Wed Oct 28 // Katherine Dunning // MSc Candidate, Queen's University

Katherine Dunning
MSc Candidate, Monaghan & Snedden Labs

Checkmate: You've been ubiquitinated! Investigating ubiquitin-mediated turnover of BIK1, a key plant immune signalling kinase

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. ​