In Vivo Imaging Of The S-Locus Receptor Kinase In Transgenic Self-Incompatible Arabidopsis Thaliana

Self-incompatibility (SI) in the Brassicaceae commences with the haplotype-specific binding and recognition of the pollen coat-localized S-locus cysteine rich (SCR) protein by the stigma epidermal cell-localized plasma membrane-spanning S-locus receptor kinase (SRK) protein, and culminates in the inhibition of germination of genetically-related pollen at the stigma surface. This self-recognition system allows the cells of the stigma to screen pollen that has landed on its surface for genetic relatedness, which both prevents self-fertilization and increases genetic diversity in the population. The events downstream of the ligand-receptor interaction are not well understood. However, the transgenic self-incompatible Arabidopsis thaliana model system, made possible by the transfer of SRK and SCR genes from self-incompatible A. lyrata, has proven useful in addressing questions related to SI. For example, recent results, including those from this dissertation, challenge the current mechanistic view of SI put forth from studies with Brassica. A candidate gene approach that included the use of T-DNA insertional and point mutants, targeted down-regulation, and yeast two-hybrid interaction techniques, was employed for identifying potential downstream signaling components. Candidate genes were selected based on their sequence similarity to genes identified in Brassica as being involved in SI, the commonalities between SI and plant immunity, and their high and specific expression in the stigma. In order to gain clues about the intracellular events that occur prior and subsequent to SKR-SCR recognition, SRK was successfully tagged with fluorescent protein and transformed into A. thaliana, where it is expressed and functional in stigma epidermal cells. Confocal microscopy was used to characterize the localization and dynamics of SRK in live cells using several different chimeric genes designed to highlight the localization of the different native protein species produced from the SRK gene driven by either its native or a stigma-specific promoter. Results presented in this dissertation provide insight into SRK localization in the context of pollination and floral development, and reveal characteristic architectural features of stigma epidermal cells that will be useful in future studies of this unique cell type