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 regulator of cell death-1 (rcd-1), a gene controlling PCD in germinated asexual spores in the filamentous fungus Neurospora crassa. rcd-1 alleles are highly polymorphic and fall into two haplogroups in N. crassa populations. Coexpression of alleles from the two haplogroups, rcd-1–1 and rcd-1–2, is necessary and sufficient to trigger a cell death reaction. Here, we investigated the molecular bases of rcd-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 incompatible rcd-1–1/rcd-1–2 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 rcd-1. This report documents an ancient transkingdom relationship of cell death execution modules involved in organismal defense.

丝状真菌中的程序性细胞死亡 (PCD) 可防止同种遗传不同个体之间融合后的细胞质混合（同种异体识别），并作为一种防御机制来对抗支原体、基因组利用和有害的细胞质成分（即衰老质粒）。最近，我们发现了细胞死亡调节因子 1 ( rcd-1 )，它是控制丝状真菌粗糙脉孢菌中萌发的无性孢子中 PCD 的基因。rcd-1等位基因具有高度多态性，在粗糙猪群中分为两个单倍群。来自两个单倍群rcd-1-1和rcd-1-2的等位基因的共表达, 是触发细胞死亡反应的必要和充分条件。在这里，我们研究了rcd-1的分子基础依赖性细胞死亡。基于计算机分析，我们发现 RCD-1 是 gasdermin 的 N 末端成孔结构域的远程同源物，gasdermin 是高度炎症性细胞死亡反应的刽子手蛋白，称为 pyroptosis，在哺乳动物先天免疫中起关键作用. 我们表明 RCD-1 定位于细胞外围，并且 RCD-1 的细胞定位与预测的两亲性 α-螺旋上保守的带正电残基相关，如鼠 gasdermin-D 所示。与gasdermin 类似，RCD-1 在体外结合酸性磷脂，特别是心磷脂和磷脂酰丝氨酸，并与含有此类脂质的脂质体相互作用。RCD-1 不相容系统在人类 293T 细胞中重建，其中不相容rcd-1-1/rcd-1-2 的共表达等位基因引发细胞焦亡样细胞死亡。RCD-1 的寡聚体与细胞死亡反应有关，进一步支持了 gasdermin 和rcd-1之间的进化关系。该报告记录了参与有机体防御的细胞死亡执行模块的古老跨界关系。