Molecular characterization of a fungal gasdermin-like protein

Abstract

Programmed cell death (PCD) in filamentous fungi prevents cytoplasmic mixing following fusion between conspecific genetically distinct individuals (allorecognition) and serves as a defense mechanism against mycoparasitism, genome exploitation, and deleterious cytoplasmic elements (i.e., senescence plasmids). Recently, we identified <i>regulator</i><i>of cell death-1</i> (<i>rcd-1</i>), a gene controlling PCD in germinated asexual spores in the filamentous fungus <i>Neurospora crassa</i><i>rcd-1</i> alleles are highly polymorphic and fall into two haplogroups in <i>N. crassa</i> populations. Coexpression of alleles from the two haplogroups, <i>rcd-1-1</i> and <i>rcd-1-2</i>, is necessary and sufficient to trigger a cell death reaction. Here, we investigated the molecular bases of <i>rcd-1</i>-dependent cell death. Based on in silico analyses, we found that RCD-1 is a remote homolog of the N-terminal pore-forming domain of gasdermin, the executioner protein of a highly inflammatory cell death reaction termed pyroptosis, which plays a key role in mammalian innate immunity. We show that RCD-1 localizes to the cell periphery and that cellular localization of RCD-1 was correlated with conserved positively charged residues on predicted amphipathic α-helices, as shown for murine gasdermin-D. Similar to gasdermin, RCD-1 binds acidic phospholipids in vitro, notably, cardiolipin and phosphatidylserine, and interacts with liposomes containing such lipids. The RCD-1 incompatibility system was reconstituted in human 293T cells, where coexpression of incompatible <i>rcd-1-1/rcd-1-2</i> alleles triggered pyroptotic-like cell death. Oligomers of RCD-1 were associated with the cell death reaction, further supporting the evolutionary relationship between gasdermin and <i>rcd-1</i> This report documents an ancient transkingdom relationship of cell death execution modules involved in organismal defense.