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# The Expanding Scope of Amyloid Signalling

Asen Daskalov, Sven J. Saupe

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Asen Daskalov, Sven J. Saupe. The Expanding Scope of Amyloid Signalling. Prion, 2021, 15 (1), pp.21-28. 10.1080/19336896.2021.1874791. hal-04833195

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# The expanding scope of amyloid signalling

Asen Daskalov & Sven J. Saupe

**To cite this article:** Asen Daskalov & Sven J. Saupe (2021) The expanding scope of amyloid signalling, Prion, 15:1, 21-28, DOI: 10.1080/19336896.2021.1874791

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# The expanding scope of amyloid signalling

Asen Daskalov and Sven J. Saupe

Institut de Biochimie et de Génétique Cellulaire (CNRS UMR 5095, Université de Bordeaux), France

## ABSTRACT

Formation of higher-order supramolecular complexes has emerged as a common principle underlying activity of a number of immune and regulated cell-death signalling pathways in animals, plants and fungi. Some of these signalosomes employ functional amyloid motifs in their assembly process. The description of such systems in fungi finds its origin in earlier studies on a fungal prion termed [Het-s], originally identified as a non-Mendelian cytoplasmic infectious element. Janine Beisson has been a key contributor to such early studies. Recent work on this and related systems offers a more integrated view framing this prion in a broader picture including related signalling systems described in animals. We propose here an auto-commentary centred on three recent studies on amyloid signalling in microbes. Collectively, these studies increase our understanding of fold conservation in functional amyloids and the structural basis of seeding, highlight the relation of fungal amyloid motifs to mammalian RHIM (RIP homotypic interaction motif) and expand the concept of Nod-like receptor-based amyloid signalosomes to the prokaryote reign.

## ARTICLE HISTORY

Received 9 December 2020
Revised 7 January 2021
Accepted 8 January 2021

## KEYWORDS

NLR; prion; amyloid;
regulated cell death; Het-s;
RHIM

## Introduction

On February 24th of 1958, Janine Beisson presented a paper at the *Académie des Sciences* reporting that the transformation of *Podospora anserina* sS strains to the s phenotype (now written respectively [Het-s*] and [Het-s]) is induced by a single cytoplasmic contact and that this transformation invades the mycelium at a rate of up to 7 cm per day (that is about an order on magnitude faster than the radial growth rate of the mycelium). In this paper, she proposed that this invasive [Het-s*] to [Het-s] transformation results 'from multiplication in the [Het-s*] strains of particles brought in by the [Het-s] strain' ('résulte de la multiplication dans la souche s de particules apportées par la souche s') [1]. It is now known that these particles are prions [2]. This study was based on micromanipulation techniques allowing for controlled anastomoses of individual fungal filaments and their re-isolation. Beisson would use these micromanipulation and microsurgery techniques anew in Tracy Sonneborn's group in Bloomington in the context of their now classic common work on cortical inheritance in Paramecia [3]. Janine Beisson who sadly passed away last august once explained that her initial intent as a young student was to study the 'transforming principle' under the supervision of Harriett Ephrussi-Taylor who had been a co-worker of Avery. Instead, she was re-directed by

Boris Ephrussi to Georges Rizet. Rizet a founding father of French genetics had introduced *Podospora* as a genetic model organism. He described in 1950, the peculiar genetics and cytology of s/S incompatibility as 'un phénomène tout à fait original', a thoroughly original phenomenon [4]. It is thus delightful enough that this initial impetus to pursue the 'transforming principle' led Janine Beisson to develop a body of work completing the DNA heredity paradigm, in the study of epigenetics of structural inheritance in both fungi and protists. The seminal studies by Rizet and Beisson in the 1950s and 1960s lay the groundwork for ongoing developments in the characterization in *Podospora* and beyond, of genetic systems related to 'petit s' aka [Het-s]. After a few years of practising experimental biology, one is likely to have faced at least once – when exposed to biological unorthodoxy – the choice between engaging the risky path of additional experimentation and autoclaving the whole business. In retrospect, one can only marvel at the insight and dedication of these scientists who simply would not let the 'petit s' genetic oddity rest unquestioned and who thus established the solid theoretical and experimental foundations, on which recent studies of such systems are based on. We will here very rapidly describe the biology of the [Het-s] prion and the path leading from the [Het-s] prion to the characterization of amyloid signalling in
<figure><img src="image_16.png" /></figure>

fungi. We then discuss three recent papers on [Het-s]-related systems that collectively expand the evolutionary span of amyloid signalling and attempt to integrate these systems into the broader picture of regulated cell death in animal, plant and prokaryotic organisms.

