Production and characterization of the self-incompatibility locus glycoprotein and receptor kinase

Self-incompatibility (SI) is a phenomenon that involves recognition of self versus non-self pollen, leading to the rejection of self-related pollen and preventing self-fertilization. The effects of inbreeding versus outbreeding on offspring quality have been studied since Darwinian times. The block to self-fertilization results in outcrossing and an increase in genetic diversity. In Brassica, SI is genetically controlled by haplotypes of a complex locus, designated the S locus. When pollen and stigma share an active S haplotype, an incompatible response occurs resulting in the failure of pollen hydration, germination, and pollen tube ingress into the cell wall of the stigmatic papillae. Two Brassica oleracea stigma-specific proteins are required for SI: SLG, a soluble cell wall-localized glycosylated protein, and SRK, a receptor-like integral plasma membrane glycoprotein with serine/threonine kinase activity. In a proposed model of SI, the extracellular, SLG-like domain (S-domain) of SRK, separately or complexed together with SLG, could interact with an as yet unknown pollen ligand after a self-pollination event to trigger the SRK kinase domain to become activate and act upon downstream elements responsible for blocking pollen tube growth. The soluble and abundant SLG protein may even act as a shuttle to carry a pollen ligand through the cell wall to the membrane-bound SRK. The work presented here involves the production of these two proteins, their use to find interacting proteins, and to characterize their functions. The first section details the production of the extracellular domain of SRK in E. coli and its use in an interaction cloning scheme to identify interacting proteins and the purification and use of native SLG to find interacting proteins. Next, a baculovirus-infected insect cell culture system was used to express SLG and SRK. Insect cells expressing SRK were used in conjunction with immunofluorescence and a whole cell enzyme-linked immunosorbant assay (CELISA) to demonstrate that the receptor is targeted to the cell surface and is oriented with its N-terminal S domain towards the outside of the cell. Attempts to characterize SRK protein oligomerization, interaction with SLG, induction of autophosphorylation, and phoshorylation of stigma substrates were made.