PhD Exit Seminar - Derek Seto (Desveaux Lab)
Recognition of the Pseudomonas syringae type III effector HopF1r by the Arabidopsis NLR ZAR1
Abstract
The plant pathogen Pseudomonas syringae uses the needle-like type III secretion system (T3SS) to inject virulence factors known as type III secreted effector proteins (T3SEs) directly into the plant cell. T3SEs disrupt plant immune pathways, allowing for successful colonization. However, plants have evolved resistance (R) genes that encode for nucleotide-binding leucine-rich repeat (NLR) proteins that detect the presence of some effectors, and subsequently trigger an immune response that suppresses pathogen growth. Recognition of effectors by NLRs often involves monitoring another host protein for effector-induced molecular perturbations; upon detection of these modifications, an immune response is triggered. For example, the NLR ZAR1 senses effector-mediated perturbations of associated kinases to detect at least 6 families of T3SEs. The effector HopF2a/HopF1r from P. syringae pv. aceris M302273PT has been found to trigger immunity in Arabidopsis. The objective of my research was 1) to identify other potential genetic requirements for HopF2a/HopF1r recognition in Arabidopsis, and 2) to understand how these components are involved in the HopF2a/HopF1r recognition mechanism. In this thesis, I demonstrate that recognition of HopF2a/HopF1r requires the R gene ZAR1, as well as the kinases ZRK3 and PBL27. The results of this project provide insight into how a single NLR is able to recognize several unrelated effectors by associating with different members of a diverse family of kinases, providing plants with the potential to defend themselves against a wide variety of pathogens.
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https://utoronto.zoom.us/j/87272098970
Meeting ID: 872 7209 8970
Host: Darrell Desveaux (darrell.desveaux@utoronto.ca)
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PhD Exit Seminar - Nawar Alwash (Levine Lab)
The role of the foraging gene (for) on Social Interaction Networks of Drosophila melanogaster
Abstract
Social interactions are prevalent in the lives of many animals, including Drosophila melanogaster. D. melanogaster aggregate on fermenting fruit, creating complex social environments within which they display various social behaviours. Deciphering the genetic underpinnings of these social behaviours is a difficult task, and few studies have attempted to address this. Here, I investigate how the foraging gene (for) influences social networks in D. melanogaster. for is a well-established example of a pleiotropic gene that modifies behavioural phenotypes. In fruit flies, there are two naturally occurring alleles known as rover and sitter, that differ in their foraging behaviour. for regulates multiple phenotypes, the best known of these involves larval food-related behaviours. Recent studies suggest that for’s influence extends to social behaviours across a variety of taxa. In my thesis, I report that for plays a role in influencing both behavioural elements of social networks and social network measures. Rover flies are characterized by having greater interaction rates, moving more during the trial, and having higher global efficiency values. While sitter flies spend more time interacting, are more likely to reciprocate an interaction, and create more homogeneous networks in which they display higher clustering coefficient values. My initial findings establish that this natural polymorphism of for influences the behavioural elements and social network measures. Using gene dosage manipulations, I show that differences in behavioural and social network phenotypes are mainly due to differences in the for locus. To address this further, I attempt to investigate the critical period of for expression on behavioural elements and social network measures. I separately knockdown for in the adult stage, as well as from the embryonic until the larval wandering stage. I did not find an effect of these developmental manipulations on behavioural elements and social network measures. These results suggest that the critical period of for expression in relation to behavioural elements and social network phenotypes is likely within the pupal stage, during metamorphosis. I also use promoter-driven rescues of for and show that the different for promoters may independently regulate different phenotypes within the analysis of social networks. Additionally, I report that for's influence on social behaviour exhibits plasticity to the environment. I report that even though network measures are resilient to social isolation, food, and sleep deprivation, the behavioural elements of a network are responsive to these stressors. Finally, I examine networks of mixed groups of the rover and sitter flies to shed light on how these strains may interact when they are in the same group. In summary, this thesis characterizes the effects of a specific gene on social networks. My findings emphasize the complexity of for’s influence on Drosophila social behaviour, and support the theory of genetic effects on social behaviour.
