The effector FEP3/IRON MAN1 modulates interaction between BRUTUS-LIKE1 and bHLH subgroup IVb and IVc proteins

Plants use the micronutrient iron (Fe) efficiently to balance the requirements for Fe during growth with its potential cytotoxic effects. A cascade of basic helix-loop-helix (bHLH) transcription factors is initiated by bHLH proteins of the subgroups IVb and IVc. This induces more than 50 genes in higher plants that can be grouped in co-expression clusters. Gene co-expression networks contain information on functional protein interactomes. We conducted a targeted yeast two-hybrid screen with pairwise combinations of 23 proteins stemming from previously characterized Fe-deficiency-induced gene co-expression clusters and regulators. We identified novel and described interactions, as well as interaction hubs with multiple interactions within the network. We found that BRUTUS-LIKE E3 ligases (BTSL1, BTSL2) interacted with basic helix-loop-helix (bHLH) transcription factors of the subgroups IVb and IVc including PYE, bHLH104 and ILR3, and with small FE UPTAKE-INDUCING PEPTIDE3/IRON MAN1 (FEP3/IMA1). Through deletion studies and with support of molecular docking, we mapped the interaction sites to three-amino-acid regions in BTSL1 and FEP3/IMA1. The FEP3/IMA1 active residues are present in interacting sites of the bHLH IVc factors. FEP3/IMA1 attenuated interaction of BTSL1 with bHLH proteins in a quantitative yeast three-hybrid assay suggesting that it is an inhibitor. Co-expression of BTSL1 and bHLH IVb and IVc factors uncovered unexpected patterns of subcellular localization. Combining deletion mapping, protein interaction and physiological analysis, we discuss the model that FEP3/IMA1 is a small effector protein inhibiting BTSL1/BTSL2-mediated degradation of bHLH subgroup IVb and IVc proteins. Highlights A targeted yeast two-hybrid screen of Fe deficiency-regulated proteins reveals a regulatory protein interactome consisting of E3 ligases BTS/BTSL, bHLH transcription factors of subgroups IVb and IVc and small protein FEP3/IMA1. Interaction sites between BTSL1, FEP3/IMA1, and bHLH IVc transcription factors were fine-mapped. FEP3/IMA1 is as a small effector protein that selectively attenuates the bHLH interaction with BTSL1 to regulate Fe deficiency responses. One sentence summary A targeted protein interaction screen uncovered a interactions of E3 ligase BTSL1, bHLH proteins of subgroup IVb and IVc and effector protein FEP3/IMA1 to regulate Fe deficiency responses.