COPT4 interacts with COPT1 or COPT3 to manipulate the signaling response for copper-triggered immunity in Arabidopsis

Copper transporters (COPTs) and chaperones (CCs) regulate root Cu⁺ transport and homeostasis. Basic helix-loop-helix107 (bHLH107)-mediated ACC synthesis 8 (ACS8) activation is crucial for Cu⁺-triggered immunity (CuTI) in Arabidopsis leaves. Here, we aimed to identify the components of COPTs and CCs and elucidate their roles in CuTI. Six copt mutants were screened for Cu⁺-mediated resistance to Pst DC3000 and ACS8 activation. Protein interactions were evaluated using split luciferase complementation, membrane-based yeast two-hybrid, and pull-down assays. Cu⁺-triggered defense responses were analyzed with comparative RNA-seq profiling and reverse transcription quantitative polymerase chain reaction. Among copt1 to copt6, only copt4 attenuated Cu⁺-mediated immune responses, while COPT4 abolished the transport of Cu⁺ alone. Intriguingly, COPT4 interacts with the complete transporter proteins COPT1 and COPT3. Similar to copt4 and bhlh107 copt4 plants, copt1 copt3 plants exhibited impaired Cu⁺-mediated bacterial load reduction and ACS8 induction. Compared with the wild-type, it also attenuated Cu⁺-mediated differentially expressed genes and copper accumulation, as did the copt4 plants. Furthermore, we revealed that the CCs of Arabidopsis homolog of anti-oxidant 1 (ATX1) interact with COPT1 or COPT3 but not with COPT4, which is involved in CuTI. Collectively, our findings suggest that COPT4 manipulates two redundant heterodimers to interact with ATX1, subsequently activating the Cu⁺-responsive module of bHLH107-ACS8 inside plant cells.