Iron is a biochemically critical metal cofactor in enzymes involved in photosynthesis, cellular respiration, nitrate assimilation, nitrogen fixation, and reactive oxygen species defense. Marine microeukaryotes have evolved a phytotransferrin-based iron uptake system to cope with iron scarcity, a major factor limiting primary productivity in the global ocean. Diatom phytotransferrin is endocytosed, however proteins downstream of this environmentally ubiquitous iron receptor are unknown. We applied engineered ascorbate peroxidase APEX2-based subcellular proteomics to catalog proximal proteins of phytotransferrin in the model marine diatom Phaeodactylum tricornutum. Proteins encoded by poorly characterized iron-sensitive genes were identified including three that are expressed from a chromosomal gene cluster. Two of them showed unambiguous colocalization with phytotransferrin adjacent to the chloroplast. Further phylogenetic, domain, and biochemical analyses suggest their involvement in intracellular iron processing. Proximity proteomics holds enormous potential to glean new insights into iron acquisition pathways and beyond in these evolutionarily, ecologically, and biotechnologically important microalgae.

Active DNA demethylation has emerged as an important regulatory process of plant and mammalian immunity. However, very little is known about the mechanisms by which active demethylation controls transcriptional immune reprogramming and disease resistance. Here, we first show that the Arabidopsis active demethylase ROS1 promotes basal resistance towards Pseudomonas syringae by antagonizing RNA-directed DNA methylation (RdDM). Furthermore, we demonstrate that ROS1 facilitates the flagellin-triggered induction of the disease resistance gene RMG1 by limiting RdDM at the 3' boundary of a transposable element (TE)-derived repeat embedded in its promoter. We further identify flagellin-responsive ROS1 putative primary targets and show that at a subset of promoters, ROS1 erases methylation at discrete regions exhibiting WRKY transcription factors (TFs) binding. In particular, we demonstrate that ROS1 removes methylation at the orphan immune receptor RLP43 promoter, to ensure DNA binding of WRKY TFs. Finally, we show that ROS1-directed demethylation of RMG1 and RLP43 promoters is causal for both flagellin responsiveness of these genes and for basal resistance. Overall, these findings significantly advance our understanding of how active demethylases shape transcriptional immune reprogramming to enable antibacterial resistance.