## [Het-s]/HET-S

Strains of the fungus *P. anserina* exist as two genotypes termed *het-s* and *het-S* and the strains of these genotypes are incompatible, meaning that cell fusions between them result in regulated cell death [5]. This occurs however only when the *het-s*-encoded protein is in its [Het-s] prion form. When in the soluble state-designated [Het-s*], the protein is inactive in incompatibility. [Het-s*] strains eventually all acquire the [Het-s]-prion state and as mentioned above, cytoplasmic contact between a prion-infected [Het-s*] strain induces rapid and invasive transformation of the [Het-s*] to [Het-s] phenotype. The HET-s protein displays a C-terminal prion-forming domain (PFD or HET-s (218–289)) comprising two 21 amino acid long pseudo-repeats (R1 and R2) adopting a specific β-solenoid amyloid fold, in which each repeat occupies one rung of β-strands [6,7]. Incompatibility is triggered when [Het-s] interacts with HET-S ('large S', which is an allelic variant of the same protein differing from HET-s by just a few residues). Incompatibility (*i.e.* cell death) occurs because the PFD region induces β-solenoid folding of the corresponding region in HET-S, leading to a conformation change in the N-terminal globular domain of HET-S (termed HeLo), which turns it into a pore-forming toxin by exposing a hydrophobic N-terminal α-helix [8–10]. In HET-s in contrast, amyloid conversion of the PFD does not lead to toxicity. This is because amino acid changes in the N-terminal α-helix region abolish activity of the HeLo domain. As a result, HET-s is a prion while HET-S is not. In a nutshell, in this incompatibility system, the [Het-s] prion acts as an activation switch of HET-S pore-forming activity.

**Remarka**

Remarka, the HeLo domain shows homology to other pore-forming domains involved in regulated cell death, both in plants and animals, the four helix bundle domain of the mixed lineage kinase domain-like protein (MLKL) involved in necroptosis execution and coiled coil-related domains found in Nod-like receptors and other plant immune response proteins [11–14].

## NWD2/HET-S

The allorecognition role of the *het-s* and *het-S* alleles appears however to be a derived function [15]. A distinct, evolutionarily more ancient mode of activation of HET-S is dependent on NWD2, the protein encoded by the gene immediately adjacent to *het-S* in the genome. NWD2

encodes a Nod-like receptor (NLR) with a nucleotide-binding and oligomerization domain of the NACHT type and C-terminal WD-repeats (Figure 1). NLRs constitute a diverse family of immune receptors involved in host defence both in plants and animals [16]. NLR homologs also control regulated cell death in the context of non-self recognition in fungi [17–19]. NLRs form supramolecular signalosomes (also designated supramolecular organizing centres or SMOCs [20]) when recognizing specific non-self cues or danger signals. The N-terminus of NWD2 displays a short region termed R0, homologous to the elementary repeats of the HET-S PFD [21]. This observation suggested an amyloid signalling process by which specific NLRs would engage cognate downstream cell-death execution proteins by heterotypic amyloid templating. It was found that indeed chimeric variants of NWD2 are able to induce HET-s priogenesis in a ligand-dependant manner [22]. The interaction between the NWD2 R0 and the HET-s PFD modules can be recapitulated in mammalian cells. These two modules can substitute for the Pyrin domains in the pathway leading to ASC (apoptosis-associated speck-like protein containing a CARD domain) activation by NLRP3 (nucleotide-binding domain leucine-rich repeat and pyrin-containing receptor 3) [23], stressing the analogy between amyloid signalling and prion-like activation in mammalian NLR-signalosomes. While the exact function of HET-S/NWD2 is currently unclear (in particular the cognate ligand of NWD2 is not known), it is proposed that this gene pair functions in fungal immunity to induce cell death in response to parasites or pathogens. While many open questions remain regarding the biology of the NWD2/HET-S pathway, it can be considered established that fungi possess amyloid NLR-signalosomes [24]. The NWD2/HET-S system thus represents, after the mammalian necrosome (assembled through the interaction of the RHIM amyloid motifs), a second example of an amyloid signalosome [25].

## More of the same

Bioinformatic surveys suggest that a number of genetic systems analogous to *Podospora anserina* nwd2/het-S exist in fungal genomes; gene clusters comprising a gene encoding an NLR and one or several genes encoding predicted cell death execution proteins [21] (Figure 1). These systems can be classified based on the sequence of the predicted amyloid motif, shared between the NLR and the execution proteins and have been grouped so far into three superfamilies termed HRAM (for *HET-s* related amyloid motifs), PP (pseudo-palindrome) and sigma [21,26]. Although the evidence is yet indirect, the sigma motif is thought to be responsible for infectious propagation of a cytoplasmic element termed σ, identified in the fungus *Nectria haematosocca* [21,27]. The PP-motif in turn was functionally
<figure><img src="image_17.png" /></figure>

characterized as amyloid signalling motif as part of a study - by heterologous expression in *P. anserina* - of a three-gene cluster from the fungus *Chaetomium globosum* [12]. The PP-motif stands out among fungal amyloid signalling motifs because it shows homology to the RHIM motif involved in amyloid necrosome assembly in mammals [12,25,28]. As we shall see, recent studies have confirmed these bioinformatic predictions and support the existence of other amyloid signalosomes in fungi and also in filamentous bacteria.

