Analysis of E3 ligases and identification of ubiquitination sites regulating turnover of the plant immune signaling kinase BIK1

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Grubb, Lauren

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thesis

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eng

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plant immune homeostasis , ubiquitination

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Plants possess sophisticated mechanisms for responding to the threat of pathogens. Microbial patterns such as bacterial flagellin are perceived by receptor complexes located at the plant plasma membrane. This perception initiates a signaling cascade which culminates in a robust immune response including an influx of Ca2+ into the cytosol, an apoplastic burst of reactive oxygen species, and up-regulation of defence genes. The receptor-like cytoplasmic kinase BIK1 is activated by many different receptor complexes and is a key component of the immune system. Although necessary for survival, the plant immune response must be tightly regulated to avoid fitness costs and maintain normal growth and development. As an important component in several immune responses, BIK1 must be maintained at optimal levels; however, the mechanism for this is poorly understood. Previous work identified the calcium-dependent protein kinase CPK28 to be a negative regulator of immunity and of BIK1 accumulation. It also provided evidence for proteasome-mediated turnover. Our collaborators identified the plant U-box proteins PUB25 and PUB26 as E3 ligases responsible for destabilization of BIK1 through polyubiquitination. The major objectives of my thesis were to determine which residues on BIK1 were polyubiquitinated, and to determine how CPK28, PUB25 and PUB26 regulate BIK1 turnover. I found that PUB25 and PUB26 associate with CPK28 and used epistasis analysis to place CPK28 upstream of PUB25 and PUB26 in this immune signaling pathway. This thesis also presents work toward determining CPK28- and PUB25- or PUB26-mediated BIK1 ubiquitination sites, as well as providing a dataset of ubiquitinated plasma membrane proteins through generation of a ubiquitome. Overall, the work presented in this thesis provides insight into the regulation of BIK1 turnover and contributes to our understanding of plant immune homeostasis.

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