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journal homepage: www.elsevier.com/locate/fbr

Review

Vegetative incompatibility in fungi: From

recognition to cell death, whatever does the trick

Mathieu PAOLETTI
Non self recognition in fungi, Institut de Biochimie et G
en
etique Cellulaire, UMR5095 CNRS-Universit
e de Bordeaux,
1 rue Camille Saint-Sa€ens, 33077 Bordeaux Cedex, France

article info abstract

Article history: Allorecognition in fungi takes the form of vegetative incompatibility (VI), a process leading
Received 28 April 2016 to the programmed cell death of heterokaryotic cells formed after anastomosis between
Accepted 4 August 2016 hyphae of genetically incompatible isolates, thereby keeping different genotypes sepa-
rated. VI is ubiquitous amongst ascomycetes and basidiomycetes, determined by loci
Keywords: named het or vic, and responds to both promoting and limiting selective constraints. While
Cell death VI has been widely used to analyze fungal populations, genes controlling VI systems have
Fungi only been characterized at the molecular level in three ascomycete species. VI systems can
Non self recognition be considered as having a modular organization, comprised of a polymorphic component
Polymorphism for recognition associated with a cell death inducing component often (but not exclusively)
including a HET domain protein. However, the actual genes involved differ in sequence and
properties. Some VI genes display a patchy phylogenetic distribution, whereas others
appear widely conserved in fungal genomes, but their function in controlling VI is
restricted to a single or a few related species. It also appears that evolutionary trajectories
generating and maintaining polymorphism at these loci differ. Some het genes show low
allelic diversity and signs of long term balancing selection and may be specifically selected
for allorecognition. Others show high allelic diversity, evidence of positive selection and
fast evolution and their products are believed to correspond to immune receptors whose
functions have been coopted for allorecognition. Finally, where known, mechanisms for
initiating cell death and the cell death reaction itself display similarities and differences
between different model species. All these data support the hypothesis that VI is a ubiqui-
tous phenomenon acquired time and time again independently in different fungal
lineages.
ª 2016 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

1. Introduction incompatibility during reproduction in plants (Iwano and

Takayama, 2012), sexual (Dyer et al., 2016) and vegetative in-
Allorecognition mechanisms, the ability to discriminate self compatibility in fungi (Glass and Dementhon, 2006; Saupe,
from non self within a species, exist in all kingdoms of life, 2000), tissue fusion in marine colonial invertebrates
and include kin recognition in amoebas (Buss, 1982), self- (Rosengarten and Nicotra, 2011), and the highly refined Major

E-mail address: paoletti@ibgc.cnrs.fr

http://dx.doi.org/10.1016/j.fbr.2016.08.002
1749-4613/ª 2016 British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Vegetative incompatibility in fungi 153

