Rui Huang, PhD Candidate, diCenzo Lab and Snedden Lab Characterizing the functional impact of Sinorhizobium meliloti bacA allelic variation on symbiosis with legume hosts The symbiosis between legume plants and rhizobial bacteria are highly specific and require both partners to be compatible with each other during the process of symbiotic development. A range of host and symbiont properties regulate the specificity of these relationships. In certain clades of legume plants, the rhizobial BacA transporter and legume-expressed NCR peptides are thought to play a role in defining host/symbiont compatibility. The inverted repeat-lacking clade (IRLC) of legumes expresses NCR peptides to initiate and maintain terminal differentiation of rhizobia, a developmental process that leads to mature, nitrogen-fixation bacteroids. The BacA transporter is essential in transporting NCR peptides and protecting the rhizobia from the antimicrobial activity of these peptides. A previous study found that replacing the bacA allele of Sinorhizobium meliloti with the ortholog from Rhizobiumleguminosarum allowed symbiosis with the host Melilotus officinalis but not with Medicago sativa. In this thesis, I investigated the mechanisms underlying the symbiotic specificity of bacA alleles. On the host legume side, the nodule transcriptomes of M. sativa and M. officinalis were assembled, and their NCR peptide transcripts were identified. The transcriptomic data revealed an approximately 2-fold greater abundance of transcripts encoding highly cationic NCR peptides (isoelectric point > 9.5), which are known to have antimicrobial properties, in M. sativa vs M. officinalis. I hypothesize that the difference in the abundance of highly cationic NCR peptide contributes to why the R. leguminosarum BacA can support symbiosis with M. officinalis but not M. sativa. In addition, I hypothesize that unlike the S. meliloti BacA, the R. leguminosarum BacA is unable to efficiently transport all of the highly cationic NCR peptides of M. truncatula, resulting in a build-up in the bacterial periplasm, leading to cell death. On the rhizobia side, I combined genetic analysis and computational modelling to better understand the BacA transporter. Site-directed mutagenesis analyses showed that S. meliloti
Q193G and N312G BacA mutants recreated the symbiotic phenotypes of S. meliloti carrying the R. leguminosarum bacA allele when paired with hosts M. sativa and M. officinalis. In addition, I generated mutant libraries of S. meliloti and R. leguminosarum bacA through error-prone PCR that identified amino acid residues important for BacA function. Together with BacA structural models that I generated, these data provide novel insight and hypotheses into the functional differences between S. meliloti and R.leguminosarum BacA and their ability to transport NCR peptides. Collectively, the data presented in this thesis provides a deeper understanding of how BacA and NCR peptides may contribute to host/symbiont compatibility in rhizobium – legume symbioses. Dr. Catherine Cullingham, Assistant Professor, Carleton University Using the mountain pine system to demonstrate the use of genomics to understand risk and resiliency of forests to large scale disturbances Mountain pine beetle has had significant impacts in western Canadian pine forests over the past two decades. Over 20 million hectares of pine in Canada has been affected impacting industry, communities, carbon cycling and ecosystem function. In 2006 the beetle expanded into central Alberta where it has encountered a novel host, jack pine. Many questions have arisen since then including, is jack pine an appropriate host for mountain pine beetle? What is the potential for continued spread across the Boreal forest? What genes underlie host susceptibility? Using population genetics, spatial ecology, and molecular biology my lab helps to answer some of these important questions, and provide useful outputs for management and predictive modelling. Through this integrated approach we provide insights into whether jack pine is a suitable host for mountain pine beetle which is an important step towards understanding the spread-risk potential of the beetle. We also have begun to identify the genetic component of pine host resilience to mountain pine beetle. Given the increasing frequency and intensity of biological invasions in forest ecosystems, approaches that consider interactions from the landscape to the individual will be critical for ensuring forest resiliency in the future.
AAKANX PANCHAL - MSc Candidate Tackling the rhizobium competition problem by engineering exclusive compatibility between legumes and inoculant rhizobia Legume plants can benefit tremendously from their relationship with nitrogen-fixing soil bacteria called rhizobia. Within structures rhizobia induce along legume roots called nodules, rhizobia fix atmospheric dinitrogen into ammonia. High nitrogen-fixing rhizobium inoculants can provide legume crops with enough bioavailable nitrogen under ideal conditions to limit nitrogen fertilizer use and its negative environmental impacts. However, low nitrogen-fixing rhizobia native to the soil can outcompete high nitrogen-fixing rhizobia for nodule occupancy, reducing the benefit to the legumes and ultimately perpetuating dependence on nitrogen fertilizer. A potential solution to this "rhizobium competition problem" is engineering exclusive compatibility at the level of nodule occupancy between high nitrogen-fixing rhizobium inoculants and their associated legume crops. Nodule occupancy begins with rhizobia producing Nod factors specific to legume Nod factor receptors, where Nod factors and Nod factor receptors are like keys and locks. It is the ability of low nitrogen-fixing rhizobia native to the soil to produce the same Nod factors as high nitrogen-fixing rhizobium inoculants that allows low nitrogen-fixing rhizobia to nodulate the legume. Exclusive compatibility between the rhizobium inoculant and legume crop would involve inoculant rhizobia producing Nod factors that are unique in the target environment and legumes producing matching Nod factor receptors, such that low nitrogen-fixing rhizobia producing the original Nod factors should no longer be able to nodulate the legume. To test the feasibility of using this strategy, we have been working towards genetically engineering a model rhizobium-legume pair, Sinorhizobium meliloti and Medicago truncatula, to use Nod factors and Nod factor receptors from a different rhizobium-legume pair, Mesorhizobium japonicum and Lotus japonicus. In my seminar, I will present our progress in bringing the Lotus japonicus Nod factor receptor genes together into a single construct for their eventual expression in Medicago truncatula.