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https://utoronto.zoom.us/j/83847112654
Meeting ID: 838 4711 2654
Host: Joel Levine (joel.levine@utoronto.ca)
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PhD Exit Seminar - Morley Willoughby (Bruce Lab)
The recycling endosome protein Rab25 coordinates collective cell movements in the zebrafish surface epithelium during epiboly
Abstract
The surface epithelium of the zebrafish gastrula spreads to completely enclose an underlying yolk cell in a process known as epiboly. Epithelial epiboly is coordinated by oriented cell division and intercalation events, as well as the transmission and modulation of global tissue tensions. Currently, the molecular mechanisms underpinning zebrafish epithelial epiboly movements are unknown. Here I present the findings of a poorly characterized Rab protein, Rab25, during zebrafish epithelial tissue spreading and show that Rab25 is a recycling endosome protein that coordinates collective cell movements through endomembrane trafficking. Spatiotemporal analysis showed rab25a and rab25b transcripts were restricted to the outer epithelium upon epiboly initiation. Rab25 fluorescent fusion proteins distributed near cytokinetic midbodies and cell vertices during cytokinesis and cell intercalations, respectively. In maternal-zygotic Rab25a and Rab25b embryos, cytokinetic abscission and vertex remodelling was impaired, leading to anisotropic shaped multinucleate cells and uncoordinated cell rearrangements. At the tissue scale, global reductions in contractile actomyosin networks were associated with altered viscoelastic responses of the tissue. Taken together the slow cell rearrangements and defective tissue material properties likely contribute to delayed epiboly. I present a model in which Rab25 vesicle transport coordinates timely epiboly movements by regulating local cell behaviours and tissue scale forces via cytokinetic bridge abscission, cell intercalation and junctional actomyosin maintenance.
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https://utoronto.zoom.us/j/81741541128
Meeting ID: 817 4154 1128
Host: Ashley Bruce (ashley.bruce@utoronto.ca)
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PhD Exit Seminar - Bradley Laflamme (Desveaux and Guttman Labs)
A Systems-Level Study of Effector-Triggered Immunity in Arabidopsis thaliana
Abstract
Despite the continued prevalence of agricultural crop failures due to pathogen attack, any given plant host is resistant to most pathogen diversity. This phenomenon, often described as “broad-spectrum disease resistance,” is well-documented though poorly understood in terms of how various facets of defense contribute to its overall potency. Effector Triggered Immunity (ETI) is a major branch of plant innate immunity which entails the recognition of pathogen virulence factors, termed effectors, by intracellular Nucleotide-Binding Leucine Rich Repeat (NLR) proteins. We used the Arabidopsis thaliana-Pseudomonas syringae pathosystem to explore the contribution of ETI to broad-spectrum resistance. Though most P. syringae strains utilize a type III secretion system to translocate effectors into host cells, there is extensive genetic diversity across strains at effector-coding loci. We hypothesized that the effector sequence diversity of P. syringae, most of which has never been cloned and studied, contains hitherto unidentified ETI responses. We thus developed PsyTEC (P. syringae Type III Effector Compendium), a collection of 529 effectors from across 494 genetically diverse P. syringae strains, to screen for interactions between P. syringae effectors and the A. thaliana immune system, ultimately identifying 59 effectors spanning 19 protein families which elicit ETI. We found that the majority of P. syringae strains have the capacity to elicit ETI on A. thaliana, and that this broad-spectrum resistance to P. syringae diversity is conferred by a small repertoire of NLRs. Two NLRs, ZAR1 and CAR1, can potentially recognize 94.7% of strains due to their recognition of multiple broadly distributed effectors. We further explored ZAR1’s recognition of 5 P. syringae effectors and identified other genetic requirements for these ETI responses. We propose that ZAR1’s capacity for broad effector recognition is achieved by monitoring several cytoplasmic kinases for perturbation by effectors. This indirect monitoring enables a single NLR to guard against many virulence factors from diverse pathogen backgrounds. These systems-level insights into the ETI landscape of A. thaliana-P. syringae highlight its importance to the plant host’s broad-spectrum resistance strategy: ETI is ubiquitous in this system, and sophisticated mechanisms of indirect recognition enable immune responses to be disseminated through a small repertoire of NLRs.