Histocompatibility Complex governing graft rejection in meta- crassa, Podospora anserina and C. parasitica (Table 1). Conse-
zoans (Muraille, 2014). These organisms span the whole quently the present review will focus on data gained from
eukaryotic kingdom and although their allorecognition mech- these three models with input from other species where
anisms are quite diverse, they all rely on one common feature possible.
to ensure recognition, a set of loci polymorphic between indi-
viduals (Nydam and De Tomaso, 2011). In consequence, inde-
2. Selective forces acting on vegetative
pendent of the actual mechanisms specific to each
incompatibility
recognition systems, an understanding of how polymorphism
emerges and is maintained at these loci is fundamental to
VI is selectively advantageous as it restricts the horizontal
explaining non self recognition. Indeed, it appears that
propagation of deleterious cytoplasmic elements. For
depending on the organisms and systems investigated, allore-
example, there are numerous demonstrations that virus
cognition might be the driving force for the evolution of these
transmission is restricted to various degrees by the different
recognition systems, while in other circumstances allorecogni-
VI systems in C. parasitica (Brusini et al., 2011; Cortesi et al.,
tion might be an undesirable consequence of other recognition
2001; Nuss, 2005). Consequently a C. parasitica strain lacking
mechanisms (Chae et al., 2014; Nydam and De Tomaso, 2011;
all incompatibility systems has been engineered as an univer-
Ostrowski and Shaulsky, 2009; Rosengarten and Nicotra, 2011).
sal donor to help propagate hypovirulent viruses in popula-
In ascomycete and basidiomycete fungi allorecognition oc-
tions as a tool for biocontrol (Zhang and Nuss, 2016). In O.
curs during the sexual cycle or after anastomosis between
novo-ulmi populations decrease in virus prevalence was asso-
vegetative hyphae that results in the formation of a hetero-
ciated with a parallel increase in vic genotype diversity
karyon. Sexual reproduction is governed by alternative
(Brasier, 1988). In Podospora anserina, distribution of the delete-
mating-type loci and has been largely reviewed recently
rious plasmid pAL2.1 depends on het genotypes (Bastiaans
(Dyer et al., 2016; Heitman, 2015). Vegetative incompatibility
et al., 2014a; Debets et al., 2012; van Diepeningen et al., 2008).
(VI; also known as heterokaryon incompatibility) manifests it-
VI can also restrict resource plundering by aggressive geno-
self when incompatible alleles of specific loci called het (het-
types (Debets and Griffiths, 1998), or the propagation of
erokaryon) or vic (vegetative) are co-expressed in a common
cheater genotypes thereby facilitating emergence of multi-
cytoplasm, usually after anastomosis between genetically
cellularity (Bastiaans et al., 2016).
different hyphae, and induces a programmed cell death
However, selection also limits VI. For instance, it was
(PCD) reaction. VI thus contributes to define individuals by
recently demonstrated by in vitro evolutionary studies that un-
maintaining the separation of genetically different entities.
restricted fusion between isolates was favorable for the devel-
In some model species VI is easy to visualize on culture
opment of N. crassa (Aanen et al., 2008; Bastiaans et al., 2015)
plates as it results in an altered contact zone between incom-
and VI decreased cooperation between isolates. In a P. anserina
patible isolates that is not detected upon fusion between
population, Pahet-c alleles producing stronger incompatibility
compatible isolates (Fig. 1A). VI can also be visualized by forc-
reactions with a set of tester strains were less frequent in a
ing the formation of heterokaryons by growing compatible
population (Bastiaans et al., 2014b). VI also potentially restricts
complementary auxotrophic mutants on minimal media, or
parasexuality, a mechanism allowing mitotic recombination
by genetic transformation of an allele into a recipient strain,
after vegetative fusion (Pontecorvo, 1956). Parasexuality might
the resulting phenotype depends on the het alleles expressed
be important for generating genetic variation in asexually
by the recipient heterokaryon or strain. One can define Vege-
reproducing fungi, and has even been reported to occur be-
tative Compatibility Groups (VCG) as being constituted of
tween incompatible isolates (McGuire et al., 2005) suggesting
compatible isolates that are genetically related. The ease of
that certain conditions might suppress VI to allow parasexu-
testing sexual and vegetative compatibility was used to great
ality. It is also noted that VI can be suppressed in certain con-
length and is still a tool of choice to study fungal populations
ditions such as during sexual reproduction or during Conidial
(Chang et al., 2014; Hoekstra, 1994). These approaches have
Anastomosis Tube (CAT) fusion occurring between incompat-
been instrumental in analyzing population structure (Cortesi
ible germlings (Ishikawa et al., 2012). Thus, the acquisition and
and Milgroom, 1998), in demonstrating the fast evolution of
maintenance of non-self recognition loci will involve a bal-
invasive pathogens such as Ophiostoma novo-ulmi (Brasier,
ance of opposing evolutionary constraints.
1988), and in analyzing selective constraints acting on VI
(see below). These approaches are also central to the develop-
ment of biocontrol strategies of plant pathogens such as Cry- 3. Genetic control of VI
phonectria parasitica (Milgroom and Cortesi, 2004) or to
control toxin producers such as Aspergillus flavus (Atehnkeng VI is genetically controlled by about ten different loci in asco-
et al., 2016; Grubisha and Cotty, 2015; Moore et al., 2013). VI mycete fungi and less than five in basidiomycetes. Two forms
testing has been used for analyzing populations of numerous of VI have been reported. First ‘allelic interactions’ where co-
fungal species and revealed that ascomycete usually contain expression of incompatible alleles from a single gene results
about 10 het loci and basidiomycetes less than five generally in VI; second, ‘non-allelic’ interactions where co-expression
multi-allelic het loci (Van der Nest et al., 2014). Basidiomycetes of incompatible alleles from different genes results in VI. Ef-
are not the most amenable models for studying vegetative in- forts to clone het genes started in the late 80’s in N. crassa
compatibility because of the essentially heterokaryotic life cy- and P. anserina using combinations of positional cloning,
cle. The molecular-genetic basis of VI has been studied in phenotypic expression and functional analysis (Espagne
great details in only three ascomycete species, Neurospora et al., 2002; Glass et al., 1988; Saupe et al., 1994, 1995, 1996;
154 M. Paoletti

