and endophytic species (Kubicek et al. 2011;
Martino et al. 2018; Peter et al. 2016; Wang
et al. 2014). This observation is mirrored in
Table 6.1 where many species with very high
numbers of NLR genes show complex symbiotic/parasitic lifestyles. It is tempting to propose, as was suggested in the case of the coral
A. digitifera (Hamada et al. 2013), that at least a
part of the extensive NLR sets in these fungi has
a role in the establishment and maintenance of
a symbiotic lifestyle.
V. Prion-Forming Domains in NLR
Signaling
A subset of N-terminal signaling domains of the
fungal NLRs forms b-sheet-rich amyloid structures, whose inherent prion properties play a
crucial role in the signaling process (Daskalov
et al. 2012, 2015a, b, 2016; Loquet and Saupe
2017). Prion amyloids are strictly cooperative
protein aggregates (fibers) exposing a supersecondary cross-b conformation, in which the bsheets are perpendicular to the axis of the amyloid fiber. The high cooperativity and repetitiveness of the amyloid fold bring forth a
remarkable feature of “self-propagation” (the
prion aspect), where the amyloid serves as a
molecular template for the unstructured primary sequence of a protein able to adopt a
similar amyloid fold (Riek and Eisenberg
2016). The amyloid signal transduction is
based on the controlled emergence and transmission (as templating) of structural information from the activated NLR receptor to a
downstream executioner protein. In the current
model, the oligomerization of an activated NLR
protein brings the N-terminal unstructured
amyloid motifs of the monomeric NLR molecules in close proximity (Daskalov et al. 2015b).
The spatial clustering of these motifs induces
the cooperative folding of an amyloid structure, which then templates the amyloid folding
of an unstructured amyloid domain situated on
the executioner protein. The NLR and its downstream effector protein represent a functional
unit and are frequently encoded by adjacent
genes in the genomes of various filamentous
ascomycetes (Daskalov et al. 2012, 2015a).
The first NLR protein identified to carry an accessory
amyloid domain was NWD2 (NACHT-WD) from P.
anserina (Daskalov et al. 2015b). The nwd2 gene is
located adjacent to the het-S gene, which encodes the
downstream effector of the NLR (Fig. 6.3a). HET-S is a
289 amino acids pore-forming protein consisting of a
cytotoxic N-terminal HeLo domain and C-terminal
prion forming domain (PFD) (Seuring et al. 2012). The
PFD, or HET-S (218–289), transitions from an unstructured state in the inactive monomer to a left-handed bhelix (or b-solenoid) in the activated cytotoxic state. The
adoption of the amyloid fold by the PFD triggers a
conformational change in the HeLo domain resulting
in the release of an N-terminal transmembrane a-helix,
which induces plasma membrane damage and causes
cell death. The HET-S PFD is used as a model to study
the fundamental properties of amyloids and a solid-state
NMR structure of the b-solenoid fold has been obtained
more than a decade ago (Wasmer et al. 2008). The PFD
consists of two 21 amino acid pseudo-repeats (termed
R1 and R2) connected by a flexible 15 amino acid long
loop (Fig. 6.3b, c). The repeats are alternately stacked
along the axis of the b-solenoid, each contributing with
four b-strands per ring (Fig. 6.3c). The resulting bsheets delimit a highly hydrophobic triangular b-solenoid core. The core is tightly packed with predominantly hydrophobic residues while polar and charged
residues are found on the exterior, solvent-facing side of
the amyloid (Fig. 6.3d). Three salt bridges and two
asparagine ladders are part of the b-solenoid and impact
its stability and prion properties. While the PFD of HETS contains two pseudo-repeats, NWD2 contains only
one repeat, termed R0 (Fig. 6.3b). This R0 motif
(NWD2(3-24)) is essential for the NLR signaling process, shows sequence homology to the R1 and R2
pseudo-repeats and adopts a related amyloid fold (Daskalov et al. 2015b) (Fig. 6.3b, d). It is proposed, based on
the accumulated experimental evidence, that the oligomerization of NWD2 brings the R0 motifs of different
NWD2 molecules in proximity so that these motifs
cooperatively adopt a HET-S-like amyloid fold and subsequently template the PFD of HET-S to trigger the
cytotoxic activity of the HeLo domain (Fig. 6.3a).
