hosts. Three other genes were selected on the basis on RNA-Seq data that showed high expression levels on bean roots; these encoded a hydrophobin (Hyd3), a subtilisin-like serine protease (Pr1A) and a hypothetical protein. Root colonization assessment revealed that the loss of Hyd3, Pr1A, or the hypothetical protein gene from M. robertsii had no influence on establishing association with barley roots. We also assessed ten Metarhizium generalist and specialist strains and a related endophytic fungus Pochonia, for insect pathogenicity and their ability colonize plants; however, regardless of whether the Metarhizium species was a generalist or specialist insect pathogen all strains tested showed some ability to associate with plants. Moreover, Metarhizium spp. were able to colonize monocots better than dicots. Our data indicates that even after divergence as generalist or specialist insect pathogens, Metarhizium spp. maintain their ancestral ability to colonize plants. Overall, this study provides useful insights into the genes involved in EIPF-root interactions and also highlights the impact of gene deletion in triggering compensatory pathways.Item Open Access Transmission Dynamics and Epidemiology of West Nile Virus in Ontario, Canada(Brock University) Giordano, Bryan; Centre for BiotechnologyThe resurgence of West Nile virus (WNV; Family Flaviviridae, genus Flavivirus) in Ontario, Canada in 2012 demonstrated that there is a great need for a reassessment of the local mosquito fauna, estimation of risk of WNV transmission, and the creation of effective arboviral awareness campaigns. A review of the current literature and collection databases revealed that there are 68 mosquito species known from Ontario (Chapter 2). Ten species were added to the list of species including Culex erraticus (Chapter 3) and Aedes albopictus (Chapter 4), both of which are capable of transmitting West Nile virus. Ae. albopictus was repeatedly collected from Windsor, Ontario in 2016 (Chapter 4). Immatures (n=78) were collected from tires, StyrofoamTM containers, and discarded garbage. Adult female (n=17) and male (n=2) specimens were collected from light traps (n=7) and Biogents-Sentinel traps (n=10). Additional specimens were obtained from Franklin County, Ohio. The generated gene tree and Bayesian cluster analysis grouped sequences described from Ohio and Windsor together on the same branches. Together these data suggest that the population in Windsor originated as a founder population of North American origin by means of human-aided dispersal. Mosquito abundance predication surfaces and seasonal distributions were attempted for each vector species to identify where and when vector species are most abundant in southern Ontario (Chapter 5). Spatial prediction surfaces using kriging were created for Aedes vexans, Aedes japonicus, Culex pipiens, Culex restuans, and Ochlerotatus trivittatus. Proximity to landscape variables was observed to improve model prediction. An epidemiological analysis of WNV human case prevalence and mosquito infection was conducted (Chapter 6). A strong quadratic relationship between the number of human cases and positive mosquito pools at the end of each year was observed (R2=0.9783, p < 0.001). Spearman rank correlation tests identified mosquito infection rates as the strongest predictors of human case prevalence at a one-week lag period. Average temperature was a strong predictor of mosquito infection rates. Cumulative positive Culex pools recorded by epidemiological week 34 is a sufficient action threshold for West Nile virus epidemics. These data have the potential to contribute to a more efficient West Nile virus awareness campaign.Item Open Access A study of biting midge populations and an assessment of Bluetongue virus presence in southern Ontario, with a visual dichotomous key to the North American genera of Ceratopogonidae(Brock University) Jewiss-Gaines, Adam; Centre for BiotechnologyBiting midges (Diptera: Ceratopogonidae) are flying insects that feed on blood in order to produce eggs. Due to their bloodfeeding habits, some species act as vectors for viruses, many of which affect ruminant animals. This establishes ceratopogonids as possible threats to livestock farmers, and therefore it is important to understand their distribution and habits. This study focussed on collecting biting midge specimens from localities across Ontario where livestock farms are present, and determining if they were carrying Bluetongue virus (BTV). Fourteen livestock farms were sampled to assess presence of ceratopogonid vectors. Captured Culicoides specimens were