Recent findings have revealed the role of prion-like mechanisms in the control of host defense and programmed cell death cascades. In fungi, HET-S, a cell death-inducing protein containing a HeLo pore-forming domain, is activated through amyloid templating by a Nod-like receptor (NLR). Here we characterize the HELLP protein behaving analogously to HET-S and bearing a new type of N-terminal cell death-inducing domain termed HeLo-like (HELL) and a C-terminal regulatory amyloid motif known as PP. The gene encoding HELLP is part of a three-gene cluster also encoding a lipase (SBP) and a Nod-like receptor, both of which display the PP motif. The PP motif is similar to the RHIM amyloid motif directing formation of the RIP1/RIP3 necrosome in humans. The C-terminal region of HELLP, HELLP(215-278), encompassing the motif, allows prion propagation and assembles into amyloid fibrils, as demonstrated by X-ray diffraction and FTIR analyses. Solid-state NMR studies reveal a well-ordered local structure of the amyloid core residues and a primary sequence that is almost entirely arranged in a rigid conformation, and confirm a β-sheet structure in an assigned stretch of three amino acids. HELLP is activated by amyloid templating and displays membrane-targeting and cell death-inducing activity. HELLP targets the SBP lipase to the membrane, suggesting a synergy between HELLP and SBP in membrane dismantling. Remarkably, the HeLo-like domain of HELLP is homologous to the pore-forming domain of MLKL, the cell death-execution protein in necroptosis, revealing a transkingdom evolutionary relationship between amyloid-controlled fungal programmed cell death and mammalian necroptosis.amyloid | prion | programmed cell death | incompatibility | necroptosis

Nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) are intracellular receptors that control innate immunity and other biotic interactions in animals and plants. NLRs have been characterized in plant and animal lineages, but in fungi, this gene family has not been systematically described. There is however previous indications of the involvement of NLR-like genes in nonself recognition and programmed cell death in fungi. We have analyzed 198 fungal genomes for the presence of NLRs and have annotated a total of 5,616 NLR candidates. We describe their phylogenetic distribution, domain organization, and evolution. Fungal NLRs are characterized by a great diversity of domain organizations, suggesting frequently occurring combinatorial assortments of different effector, NOD and repeat domains. The repeat domains are of the WD, ANK, and TPR type; no LRR motifs were found. As previously documented for WD-repeat domains of fungal NLRs, TPR, and ANK repeats evolve under positive selection and show highly conserved repeats and repeat length polymorphism, suggesting the possibility of concerted evolution of these repeats. We identify novel effector domains not previously found associated with NLRs, whereas others are related to effector domains of plant or animals NLRs. In particular, we show that the HET domain found in fungal NLRs may be related to Toll/interleukin-1 receptor domains found in animal and plant immune receptors. This description of fungal NLR repertoires reveals both similarities and differences with plant and animals NLR collections, highlights the importance of domain reassortment and repeat evolution and provides a novel entry point to explore the evolution of NLRs in eukaryotes.