Recent Submissions

  • A study on the dynamics of the symbiosis between Metarhizium and plants

    Barelli, Larissa Ruth; Centre for Biotechnology
    Members 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.
  • Considerations for the Development and Optimization of Wine made from Partially Dehydrated Grapes in Ontario, Canada

    Kelly, Jennifer; Centre for Biotechnology
    The 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.
  • Resveratrol slows cell growth by targeting the Warburg effect and stimulating mitochondria metabolism

    Fonseca, Joao; Centre for Biotechnology
    Resveratrol (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.
  • Investigation of the effect of microwave irradiation (2.45 GHz) on biological systems at constant bulk temperature

    MAZINANI, SINA; Centre for Biotechnology
    In 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.
  • Evaluation of genetic and strain specific factors on root colonization in endophytic insect pathogenic fungi (EIPF) Metarhizium and Beauveria, with special emphasis on hydrophobins

    Moonjely, Soumya; Centre for Biotechnology
    The 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.
  • Transmission Dynamics and Epidemiology of West Nile Virus in Ontario, Canada

    Giordano, Bryan; Centre for Biotechnology
    The 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.
  • 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

    Jewiss-Gaines, Adam; Centre for Biotechnology
    Biting 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.
  • Large-scale studies and biophysical analysis of systems involved in plant immunity.

    Kuai, Xiahezi; Centre for Biotechnology
    The 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.
  • A study of lipid recognition and membrane binding by the human oxysterol-binding protein (OSBP).

    Mukherjee, Parthajit; Centre for Biotechnology
    Recent 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.
  • The production and synthetic utility of the dioxygenase-derived metabolites of substituted aromatics

    Froese, Jordan; Centre for Biotechnology
    The 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.
  • Functional genomics of monoterpenoid indole alkaloid biosynthesis in Rauvolfia serpentina

    Cazares, Paulo; Department of Biological Sciences
    Monoterpenoid 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.
  • Novel plant DNA binding protein: Non-expresser of pathogenesis related 1 gene (NPR1) involved in disease resistance

    Pirnia, Saba; Centre for Biotechnology
    For 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.
  • The Ligand and Membrane-binding Behaviour of the Phosphatidylinositol Transfer Proteins (PITPa & PITPb)

    Baptist, Matilda; Centre for Biotechnology
    Human 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.
  • The Arabidopsis NPR1 Protein Is a Receptor for the Plant Defense Hormone Salicylic Acid

    Wu, Yue; Department of Biological Sciences
    Systemic 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.
  • Trading nitrogen for carbon: Nitrogen and carbon translocation in a plant/fungal (Metarhizium spp.) symbiosis

    Behie, Scott William; Centre for Biotechnology
    While nitrogen is critical for all plants, they are unable to utilize organically bound nitrogen in soils. Therefore, the majority of plants obtain useable nitrogen through nitrogen fixing bacteria and the microbial decomposition of organic matter. In the majority of cases, symbiotic microorganisms directly furnish plant roots with inorganic forms of nitrogen. More than 80% of all land plants form intimate symbiotic relationships with root colonizing fungi. These common plant/fungal interactions have been defined largely through nutrient exchange, where the plant receives limiting soil nutrients, such as nitrogen, in exchange for plant derived carbon. Fungal endophytes are common plant colonizers. A number of these fungal species have a dual life cycle, meaning that they are not solely plant colonizers, but also saprophytes, insect pathogens, or plant pathogens. By using 15N labeled, Metarhizium infected, wax moth larvae (Galleria mellonella) in soil microcosms, I demonstrated that the common endophytic, insect pathogenic fungi Metarhizium spp. are able to infect living soil borne insects, and subsequently colonize plant roots and furnish ts plant host with useable, insect-derived nitrogen. In addition, I showed that another ecologically important, endophytic, insect pathogenic fungi, Beauveria bassiana, is able to transfer insect-derived nitrogen to its plant host. I demonstrated that these relationships between various plant species and endophytic, insect pathogenic fungi help to improve overall plant health. By using 13C-labeled CO2, added to airtight plant growth chambers, coupled with nuclear magnetic resosnance spectroscopy, I was able to track the movement of carbon from the atmosphere, into the plant, and finally into the root colonized fungal biomass. This indicates that Metarhizium exists in a symbiotic partnership with plants, where insect nitrogen is exchanged for plant carbon. Overall these studies provide the first evidence of nutrient exchange between an insect pathogenic fungus and plants, a relationship that has potentially useful implications on plant primary production, soil health, and overall ecosystem stability.
  • UNDERSTANDING THE RELATIONSHIP BETWEEN ONCOLYTIC AD5 DELETED E1b55KDA LYTIC INFECTION AND P53 IN MAMMALIAN CELLS