subjected to RT-qPCR analysis to test for BTV. The North American vector for BTV (Culicoides sonorensis) was collected at multiple localities, constituting the first record of this species in Ontario. Identity of C. sonorensis specimens was verified using molecular analysis of three gene regions: CO1, ITS1, and EF1α. Gene sequences for Culicoides variipennis, a species easily confused with C. sonorensis, were also analyzed. Analysis revealed that EF1α introns differed between the species and may be useful as molecular identifiers. Sequences for all three gene regions were submitted to the National Center for Biotechnology Information gene database. The Brock University Rothamsted Trap operated during the summer seasons of 2013 to 2017, collecting insects at ~12m above ground on a daily basis. Collected biting midges were identified to genus, and genus tallies were compared with five climate variables using redundancy analysis (RDA) to determine factors that affect the activity of collected genera. Phenological patterns of Bezzia, Culicoides, and Probezzia were compared in detail to temperature values. A literature search was performed to evaluate progression of BTV and epizootic hemorrhagic disease virus (EHDV) across North America since their initial detections in the 1950s. Records detailing virus outbreaks were analyzed and maps displaying the chronological progression of BTV and EHDV were created, providing a visual representation of their dispersal patterns. Finally, a dichotomous key to the Nearctic biting midge genera was constructed and illustrated with high-definition photography to show key characters. This key aids with taxonomic identification of the 35 recognized genera occurring north of Mexico.Item Open Access Large-scale studies and biophysical analysis of systems involved in plant immunity.(Brock University) Kuai, Xiahezi; Centre for BiotechnologyThe field of plant immunity has progressed significantly in the last decade, driven primarily by both forward and reverse genetics and to a lesser extent by molecular biology techniques. However, many unknowns still remain before a more complete picture of this system can be achieved, which hinders our capacity to develop biotechnological solutions to ensure food safety for our growing population. Some of the problems that still need to be tackled relate to the multi-system involvement of some proteins, the interrelation of the different hormones, such as in trade-off systems, and the challenges of translating existing molecular knowledge into crop protection strategies. The goal of this thesis was to develop new methods and to adapt existing ones to address the challenges and push the boundaries of our knowledge of plant immunity as a system. We have adapted ClueGO analyses to visualize functionally grouped Gene Ontology (GO) terms specific to Arabidopsis. We developed a transcription factor- coregulator identification strategy based on double-transcriptome analyses. Finally, we have adapted a biophysical method, differential scanning fluorimetry (DSF). We tested the usefulness of these methods by interrogating different immune proteins/genes of the model plant Arabidopsis thaliana. Here is a summary of the major results obtained. In the realm of basal immunity, we discovered that clade I TGA transcription factors positively regulate this system by repressing WRKY transcription factors, which are negative regulators of the process. Furthermore, we have demonstrated that clade I TGA integrates into the growth- immunity trade-off system regulated by brassinosteroids by antagonizing the brassinosteroids-dependent suppression of basal immunity. In the realm of systemic acquired resistance (SAR), we have demonstrated that clade I TGA recruits a specific novel glutaredoxin as a corepressor to dampen the expression of a set of SAR-regulated genes controlled by salicylic acid (SA) and the SAR-orchestrator, NPR1. Finally, we demonstrated that NPR1 binds SA and that this interaction leads to the destabilization of NPR1. More importantly, the method used to show the latter is scalable and can be used to develop novel chemistries capable of deploying plant immunity in the field.Item Open Access A study of lipid recognition and membrane binding by the human oxysterol-binding protein (OSBP).