Fig. 1 e Various aspects of the vegetative incompatibility reaction: A. Formation of a barrage in the contact region between
incompatible isolates (red arrow) that is not visible in the contact zone between compatible isolates, illustrated here by
confrontations between several Cryphonectria parasitica isolates (from Choi et al., 2011). BeD. Membrane localization of
incompatible HET proteins: Incompatible transformants of Neurospora crassa expressing a NcHET-CPA (B, green channel) and
NcHET-COR (C: red channel). Both proteins colocalize in the plasma membrane (D: overlay) (from Sarkar et al., 2002). EeG.
Vacuolization and autophagosome formation during cell death associated with VI: Images extracted from a time lapse video
of an auto-incompatible strain undergoing cell death. Arrowheads indicate septa present before (black) or appearing after
(white) induction of the VI reaction. White arrows indicate collapsus of the hyphae (E). During the VI reaction, the GFP-
PaATG8 protein localizes to the autophagosome membrane (F). Autophagosome visualized by Transmission Electron Mi-
croscopy (G) (from Pinan-Lucarre et al., 2007).

Smith et al., 2000; Turcq et al., 1991). From this pioneering work
a modular concept of vegetative incompatibility systems with
two key characteristics emerged. First, alternative alleles at
het loci displayed a high level of polymorphism; and second
the alleles contributing to VI often included a gene encoding
for a protein with a so called HET domain (Smith et al., 2000)
frequently found in fungal genomes (Fedorova et al., 2005).
Subsequently these characteristics were exploited, along
with opportunities arising from advances in genomics and
population genomics, to identify additional het genes. P. anser-
ina het-r was identified as a paralogue of the het-e and het-
d genes as a member of the nwd gene family (Chevanne
et al., 2009). Analysis of allele co-segregation with VI pheno-
type and loss of function mutant selection confirmed this
gene functions in VI. In C. parasitica considerable efforts to
clone vic genes combined with development of near isogenic
tester strains for each vic locus (Cortesi and Milgroom, 1998)
and classical genetic mapping (Kubisiak and Milgroom,
2006), ultimately benefited from comparative genomics and
led to the identification of regions of high polymorphism
between genomes from selected tester strains of defined vic
genotypes (Choi et al., 2011; Zhang et al., 2014). Functional
characterization of candidate genes present in these regions
confirmed that they indeed control VI in this organism. In N.
crassa, comparative population genomic analysis identified
34 out of 73 HET domain encoding genes to be highly polymor-
phic between 25 isolates from a single population, including
two already known het genes (Nchet-c and het-6) and one that
further functional characterization confirmed to be a gene
controlling VI, namely Nchet-e (Zhao et al., 2015). A role of the
remaining HET domain encoding genes in controlling VI re-
mains to be investigated. These fruitful efforts have so far
led to the molecular characterization of 22 genes mediating
VI included in 16 incompatibility systems as depicted in
Table 1. What can be learnt from this list?
4. Modular organization of VI systems
As mentioned, the modular organization of VI systems has
been suspected for a longtime, and newly characterized genes
Vegetative incompatibility in fungi
involved with VI further support this initial concept. A VI sys-
tem includes a recognition domain and a cell death inducing
domain that may be associated on the same protein or be pre-
sent on separate proteins (Fig. 2, Table 1). The recognition
module includes at least one polymorphic component
defining allele specificity. These polymorphisms can take
multiple forms from point mutations (P. anserina het-s)
(Turcq et al., 1991), insertions and deletions (Wu et al., 1998)
to highly divergent idiomorphic organizations as seen in the
mata/matA system in N. crassa (Glass et al., 1988) or the vic4 lo-
cus in C. parasitica (Choi et al., 2011). Polymorphism at het loci
can also involve several genes maintained in tight linkage
disequilibrium because of suppressed recombination as
described for the un-24/het-6 system in N. crassa (Smith et al.,
2000), or because of physical proximity as for the N. crassa
Nchet-c/pin-c (Kaneko et al., 2006) or the C. parasitica vic6/pix6
(Zhang et al., 2014) gene associations. When carefully exam-
ined it appears that induction of a full VI reaction by these sys-
tems requires a complex interplay between allelic and non
allelic interactions between the different components of these
gene complexes (Table 1).
The second module of a VI system consists of a cell death
activating domain. It is often associated with protein contain-
ing the almost fungal specific but frequent HET domain
(Fedorova et al., 2005; Smith et al., 2000; Zhao et al., 2015).
The HET domain (Pfam: PF06985) is characterized by three
conserved blocks of about 50 amino acids comprised within
about 220 amino acids stretch. Overexpression of this HET
domain has been shown to initiate a VI like cell death reaction
in P. anserina (Paoletti and Clave, 2007). In addition mutations
altering the HET domain of N. crassa pin-c (Kaneko et al., 2006)
