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Abstract
There are 25,000 protein-coding genes in Arabidopsis thaliana. About 11% of these have been reported as essential genes. Essential genes are required for normal growth and development and are associated with a loss-of-function phenotype in a standard genetic background. Many of the genes that encode for cell division components are essential, such as the kinetochore foundation genes. A kinetochore is a large protein complex that assembles at the centromere to ensure faithful and accurate chromosome movement and segregation during mitosis and meiosis. The foundation genes are constitutively expressed in every cell throughout the cell cycle and are required to build a functional kinetochore at the centromere. Traditional forward and reverse genetic techniques are limited in the study of these genes because complete loss-of-function results in embryo or seedling lethality in Arabidopsis. I developed a tissue-specific RNAi interference (tsRNAi) system to study the function of CENPC and MIS12, two essential kinetochore foundation components. These genes were successfully targeted in petals and anther tissue, both non-essential plant organs. CENPC is required at all stages of mitosis while MIS12 functions in chromosome alignment and movement towards the spindle poles during metaphase and anaphase. Our tsRNAi system was also applied to understand the role of MAD2 and BUBR1, two non-essential genes involved in the mitotic checkpoint that controls cell cycle progression. Finally, we applied the tsRNAi system to investigate whether the small RNAs being generated in somatic cells can move into the germ line and consequently have a phenotypic effect at the next generation. The different applications of tsRNAi show that this system can potentially serve as a valuable tool in plant biology for the study of essential genes as well as small RNA inheritance. In addition to the development of a tsRNAi system, we also report our attempts to characterize seven homologues of CENP-E in Arabidopsis. CENP-E is a motor protein thought to have a role in chromosome movement and segregation. Although we were not able confirm the kinetochore localization of CENP-E, we found that plant CENP-Es have evolved diverse roles in cytokinesis and plant development.ill