## HELLF is like HET- Syet different

Accumulation of sequenced fungal genomes allowed for the identification of hundreds of HeLo domain-containing proteins, homologous to HET-S/HET-s.

The *in silico* analyses resulted in the discovery of 155 homologs carrying PFDs constituted of two pseudo-repeats (R1 and R2) - an organization similar to the HET-s PFD. The pseudo-repeats (of about 21 amino acids of length) of these 155 PFDs shared sequence homology with one strongly conserved glycine residue, forming a functionally important β-arch for the [Het-s] prion, and a general pattern of hydrophobic-polar residues [26]. The putative PFD sequences were termed HET-s-related amyloid motifs (HRAMs) and grouped into five distinct families. Although the HRAMs are defined by a small number of strongly conserved positions - carrying HRAM-specific residues - it has been hypothesized that the motifs adopt a common amyloid fold, structurally related to the HET-s β-solenoid.

<figure><img src="image_18.png" /><figcaption>**Figure 1.** A generic scheme of NLR amyloid signalling.</figcaption></figure>

This simplified sketch summarizes key features common to the *het-s/nwd2* system of *Podospora anserina* and to analogous signalosome pathways, described or predicted in other fungal and bacterial species. The signalosome components include an NLR (Nod-like receptor) (for instance NWD2) and a cell-death execution protein (for instance HET-S). The NLR and execution proteins are encoded by adjacent (or even overlapping) genes. The N-terminal part of the NLR and the C-terminal part of the execution protein share a homologous amyloid-forming region (symbolized as a black curved segment). The cell-death execution domain can be of the HeLo or HELL (HeLo-like) type in fungi or BELL type in bacteria (bacterial domain analogous to HELL). The NLR can display a range of architectures such as NACHT/WD, NACHT/ANK or NB-ARC/TPR. Upon binding of a ligand (symbolized as star-shape) in the C-terminal repeat domain, the NLR oligomerizes. This oligomerization induces cooperative folding of the N-terminal motifs into an amyloid fold. This fold is used as template to induce amyloid formation in the homologous region of the execution protein. Amyloid folding triggers activation of the pore-forming activity of the execution protein, which is dependent on the formation of a membrane-targeting α-helix and leads to induction of cell death. Many steps in this pathway remain hypothetical in some or all of the systems described in the text.
<figure><img src="image_19.png" /></figure>

This hypothesis has been recently explored with the
functional and structural characterization of a protein
named HELLF, endogenous to *Podospora anserina* [29].
The *hellf* gene is adjacent to a gene encoding an NLR
protein, *fnt1*. HELLF is a distant HET-S/s homolog
constituted of 277 amino acids forming an N-terminal
HeLo-like (HELL) domain and a C-terminal PFD. The
pseudo-repeats comprising the PFD of HELLF belong
to the HRAM5 family, which is the most diverged
HRAM family from HET-s (HRAM1 family, respec-
tively). The PFD of HELLF (HELLF(209–277)) propa-
gated *in vivo* as a prion termed [Φ], representing a cell-
death trigger for the full-length HELLF protein. The
sequence identity between the pseudo-repeats of the
two distant homologs was found to be as low as 17%,
nevertheless it was found that HELLF(209–277) formed
amyloid fibres with a β-solenoid fold virtually identical
to the backbone structure of HET-s. This discovery
establishes that specific amyloids folds can be main-
tained well into the twilight zone of sequence identity
and that (as established for globular and membrane
proteins) very little sequence conservation is required
to maintain structural relatedness. In spite of the very
strong similarity of the amyloid backbones of the β-
solenoids formed by HET-s and HELLF, no cross-
seeding was observed between [Φ] and [Het-s]
in vivo. These findings demonstrate that amyloid back-
bone similarity does not determine alone the cross-
seeding ability of prion amyloids and suggested that
key side-chain residues can impact the cross-seeding
between amyloidogenic sequences. The latter statement
finds additional support in the observation that
a designed HELLF-derived variant (termed HEC), pro-
duced by substitutions of HRAM5-specific residues in
HELLF with HRAM1-specific residues from HET-s,
breaches the cross-seeding barrier in vivo with [Het-
s]. Structural examination of HEC showed that the
chimeric PFD adopts a HELLF/HET-s-like amyloid
fold in vitro, highlighting the importance of the side-
chains of the HRAM-specific residues for the breach of
the cross-seeding barrier between HELLF and HET-s.
The designed synthetic prion HEC shows some level of
structural promiscuity both in vivo and in vitro and this
type of plasticity (not observed in natural HET-
s-related amyloids) might be critical for the breaching
of the seeding barriers. The characterization of HELLF
shows that the HRAM families have diversified into
signalling domains with distinct signalling specificities
integrated into independent cell death pathways. The
establishment of the HRAMs as a structurally unified
superfamily provides a powerful experimental set-up
for future investigations, because determinants specify-
ing the amyloid signalling (ability to seed and cross-

seed) must be reflected in the catalogues of natural
variation found for these domains, which share
a common β-solenoid fold.

