Exploring a diverse world of effector domains and amyloid signaling motifs in fungal NLR proteins

NLR proteins are intracellular receptors constituting a conserved component of the innate immune system of cellular organisms. In fungi, NLRs are characterized by high diversity of architectures and presence of amyloid signaling. Here, we explore the diverse world of effector and signaling domains of fungal NLRs using state-of-the-art bioinformatic methods including MMseqs2 for fast clustering, probabilistic context-free grammars for sequence analysis, and AlphaFold2 deep neural networks for structure prediction. In addition to substantially improving the overall annotation, especially in basidiomycetes, the study identifies novel domains and reveals the structural similarity of MLKL-related HeLo- and Goodbye-like domains forming the most abundant superfamily of fungal NLR effectors. Moreover, compared to previous studies, we found several times more amyloid motif instances, including novel families, and validated aggregating and prion-forming properties of the most abundant of them in vitro and in vivo. Also, through an extensive in silico search, the NLR-associated amyloid signaling was identified in basidiomycetes. The emerging picture highlights similarities and differences in the NLR architectures and amyloid signaling in ascomycetes, basidiomycetes and other branches of life.Author summary: All living organisms possess an immune system allowing them to cope with pathogens and, more broadly, to manage interactions with other organisms. One of its conserved components are the so-called NLR proteins, which are found in bacteria, plants, animals and fungi. NLRs are intracellular sensors that trigger a host response upon the detection of non-self markers, which is typically performed by effector domains of NLRs. We investigate the repertoire of these domains in almost 500 fungal strains. We identify several major effector classes, most of which are involved in regulated cell death. Some NLRs do not have built-in effector domains but instead activate separate effector proteins via prion-like signal propagation. This activation is triggered by passing the amyloid fold from a short signaling domain on the NLR to its counterpart on the effector. Using innovative computational approaches, we identify new amyloid signaling motifs and find them overall several times more common in fungal NLRs than previously reported, including the evidence of amyloid signaling in basidiomycetes. Our results describe the global ensemble of NLRs effector domains in fungi and thus enhance our comparative view of this nearly-universally conserved immune receptor family.