Autophagic regulation of ferroportin 1 induces developmental ferroptosis in rice blast

The rice blast fungus, <i>Magnaporthe oryzae</i>, imposes a great threat to global food security. Autophagic cell death of conidium is essential for appressorium-mediated host invasion during pathogenesis. Our recent study revealed that ferroptosis, potentially regulated by macroautophagy/autophagy, is responsible for <i>M. oryzae</i> conidial death during appressorium formation and maturation. Here, we characterized the role of the iron exporter MoFpn1 (ferroportin 1) and showed that its loss led to increased intracellular iron levels, accelerated conidial death, and reduced sensitivity to liproxstatin-1, suggesting that MoFpn1 negatively regulates ferroptosis as an iron exporter in <i>M. oryzae</i>. In conidia, MoFpn1-mCherry fusion protein localized on punctate/vesicular organelles, largely overlapping with CMAC-stained vacuoles, and is subject to regulation by iron availability and autophagy. MoFpn1 was associated with Atg8, based on yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. MoFpn1-mCherry partially colocalized with GFP-Atg8-tagged autophagosomes or autophagic vacuoles in developing conidia. Upon appressorium formation, MoFpn1-mCherry localized to the plasma membrane of appressoria. In mature appressorium, plasma membrane-localized MoFpn1-mCherry transferred to the vacuolar lumen. MoFpn1 also directly interacted with components of the vesicular sorting complex, including the vacuolar SNARE Vam7 that mediates autophagosome-vacuole fusion. Individual deletion of <i>ATG8</i> or <i>VAM7</i> resulted in mislocalization of MoFpn1-mCherry to the vacuolar membrane or multivesicular bodies (MVBs) instead of the vacuolar lumen, under autophagy inducing conditions, or remained on the plasma membrane of the mature appressorium. Overall, our study demonstrates that regulation of the intracellular level of iron by Atg8- and Vam7-mediated autophagy-dependent degradation of MoFpn1 is crucial for conidial death and pathogenicity. <b>Abbreviations:</b> BiFC: bimolecular fluorescence complementation; CMAC: 7-amino-4-chloromethylcoumarin; Fpn1: ferroportin 1; Lip-1: liproxstatin-1; MDA: malondialdehyde; MVBs: multivesicular bodies; SNARE: soluble NSF attachment protein receptor; Y2H: yeast two-hybrid.