Determination of the motif responsible for interaction between the rice APETALA1/AGAMOUS-LIKE9 family proteins using a yeast two-hybrid system

A MADS family gene, OsMADS6, was isolated from a rice (Oryza sativa L.) you ng flower cDNA library using OsAMDS1 as a probe. With this clone, various M ADS box genes that encode far protein-to-protein interaction partners of th e OsMADS6 protein were isolated by the yeast two-hybrid screening method. O n the basis of sequence homology, OsMADS6 and the selected partners can be classified in the APETALA1/AGAMOUS-LIKE9 (AP1/AGL9) family. One of the inte raction partners, OsMADS14, was selected for further study. Both genes bega n expression at early stages of flower development, and their expression wa s extended into the later stages. In mature flowers the OsMADS6 transcript was detectable in lodicules and also weakly in sterile lemmas and carpels, whereas the OsMADS14 transcript was detectable in sterile lemmas, paleas/le mmas, stamens, and carpels. Using the yeast two-hybrid system, we demonstra ted that the region containing of the 109th to 137th amino acid residues of OsMADS6 is indispensable in the interaction with OsMADS14. Site-directed m utation analysis revealed that the four periodical leucine residues within the region are essential for this interaction. Furthermore, it was shown th at the 14 amino acid residues located immediately downstream of the K domai n enhance the interaction, and that the two leucine residues within this re gion play an important role in that enhancement.