HELLP and the cross-interaction with mammalian RHIM

In addition to HELLF and HET-S, HELLP constitutes
a third amyloid-activated cell-death execution protein
of *Podospora anserina*. The *hellp* gene occurs in a two-
gene cluster immediately adjacent to a gene encoding
an NLR termed PNT1 displaying an N-terminal PP-
motif, a central nucleotide-binding and oligomerisation
domain of the NB-ARC type and a C-terminal TPR
domain (tetratricopeptide repeats) [30]. HELLP func-
tions in all respects analogously to HET-S and HELLF
(Figure 1). The PP-motif region of HELLP forms
a prion termed [π] and induces toxicity of full-length
HELLP. The N-terminal PP-motif region of PNT1 also
forms a prion and similarly induces toxicity and mem-
brane localization of HELLP. Importantly, there is no
cross-seeding between the respective prions formed by
the HET-S, HELLF and HELLP PFDs (respectively
[Het-s], [φ] and [π]). This absence of cross-seeding
would indicate that NWD2/HET-S, FNT1/HELLF and
PNT1/HELLP correspond to three parallel indepen-
dently regulated cell death pathways, in which each
NLR induces activation of only its cognate execution
protein, as opposed to branched pathways where
a given NLR could also induce other cell-death execu-
tion proteins. This last situation, at least based on
bioinformatic predictions, seems however to occur in
other species for which NLRs displaying similar
N-terminal motifs are predicted to activate several dis-
tinct cell-death execution proteins (encoded by
unlinked genes) [31]. While, the similarity of the PP-
motif with the RHIM motif was noted previously this
new study establishes now a functional link between
RHIM and PP. Indeed, RHIM motifs of the human
RIP1 and RIP3 kinases were found to behave as prions
when expressed in Podospora and these prions termed
[Rhim] show partial cross-seeding with [π] prions.
Cross-infections are however not as efficient as in the
case of homotypic [π]/[π] interactions or interactions
between different [π] prions of fungal origin. These
observations (in agreement with the fact that the
sequence signatures of the PP and RHIM-motif differ)
suggest that while there exists some level of structural
relation between RHIM and PP-amyloids there must
also exist marked differences lowering the cross-seeding
ability. Nevertheless, the partial cross-seeding between
RHIM and PP strengthens the hypothesis that these
two motifs are evolutionarily related and that this
<figure><img src="image_20.png" /></figure>

type of motif has a long evolutionary history as amyloid
signalling motif, involved in regulated cell death. The
alternate explanation for this sequence similarity would
be convergent evolution. But this hypothesis is chal-
lenged by the sequence diversity of amyloid motifs as
found in fungi and bacteria (as developed below) which
indicates that there are multiple evolutionary solutions
to the problem of generating a short prion amyloid
signalling motif (rather than a unique motif onto
which different evolutionary paths would necessarily
converge).

Amyloid signalling, also in filamentous bacteria

It is important to note that the fungal amyloid NLR-
signalosomes (and NLRs in general in fact) appear as
far as it can be estimated from current genomic cover-
age limited to filamentous fungi [17,32]. Genomes of
unicellular (ascomycete and basidiomycete) yeasts spe-
cies do not display genes encoding amyloid signalling
motifs or NLRs as identified in filamentous fungi. This
is consistent with the proposed role of these NLRs in
regulated cell death in the context of host-defence.
While it is clear that unicellular species may also dis-
play regulated cell death pathway, immune cell death
makes immediate sense in the context of multicellu-
larity. In this context, it is of note that some bacterial
proteins received a PROSITE annotation with RHIM
motifs. These annotations due to the small size of the
motif signature could represent false positives or bona
fide hits oras sequences analogous to animal or viral
RHIM motifs. This observation primed a bioinforma-
tic quest for related sequences in prokaryotic genomes
[33]. Specifically, it was noted that the RHIM-related
motif occurs in a genomic setting reminiscent of the
gene organization of fungal amyloid signalosomes,
that is in gene clusters associating an NLR-encoding
gene and a second gene, encoding a potential effector
protein (Figure 1). Therefore, tentative amyloid signa-
losomes were identified based on these criteria of
resemblance in overall architecture to fungal amyloid
signalosomes. Amyloid motifs were clustered accord-
ing to sequence similarity into 10 families designated
BASS (for bacterial amyloid signalling sequences).
These BASS motifs occur in proteins encoded by adja-
cent or overlapping genes at the N-terminus of NLRs
and at the C-terminus of a protein domain termed
BELL (for bacterial domain analogous to HELL).
This small domain predicted to form an α-helical bund
le with a hydrophobic transmembrane helix shares
some sequence features with the fungal HeLo and
HeLo-related domains and homologous domains
involved in the control of regulated cell death in

