Identification and Characterization of IBR5 Interacting Protein 1(IIP1) in Arabidopsis
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Auxin is a vital plant hormone that regulates growth and development throughout the life cycle. Recently, IBR5, a gene that encodes a dual specificity phosphatase, was identified as protein that regulates plant auxin signaling. However, the exact molecular mechanism of IBR5’s function in plant auxin response is unknown. One approach to understand the molecular mechanism is to identify IBR5 interacting proteins. To this end we performed a yeast two hybrid screen using an Arabidopsis cDNA library. Among several IBR5 interacting protein (IIP), IIP1 was selected for further studies. While yeast two hybrid screen is a powerful technique to identify new interacting proteins, results of this assay have to be validated by other biochemical and/or genetic techniques. We hypothesized that IIP1 is a true IBR5 interacting protein and used other molecular techniques to validate IBR5-IIP1 interaction. First, In vitro pull-down assay was used to confirm the interaction between IBR5 and IIP1. IBR5 was expressed in Arabidopsis plants as a Myc-tagged fusion protein (IBR5-Myc), and IIP1 was expressed in E. coli as GST-tagged recombinant protein (GST-IIP1). GST-IIP1 protein was purified from E. coli and added to Arabidopsis crude protein extract isolated from 35S::IBR5-Myc seedlings. Proteins interacted with GST-IIP1 were recovered and separated by SDS-PAGE. Interacting proteins were detected by western blot using anti-Myc antibody. Results indicate that IIP1 is a true IBR5 interacting protein. This interaction has further been confirmed in vivo using co-immunoprecipitation (Co-IP) assay. To understand the genetic interaction between IBR5 and IIP1, Arabidopsis T-DNA insertion mutant of iip1 was obtained from Arabidopsis biological resource center (ABRC). PCR analysis was performed to identify iip1 homozygous mutant.