Plants are susceptible to infection from bacterial, fungal, and viral diseases; although they cannot run away, they are far from defenseless! Plants have complex immune systems that are responsible for the detection of pathogens and initiating signal transmission in order to respond to and fight off infections. BOTRYTIS INDUCED KINASE 1 (BIK1) is a key regulator of immune signaling that is required for protection against multiple types of pathogens. BIK1 is activated through phosphorylation by plasma-membrane receptor kinases and in turn phosphorylates and activates other downstream proteins. To prevent overactive immune responses, accumulation of BIK1 is tightly regulated through ubiquitination by PLANT U-BOX 25 and subsequent degradation by the proteasome. This thesis investigates the functional importance of ubiquitination on BIK1 and how ubiquitination impacts BIK1 turnover. We identified nine BIK1 ubiquitination sites using highly sensitive mass spectrometry. Higher order ubiquitin-ablative mutants were generated to determine which terminal residues are important for proteasomal turnover. We verified that BIK1 ubiquitin-ablative mutants were catalytically active and appropriately localized to the plasma membrane and nucleus when transiently expressed in tobacco. Using a semi-in vivo approach, I demonstrate that ablating all nine of these BIK1 ubiquitination sites slows the rate of degradation and therefore may be important for regulating BIK1 levels in plants. Genetic analyses were explored using mutated BIK1 variants in a bik1 knockout or WT background but yielded inconclusive data. This work contributes to the growing body of literature on post-translational modification by ubiquitination and highlights that, unlike certain post-translational modifications which are highly residue specific (ie phosphorylation), ubiquitination substrate specificity is likely less critical at the residue level. ​

Most animals harbor a gut microbiota that consists of potentially pathogenic, commensal and mutualistic microorganisms. In insects, antimicrobial peptides (AMPs) and the reactive oxygen species (ROS)-producing enzyme “Dual oxidase” (Duox) are the central regulators of gut mucosal immunity. They antagonize pathogenic bacteria and maintain gut homeostasis. However, the non-specific harmful activity of AMPs and ROS on microorganisms raises the question about the role of AMPs and Duox in the maintenance of mutualistic gut symbionts. In my seminar, I will highlight how AMPs and Duox control the establishment of a specific gut symbiosis in the bean bug Riptortus pedestris, which harbors in a symbiotic compartment of the midgut the mutualistic bacterium Burkholderia insecticola.
We have demonstrated that symbiosis-specific as well as immunity-specific AMPs are produced massively during B. insecticola infection of the gut and that resistance to these AMPs is crucial for the capacity of the bacterium to colonize the gut. On the other hand, and contrarily to our initial expectation, we found that Duox-dependent ROS did not directly contribute to epithelial immunity in the midgut in response to B. insecticola or to pathogenic bacteria. Instead, we demonstrated that Duox is essential for symbiosis and the colonization of the gut by the aerobic B. insecticola by mediating the formation of a respiratory network enveloping the gut.

Irrespective of species, plant roots have remarkably similar patterning, and thus, many cell types are considered functionally homologous across species.  Despite these similarities, there is also diversity in root cell types, such as the exodermis, which is present in a reported 89% of angiosperms, but absent in the intensely studied model species Arabidopsis (Perumalla, Peterson, and Enstone 1990).  Additionally,  multiple cortical cell types exist in species like tomato and rice (Henry et al. 2015), but only a single cortical cell type exists in Arabidopsis.  To understand this diversity we profiled tomato root cell type translatomes and chromatin accessibility.  Using xylem differentiation in tomato, relative to Arabidopsis, examples of functional innovation, repurposing and conservation of transcription factors are described.  Repurposing and innovation of genes are further observed within an exodermis regulatory network and illustrate its function.  Finally, we extend these comparisons between Arabidopsis and tomato, to rice, and explore the question of homology between cell types across diverse species. 