animals and plants. However there is no direct detect-
able sequence homology between the BELL and HeLo
domains, therefore functional and structural charac-
terization of these domains are required to precisede-
fine its relation to HeLo. It is currently hypothesized
that BELL functions analogously as a membrane-
targeting cell death-inducing domain. Interestingly as
described in fungi where amyloid signalling motifs
occur most frequently but not exclusively in HeLo-
related domains (HeLo, HELL and sesA), the BASS-
motifs do not occur only in association with BELL
domains but also less frequently in association with
other domains that have been implicated in host
defence such as CHAT protease domains (Caspase
HetF Associated with Tprs) or bacterial TIR domains
(Toll/interleukin-1 receptor). The classification as 10
families is likely to represent a conservative estimate as
a cut-off for the number of occurrences has been
applied in the motif identification process. A lower
cut-off or future expansion in genome coverage
might increase the number of BASS families. Based on
the current estimates however, there appears to be
a greater diversity of amyloid signalling motifs in
bacteria than in fungi, which in turn display more mot
if families than metazoans. This distribution is not
surprising considering the differences in global phylo-
genetic diversity in these lineages and should probably
not be taken as an indication that this form of signal-
ling has experienced greater evolutionary success in
multicellular bacteria than in fungi or metazoans.

The present suggestion of the existence of fungal-
like amyloid NLR signalosomes points at a more gen-
eral aspect of NLR function and evolution in prokar-
yotes and fungi. There is experimental support for the
role of NLRs in fungi in the control of regulated cell
death and host defence (at least in the context of
allorecognition) [17]. If these putative bacterial amy-
loid signalosomes, but also other proteins with NLR
architectures (which are also overrepresented in multi-
cellular bacteria), function in host defence processes
then a re-evaluation of the evolutionary trajectory of
NLRs in the control of immunity would be required.
Instead of explaining the common occurrence of NLRs
in plant and vertebrate immunity as a result of con-
vergent evolution [34], a scenario of long-term conser-
vation of this architecture for the regulation of biotic
interactions across the tree-of-life would have to be
favoured [17].

Outlook and open questions

We discuss here recent findings concerning the mole-
cular mechanisms of NLR-associated amyloid signal
<figure><img src="image_21.png" /></figure>

transduction [29] and the evolutionary history of this
class of functional amyloids [30,33]. We reflect on the
origin of a signal-transducing paradigm, where signal
transmission is achieved through ‘structural templating’
(or replication of a protein fold), from the early obser-
vation of non-Mendelian inheritance phenomena in
P. anserina [1], through the discovery and characteriza-
tion of the [Het-s] prion [35], to the very recent iden-
tification of signal-transducing amyloids in multicellu-
lar prokaryotes [33]. Parallel important work on RHIM
and RHIM-like amyloid signalling motifs in mammals,
viruses and flies further stresses the general importance
of these mechanisms of signal transduction in immune
pathways [36,37].

Functional amyloids in bacteria have been foremost
described as constituents of biofilms [38]. Yet, our
recent analyses uncovered that filamentous bacteria,
mainly actinobacteria and cyanobacteria, carry two-
gene clusters encoding for an NLR-like sensor and its
downstream effector protein, which share a common
signal-transducing amyloid motif. These findings sug-
gest that amyloid NLR signalosomes occur in both
eukaryotes (fungi) and prokaryotes (bacteria). While
in fungi the amyloid signalling controls regulated cell
death, which appears evolutionary related to mamma-
lian necroptosis [12,22], the outcome of the signalling
process in filamentous bacteria remains to be explored.
However, it has been hypothesized that similar to the
role it plays in fungi, NLR-dependent amyloid signal-
ling represents or is integral to programmed cell death
pathways in prokaryotes [33]. Future work will eluci-
date the extent of similarity in regards to the down-
stream responses of amyloid-dependent signalling
between distant phylogenetic groups and likely provide
additional support for a common and extremely ancie-
nt evolutionary origin of amyloid-controlled cell death.
In this context, the RHIM-related amyloid motifs hold
a central stage as a signalling amyloid domain common
to mammals and their viruses [25,37], flies [36], fungi
[12] and prokaryotes [33]. The structure of the necro-
some – a heterotypic RHIM-based amyloid formed by
the RHIM domains of the RIPK1 and RIPK3 kinases –
has recently been resolved [39]. Our recent discovery
that mammalian RHIM motifs can propagate as prions
and cross-seed with the fungal PP-based prion amy-
loids and BASS3 bacterial amyloids [30] indicates
a conserved signalling specificity and suggests, in the
frame of the amyloid templating paradigm, that
a degree of structural similarity exists between these
signalling amyloids. Further structural exploration
between these signalling amyloids would be of great
interest considering that the short amyloidogenic
domains are not found in similar protein architectures,