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https://utoronto.zoom.us/j/89335412013
Meeting ID: 893 3541 2013
Host: Darrell Desveaux (darrell.desveaux@utoronto.ca)
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PhD Exit Seminar - Luka Sheppard (Tepass Lab)
The M-Region of α-Catenin Cooperates with Canoe, Ajuba, Vinculin and α-Actinin to Support Adhesion During Drosophila Embryonic Morphogenesis
Abstract
α-Catenin is a core component of the cadherin-catenin adhesion complex that couples cadherin to the actin cytoskeleton. The mechanosensitive α-Catenin M-region is thought to recruit F-actin binding partners such as Vinculin under application of force to support adherens junctions (AJs). However, the function of M-region mechanosensing and its cooperation with binding partners to support dynamic adhesion in vivo remains unclear. Actomyosin contraction stretches the M-region into an open conformation, revealing cryptic binding sites and recruiting Vinculin. Whereas the loss of α-Catenin compromises adhesion and morphogenesis, null mutants for the α-Catenin-binding partners Vinculin, α-Actinin and Ajuba complete embryogenesis with no or minimal defects. Furthermore, the contribution of the M-region to recruitment of the only reported binding partner with any strong adhesion phenotype, Afadin/Canoe, has not been explored in vivo. Here we report an essential requirement for the M-region to epithelial integrity during Drosophila morphogenesis. We determine the distinct contributions of the M-region subdomains, M1, M2 and M3, with M2 making the major contribution to adhesion. M2 is required for the mechanosensitive enrichment of Ajuba, and together with M3 supports Canoe recruitment to tricellular junctions and reinforces AJs. In contrast, M1 negatively regulates adhesion through its inhibition of Ajuba recruitment and possibly through recruitment of α-Actinin. M1 also recruits Vinculin, which supports E-cadherin and adhesion at high tension edges. However, we find this role to be subtle - ultimately dispensable for adhesion. We also found that the M1-Vinculin interaction is redundant with Canoe, and a striking genetic interaction between the M-region and Canoe, together suggesting that Canoe and the M-region act in parallel to support AJs. Our data clarify the function of the cadherin-actin interface and argue that α-Catenin and its interaction partners are part of a cooperative and redundant network that supports AJs remodelling in embryogenesis.
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https://us02web.zoom.us/j/85720950715?pwd=ZnJjUXdWMnBCcm4wTldPdU9Zcm5pQT09
Meeting ID: 857 2095 0715
Host: Ulrich Tepass (u.tepass@utoronto.ca)
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PhD Exit Seminar - Sonhita Chakraborty (Yoshioka Lab)
Investigation of the biological function of plant Cyclic Nucleotide Gated Ion Channel 2 in auxin signaling
Abstract
Calcium (Ca2+) is a ubiquitously important signaling molecule in eukaryotic cells. For plants, developmental cues, and signals from the environment lead to changes in cellular Ca2+ concentrations that are required to activate appropriate responses. CYCLIC NUCLEOTIDE GATED CHANNELS (CNGCs) are ion channels that are thought to conduct cations like Ca2+ in plant cells. Of the 20 members of the Arabidopsis CNGC family, the isomer CNGC2 is of special interest as the loss-of-function of CNGC2 leads to defects in multiple physiological processes.
In this thesis, I investigated the biological function of CNGC2 and propose that CNGC2 is important in auxin homeostasis in addition to its prescribed role in immunity.
The first suppressor of the autoimmune CNGC2 null mutant was found to carry a mutation in the auxin biosynthesis gene YUCCA6 (YUC6). An investigation into alterations in auxin signals revealed that the CNGC2 knockout mutant dnd1/cngc2 exhibits abnormalities in auxin signaling and hyper-accumulates endogenous auxin, indole-3-acetic acid (IAA). This is the first report of CNGC2 being potentially involved in auxin signals through auxin biosynthesis. I next showed that a loss-of-function of another IAA biosynthesis gene, TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1 (TAA1) also suppresses phenotypes associated with cngc2. This indicated that hyper-accumulation of IAA is a source of cngc2 phenotypes. IAA-induced Ca2+ influx is impaired in cngc2, and this phenotype is alleviated in the absence of YUC6. This indicates that CNGC2 functions in auxin-induced Ca2+ signals that can influence auxin homeostasis. The hyper accumulation of auxin in cngc2 positions CNGC2 in a negative feedback loop of auxin biosynthesis.