(Brock University) Mukherjee, Parthajit; Centre for BiotechnologyRecent studies have established oxysterol-binding protein (OSBP) and members of the OSBP-related protein (ORP) family as global cellular sterol sensors that participate in non-vesicular anterograde transport of monomeric sterols from the endoplasmic reticulum to other organelles such as the Golgi and the plasma membrane. By exchanging sterols for phosphoinositides, these multi-domain proteins change the bilayer composition at membrane contact sites and thus, regulate various signaling pathways. Despite the wealth of knowledge garnered from the study of fluorescent/radiolabeled ligand-protein interactions and inter-vesicular lipid transfer assays in vitro, the precise nature of the association of ORPs with organellar membranes and the factors modulating such interactions have remained largely enigmatic. The goal of my project was to characterize the behaviour of human OSBP using a label-free analytical technique called dual polarization interferometry (DPI). This technique enables surface-immobilization of phospholipid vesicles to observe and analyze the behaviour of proteins towards adsorbed bilayers. From my investigation, I found that OSBP prefers binding to membranes containing anionic phospholipids, such as phosphatidylinositol-4-phosphate (PI(4)P), over membranes made up of neutral phosphatidylcholine (PC). In the presence of PI(4)P, the wild-type protein clearly demonstrated a rapid bilayer association, followed by PI(4)P extraction and a slower dissociation, in a dosage-dependent fashion. The OSBP-related domain (ORD) mutant, OSBP-HH/AA, due to its impaired ability to extract PI(4)P, failed to dissociate from the membrane while the pleckstrin homology domain (PHD) mutant, OSBP-RR/EE, could not associate with membranes at all. The presence of sterols did not alter OSBP’s affinity for PC membranes despite a two-fold increase in protein adsorption per unit area in the presence of cholesterol in the membrane, compared to 25-hydroxycholesterol. Both cholesterol and 25-hydroxycholesterol competed with 22-NBD-cholesterol for the binding site in the ORD of OSBP, with resulting EC50 values of 15.6 ± 0.7 nM for the former and 5.0 ± 0.5 nM for the latter. OSBP also transferred ORD-bound fluorescent cholesterol to acceptor vesicles, but the rate remained unaltered upon incorporation of PI(4)P in those membranes. These results provide useful insight into the preferential association of OSBP with membranes containing specific recognizable ligands, such as sterols and PI(4)P, and help build a molecular level description of the mechanism of this protein.Item Open Access The production and synthetic utility of the dioxygenase-derived metabolites of substituted aromatics(Brock University) Froese, Jordan; Centre for BiotechnologyThe substrate scope and selectivity of toluene dioxygenase overexpressed in E.coli JM109 (pDTG601A) was investigated with series of ortho-halobenzoates and para-substituted arenes. Palladium-catalyzed carbonylation methodology was developed to convert halogenated cis-dihydrodiol metabolites to the corresponding carboxylates and a comparison of the overall efficiency between the enzymatic and chemical methods of access was made. Some of the metabolites produced by toluene dioxygenase were employed in a synthetic approach toward tetrodotoxin. Enzymatic dihydroxylation of benzoic acid with R. eutropha B9 provided the corresponding ipso-diol that was used in the first total synthesis of pleiogenone A, a bioactive natural product. Experimental and spectral data are provided for all new compounds.Item Open Access Functional genomics of monoterpenoid indole alkaloid biosynthesis in Rauvolfia serpentina(Brock University) Cazares, Paulo; Department of Biological SciencesMonoterpenoid indole alkaloids (MIAs) are a large and heterogeneous group of nitrogen-containing specialized metabolites produced by plants belonging to the Apocynaceae, Loganiaceae and Rubiaceae families. Many of these MIAs exhibit interesting biological activities and are currently used as pharmaceutical drugs to treat several medical conditions. Thus, the biosynthetic pathways responsible for their production have been extensively investigated. Recent advancements in large-scale DNA-sequencing technologies have provided access to a vast collection of genes. Here we have used bioinformatics guided screen to identify candidate genes involved in MIA biosynthesis in Rauvolfia serpentina. We utilized our large annotated transcriptome databases (www.phytometasyn.ca) as a source to mine genes. The identification of a Catharanthus roseus enzyme responsible for indole nitrogen methylation of an MIA intermediate of the vindoline pathway provided us with a query sequence to mine candidate genes responsible for N-methylation of other MIAs in R. serpentina. This led to the identification, molecular cloning and biochemical characterization of