specifically in mammalian signalling compared to fungi
and prokaryotes. It is conceivable that some amyloid
domains, like RHIM, had have higher evolutionary
success due to their adaptability to form signalling
complexes in a variety of protein architectures and are
not strictly limited to the adaptor N-terminal domains
of NLRs or the C-terminus of execution proteins. In
particular, RHIM might be able in contrast to the other
motifs to function in a central position as opposed to
N or C-terminal positions. The fact that RHIM-like
motifs also occur in Drosophila and viruses, in different
protein architectures and in a central position, tends to
support this view [28,36]. In turn, it must be realized
that the bioinformatics approaches used to identify
amyloid motifs in fungi and bacteria are biased to
identify motifs occurring in the NLR/executor architec-
tures. Further genome mining approaches to identify
amyloidogenic motifs with redefined criteria would be
welcome and could help answer three key questions
about the phylogenetic distribution of the signalling
amyloids. Are there signalling amyloids in fungi and
bacteria, in proteins and signalling pathways not
dependent on NLRs? Is the RHIM motif the only sig-
nalling amyloid in metazoans (and their viruses) and
can the amyloid signalling paradigm be extended to
other taxa, notably plants and Archaea? While experi-
mental validation is required, it is of note that some of
the amyloid motifs occurring in bacteria also occur in
Archaea genomes and specifically in multicellular spe-
cies [33]. The answers to these questions would bring
more clarity on where we stand now in regards to
diversity, abundance and distribution for this class of
functional amyloids.

Aside from these structural and phylogenetic aspects,
a major blind spot in these studies is the question of the
biological role of the identified NLR signalling cascades.
This is true for the microbial NLRs involving amyloid
motifs but equally for the microbial NLRs in general. In
particular, with the exception of a handful of NLRs
involved in allorecognition in fungi, for none of these
systems are the triggering cues known [17]. While the
central connection point (the amyloid signalling motif
per se) begins to be well defined, both the downstream
events (how exactly do HeLo-related proteins bring about
membrane damage?) and foremost the upstream activa-
tion steps (what are the ligands?) remain for the time
being, at best, highly hypothetical. Then, an important
knowledge gap resides in the definition of the actual
structure of the NLR amyloid signalosomes, notably
regarding the actual subunit stoichiometry of these com-
plexes. In addition, what is unclear at present is whether
the execution domains (such as HeLo) remain associated
to the NLR moiety as a heterotypic amyloid assembly or
<figure><img src="image_22.png" /></figure>

whether the PFD regions receive the structural template information and then detach from the NLR amyloid hub. Thus, while some structural aspects of amyloid signalling in fungi have been explored in atomic detail [7,29], the global structural and biological understanding of these nanomachines is still in its very infancy. Important also is to articulate precisely, the relation of amyloid signalling to prion propagation. While several of the amyloid signaling components from fungi, bacteria and mammals (HELLP, HELLF, BASS and RHIM) could be artificially turned into prions, none of these systems, with the notable exception of [Het-s] (and σ in *Nectria*), occurs as natural prions. Only the latter two behave as infectious entities and cytoplasmic genetic elements transmitted between organisms. This is the very property that ignited in early studies the interest of pioneering scientists such as Janine Beisson.

## Acknowledgments

Work in the Saupe group is supported by the CNRS, the University of Bordeaux and a grant from ANR (ANR SFAS AAP CE11).

## Disclosure statement

No potential conflict of interest was reported by the authors.

## Funding

This work was supported by the Agence Nationale de la Recherche Scientifique [ANR SFAS AAP CE11].

## References

[1] Schecroun J. Sur la réversion provoquée des souches $s^S$ en souche s chez *Podospora anserina*. 1958;246:1268-1270. C R Acad Sci Hebd Seances Acad Sci.

[2] Coustou V, Deleu C, Saupe S, et al. The protein product of the het-s heterokaryon incompatibility gene of the fungus *Podospora anserina* behaves as a prion analog. Proc Natl Acad Sci U S A. 1997;94(18):9773-9778.

[3] Beisson J, Sonneborn TM. cytoplasmic inheritance of the organization of the cell cortex in paramecium Aurelia. Proc Natl Acad Sci U S A. 1965 Feb;53 (2):275-282. PubMed PMID: 14294056.

[4] Rizet G, Sichler G. Sur le démasquage du génotype des souches $s^S$ chez *Podospora anserina*. 1950;231:719-721. C R Acad Sci Hebd Seances Acad Sci.

