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 identifiedregulatorof cell death-1(rcd-1), a gene controlling PCD in germinated asexual spores in the filamentous fungusNeurospora crassa.rcd-1alleles are highly polymorphic and fall into two haplogroups inN. crassapopulations. Coexpression of alleles from the two haplogroups,rcd-1–1andrcd-1–2, is necessary and sufficient to trigger a cell death reaction. Here, we investigated the molecular bases ofrcd-1-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 incompatiblercd-1–1/rcd-1–2alleles triggered pyroptotic-like cell death. Oligomers of RCD-1 were associated with the cell death reaction, further supporting the evolutionary relationship between gasdermin andrcd-1. This report documents an ancient transkingdom relationship of cell death execution modules involved in organismal defense.