NLR-mediated amyloid signaling appears
to be widespread in the fungal kingdom, and
several other putative signal-transducing amyloid domains have been identified (Daskalov
et al. 2012, 2015a, 2016). Noteworthy, some of
these signal-transducing amyloids are evolutionarily related to the PFD of HET-S/s and
are named HRAMs (HET-s-related amyloid
motifs). The HRAMs have diversified into
132
A. Daskalov et al.
Martino et al. 2018; Peter et al. 2016; Wang
et al. 2014). This observation is mirrored in
Table 6.1 where many species with very high
numbers of NLR genes show complex symbiotic/parasitic lifestyles. It is tempting to propose, as was suggested in the case of the coral
A. digitifera (Hamada et al. 2013), that at least a
part of the extensive NLR sets in these fungi has
a role in the establishment and maintenance of
a symbiotic lifestyle.
V. Prion-Forming Domains in NLR
Signaling
A subset of N-terminal signaling domains of the
fungal NLRs forms b-sheet-rich amyloid structures, whose inherent prion properties play a
crucial role in the signaling process (Daskalov
et al. 2012, 2015a, b, 2016; Loquet and Saupe
2017). Prion amyloids are strictly cooperative
protein aggregates (fibers) exposing a supersecondary cross-b conformation, in which the bsheets are perpendicular to the axis of the amyloid fiber. The high cooperativity and repetitiveness of the amyloid fold bring forth a
remarkable feature of “self-propagation” (the
prion aspect), where the amyloid serves as a
molecular template for the unstructured primary sequence of a protein able to adopt a
similar amyloid fold (Riek and Eisenberg
2016). The amyloid signal transduction is
based on the controlled emergence and transmission (as templating) of structural information from the activated NLR receptor to a
downstream executioner protein. In the current
model, the oligomerization of an activated NLR
protein brings the N-terminal unstructured
amyloid motifs of the monomeric NLR molecules in close proximity (Daskalov et al. 2015b).
The spatial clustering of these motifs induces
the cooperative folding of an amyloid structure, which then templates the amyloid folding
of an unstructured amyloid domain situated on
the executioner protein. The NLR and its downstream effector protein represent a functional
unit and are frequently encoded by adjacent
genes in the genomes of various filamentous
ascomycetes (Daskalov et al. 2012, 2015a).
The first NLR protein identified to carry an accessory
amyloid domain was NWD2 (NACHT-WD) from P.
anserina (Daskalov et al. 2015b). The nwd2 gene is
located adjacent to the het-S gene, which encodes the
downstream effector of the NLR (Fig. 6.3a). HET-S is a
289 amino acids pore-forming protein consisting of a
cytotoxic N-terminal HeLo domain and C-terminal
prion forming domain (PFD) (Seuring et al. 2012). The
PFD, or HET-S (218–289), transitions from an unstructured state in the inactive monomer to a left-handed bhelix (or b-solenoid) in the activated cytotoxic state. The
adoption of the amyloid fold by the PFD triggers a
conformational change in the HeLo domain resulting
in the release of an N-terminal transmembrane a-helix,
which induces plasma membrane damage and causes
cell death. The HET-S PFD is used as a model to study
the fundamental properties of amyloids and a solid-state
NMR structure of the b-solenoid fold has been obtained
more than a decade ago (Wasmer et al. 2008). The PFD
consists of two 21 amino acid pseudo-repeats (termed
R1 and R2) connected by a flexible 15 amino acid long
loop (Fig. 6.3b, c). The repeats are alternately stacked
along the axis of the b-solenoid, each contributing with
four b-strands per ring (Fig. 6.3c). The resulting bsheets delimit a highly hydrophobic triangular b-solenoid core. The core is tightly packed with predominantly hydrophobic residues while polar and charged
residues are found on the exterior, solvent-facing side of
the amyloid (Fig. 6.3d). Three salt bridges and two
asparagine ladders are part of the b-solenoid and impact
its stability and prion properties. While the PFD of HETS contains two pseudo-repeats, NWD2 contains only
one repeat, termed R0 (Fig. 6.3b). This R0 motif
(NWD2(3-24)) is essential for the NLR signaling process, shows sequence homology to the R1 and R2
pseudo-repeats and adopts a related amyloid fold (Daskalov et al. 2015b) (Fig. 6.3b, d). It is proposed, based on
the accumulated experimental evidence, that the oligomerization of NWD2 brings the R0 motifs of different
NWD2 molecules in proximity so that these motifs
cooperatively adopt a HET-S-like amyloid fold and subsequently template the PFD of HET-S to trigger the
cytotoxic activity of the HeLo domain (Fig. 6.3a).
NLR-mediated amyloid signaling appears
to be widespread in the fungal kingdom, and
several other putative signal-transducing amyloid domains have been identified (Daskalov
et al. 2012, 2015a, 2016). Noteworthy, some of
these signal-transducing amyloids are evolutionarily related to the PFD of HET-S/s and
are named HRAMs (HET-s-related amyloid
motifs). The HRAMs have diversified into
132
A. Daskalov et al.