four enzymes catalyzing N-methylation. Two separate genes cloned from R. serpentina and V. minor, both encoded enzymes displaying high affinity and specificity for picrinine converting it to N-methyl-picrinine (ervincine) in the presence of S-adenosyl-L-methionine. The other two genes, cloned from R. serpentina, encoded enzymes involved in final steps of ajmaline biosynthesis. Norajmaline N-methyltransferase catalyzed the indoline N-methylation of norajmaline to generate ajmaline, while ajmaline Nβ-methyltransferase catalyzed the side chain N-methylation of ajmaline to generate Nβ-methylajmaline, an unusual positively charge MIA molecule found in Rauvolfia.Item Open Access Novel plant DNA binding protein: Non-expresser of pathogenesis related 1 gene (NPR1) involved in disease resistance(Brock University) Pirnia, Saba; Centre for BiotechnologyFor the first time, through validation of the modified Chromatin Immunoprecipitation (ChIP) method (in vitro ChIP), the direct binding of NPR1 to the PR1 promoter was demonstrated. This is a novel advancement on plant systemic acquired resistance (SAR)-mediated disease responses. The NPR1 protein (nonexpressor of pathogenesis related gene 1) is a transcriptional co-activator and positive regulator of SAR, a long-lasting mobile defense signal found in plants. The pathogenesis-Related gene 1 (PR1) is particularly induced during defense response, and as such, is typically used as a marker for establishment of SAR in plants. Salicylic acid (SA) is a phytohormone required for SAR-mediated defense responses against pathogens. Recently, the role of NPR1 as a SA receptor was demonstrated; SA has been shown to directly bind to NPR1 through Cysteine 521 and 529 on the Cterminus region of NPR1 via the transition metal copper. The binding of SA to NPR1 results in disruption of the interaction between BTB/POZ and the C-terminus domains of NPR1. Upon SA-NPR1 binding, the C-terminus transactivation domain is released from the auto-inhibitory BTB/POZ domain, resulting in activation of the NPR1 transcription co-activator function, followed by PR1 transcription in Arabidopsis thaliana. Arabidopsis thaliana has an inducible defense system and is considered a model plant for studying disease resistance responses. In the current research, NPR1 was demonstrated to bind to the PR1 promoter at two distinct regions, in the presence and absence of SA. In the presence of SA, the binding site of NPR1 was determined to be localized at the -636 to -646 base pair sequence; however, in the absence of SA, NPR1 was found to bind around the -790 to -833 base pair sequence. In addition, two distinct DNA binding domains were identified within NPR1, localized on the C and N-terminus regions. In the absence of SA, the DNA binding domain within the N-terminus region, located between amino acids 110-190, was shown to facilitate the binding of NPR1 to the PR1 promoter through the amino acid cysteine 150 (Cys150) via transition metal. The DNA binding domain on the C-terminus region, located between amino acids 513-535, was demonstrated to allow the binding of NPR1 to the promoter of PR1 in the presence of SA. Two amino acids, cysteine 521 and 529 (Cys521/529), were shown to be essential for SA binding to NPR1 and subsequent NPR1 binding to the PR1 promoter. Furthermore, 4hydroxy benzoic acid (4-OH-BA), the inactive analogue of SA, has been demonstrated to be a potent inhibitor of NPR1-PR1 promoter interaction, both in vivo and in vitro, by competing with SA for NPR1 binding. Moreover, we demonstrated that other analogues of the NPR1 protein, NPR2, NPR3, and NPR4, are also recruited to the PR1 promoter. NPR4 showed a similar binding profile to NPR1, both in the presence and absence of SA. NPR2 and NPR3 were observed to only interact with the PR1 promoter in the absence of SA. Both NPR5 and NPR6 were shown to forgo binding to the PR1 promoter, further confirming their role in plant developmental processes other than defense. In addition, the binding of NPR1 to the PR1 promoter was demonstrated to be conserved among other plant species, including rice and maize. Both rice and maize NPR1 proteins were observed to bind to the PR1 promoter in the presence of SA and a metal co-factor, similar to Arabidopsis NPR1. Our results expand our understanding of how NPR1 interacts with the PR1 promoter to regulate gene expression during SAR establishment. This study also revealed that NPR1-mediated SAR defense signaling is conserved among other crop species, which can potentially facilitate the identification of novel plant-priming compounds through high-throughput chemical