Investigating ubiquitin-mediated turnover of the key immune signaling protein kinase BOTRYTIS INDUCED KINASE1
Plants are susceptible to infection from bacterial, fungal, and viral diseases; although sessile, they are far from defenseless. Plants have evolved complex immune systems for the detection of pathogens and initiation of signal transmission 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 polyubiquitination by PLANT U-BOX 25 and subsequent degradation by the proteasome. My thesis investigates the functional importance of ubiquitination of BIK1 and how this process impacts BIK1 turnover. We identified nine in vivo BIK1 ubiquitination sites using highly-sensitive mass spectrometry. Higher order ubiquitin-ablative mutants were generated to determine which lysine residues are important for proteasomal turnover. Using an in vitro approach, I demonstrate that ablating all nine of these BIK1 ubiquitination sites slows the rate of degradation and therefore that ubiquitination may be important for regulating BIK1 levels in plants. Genetic analyses were explored using site-directed BIK1 mutants in a bik1 knockout or Col-0 background but yielded inconclusive data. This work contributes to the growing body of literature on ubiquitination regulation 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.