or tol (Shiu and Glass, 1999), or P. anserina Pahet-e or het-r
(Chevanne et al., 2010) abolish VI. Ten of the sixteen character-
ized incompatibility systems include a gene encoding a HET
domain protein (Table 1). However, cell death can be initiated
through other means i.e. VI does not necessarily require the
presence of a HET domain protein. For example, in P. anserina
interaction with the antagonistic HET-s protein leads to the
release of a pore forming transmembrane segment in the
HeLo domain of the HET-S counterpart protein, which proves
toxic (Mathur et al., 2012). The patatin domain of the C. para-
sitica vic2 system is similarly likely to initiate cell death, as a
patatin domain initiates cell death in Arabidopsis to promote
resistance to cucumber mosaic virus (La Camera et al., 2009).
In N. crassa un-24 VI system, cell death is thought to be the
consequence of the abolition of the essential ribonuleotide
reductase activity during interactions of incompatible vari-
ants of the proteins (Smith et al., 2013a). Only in the C. para-
sitica vic3 system has molecular identification of the genes
failed to suggest a mechanism for cell death induction,
although both genes of this locus are required for induction
of VI in the form of a barrage (Zhang et al., 2014).
5. VI functions correspond to phylogenetically
restricted acquisitions
Blastp searches with VI controlling genes reveal three situa-
tions regarding the phylogenetic distribution of VI genes
(Table 1). Some het genes such as Nchet-c encoding a
155
membrane protein, un-24 encoding a ribonucleotide reduc-
tase, or Pahet-c encoding a glycolipid transfer protein yield a
high number of hits even in distantly related species, and
display expected orthologous relationships. Comparing se-
quences of homologues of the Nchet-c gene between different
isolates in Fusarium proliferatum (Kerenyi et al., 2006), Botritys
cinerea (Fournier et al., 2003), A. niger (van Diepeningen et al.,
2009) and Tuber melanospora (Iotti et al., 2012) revealed little
or no polymorphism. Interestingly however, heterologous
expression of N. crassa alleles in A. niger, or mutagenesis of
endogenous sequences aimed at creating variants mimicking
N. crassa polymorphisms, resulted in phenotypes akin to VI re-
action with reduced growth and altered cell morphology (van
Diepeningen et al., 2009). Similar results were obtained with
hch, the P. anserina homologue of Nchet-c (Saupe et al., 2000).
These results indicate that Nchet-c homologues in these spe-
cies are also likely to have the potential to control VI, although
this has not yet been exploited to this effect in these species.
Similarly, N. crassa un-24 encodes a ribonucleotide reductase
conserved in eukaryotes. However, the utilization of Ncun-24
in VI appears to be restricted to N. crassa and related species
where it is linked to the insertion of a sequence of about 300
bp depending on the alleles (Smith et al., 2000). Thus it appears
that un-24 homologues are not functioning as het genes in
other species. Finally, a high level of polymorphism has
been observed in the Pahet-c gene only in P. anserina but not
in orthologues in other species (Bastiaans et al., 2014b). There-
fore these data indicate that certain widespread genes have
been co-opted for use in VI only within a limited number of
species e an important insight into the evolutionary origins
of VI.
In contrast other het genes seem to display a patchy phylo-
genetic distribution with homologues in a reduced number of
species, as for example N. crassa tol. Such genes often encode
proteins with a HET domain, the homology being restricted to
this domain. It has been long known that fungal genomes
include a great number of HET domain encoding genes
(Fedorova et al., 2005). However the corresponding genes are
usually not conserved between species and orthology rela-
tionships are difficult to delineate (Dyrka et al., 2014;
Fedorova et al., 2005). It is important to be aware that not all
HET domain encoding genes control VI, but their functions
remain unknown (Zhao et al., 2015). P. anserina het-s homo-
logues also have a patchy distribution. In P. anserina two al-
leles are found at the het-s locus. The het-s (small s) allele
encodes a 289 amino acid protein with a HeLo and a prion
forming domain (pfd) with prion properties (Coustou et al.,
1997). The het-S (big S) allele differs by just 13 amino acids,
one difference being sufficient to alter allelic specificity
(Deleu et al., 1993). In addition het-S is linked to the nwd2
gene, which is absent from the het-s locus. All identified ho-
mologues in ascomycetes display the het-S characteristic
sequence (Daskalov et al., 2012) and are associated with a
nwd2 like gene suggesting that they are not involved in VI con-
trol (Daskalov and Saupe, 2015).
Finally, six het genes (four in P. anserina, two in C. parasitica)
encode for NOD Like Receptors (Dyrka et al., 2014) of the
STAND family of proteins characterized by a tripartite organi-
zation and involved in signal transduction (Leipe et al., 2004)
for which numerous homologues can be found in distantly
 156
Table 1 e Components of the VI systems characterized in Neurospora crassa, Podospora anserina and Cryphonectria parasitica.
Species System Protein domains Phylogeny Genetic interactions References

