The CPK3-mediated cytosolic–nuclear translocation of bHLH107 recruits HY5 to regulate the Cu2+-triggered upregulation of ACS8

Copper serves as a key component of plant micronutrients and copper-based antimicrobial compounds (CBACs), and it has been used to protect plants against diseases for more than 130 years. We previously revealed that low concentrations of Cu2+ elicit plant immune responses by activating the expression of the ethylene synthesis rate-limiting enzyme 1-aminocyclopropanecarboxylic acid (ACC) synthesis 8 (ACS8), which depends on the copper response element (CuRE) in the promoter. However, the transcription regulatory mechanism of Cu2+-triggered immunity upstream of ACS8 remains unclear. Here, we aimed to identify the CuRE-binding transcription factor (CuTF) and elucidate it roles in activating the Cu2+-induced expression of ACS8. To identify the CuTF and its interactors, we performed DNA-pull-down and mass spectrometry and protein phosphorylation proteomics assays. Cu2+-induced plant immune responses were conducted assays measuring the ACS8 expression, bacterial populations and RNA-seq. Additionally, protein-DNA and protein-protein interactions were performed using yeast one hybridization, electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR), pull-down, luciferase complementation assay, and coimmunopreciptitation assay. We identified a CuTF of basic helix-loop-helix 107 (bHLH107) which is required for Cu2+-triggered activation of ACS8 expression and resistance to Pseudomonas syringae pv. tomato (Pst) DC3000. Calcium-dependent protein kinase 3 (CPK3) interacts with and phosphorylates bHLH107 at Ser62 and Ser72 to mediate bHLH107 translocation from the cytoplasm into the nucleus, leading to an increase in the transactivation activity of bHLH107. Moreover, our findings revealed that bHLH107 interacts with Arabidopsis ELONGATED HYPOCOTYL5 (HY5) in nucleus. HY5 directly binds to the ACGT-containing elements (ACE-box) and acts as a coactivator to promote bHLH107 binding to the CuRE cis-element and to increase transcription of ACS8 upon Cu2+ treatment. Overall, we revealed a CPK3-bHLH107-HY5 module that regulates the network of Cu2+-triggered plant immunity upstream of ACS8 that is involved in the cytosolic-nuclear translocation of bHLH107.