MADS-box protein interactions in Arabidopsis thaliana
Reader, Laura Jeanne Voll
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Arabidopsis thaliana, a member of the Cruciferae family, is widely used as a model system of plant development. In this study the structure - function relationships between AG and AGL2 (AGAMOUS Like) and AG and AGL4 were analyzed for their interaction capabilities at the protein level using the yeast two-hybrid system. Five ag12 loss-of-interaction mutants were identified that had lost the ability to interact with AG. All five mutations mapped to the K-box region of the deduced protein sequence of AGL2, and resulted in a substitution of a proline for either a leucine or serine. These mutants had also lost the ability to activate transcription in the two-hybrid system when cloned as GAL4 DNA Binding (DB) protein fusions. The screens for ag mutants inactivated for AGL2 interaction recovered uninformative frameshifts, nonsense mutations, and silent mutations within the first one-third of the coding region. The screen for agl4 mutants that had gained the ability to interact with AG yielded phenotypic gain-of-interaction mutants, but no mutation events could be defined. The position and nature of the agl2 loss-of-interaction mutations suggested that disruption of the coiled-coil structure of the K-box domain was deleterious for interaction between the AGL2 and AG proteins. The results further suggest that the C terminus of the AGL2 protein contains a domain(s) responsible for the transcription activation properties exhibited by wild type AGL2 expression constructs in the yeast two-hybrid system.