Neurospora het-c/pin-c NCHET-C: Transmembrane W Kaneko et al. (2006)

het-c1 pin-c1
crassa (Nchet-c) protein R
het-c3
PIN-C: HET
het-c2 pin-c2
het-c1
het-c3 pin-c3
mat/tol MATa: HMG box W mat-a Glass and Smith
MATA: a box W tol (1994),
TOL: HET R mat-A Shiu and Glass (1999)
un24 UN24: Ribonucleotide W PA PA Lafontaine and
un-24 het-6
het-6 reductase W Smith (2012),
HET-6: HET OR OR
Smith et al. (2000)
un-24 het-6
het-e NcHET-e: HET W het-e1 Zhao et al. (2015)
(Nchet-e)
het-e2

het-e3
Podospora het-s HET-s: HeLo þ pfd R het-s Daskalov et al. (2015),
anserina HET-S: HeLo þ pfd W Turcq et al. (1991)
NWD2: pfd þ NACHT þ WD S het-S nwd2
het-c/het-e PaHET-c: Glycolipid W het-e Espagne et al. (2002),
(Pahet-c, Pahet-e) transfer protein S het-c Saupe et al. (1994),
het-c/het-d PaHET-e: HET þ NACHT þ WD S Saupe et al. (1995)
het-d
(Pahetc, het-d ) HET-d: HET þ NACHT þ WD
het-R/het-V HET-r: HET þ NACHT þ WD S het-V het-R Chevanne et al.
het-v/het-V HET-v: unk - (2009)
het-v
Cryphonectria vic2 VIC2: Patatin þ NB þ TPR S Choi et al. (2011)
vic2-1 vic2a-1
parasitica VIC2a: Sec9 SNARE W

vic2-2 vic2a-2
vic4 VIC4-1: Protein kinase c-like W vic4-1 Choi et al. (2011)
VIC4-2: A/B þ NACHT þ WD S
vic4-2
Cryphonectria vic6 VIC6: HET R Choi et al. (2011)
vic6-1 pix6-1
parasitica PIX6: DUF1014 W

vic6-2 pix6-2

vic7 VIC7: HET W vic7-1 Choi et al. (2011)

M. Paoletti
vic7-2
Vegetative incompatibility in fungi 157

related species. Searching for homologues of the P. anserina

Incompatibility systems lists genes involved under their names at the time of their description, and names in parenthesis are used in this paper to avoid confusion between P. anserina and N. crassa
when necessary. The protein domains column lists functional domains of the proteins. The phylogenetic distribution of het genes are summarized as widespread (W) when blastp hits are numerous

column, each gene is represented by a horizontal bar. Polymorphic components of VI systems are represented by colored bars (red, green, blue) and non polymorphic components are depicted by black
bars. Incompatible genetic interactions are indicated by colored arrows (blue for allelic interactions, red for non allelic interactions, and green for interactions between idiomorphs). Wide arrows
and genes seem to display orthologous relationships or Reduced (R) when distribution is patchy, while (S) indicate STAND proteins likely to have arisen by domain shuffling. In the Genetic Interactions
nwd family members resulted in the identification of
Zhang et al. (2014)

Zhang and Nuss

numerous candidates in Aspergillus and Tuber species (Iotti
et al., 2012; Pal et al., 2007). However, as these genes evolve
mostly by domain shuffling (Dyrka et al., 2014), the relation-
(2016) ship between these homologues is difficult to establish. They
could either correspond to orthologues maintained over long
periods of time in these species, or result from convergent
evolution by assortment of homologous functional domains
together several times in different lineages. Additional inves-
tigation is therefore needed before it can be assumed that
these genes control VI in the other respective species.
vic1d-1

Overall these data suggest that whether het genes are

vic1b-1

vic1b-2

vic3b-1

vic3b-2

conserved or not, their function in controlling VI is most often

vic1c-1

likely to correspond with lineage specific acquisition of this

indicate prevalent interactions in nature. Names of proteins harboring a HET domain are written in red while STAND proteins are highlighted in grey.

