Characterization of the Key Region and Phosphorylation Sites of EcaICE1 for its Molecular Interaction with EcaHOS1 Protein from Eucalyptus camaldulensis

ICE1 (inducer of CBF expression 1), a MYC-like bHLH transcriptional activator, plays an important role in plant under cold stress via regulating transcriptional expression of downstream cold-responsive genes. Ubiquitination-proteasome pathway mediated by high expression of osmotically responsive gene1 (HOS1) can effectively induce the degradation of ICE1 and decrease the expression of expression of CBFs and their downstream genes under cold stress response in Arabidopsis, but the knowledge about ubiquitination regulation of ICE1 by HOS1 is still limited in woody plants. In this study, a complete open reading frame of EcaICE1 gene and EcaHOS1 was amplified from the tissue culture seedlings of Eucalyptus camaldulensis. Yeast two-hybrid (Y2H) and BiFC assay results showed that EcaICE1 can interact with EcaHOS1 protein in the nucleus, and further Y2H assay demonstrated that the 126-185 amino acid region at the N-terminus of EcaICE1 protein was indispensable for its interaction with EcaHOS1 protein. Moreover, we found that the amino acids at positions 143, 145, 158 and 184 within the key interaction region were the potential phosphorylation sites of EcaICE1 based on bioinformatics analysis, and that only the substitution of Serine (Ser) 158 by Alanine (Ala) blocked the protein-protein interactions between EcaICE1 and EcaHOS1 by Y2H and β-galactosidase assays using site-direct mutagenesis. In summary, we first reported that EcaICE1 could interact with EcaHOS1 protein in Eucalyptus, and identified Ser 158 of EcaICE1 as the key phosphorylation site for its interaction with EcaHOS1 protein.