Since all 11 members of the YUC gene family are thought to be involved in auxin biosynthesis, I asked whether a loss-of-function in any YUC gene could suppress cngc2 phenotype. Of all the yuc mutants tested, only yuc6 could suppress cngc2 phenotypes. YUC6 and TAA1 have been shown to exhibit similar tissue-specific expression patterns at the vasculature, a pattern not observed with other YUCs. This indicates a specific role of YUC6 in the CNGC2-mediated auxin feedback loop, which may be governed by its unique expression pattern.
Collectively, these findings cast a new light of CNGC2’s complex regulation by auxin signaling and immune responses.
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https://utoronto.zoom.us/j/81354513761
Meeting ID: 813 5451 3761
Host: Keiko Yoshioka (keiko.yoshioka@utoronto.ca)
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PhD Exit Seminar - Tim Jiang (Zhao Lab)
The Role of HSP90C in Chloroplast Maturation and Thylakoid Protein Transport
Abstract
The chloroplast is an organelle that has a primary role in performing photosynthesis in green plants and contains thousands of proteins. Since most chloroplast proteins are synthesized outside of the chloroplast on cytosolic ribosomes, they are imported into chloroplasts in mostly unfolded states. A protein quality control system in the chloroplast is then responsible for their correct folding, assembly, and repair, or facilitate their removal if terminally damaged. The composition and translocation process of the protein import machineries on the chloroplast envelopes and the thylakoid membranes have been studied in-depth; however, how stromal factors are involved in bridging chloroplast membrane import and thylakoid transport of thylakoid proteins is still poorly understood. In this thesis, I elucidated a role and mechanism of action for the essential plastid HSP90 family heat shock protein HSP90C in this process. Using a thylakoid lumen-localized subunit of the oxygen evolving complex, I demonstrated that PsbO1 is tightly regulated by the HSP90C complex in the stroma. By analyzing the chloroplast maturation process, the essential role of HSP90C and its ATPase activity in regulating and balancing the influx and efflux of PsbO1 fusion proteins within the stroma was demonstrated. A specific role of HSP90C in forming a guiding complex that interacts with the SEC translocation system and facilitates ATP-dependent thylakoid protein targeting and transport was proposed. These studies reveal critical roles of HSP90C in the chloroplast and thus provide new insights into the molecular mechanism of HSP90 family molecular chaperones in regulating organellar protein homeostasis.
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https://us02web.zoom.us/j/89416221060?pwd=SlhERlJNZzZGRGcxc3pkczRYQm1uQT09
Meeting ID: 894 1622 1060
Host: Rongmin Zhao (rongmin.zhao@utoronto.ca)
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PhD Exit Seminar - Eliana Vonapartis (Gazzarrini Lab)
The Transcriptional Regulation and Role of XERICO in Arabidopsis thaliana Development and Stress Responses
Abstract
Every year, periods of agricultural drought devastate crop yields globally, limiting food production and leading to significant economic losses. The phytohormone abscisic acid (ABA) accumulates during drought and is crucial for the plant to mount an effective stress response. Arabidopsis thaliana XERICO (XER) is a stress-inducible E3 ubiquitin ligase that plays a role in regulating ABA levels, stomatal closure, and drought tolerance. While the function XER in ABA homeostasis and drought tolerance has been substantiated in various monocot and dicot species, other roles for XER in controlling plant stress responses remain unknown. In addition, knowledge of the mechanisms governing XER transcriptional regulation under stress conditions is limited. Thus, this work aimed to further characterize the function of XER in Arabidopsis stress responses and to shed light on the regulation of its spatiotemporal expression pattern by stress stimuli. The data in this thesis demonstrate that XER expression is repressed by stress-responsive CBF4/DREB1D, which was identified by yeast one-hybrid screening and established as a negative regulator of ABA responses and drought tolerance through CRISPR/Cas9 mutant analysis. Furthermore, by characterizing loss-of-function xer mutants and transgenic lines overexpressing a RING-mutated XER variant, XER was found to inhibit stomatal development and gibberellin (GA) signaling. Lastly, 88 ABA-regulated putative XER interactors were identified by high-throughput yeast two-hybrid screening, unveiling potential mechanisms and pathways through which XER may modulate ABA and stress responses. Overall, the results presented in this thesis reveal that XER contributes to stress responses by enhancing ABA and repressing GA signaling, as well as by restricting stomatal development, and that tight transcriptional control of XER expression by CBF4 is important for the plant to balance plant growth and survival under adverse environmental conditions such as drought.