function. After acquisition some systems can be maintained

for long periods of time even through speciation events,
resulting in trans-species polymorphism (Kaneko et al., 2006;
Smith et al., 2013b; Wu et al., 1998; Zhao et al., 2015). Thus, ho-
mology based identification of candidates genes alone is likely
vic3a-1

vic3a-2
vic1a-1

vic1a-2

to fail to identify het genes. Instead, candidate based ap-

proaches will benefit from new sequencing methods that
allow population genomic screens for polymorphism to be un-
dertaken rapidly and at relatively low cost. If it can be shown
that certain genes previously linked to VI show high levels of
polymorphism within populations, then there is a good likeli-
hood that they may be involved with VI in the study species,
which can lead onto functional characterization of candidate
polymorphic genes (Zhao et al., 2015).
R
R
R
R

R
R

6. Acquisition and maintenance of polymor-

phism of het genes

Modelling of the evolution of polymorphism at vic/het loci pre-

associated lifeguard-1-like
VIC3b: glutamate receptor

dicts that selective pressure exerted by deleterious cyto-

plasmic elements might be enough to explain how
polymorphisms arise and are maintained at the recognition
VIC1b: DUF1909
VIC1c:TY1/LTR

loci (Brusini et al., 2011; Czaran et al., 2014; Muirhead et al.,

VIC1-d: HET
VIC1a: HET

2002). This process would result in negative frequency depen-

VIC3a: nd

dent selection. In accordance, in N. crassa, alleles at the Nchet-

c/pin-c, Nchet-e, and het-6/un-24 systems have been shown to
be maintained in equal proportions in populations by
balancing selection (Powell et al., 2007; Wu et al., 1998; Zhao
et al., 2015). This selection has maintained allelic variants at
equilibrium for long period of time and resulted in trans-
species polymorphism. In other words, these polymorphisms
have been maintained through speciation events leading to
extant Neurospora/Sordaria species. In C. parasitica populations
only vic2 alleles appeared to be under balancing selection
vic1

