Identification of dynamin as an interactor of rice GIGANTEA by tandem affinity purification (TAP)

GIGANTEA (GI), CONSTANS (CO) and FLOWERING LOCUS T (FT) regulatephotoperiodic flowering in Arabidopsis. In rice, OsGI, Hd1 andHd3a were identified as orthologs of GI, CO and FT, respectively,and are also important regulators of flowering. Although GIhas roles in both flowering and the circadian clock, our understandingof its biochemical functions is still limited. In this study,we purified novel OsGI-interacting proteins by using the tandemaffinity purification (TAP) method. The TAP method has beenused effectively in a number of model species to isolate proteinsthat interact with proteins of interest. However, in plants,the TAP method has been used in only a few studies, and no novelproteins have previously been isolated by this method. We generatedtransgenic rice plants and cell cultures expressing a TAP-taggedversion of OsGI. After a two-step purification procedure, theinteracting proteins were analyzed by mass spectrometry. Sevenproteins, including dynamin, were identified as OsGI-interactingproteins. The interaction of OsGI with dynamin was verifiedby co-immunoprecipitation using a myc-tagged version of OsGI.Moreover, an analysis of Arabidopsis dynamin mutants indicatedthat although the flowering times of the mutants were not differentfrom those of wild-type plants, an aerial rosette phenotypewas observed in the mutants. We also found that OsGI is presentin both the nucleus and the cytosol by Western blot analysisand by transient assays. These results indicate that the TAPmethod is effective for the isolation of novel proteins thatinteract with target proteins in plants.