MSc Candidate, Aristizabal Lab Understanding the regulation and function of Cdk8 Cdk8 is a conserved protein kinase and a member of the Mediator transcription co-activator complex. Accumulating evidence highlights Cdk8 as a crucial oncogene in colorectal cancer, emphasizing the need to understand its function and regulation. Notably, CDK8 is amplified or overexpressed in approximately 60% of colon cancer tumors, and elevated CDK8 levels correlate with tumor stage and reduced patient survival. Supporting its oncogenic role, knockdown of CDK8 diminishes the proliferation of colon cancer cell lines, while overexpression induces tumor formation in immunodeficient mice, effects that depend upon Cdk8's kinase activity. Beyond colorectal cancer, growing research also implicates CDK8 in melanoma, leukemia, as well as breast, pancreatic, and prostate cancer, evidence that has stimulated the development of Cdk8 inhibitors for cancer therapy. Despite a growing interest to target Cdk8 for anti-cancer treatment we have limited information about Cdk8 regulation, a knowledge gap that may complicate efforts to block its activity. For my exit seminar, I will discuss my work using the budding yeast model system to understand the function and regulation of Cdk8 by leveraging disease-associated variability and recent insight from structural and biochemical analyses. Specifically, I have leveraged data from The Cancer Genome Atlas and ClinVar to identify CDK8 missense mutations that are likely to affect function. This work led me to find a cluster of mutations that localize to the kinase ATP binding pocket or sites of interaction with CycC and Med12, proteins known to regulate Cdk8 kinase activity. My findings so far suggest that most of the mutants disrupt Cdk8 function, resulting in a feedback mechanism that leads to an increase in Cdk8 protein and mRNA levels. In addition, my work examining the role of Med12 in regulating Cdk8’s activity in vivo showed that this interaction contributes to Cdk8 function in a condition specific manner. Collectively, this research sheds light on the function and regulation of Cdk8, work that will inform the development of targeted therapies and enhance our understanding of the mechanisms by which Cdk8 contributes to cancer development.
Dr. Paulo Teixeira, Professor at "Luiz de Queiroz" College of Agriculture, University of São Paulo Nonhost plants as a source of immune receptors against pathogens of agricultural importance Plants use NLR receptors to detect effectors from pathogens and activate defense responses. However, most immune receptors in the plant kingdom remain uncharacterized, especially those in nonhost plants that are resistant to a wide range of pathogens of agricultural importance. Here, we examined whether nonhost Solanaceae species can recognize effectors from the citrus pathogen Xanthomonas citri subsp. citri. We transiently expressed 22 effectors in tomato, eggplant, Nicotiana benthamiana and N. tabacum and found that these nonhost plants recognized a distinct, but overlapping, set of effectors. Interestingly, XopAZ was recognized by all species, indicating the presence of conserved NLR receptors that respond to this effector. We subsequently identified a novel pair of TIR-NLRs in Solanaceae that is required for XopAZ recognition. I will discuss how this approach may facilitate the identification of immune receptors that could be transferred to crops as a strategy to combat diseases.
MSc Candidate, Lougheed Lab Mapping the Contact Zone of Two Chorus Frog Mitochondrial Lineages in Southern Ontario Contact zones facilitate the study of diverging evolutionary lineages in primary (evolved in situ) or secondary (evolved in allopatry) contact; outcomes of contact zone dynamics have implications for species range limits, can clarify questions regarding species delineation and speciation itself, and may be relevant for conservation. Chorus frog populations within Ontario and Quebec (currently collectively classified as Pseudacris triseriata) possess distinct mitochondrial haplotypes representing Boreal (P. maculata) and Western Chorus Frogs (P. triseriata). These diverging mitochondrial lineages are in secondary contact in Southern Ontario, yet the exact location of contact is uncertain, lying somewhere between Toronto and southern Georgian Bay (a linear span of ~150km). I used environmental DNA (eDNA) from water sampled at ~50 chorus frog breeding ponds to better delineate the chorus frog contact zone. Environmental DNA facilitates extensive geographic sampling, allows for the identification of single wetlands with co-occurring populations, and is less invasive than tissue sampling (e.g. toe clipping, buccal swabbing, lethal sampling). I used droplet digital PCR with custom primer-probe mitochondrial cytochrome b sets that target each lineage (one primer-probe set for each lineage). My data refine the location of the contact zone and identify at least three ponds with both lineages present. My findings set the stage for future work in hybridization and speciation, and have implications for conservation and designations by the Committee on the Status of Endangered Wildlife in Canada (COSEWIC). My research also showcases the effectiveness of eDNA in mapping the distributions of cryptic or secretive species or evolutionary lineages.
Maureen Buchanan is an Anishinaabe Kwe, a member of the local urban Indigenous community of Kingston and a member of the Batchewana First Nation near Sault Ste. Marie. Maureen has lived in Kingston for 30 years and raised her family. She, like many others in her community, through volunteerism, has worked to create small spaces of urban indigenous visibility, community connectedness, cultural sharing and language learning. She is a founder of the Kingston Indigenous Languages Nest and a founding director of the All Our Relations Land Trust. The talk will describe a local land-based project in which urban Indigenous people and allies have put into practice an evolving practice of land stewardship based on a worldview of sacred relations with our ecological kin and informed by Western science. Given the twin crises of biodiversity loss and climate change, Maureen will discuss the actions of this group of volunteers over the last three years and what actions are on the horizon.
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