vic3

(Milgroom and Cortesi, 1999), and interestingly vic2 is the

most efficient locus at preventing the propagation of the
hypovirulent virus CHVI in lab testing conditions (Cortesi
et al., 2001), although these conditions might underestimate
virus transmission (Brusini and Robin, 2013). In a P. anserina
population from Wageningen in the Netherlands (van der
Gaag et al., 1998) about 70% of alleles were found to be of the
het-s genotype, while the remaining 30% were of the het-S ge-
notype. This may reflect the fact that het-s acts as a meiotic
drive element, distorting normal Mendelian segregation to
158
its advantage (Dalstra et al., 2003, 2005). However, het-S iso-
lates appear to be infected less by the deleterious PAL2.1
plasmid (Debets et al., 2012).
An alternative but not exclusive hypothesis proposes that
polymorphism may be acquired at het loci for reasons inde-
pendent from VI before being co-opted for this function
(Paoletti and Saupe, 2009). In P. anserina, the Pahet-c/het-d and
Pahet-c/Pahet-e incompatibility systems appear to be
extremely dynamic. The nwd family members, including het-
d, Pahet-e and het-r, are undergoing concerted evolution of
the WD40 repeat sequence encoding regions, with evidence
for positive selection acting on codons specifying amino acids
located at the interaction surface of the b-propeller structure
adopted by the WD repeats (Paoletti et al., 2007). These se-
quences have been shown to be genetically unstable, resulting
in the frequent production of allelic variants (Chevanne et al.,
2010) which is reflected by the high heterogeneity of alleles in
a single population (Daskalov et al., 2015). In addition the
antagonist Pahet-c is also subject to positive selection, result-
ing in high allele diversity in a single population (van der
Gaag et al., 1998), with unbalanced allele distribution
(Bastiaans et al., 2014b). It has been proposed that these pro-
teins are involved in pathogen recognition and are evolving
under the selective pressure of pathogenic effectors following
a model known as guard/guardee (Jones and Dangl, 2006). This
evolutionary arms race would on occasion be expected to lead
to the emergence of incompatible combinations between
Pahet-c and Pahet-e alleles (Paoletti and Saupe, 2009). However,
testing for VI interactions of these het-c alleles revealed a
balanced distribution of het-c alleles incompatible with
Pahet-e1 or Pahet-e2 tester strains (Bastiaans et al., 2014b). To
summarize, multiple alleles at het-c and hnwd loci are pro-
posed to have been generated under pathogen pressure and
incompatible allele combinations then maintained at a
balanced frequency in the population to control VI. This
would be a clear case of exaptation, defined as the process
by which a selected function (in this case recognition of path-
ogens) is exploited for other purposes (VI) (Gould SJ and Erba
ES, 1982). Another example of exaptation linking VI to im-
mune response comes from the het-s/het-S incompatibility
system. The HET-S protein is the cell death effector of the
innate immune receptor NWD2 in a signal transduction
mechanism requiring amyloid fibril formation (Daskalov
et al., 2015). The HET-S cell death activating function was
lost in HET-s through mutations that concomitantly gener-
ated the het-s/het-S incompatibility system (Daskalov and
Saupe, 2015). Similarly, a survey of N. crassa populations
revealed two types of polymorphism in HET domain proteins.
Some showed little allelic diversity linked to long term main-
tenance, providing evidence for balancing selection acting on
these genes, while the other displayed higher allelic diversity
with and restricted phylogenetic distribution. It was hypothe-
sized that this latter group may encode products involved in
pathogen recognition (Zhao et al., 2015), and it is tempting to
speculate that some of these may generate incompatibility
systems.
Beside mutagenic processes, allelic diversity can be ac-
quired by other less well described processes. For instance
in the invasive O. novo-ulmi species new vic alleles are acquired
by introgression from the related resident O. ulmi species
M. Paoletti
(Paoletti et al., 2006). It appears from these observations that
situations differ from the models and the loci considered.
Although the number of species where het genes have been
characterized is still limited, one can speculate on the impact
of the species lifestyle on the nature of the het genes. N. crassa
propagates essentially through wind dispersal of conidio-
spores so encounter with pathogens may be rare and het genes
appear relevant to restrict cheating (Bastiaans et al., 2016). In
contrast P. anserina propagates through ascospores germi-
nating on herbivore dungs, so pathogens encounter may be
a greater threat and immune receptors more prone to co-
option for VI purposes. Characterizing het gene diversity and
their distribution in populations for different loci and different
model organisms may help draw a clearer picture of forces
acting on these loci. For instance, investigating C. parasitica
genomic diversity at vic loci in populations in relation with
their ability to restrict virus transmission and with plant path-
ogenicity may be informative in this respect.
7. Initiating cell death
Early events leading to initiation of cell death have been eluci-
dated in the N. crassa het-c and P. anserina het-s systems. In N.
crassa, interactions of alternate antagonistic NcHET-C pro-
teins occurs in the cell membrane (Sarkar et al., 2002)
(Fig. 1BeD). In P. anserina, interaction of alternate HET-s pro-
teins results in a conformational change of the HET-S HeLo
domain that unleashes a transmembrane segment acting as
a pore forming toxin after insertion in the membrane
(Mathur et al., 2012; Seuring et al., 2012). This pore forming
toxin acts autonomously in a heterologous system (Taneja
et al., 2007). Similarly, a newly identified protein domain
with the potential to generate VI in Chaetomium globosum
also involves localization of a protein complex to the mem-
brane (Daskalov et al., 2016). Finally, although direct detection
was not possible, N. crassa Western blotting after sub-cellular
fractionation revealed that the NcHET-E protein was associ-
ated with cytoplasmic or endomembrane compartments
(Zhao et al., 2015). Thus it seems that alteration of membranes
by incompatible protein complexes is a recurrent early event
in the initiation of many VI associated cell death reactions.