[5] Saupe SJ. The [Het-s] prion of *Podospora anserina* and its role in heterokaryon incompatibility. Semin Cell Dev Biol. 2011 Jul;22(5):460-468. PubMed PMID: 21334447.

[6] Ritter C, Maddelein ML, Siemer AB, et al. Correlation of structural elements and infectivity of the HET-s prion. Nature. 2005 Jun 9;435(7043):844-848. PubMed PMID: 15944710.

[7] Wasmer C, Lange A, Van Melckebeke H, et al. Amyloid fibrils of the HET-s(218-289) prion form a beta solenoid with a triangular hydrophobic core. Science. 2008 Mar 14;319(5869):1523-6. PubMed PMID: 18339938. DOI:10.1126/science.1151839

[8] Seuring C, Greenwald J, Wasmer C, et al. The mechanism of toxicity in HET-S/HET-s prion incompatibility. PLoS Biol. 2012;10(12):e1001451. PBIOLoGY-D-12-01732 [pii]. PubMed PMID: 23300377; PubMed Central PMCID: PMC3531502. eng.

[9] Mathur V, Seuring C, Riek R, et al. Localization of HET-S to the cell periphery, not to [Het-s] aggregates, is associated with [Het-s]-HET-S toxicity. Mol Cell Biol. 2012 Jan;32(1):139-153. MCB.06125-11 [pii]. PubMed PMID: 22037764; PubMed Central PMCID: PMC3255711. eng.

[10] Greenwald J, Buhtz C, Ritter C, et al. The mechanism of prion inhibition by HET-S. Mol Cell. 2010 Jun 25;38 (6):889-899. [pii] 10.1016/j.molcel.2010.05.019. PubMed PMID: 20620958; eng.

[11] Hofmann K. The evolutionary origins of programmed cell death signaling. Cold Spring Harb Perspect Biol. 2020 Sep 1;12 (9). PubMed PMID: 31818855; PubMed Central PMCID: PMCPMC7461761. DOI: 10.1101/cshperspect.a036442.

[12] Daskalov A, Habenstein B, Sabate R, et al. Identification of a novel cell death-inducing domain reveals that fungal amyloid-controlled programmed cell death is related to necroptosis. Proc Natl Acad Sci U S A. 2016 Mar 08;113 (10):2720-2725. PubMed PMID: 26903619; PubMed Central PMCID: PMC4790977.

[13] Mahdi LK, Huang M, Zhang X, et al. Discovery of a family of mixed lineage kinase domain-like proteins in plants and their role in innate immune signaling. Cell Host Microbe. 2020 Sep 30;28(6):813-824.e6. PubMed PMID: 33053377.

[14] Adachi H, Contreras MP, Harant A, et al. An N-terminal motif in NLR immune receptors is functionally conserved across distantly related plant species. Elife. 2019 Nov 27;8:e49956. doi:10.7554/eLife.49956. PMID: 31774397; PMCID: PMC6944444.

[15] Daskalov A, Saupe SJ. As a toxin dies a prion comes to life: A tentative natural history of the [Het-s] prion. PubMed PMID: 26110610 Prion. 2015;9(3):184-189.

[16] Jones JD, Vance RE, Dangl JL. Intracellular innate immune surveillance devices in plants and animals. Science. 2016 Dec 02;354(6316):aaf6395-aaf6395. PubMed PMID: 27934708.

[17] Daskalov A, Dyrka W, Saupe SJ. NLR function in fungi as revealed by the study of self/non-self recognition systems. In: Benz JP,KS, editor. Genetics and Biotechnology. The Mycota (A comprehensive treatise on fungi as experimental systems for basic and applied research). Vol. 2. Cham: Springer; 2020. p. 123-141.

[18] Heller J, Clave C, Gladieux P, et al. NLR surveillance of essential SEC-9 SNARE proteins induces programmed cell death upon allorecognition in filamentous fungi. Proc Natl Acad Sci U S A. 2018 Feb 20;115(10):E2292-E2301. PubMed PMID: 29463729.

[19] Saupe S, Turcq B, Begueret J. A gene responsible for vegetative incompatibility in the fungus *Podospora anserina* encodes a protein with a GTP-binding motif and G beta homologous domain. Gene. 1995;162 (1):135-139.
<figure><img src="image_23.png" /></figure>

[20] Wu H. Higher-order assemblies in a new paradigm of signal transduction. Cell. 2013 Apr 11;153(2):287-292. . S0092-8674(13)00335-8 [pii]. PubMed PMID: 23582320; PubMed Central PMCID: PMC3687143. eng.

[21] Daskalov A, Paoletti M, Ness F, et al. Genomic clustering and homology between HET-S and the NWD2 STAND protein in various fungal genomes. Plos One. 2012;7(4): e34854. PONE-D-11-22729 [pii]. PubMed PMID: 22493719; PubMed Central PMCID: PMC3321046. eng.