    Abbas, Basma; Centre for Biotechnology
    Adenoviruses are the most commonly used in the development of oncolytic therapy. Oncolytic adenoviruses are genetically modified to selectivity replicate in and kill tumor cells. The p53 molecule is a tumor suppressor protein that responds to viral infection through the activation of apoptosis, which is inhibited by adenovirus E1B55kDa protein leading to progressive viral lytic cycle. The non-specificity of replication has limited the use of wild type adenovirus in cancer therapy. This issue was resolved by using an E1b deleted Ad that can only replicate in cells with a deficiency in the p53 protein, a common feature of most cancer cells. Although demonstrating a moderate success rate, E1b55kDa deleted Ad has not been approved as a standard therapy for all cancer types. Several studies have revealed that E1b deleted Ad replication was independent of p53 status in the cell, as the virus replicated better in some p53 deficient cancers more than others. However, this mechanism has not been investigated deeply. Therefore, the objective of this study is to understand the relationship between p53 status, levels and functional activity, and oncolytic Ad5dlE1b55kDa replication efficiency. Firstly, five transient p53 expression vectors that contain different regulatory elements were engineered and then evaluated in H1299, HEK293 and HeLa cell lines. Data indicated that vector that contains the MARs and HPRE regulatory elements achieved the highest stability of p53 expression. Secondly, we used these vectors to examine the effect of various p53 expression levels on the replication efficiency of oncolytic Ad5dlE1b55kDa. We found that the level of p53 in the cell had an insignificant effect on the oncolytic viruses’ replication. However, the functional activity of p53 had a significant effect on its replication, as Ad5dlE1b55kDa was shown to have selective activity in H1299 cells (p53-null). In contrast, a decrease in viral replication was found in HeLa cells (p53-positive). Finally, the effect of p53’s functional activity on the replication efficiency of oncolytic Ad5dlE1b55kDa was examined. Viral growth was evaluated in H1299 cells expressing number of p53 mutants. P53-R175H mutant successfully rescued viral growth by allowing the virus to exert its mechanism of selectivity. The mechanism entailed deregulating the expression of specific genes, cell cycle and apoptosis, in the p53 pathway to promote its production leading to efficient oncolytic effect. These results confirmed that oncolytic Ad5dlE1b55kDa sensitivity is mutation-type specific. Therefore, before it is applied clinically as cancer therapy for p53 deficient tumors, the type of p53 mutation must be determined for efficient antitumor effect.
  • Towards reverse engineering of Photosystem II: Synergistic Computational and Experimental Approaches

    Mahboob, Abdullah; Centre for Biotechnology (Brock University, 2014-10-01)
    ABSTRACT Photosystem II (PSII) of oxygenic photosynthesis has the unique ability to photochemically oxidize water, extracting electrons from water to result in the evolution of oxygen gas while depositing these electrons to the rest of the photosynthetic machinery which in turn reduces CO2 to carbohydrate molecules acting as fuel for the cell. Unfortunately, native PSII is unstable and not suitable to be used in industrial applications. Consequently, there is a need to reverse-engineer the water oxidation photochemical reactions of PSII using solution-stable proteins. But what does it take to reverse-engineer PSII’s reactions? PSII has the pigment with the highest oxidation potential in nature known as P680. The high oxidation of P680 is in fact the driving force for water oxidation. P680 is made up of a chlorophyll a dimer embedded inside the relatively hydrophobic transmembrane environment of PSII. In this thesis, the electrostatic factors contributing to the high oxidation potential of P680 are described. PSII oxidizes water in a specialized metal cluster known as the Oxygen Evolving Complex (OEC). The pathways that water can take to enter the relatively hydrophobic region of PSII are described as well. A previous attempt to reverse engineer PSII’s reactions using the protein scaffold of E. coli’s Bacterioferritin (BFR) existed. The oxidation potential of the pigment used for the BFR ‘reaction centre’ was measured and the protein effects calculated in a similar fashion to how P680 potentials were calculated in PSII. The BFR-RC’s pigment oxidation potential was found to be 0.57 V, too low to oxidize water or tyrosine like PSII. We suggest that the observed tyrosine oxidation in BRF-RC could be driven by the ZnCe6 di-cation. In order to increase the efficiency of iii tyrosine oxidation, and ultimately oxidize water, the first potential of ZnCe6 would have to attain a value in excess of 0.8 V. The results were used to develop a second generation of BFR-RC using a high oxidation pigment. The hypervalent phosphorous porphyrin forms a radical pair that can be observed using Transient Electron Paramagnetic Resonance (TR-EPR). Finally, the results from this thesis are discussed in light of the development of solar fuel producing systems.
  • The Molecular Consequences of CK2-mediated Phosphorylation of the TGA2 Transcription Factor within Systemic Acquired Resistance of Arabidopsis thaliana

