Targeted Homologous Recombination in Meiotic Plant Cells.

The objective of this proposal is to develop a rapid, simple, and efficient method to perform targeted homologous recombination in plants. We will produce a specialized vector that carries the desired mutant locus, then use controlled DNA replication to produce hundreds of circular DNA copies of that vector in meiotic cells. Since homologous recombination is a normal occurrence in meiotic cells, the vector is expected to serve as a recombination substrate, resulting in replacement of the wild-type locus. Seeds will be collected and the desired mutation should appear in the progeny. The vectors will be derived from a proven geminivirus system in which vector amplification is initiated by the geminivirus Rep protein under control of a regulated promoter. The virus-encoded Rep protein initiates rolling circle replication and, through interactions with host proteins, induces DNA replication by the host. Rep protein can be expressed in trans, and it is capable of excising a pro-vector from chromosomal DNA and inducing its replication. By integrating a pro-vector harboring a desired mutant locus into the genomic DNA and inducing Rep in cells entering meiosis, many copies of substrate DNA can be made available to the meiotic recombination enzymes.