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Zogli, Prince Kudjoe

Plant Biology

2015

PhD

The Arabidopsis SON1 F-box protein was implicated in regulating a pathogen defense pathway, but its exact function in wild-type plants is unknown. As an F-box protein it was predicted that SON1 would assembles into a SON1-SCF ubiquitin ligase complex that recruits specific plant defense-related proteins for proteolysis. The yeast 2-hybrid assay was used to screen for potential substrates for a putative SON1-SCF ligase, leading to the identification of Arabidopsis LNK2 as a SON1-binding factor. Comprehensive protein-protein interaction analysis has shown that the binding of SON1 to LNK2 protein is specific, because closely related, full-length Arabidopsis F-box proteins do not interact with LNK2. However, when tested in isolation, some fragments derived from the paralog proteins did bind SON1, suggesting that higher order structure or inter-domain interference affects the ability of SON1 to recruit substrate. When analyzed for interaction domains, three regions of SON1 were identified that bind to LNK2 and a LNK2 binding region spans a conserved amino acid region. Phylogenetic analysis revealed that there is a paralogous gene called LNK1 in Arabidopsis, and both LNK1 and LNK2 are restricted to the plant kingdom. LNK2 and LNK1 are seen to be widely distributed in embryophyte seed and spore plants. Genetic analysis and complementation tests showed that LNK1 and LNK2 regulate flowering and photo-morphogenesis redundantly. Though lnk1 and lnk2 mutants look similar to wild-type plants, lnk1 lnk2 double mutant plants possess a long hypocotyls and flower late compared to wild-type plants. Because SON1 is implicated in plant defense, lnk mutants were assessed for susceptibility to a virulent oomycete pathogen, Hyaloperonospora arabidopsidis (Hpa). Interestingly, lnk mutants supported less disease development, suggesting a role of the wild-type LNK proteins in the enabling of pathogen colonization or in repressing host defenses. To confirm that each of the phenotypes described were a consequence of lnk1 and lnk2 mutations, wild type LNK1 and LNK2 were introduced into lnk1 lnk2 plants and examined. For most phenotypes, genetic complementation and thus restoration of a WT phenotype was observed. However, differences were uncovered in the ability of LNK genes to rescue the phenotypes, indicating specialization of function. The interaction of LNK2 with SON1 suggests that SON1-dependent ubiquitination and proteasomal degradation may control LNK2 abundance. I show by a cell free protein degradation assays that proteasome-based degradation of LNK2 as well as LNK1 is possible. Data showed that SON1 binds to ASK1 in-planta suggest the existence an SCFSON1 complex that targets LNK2 for polyubiquitination and its subsequent degradation by the proteasome. The data presented in this dissertation indicates a role for LNKs in flowering and plant defense and also suggest that proteasome-base regulation of LNK turnover may be utilized to regulate LNK protein abundance and proper maintenance of the circadian clock.