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https://utoronto.zoom.us/j/84827455870
Meeting ID: 848 2745 5870
Host: Sonia Gazzarrini (sonia.gazzarrini@utoronto.ca)
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PhD Exit Seminar - Norzin Shrestha (Lovejoy and Ralph Lab)
The Neural Basis of a Circadian Oscillator Underlying Time Memory
Abstract
Mice anticipate the time of day that important conditions or events are likely to occur based on their previous experiences with those conditions. They can learn to expect positive (rewarding) or negative (aversive) events. The ability to predict the coming of specific events in its environment (time memory) is crucial for its survival. Current evidence has led to three hypotheses: 1) The location of the clock(s) underlying time memory is located in the dopamine target regions, 2) The hippocampus is a site of cells that register the time of day, and 3) Subpopulations of hippocampal cells that are activated during spatial learning are also responsible for time of day learning. If the same cells that are involved in the formation of spatial memories are also oscillators, then the cells most closely involved in context learning should be the ones most readily phase shifted by a learning experience. Therefore we should be able to distinguish cells linked to specific memories by recording the phase of their circadian rhythms. Using qRT-PCR and ISH, I found that several tissues as a whole do not exhibit shifts of their clocks, particularly, the hippocampus. Instead, behavioral conditioning induced a phase shift in the circadian clock protein PERIOD 2 expression, specifically in the shell region of the nucleus accumbens (NAS) and the dorsal striatum (DS). Next, using the Tetracycline Transactivator Controlled Genetic Tagging of Active Neural Circuits (TetTag) system and ICC, my data suggests that only a few cells in the hippocampus participated in the conditioning. The hippocampal cells whose rhythms were set at the time of conditioning may be critical to the expression of craving. In human health, this research will provide a new perspective on the regulation of craving and as such, will contribute to the development of treatments for drug/substance abuse.
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https://zoom.us/j/96530805695?pwd=ZVVJZXY2cTU2aUVxT0U2S09FM0lZZz09
Meeting ID: 965 3080 5695
Host: David Lovejoy (david.lovejoy@utoronto.ca)
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PhD Exit Seminar - Roxanne Fournier (Harrison lab)
Osteocyte Form and Function in Loading and Unloading Environments
Wednesday, October 14th, 2020 at 1:10pm
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https://us02web.zoom.us/j/86106153621?pwd=WVhhUUJyZFJNc3JRVzAxd0d3d2JCUT09
Meeting ID: 861 0615 3621
Passcode: Bonecell
Abstract:
Long-term human spaceflight presents unprecedented challenges to the human body, such as microgravity-induced bone loss. Without intervention, astronauts lose between 1 – 2% bone mineral density per month in weight bearing bones and the cellular mechanisms involved in this process are still poorly understood. To date, the most abundant cell of bone, the osteocyte, has rarely been subjected to spaceflight. Yet osteocytes are known to be important regulators of bone loss in immobilized and elderly people. The first aim of this thesis was to develop novel 3D culture methods to study osteocytes using the Rotary Cell Culture System (RCCS). This device is commonly used for both suspension culture and simulated microgravity by clinorotation. The second aim was to investigate morphological and gene expression changes in MLO-Y4 cells following suspension culture or clinorotation. We found that the ideal 3D scaffold consisted of 2% type I collagen mixed with 6% synthetic hydroxyapatite (collagen-HA). The scaffold was successfully formed into droplets for suspension culture, whereas it was adhered to the RCCS vessel walls for clinorotation. After 3 days of suspension culture, MLO-Y4 cells embedded in the collagen-HA droplets showed reduced expression of the mechanosensitive genes DMP1, E11, IL-6, and RANKL and the number of cells was elevated compared to the static control. We estimated that the drag force acting on the aggregate of droplets was in the range of 2.1 – 4.4 dynes/cm2, speculated to provide a small but significant stimulatory effect on the cells located on the surface, which may de-sensitize them over the course of 3 days. Following clinorotation for 3 days, MLO-Y4 cells showed reduced expression of DMP1 and E11 genes while the cell number was unchanged compared to the static control. Since clinorotation and spaceflight cause the de-differentiation of other bone cells, we speculated that our MLO-Y4 cells were also de-differentiating during clinorotation, resulting in the lower expression of osteocyte-specific genes.