TRTech-PGR: Manipulating plant karyotypes by synthetic centromere formation

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An important goal of plant synthetic biology is to design artificial chromosomes with custom genes and pathways that can improve plant performance in areas such as drought tolerance. The overall aim of this project is to develop strategies for the introduction and inheritance of custom designed artificial plant chromosomes. This work will be communicated to the public through two “Plants by Design” research symposia, research demonstrations at the Bell Museum at the University of Minnesota, and interactive instructional events at local farmers markets in Athens Georgia. Years of research have provided a comprehensive understanding of what centromeres are made of and what proteins bind to them. This project will test the investigators' knowledge by creating synthetic centromeres with defined components. In prior work the investigators inserted long arrays of LexO binding sites on maize chromosome 4 and showed that a fusion protein containing a LexA DNA binding domain and CENH3 (LexA-CENH3) binds to LexO arrays and recruits other kinetochore proteins. In this project, they will determine whether LexA-CENH3-activated synthetic centromeres are capable of driving independent chromosome segregation. They will use several methods to break chromosome 4 and liberate the end fragments to become independent neochromosomes, which they will test for mitotic and meiotic stability. They will also create new LexO arrays and determine the minimum size necessary to activate functional centromeres. They will further extend the work to Nicotiana benthamiana, where methods exist to transform protoplasts with large molecules. They will build synthetic chromosome vectors based on the LexA-CENH3 system and test their properties in culture and regenerated plants. If successful, the results will expand knowledge of plant centromeres and provide first-generation synthetic chromosome vectors for plant genome engineering.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.