Identification of Protein Interactors of AGL15, an Embryo MADS-Domain Factor

The goal of the proposed research is to identify proteins that interact with AGL15 to regulate gene expression. AGL15 is a member of the plant MADS-domain family, a group of proteins that act as DNAbinding regulatory factors and often play central roles in critical developmental decisions. AGL15 is the only identified member of the Arabidopsis MADS-box family that is preferentially expressed during embryo development. Evidence to date is consistent with a role for AGL15 in global aspects of embryo development. To better understand AGL IS's role in development, my lab group has been identifying genes that are regulated directly by AGL 15. We have been successful in obtaining candidate targets that have already passed many of the tests to show that they are directly regulated by AGLI5. But this work has raised questions about how AGL 15 regulates these genes because expression of some genes is induced in response to AGL 15, while expression of others is repressed. We believe that protein co-regulators of AGL15 are likely to be very important to determine how AGL15 acts at different in vivo DNA-binding sites. In plants, animals, and fungi combinatorial control of gene regulation via interactions between protein co-regulators has been key in determining developmental consequences. In this proposal, we outline experiments to identify protein interactors of AGL 15 with the long-term research objective to better understand regulatory networks during seed development. We propose two complementary approaches to isolate protein interactors of AGL 15. First, the yeast 2-hybrid approach, which has become a standard for defining protein-protein interactions, will allow extensive screens for interactors. Second, a co-purification approach will allow us to isolate a complex of potentially more than two proteins that forms in situ. The work outlined in this proposal will allow us to identify and gather preliminary data on protein interactors of AGL 15. This work is particularly relevant to the USDA mission because for many crops, seeds are the harvest commodity. A better understanding of gene regulatory networks active during seed development will be essential to developing molecular strategies to increase agricultural productivity.