[22] Daskalov A, Habenstein B, Martinez D, et al. Signal transduction by a fungal NOD-like receptor based on propagation of a prion amyloid fold. PLoS Biol. 2015 Feb;13(2):e1002059. PubMed PMID: 25671553; PubMed Central PMCID: PMC4344463.

[23] Cai X, Chen J, Xu H, et al. Prion-like polymerization underlies signal transduction in antiviral immune defense and inflammasome activation. Cell. 2014 Mar 13;156(6):1207-1222. PubMed PMID: 24630723; PubMed Central PMCID: PMC4034535.

[24] Saupe SJ. Amyloid Signaling in Filamentous Fungi and Bacteria. Annu Rev Microbiol. 2020 Sep 8;74 (1):673-691. . PubMed PMID: 32689912.

[25] Li J, McQuade T, Siemer AB, et al. The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis. Cell. 2012 Jul 20;150(2):339-350. S0092-8674(12)00772-6 [pii]. PubMed PMID: 22817896; PubMed Central PMCID: PMC3664196. eng.

[26] Daskalov A, Dyrka W, Saupe SJ. Theme and variations: evolutionary diversification of the HET-s functional amyloid motif. PubMed PMID: 26219477; PubMed Central PMCID: PMC4518210 Sci Rep. 2015;5 (1):12494.

[27] Graziani S, Silar P, Daboussi MJ. Bistability and hysteresis of the 'Secteur' differentiation are controlled by a two-gene locus in Nectria haematococca. BMC Biol. 2004;2(1):18. PubMed PMID: 15312233.

[28] Kajava AV, Klopffleisch K, Chen S, et al. Evolutionary link between metazoan RHIM motif and prion-forming domain of fungal heterokaryon incompatibility factor HET-s/HET-s. Sci Rep. 2014;4(1):7436. PubMed PMID: 25500536; PubMed Central PMCID: PMC4262963.

[29] Daskalov A, Martinez D, Coustou V, et al. Structural and molecular basis of cross-seeding barriers in amyloids. Proc Natl Acad Sci U S A January. 2021 5;118(1):e2014085118.

[30] Bardin T, Daskalov A, Barrouilhet S, et al. Partial prion cross-seeding between fungal and mammalian amyloid signaling motifs. bioRxiv. 2020;20200722215483.

[31] Loquet A, Saupe SJ. Diversity of amyloid motifs in NLR signaling in fungi. Biomolecules. 2017 Apr 13;7 (2). PubMed PMID: 28406433; PubMed Central PMCID: PMC5485727. DOI: 10.3390/biom7020038.

[32] Dyrka W, Lamacchia M, Durrens P, et al. Diversity and variability of NOD-like receptors in fungi. Genome Biol Evol. 2014;6(12):3137-3158. PubMed PMID: 25398782.

[33] Dyrka W, Coustou V, Daskalov A, et al. Identification of NLR-associated amyloid signaling motifs in bacterial genomes. J Mol Biol. 2020 Oct 13;432(23):6005-6027. PubMed PMID: 33058872.

[34] Urbach JM, Ausubel FM. The NBS-LRR architectures of plant R-proteins and metazoan NLRs evolved in independent events. Proc Natl Acad Sci U S A. 2017 Jan 31;114(5):1063-1068. . PubMed PMID: 28096345; PubMed Central PMCID: PMC5293065.

[35] Riek R, Saupe SJ. The HET-S/s prion motif in the control of programmed cell death. Cold Spring Harb Perspect Biol. 2016 Sep 01;8(9):a023515. . PubMed PMID: 27352624.

[36] Kleino A, Ramia NF, Bozkurt G, et al. Peptidoglycan-sensing receptors trigger the formation of functional amyloids of the adaptor protein imd to initiate droso-phila nf-kappab signaling. Immunity. 2017 Oct 17;47 (4):635-647e6. PubMed PMID: 29045898; PubMed Central PMCID: PMCPMC5665175.

[37] Pham CL, Shanmugam N, Strange M, et al. Viral M45 and necroptosis-associated proteins form heteromeric amyloid assemblies. EMBO Rep. 2019 Feb;20(2). PubMed PMID: 30498077; PubMed Central PMCID: PMCPMC6362354. DOI:10.15252/embr.201846518.

[38] Levkovich SA, Gazit E, Laor Bar-Yosef D. Two decades of studying functional amyloids in microorganisms. Trends Microbiol. 2020 Oct 8. PubMed PMID: 33041179. DOI:10.1016/j.tim.2020.09.005

[39] Mompean M, Li W, Li J, et al. The structure of the necrosome RIPK1-RIPK3 core, a human hetero-amyl oid signaling complex. Cell. 2018 May 17;173(5):1244-1253 e10. PubMed PMID: 29681455; PubMed Central PMCID: PMC6002806.