In plants, the iron (Fe) uptake machinery is a complex regulatory cascade. Mechanistic details of Fe sensing and regulation of signalling pathways are not completely clear. We exploited Fe deficiency-induced co-expressed gene clusters to list down a set of genes for an extensive Yeast-two hybrid (Y2H) screening and uncovered several novel protein-protein interactions. Three main protein groups emerged out of this interaction map: (1) BRUTUS (BTS) and BTS-LIKE1/2 (BTSL1/BTSL2), (2) basic helix-loop-helix (bHLH) transcription factors (TFs), (3) IRONMAN1 (IMA1/FEP3). In our Y2H screening BTSL1 interacted with bHLH subgroup IVc proteins, PYE and IMA1, a possible scenario for a tripartite interaction. Hence, we designed a quantitative yeast three-hybrid assay and tested the effect of IMA1 on BTSL1-bHLH interaction strength. When IMA1 was present, some BTSL1-bHLH interactions were significantly weakened, and IMA1 expression did not affect all BTSL1-bHLH interactions equally. We pinpointed the protein interaction sites by Y2H deletion mapping and identified a conserved motif in bHLHIVc proteins that is vital for their interaction with BTS(L)s (Lichtblau et al., 2022). In further unpublished work, we can show that expression of a mutated bHLHIVc TF, having a deletion of the interaction motif, resulted in hyperaccumulation of Fe in leaves of Arabidopsis thaliana transgenic lines, which was similar to the phenotype of bts btsl1 btsl2 loss-of-function mutants. In conclusion, this study elucidated a novel protein interaction network involved in the complex regulatory system controlling iron uptake in plants, advancing our mechanistic understanding of how plants sense and respond to iron deficiency.