This direct route to alteration of cell membrane might explain
why so few suppressors of VI have been selected despite
various genetic screens (for review Loubradou and Turcq,
2000). vib-1 in N. crassa encodes a transcription factor regu-
lating het gene expression at the Nchetc/pinc, tol and het-6/un-
24 loci in N. crassa (Dementhon et al., 2006). In P. anserina
modA sequence did not reveal any functional indications
except for the presence of a SH-3 binding domain likely
involved in proteineprotein interactions (Barreau et al.,
1998). Note that if overexpression or point mutations of the
HET domain indicate that it can induce the cell death reaction
(Chevanne et al., 2010; Glass and Dementhon, 2006; Kaneko
et al., 2006; Paoletti and Clave, 2007), the mechanism of such
induction remains speculative. Distant phylogenetic relation-
ship to plant TIR domain suggest it may be involved
in proteineprotein interactions (Dyrka et al., 2014), while
Blastp searches with the HET domain reveals some homol-
ogies with proteins with lipase activities.
Vegetative incompatibility in fungi
Recognion Inducon Inducon Recognion
+ Cell death
Fig. 2 e Modular conception of vegetative incompatibility
systems: A vegetative incompatibility system includes a
recognition module (grey box) displaying high level of
polymorphism (vertical black bars), and a cell death acti-
vating domain (black box) often including a HET domain
(white box). These recognition and induction modules may
be associated on the same protein or be present on different
proteins included in the system.
8. Cell death
Despite what seems a fairly simple activation mechanism,
cell death reactions initiated by VI appear complex. Cell
death reactions have been previously described at the cyto-
logical, morphological and cellular level in both P. anserina
and N. crassa (Glass and Dementhon, 2006; Pinan-Lucarre
et al., 2007) (Fig. 1EeG). Recently transcriptional changes dur-
ing the cell death reaction have been examined in these two
models by either microarray hybridization (Bidard et al., 2013;
Hutchison et al., 2009) or RNA-seq (Lamacchia et al., 2016). In
both species VI appears to be a complex and dynamic pro-
cess, with a considerable fraction (ca 20%e30%) of the
genome up or down regulated. These analyses found that
globally down regulated genes encoded products with func-
tions related to cell development, cell division, ribosomal
components and translation, a characteristic response to
stress. Genes up-regulated appear to include an excess of
lineage specific genes or genes with limited phylogenetic dis-
tribution, often lacking an identified function. Although the
number of orthologous gene pairs upregulated in both spe-
cies is limited, the overlap between both responses is none-
theless significant, even the more so when focusing on the
top 100 most stimulated genes (Bidard et al., 2013) that
include genes involved in cell signaling or autophagy, fea-
tures common to both reactions (Fig. 1). Note that PCR based
subtractive hybridization of two cDNA libraries identified
genes related to autophagy which were induced during VI
in the basidiomycete Amylostereum areolatum (van der Nest
et al., 2011). These analyses also revealed differences. For
instance, functions related to oxidative stress response are
overrepresented in the N. crassa up-regulated gene pool
(Hutchison et al., 2009), which was not observed in the P.
anserina VI response. In contrast, genes involved in secondary
metabolite production are overrepresented in the genes up-
regulated in P. anserina (Bidard et al., 2013; Lamacchia et al.,
2016) which was not reported for N. crassa. Overall these
data indicate that while VI reactions in P. anserina and N.
crassa may share some common factors, they also include
species-specific responses. This might be related to the prop-
erties of the specific het genes controlling certain species-
specific reactions. N. crassa het genes are under long term
balancing selection and might thus represent a bona fide allor-
ecognition mechanism (Wu et al., 1998). In contrast P. anserina
159
het genes are proposed to be derived from a heterospecific
non self recognition function, and thus the response induced
may mimic a response to heterospecific non self (Paoletti and
Saupe, 2009). Indeed, recent transcriptional analyses showed
that VI in P. anserina significantly overlapped with the tran-
scriptional response to pathogenic bacteria and displayed
characteristic of an innate immune reaction (Lamacchia
et al., 2016). It would be interesting to compare in a single or-
ganism the cell death reaction initiated by het genes under
long term balancing selection to the reaction initiated by
fast evolving het genes.
9. Concluding remarks
While the ubiquity of VI testifies to its importance in fungal
biology, the diversity of genes involved, differences in selec-
tive constraints exerted on het genes (long term balancing se-
lection vs fast evolving genes) and in induced responses
indicate VI is a fungal process acquired time and time again
independently in different lineages. Various mechanisms
resulting in cell death in a timely fashion appear to have
been retained for VI, be it selected specifically for this purpose
or derived from biological processes such as immunity. None-
theless, certain recurring characteristics of VI systems can be
distinguished (prevalence of HET domain containing proteins,
STAND proteins, alterations of cell membranes.) that can be
used as helpful markers, along with population genomics
data, as a basis for the characterization of het genes in new
species. Only by investigating VI and non-self recognition in
additional models, including basidiomycetes, and contexts
(conspecific vs heterospecific recognition), will it be possible
to gain a more complete understanding of the forces and con-
straints shaping these mechanisms. From a broader perspec-
tive, fungal non-self recognition systems display evolutionary
features found in other eukaryotic non-self recognition sys-
tems and may therefore provide valuable insights into the
emergence of these peculiar loci.
Acknowledgments

and Dr S.J. Saupe for
Many thanks to Dr, F. Ness, Pr. C. Clave
helpful discussion and critical reading of the manuscript. I
also wish to thank two reviewers for their pertinent com-
ments and helpful suggestions in improving this manuscript.
This work was partly founded by ANR grant “Mykimun”
ANR11 BSV3 019 01 and by CNRS.
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