screening procedures alongside the application of the validated in vitro ChIP technique as a primary screening method.Item Open Access The Ligand and Membrane-binding Behaviour of the Phosphatidylinositol Transfer Proteins (PITPa & PITPb)(Brock University) Baptist, Matilda; Centre for BiotechnologyHuman Class I phosphatidylinositol transfer proteins (PITPs) exists in two forms: PITPα and PITPβ. PITPs are believed to be lipid transfer proteins based on their capacity to transfer either phosphatidylinositol (PI) or phosphatidylcholine (PC) between membrane compartments in vitro. In Drosophila, the PITP domain is found to be part of a multi-domain protein named retinal degeneration B (RdgBα). The PITP domain of RdgBα shares 40 % sequence identity with PITPα and has been shown to possess PI and PC binding and transfer activity. The detailed molecular mechanism of ligand transfer by the human PITPs and the Drosophila PITP domain remains to be fully established. Here, we investigated the membrane interactions of these proteins using dual polarization interferometry (DPI). DPI is a technique that measures protein binding affinity to a flat immobilized lipid bilayer. In addition, we also measured how quickly these proteins transfer their ligands to lipid vesicles using a fluorescence resonance energy transfer (FRET)-based assay. DPI investigations suggest that PITPβ had a two-fold higher affinity for membranes compared to PITPα. This was reflected by a four-fold faster ligand transfer rate for PITPβ in comparison to PITPα as determined by the FRET assay. Interestingly, DPI analysis also demonstrated that PI-bound human PITPs have lower membrane affinity compared to PC-bound PITPs. In addition, the FRET studies demonstrated the significance of membrane curvature in the ligand transfer rate of PITPs. The ligand transfer rate was higher when the accepting vesicles were highly curved. Furthermore, when the accepting vesicles contained phosphatidic acid (PA) which have smaller head groups, the transfer rate increased. In contrast, when the accepting vesicles contained phosphoinositides which have larger head groups, the transfer rate was diminished. However, PI, the favorite ligand of PITPs, or the presence of anionic lipids did not appear to influence the ligand transfer rate of PITPs. Both DPI and FRET examinations revealed that the PITP domain of RdgBα was able to bind to membranes. However, the RdgBα PITP domain appears to be a poor binder and transporter of PC.Item Open Access The Arabidopsis NPR1 Protein Is a Receptor for the Plant Defense Hormone Salicylic Acid(Brock University) Wu, Yue; Department of Biological SciencesSystemic Acquired Resistance (SAR) is a type of plant systemic resistance occurring against a broad spectrum of pathogens. It can be activated in response to pathogen infection in the model plant Arabidopsis thaliana and many agriculturally important crops. Upon SAR activation, the infected plant undergoes transcriptional reprogramming, marked by the induction of a battery of defense genes, including Pathogenesis-related (PR) genes. Activation of the PR-1 gene serves as a molecular marker for the deployment of SAR. The accumulation of a defense hormone, salicylic acid (SA) is crucial for the infected plant to mount SAR. Increased cellular levels of SA lead to the downstream activation of the PR-1 gene, triggered by the combined action of the Non-expressor of Pathogenesis-related Gene 1 (NPR1) protein and the TGA II-clade transcription factor (namely TGA2). Despite the importance of SA, its receptor has remained elusive for decades. In this study, we demonstrated that in Arabidopsis the NPR1 protein is a receptor for SA. SA physically binds to the C-terminal transactivation domain of NPR1. The two cysteines (Cys521 and Cys529), which are important for NPR1’s coactivator function, within this transactivation domain are critical for the binding of SA to NPR1. The interaction between SA and NPR1 requires a transition metal, copper, as a cofactor. Our results also suggested a conformational change in NPR1 upon SA binding, releasing the C-terminal transactivation domain from the N-terminal autoinhibitory BTB/POZ domain. These results advance our understanding of the plant immune function, specifically related to the molecular mechanisms underlying SAR. The discovery of NPR1 as a SA receptor enables future chemical screening for small molecules that activate plant immune responses through their interaction with NPR1 or NPR1-like proteins in commercially important plants. This will help in identifying the next generation of non-biocidal pesticides.