    Bosak, Jan; Centre for Biotechnology (Brock University, 2014-08-05)
    During infection, the model plant Arabidopsis thaliana is capable of activating long lasting defence responses both in tissue directly affected by the pathogen and in more distal tissue. Systemic acquired resistance (SAR) is a type of systemic defence response deployed against biotrophic pathogens resulting in altered plant gene expression and production of antimicrobial compounds. One such gene involved in plant defence is called pathogenesis-related 1 (PR1) and is under the control of several protein regulators. TGA II-clade transcription factors (namely TGA2) repress PR1 activity prior to infection by forming large oligomeric complexes effectively blocking gene transcription. After pathogen detection, these complexes are dispersed by a mechanism unknown until now and free TGA molecules interact with the non-expressor of pathogenesis-related gene 1 (NPR1) protein forming an activating complex enabling PR1 transcription. This study elucidates the TGA2 dissociation mechanism by introducing protein kinase CK2 into this process. This enzyme efficiently phosphorylates TGA2 resulting in two crucial events. Firstly, the DNA-binding ability of this transcription factor is completely abolished explaining how the large TGA2 complexes are quickly evicted from the PR1 promoter. Secondly, a portion of TGA2 molecules dissociate from the complexes after phosphorylation which likely makes them available for the formation of the TGA2-NPR1 activating complex. We also show that phosphorylation of a multiserine motif found within TGA2’s N terminus is responsible for the change of affinity to DNA, while modification of a single threonine in the leucine zipper domain seems to be responsible for deoligomerization. Despite the substantial changes caused by phosphorylation, TGA2 is still capable of interacting with NPR1 and these proteins together form a complex on DNA promoting PR1 transcription. Therefore, we propose a change in the current model of how PR1 is regulated by adding CK2 which targets TGA2 displacing it’s complexes from the promoter and providing solitary TGA2 molecules for assembly of the activating complex. Amino acid sequences of regions targeted by CK2 in Arabidopsis TGA2 are similar to those found in TGA2 homologs in rice and tobacco. Therefore, the molecular mechanism that we have identified may be conserved among various plants, including important crop species, adding to the significance of our findings.
  • Novel glutathione disulfide transferase function of CC-glutaredoxins involved in disease resistance and flower development