Carmen Gonzalez-Ferrer 
MSc Candidate, Monaghan Lab 

Initial characterization of a subgroup of Arabidopsis group VIII receptor-like cytoplasmic kinases in immune and flowering time pathways

In order to defend against disease, plants have evolved a tightly coordinated signaling
network to rapidly prevent the spread of infection. Given the severity of yearly crop loss to pathogen threats, further understanding how plants defend against pathogens is vital to the improvement of current agricultural strategies. Receptor protein kinases on the plant cell surface recognize microbes and trigger phosphorylation-dependent signaling events, ultimately leading to genetic reprogramming. Receptor-like cytoplasmic kinases are key mediators of immune signal transduction, but many remain largely understudied. This thesis focuses on the group VIII RLCKs in Arabidopsis thaliana, also known as the AtPTI1-like kinases, for which an immune function has not been shown. Using a functional genomics approach, I demonstrate that a subgroup of the group VIII RLCKs regulate flowering time and may additionally function as negative regulators of immunity by mediating the oxidative burst. Additionally, I found that one of these RLCKs, PTI1-1, interacts with the negative regulator of defense CALCIUM -DEPENDENT PROTEIN KINASE 28 (CPK28) and the positive immune regulator BOTRYTIS INDUCED KINASE 1 (BIK1), further suggesting the involvement of these RLCKs in immune signal transduction. Broadly, this work has shown two novel functions for the AtPTI1-like kinase family in both defense and development, and primes future research for this previously uncharacterized group.
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We are going virtual! Please mark your calendars and get in touch if you want to present your work. The format is flexible but will typically be 20-30 min seminar followed by 20-30 min Q&A/discussion. 

We are looking forward to it! 

Joseph Quagraine
Masters Candidate, Regan Lab
Queen's University

Anthropogenic activities have led to widespread heavy metal contaminants such cadmium and arsenic. When left untreated, they pose risk to both human and ecosystem health as well as further reduce arable lands. Phytoremediation, which is the use of plants and their associated microorganisms to clean up such contaminants, is environmentally friendly, cost effective and fast-growing trees such as Populus sp. are good candidates for phytoremediation because of their tolerance of heavy metals, high biomass and their distribution across much of the northern hemisphere. However, the molecular mechanisms underlying poplar’s phytoremediation are poorly understood. Although Populus is a model tree species, with a sequenced genome and many genetic and genomic resources, the identification of genes for important tree traits is still slower than in other model plants such as Arabidopsis. This study uses a functional genomics approach to identify genes related to bioremediation by taking advantage of a large collection of activation tagged poplars (Populus tremula x P. alba hybrid 717-1B4) created by Dr. Sharon Regan’s  Lab. After screening over 1700 independent transgenic lines for characteristics that could affect phytoremediation, seven mutants had altered root biomass whereas 15 mutants had altered response to heavy metals. Of the seven root phenotypes identified, two previously studied mutants, called rippled leaf and adventitious root were further investigated. RT-qPCR analysis showed an up-regulation of CYCLIND1;2 and E3 ubiquitin-protein ligase XBAT32/33 in the roots of rippled leaf and adventitious root mutants respectively. The upregulation of CYCLIND1;2 is suspected to increase root biomass through accelerated cell cycle division. XBAT32/33 on the other hand is suspected to promote the production of lateral roots through the regulation of ethylene biosynthesis. Altogether, this study provides a starting point in the quest to discover key genes responsible for phytoremediation and could lead ultimately to the development of biomarkers for selection of superior trees from natural population for clean-up purposes.

Dr. Matthew Andrusiak
Biological Sciences, University of California, San Diego

The genetic mutation and de-regulation of prion-like domain (PrLD) containing proteins is over-represented in human disease. Despite their role in human disease, little is known about how PrLD’s regulate protein function. PrLD-containing proteins are often capable of liquid-liquid phase separation (LLPS) resulting in the formation of non-membrane bound cellular compartments. The in vivo function and cellular mechanisms regulated by LLPS remain unknown. My work identified the PrLD coding gene tiar-2, a C. elegans member of the TIA1 family, as an intrinsic inhibitor of axon regeneration. TIAR-2 forms granules and inhibits axon regeneration in a dose-dependent manner. TIAR-2 undergoes LLPS in vitro and granules have liquid-features in vivo. Following axon injury, TIAR-2 granule number increases, and their liquid-like features are significantly reduced. Importantly, the PrLD of TIAR-2 is necessary and sufficient for its ability to inhibit regeneration and form granules. Post-translational modifications, such as phosphorylation, have been shown to act as molecular switches regulating LLPS. TIAR-2 is serine phosphorylated and this modification is required for TIAR-2 granule formation and function in axon regeneration. This work identified axonal injury as an acute cue that modulates the formation of LLPS granules and the function of the PrLD containing protein TIAR-2. Future research efforts will focus on understanding the role of prion-like domains in the regulation of biological outputs during nervous system and organismal development, as well as following neuronal injury.