Identification and functional characterization of protein components of the plant kinetochore complex

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The goal is to identify and characterize several protein components of plant kinetochore complexes for better understanding of centromere functions and later approaches to construct de novo plant artificial chromosomes. We plan to elucidate the presence an functionality of candidates for chekpoint control proteins (Bub1, Bub3, Zw10, SKP1) and proteins interacting with spindle microtubuli (CBF5, Zw10) in Arabidopsis thaliana. We have already identified the Arabidopsis genes of the putative plant kinetochore proteins (KP) in silico. Arabidopsis transformants that express the KP-GFP fusion proteins will be generated to trace their cellular localization along the mitotic an meiotic cycle. Subsequently, by an inducible RNAi approach using the KP-GFP transformants as targets, the consequences of a loss or suppression of function will be studied. Recombinant KP candidates will be expressed and isolated to test their interaction with each other and with centromeric DNA by plasmon resonance. Antibodies against the KP will be produced to verify the GFP results and to study their phylogenetic conservation in plants. An existing collection of monoclonal antibodies against plant kinetochores may help to identify KP with less homology to non-plant KP either by screening an expression library or by immunoadsorption of chromosomal/ nuclear proteins.