    Slavickova, Nina; Centre for Biotechnology (Brock University, 2014-08-05)
    Glutaredoxins are oxidoreductases capable of reducing protein disulfide bridges and glutathione mixed disulfides through the process of deglutathionylation and glutathionylation. Lately, redox-mediated modifications of functional cysteine residues of TGA1 and TGA8 transcription factors have been postulated. Namely, GRX480 and ROXY1 glutaredoxins have been previously shown to interact with TGA proteins and have been suggested to regulate redox state of these proteins. TGA1, together with TGA2, is involved in systemic acquired resistance (SAR) establishment in the plant Arabidopsis thaliana through PR1 (Pathogenesis related 1) gene activation. They both form an enhanceosome complex with the NPR1 protein (non-expressor of pathogenesis related gene 1) which leads to PR1 transcription. Although TGA1 is capable of activating PR1 transcription, the ability of the TGA1 NPR1 enhanceosome complex to assembly is based on the redox status of TGA1. We identified GRX480 as a glutathionylating enzyme that catalyzes the TGA1 glutathione disulfide transferase reaction with a Km of around 20μM GSSG (oxidized glutathione). Out of four cysteine residues found within TGA1, C172 and C266 were found to be glutathionylated by this enzyme. We also confirmed TGA1 glutathionylation in vivo and showed that this modification takes place while TGA1 is associated with the PR1 promoter enzymatically via GRX480. Furthermore, we show that glutathionylation via GRX480 abolishes TGA1's interaction with NPR1 and consequently prevents the TGA1-NPR1 transcription activation of PR1. When glutathionylated, TGA1 is recruited to the PR1 promoter and acts as a repressor. Therefore, glutathionylation is a mechanism that prevents TGA1 NPR1 interaction, allowing TGA1 to function as a repressor of PR1 transcription. Surprisingly, GRX480 was not able to deglutathionylate proteins demonstrating the irreversible nature of the reaction. Moreover, we demonstrate that other members of CC-class glutaredoxins, namely ROXY1 and ROXY2, can also catalyze protein glutathionylation. The TGA8 protein was previously shown to interact with NPR1 analogs, BOP1 and BOP2 proteins. However, unlike the case of TGA1 NPR1 interaction, here we demonstrate that TGA8-BOP1 interaction is not redox regulated and that TGA8 glutathionylation by ROXY1 and ROXY2 enzymes does not abolish this interaction in vitro. However, TGA8 glutathionylation results in TGA8 oligomer disassembly into smaller complexes and monomers. Our results suggest that CC-Grxs are unable to reduce mixed disulfides, instead they efficiently catalyze the opposite reaction which distinguishes them from traditional glutaredoxins. Therefore, they should not be classified as glutaredoxins but as protein glutathione disulfide transferases.
  • The γ-tocopherol-like family of N-methyltransferases: A taxonomically clustered gene family encoding enzymes responsible for N-methylation of monoterpene indole alkaloids

    Levac, Dylan Edward Ryan; Department of Biological Sciences (Brock University, 2013-09-19)
    The plant family Apocynaceae accumulates thousands of monoterpene indole alkaloids (MIAs) which originate, biosynthetically, from the common secoiridoid intermediate, strictosidine, that is formed from the condensation of tryptophan and secologanin molecules. MIAs demonstrate remarkable structural diversity and have pharmaceutically valuable biological activities. For example; a subunit of the potent anti-neoplastic molecules vincristine and vinblastine is the aspidosperma alkaloid, vindoline. Vindoline accumulates to trace levels under natural conditions. Research programs have determined that there is significant developmental and light regulation involved in the biosynthesis of this MIA. Furthermore, the biosynthetic pathway leading to vindoline is split among at least five independent cell types. Little is known of how intermediates are shuttled between these cell types. The late stage events in vindoline biosynthesis involve six enzymatic steps from tabersonine. The fourth biochemical step, in this pathway, is an indole N-methylation performed by a recently identified N-methyltransfearse (NMT). For almost twenty years the gene encoding this NMT had eluded discovery; however, in 2010 Liscombe et al. reported the identification of a γ-tocopherol C-methyltransferase homologue capable of indole N-methylating 2,3-dihydrotabersonine and Virus Induced Gene Silencing (VIGS) suppression of the messenger has since proven its involvement in vindoline biosynthesis. Recent large scale sequencing initiatives, performed on non-model medicinal plant transcriptomes, has permitted identification of candidate genes, presumably involved, in MIA biosynthesis never seen before in plant specialized metabolism research. Probing the transcriptome assemblies of Catharanthus roseus (L.)G.Don, Vinca minor L., Rauwolfia serpentine (L.)Benth ex Kurz, Tabernaemontana elegans, and Amsonia hubrichtii, with the nucleotide sequence of the N-methyltransferase involved in vindoline biosynthesis, revealed eight new homologous methyltransferases. This thesis describes the identification, molecular cloning, recombinant expression and biochemical characterization of two picrinine NMTs, one from V. minor and one from R. serpentina, a perivine NMT from C. roseus, and an ajmaline NMT from R. serpentina. While these TLMTs were expressed and functional in planta, they were active at relatively low levels and their N-methylated alkaloid products were not apparent our from alkaloid isolates of the plants. It appears that, for the most part, these TLMTs, participate in apparently silent biochemical pathways, awaiting the appropriate developmental and environmental cues for activity.

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