Characterization of the key region and putative phosphorylation sites of EcaICE1 in its molecular interaction with the EcaHOS1 protein in Eucalyptus camaldulensis

Inducer of CBF expression 1 (ICE1), a MYC-like bHLH transcriptional activator, plays an important role in plants under cold stress. The ubiquitination-proteasome pathway mediated by high expression of osmotically responsive gene1 (HOS1) can effectively induce the degradation of ICE1 and decrease the expression of CBFs and their downstream genes under cold stress response in Arabidopsis, but knowledge of ubiquitination regulation of ICE1 by HOS1 is still limited in woody plants. In this study, a E3 ubiquitin ligase gene EcaHOS1 were amplified from Eucalyptus camaldulensis and the protein interactions between EcaICE1 and EcaHOS1 were analysed. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assay results showed that EcaICE1 can interact with the EcaHOS1 protein in the nucleus and, further, the Y2H assay demonstrated that the 126-185 amino acid region at the N-terminus of the EcaICE1 protein was indispensable for its interaction with EcaHOS1 protein. Moreover, we found that the amino acids at positions 145, 158 and 184 within the key interaction region were the putative phosphorylation sites of EcaICE1, based on bioinformatics analysis, and only the substitution of serine (Ser) 158 by alanine (Ala) blocked the protein-protein interactions between EcaICE1 and EcaHOS1 based on Y2H and β-galactosidase activity assays using site-directed mutagenesis. We identified Ser 158 of EcaICE1 as the key putative phosphorylation site for its interaction with the EcaHOS1 protein.