Introduction
An organism’s adaptation to changing environments is fueled by its genetic variability,
which is established by mechanisms ranging from single-nucleotide polymorphisms to
large-scale structural variations, all of which affect chromosomal shape, organization,
and gene content [1]. These processes are particularly relevant for pathogens that
must respond to continual selection pressure arising from host immune systems that
evolved to detect the presence or activity of potential microbial pathogens through
a variety of invasion patterns [2]. In their adaptive response, pathogens evolve strategies,
often involving secreted effector molecules, to overcome host immunity and support
host colonization [3]. Thus, it can be anticipated that this coevolutionary arms race
leads to highly specific interactions between adapted pathogens and their specific
hosts. Paradoxically, particular pathogens successfully colonize a broad range of
hosts, yet how such pathogens cope in arms races with such a diversity of hosts remains
unknown.
A structured genome drives adaptive evolution
It has been proposed that filamentous fungal and oomycete plant pathogens often evolved
structured genomes with two distinct types of genomic regions: (1) gene-rich regions
containing slowly evolving genes that mediate general physiology and (2) gene-poor
regions that are dynamic and enriched for repetitive DNA such as transposable elements
(TEs) and virulence-related genes that often display signs of accelerated evolution
[4,5]. Extensive structural variation often occurs in these fast-evolving regions,
leading to translocation, duplication, or loss of genetic material [1,4,5]. The highly
dynamic regions can either be embedded within the core chromosomes or be located on
separate chromosomes that often display presence/absence variations within a population,
known as conditionally dispensable or accessory chromosomes [4,6]. The common occurrence
of these bipartite genomes led to the “two-speed” model for pathogen genome evolution
[4], suggesting that specific regions form sites of rapid genomic diversification
to facilitate coevolution during host interactions [1,4,5].
Transposable elements shape “two-speed” genomes
It is generally observed that the dynamic regions of two-speed genomes are enriched
in TEs, yet it remains undemonstrated how they mechanistically contribute to genome
variability [1,4,5]. Recently, the contribution of TEs towards the evolution of the
two-speed genome in the vascular wilt pathogen Verticillium dahliae was reported [7].
Extensive genome rearrangements are generated by double-strand repair pathways that
erroneously utilize stretches of homologous sequence at an unlinked locus, the majority
of which occur around TEs simply as a consequence of their abundance and sequence
similarity (Fig 1A) [7]. Genomic rearrangements often occur at dynamic regions that
are enriched for recent segmental duplications, generating genetic material subject
to evolutionary diversification, e.g., by reciprocal gene loss (Fig 1A) [7,8]. Furthermore,
these dynamic regions are enriched in in planta induced effectors [8] and evolutionary
young and “active” TEs (Fig 1B). These TEs likely contribute to accelerated genomic
diversification of dynamic effector regions [7].
10.1371/journal.ppat.1005920.g001
Fig 1
Dynamic genomic regions are associated with transposons and with distinct chromatin
landscapes.
(A) In V. dahliae strain JR2, repeat-rich dynamic effector regions that evolve by
genome rearrangements (indicated by red arrow heads) and by extensive segmental duplications
(links between highly similar duplicated regions shown in grey) are located on chromosomes
2, 4, and 5 (red highlights). Repeat density along the chromosomes is indicated by
a pink line (summarized as percent nucleotides in genomic windows of 5 kb, with a
slide of 0.5 kb). (B) Dynamic genomic regions in V. dahliae are enriched in transcriptionally
“active” and evolutionary “young” repetitive elements when compared with the core
genome [7]. (C) Different histone modifications can be associated with core (Chr 13)
and conditionally dispensable (Chr 14) chromosomes in the wheat pathogen Zymoseptoria
tritici (as previously reported [13]). Repeat density along the chromosomes is indicated
by a pink line (summarized as percent nucleotide in genomic windows of 5 kb, with
a slide of 0.5 kb). Publicly available chromatin immunoprecipitation sequencing (ChIP-seq)
samples [13] were mapped to the Z. tritici genome, and enriched regions were subsequently
identified using RSEG [28]. DNA associated with euchromatic (H3K4me2, green) and heterochromatic
(H3K27me3, orange; H3K9me3, red) marks are indicated, and significantly enriched genomic
regions are marked with a solid line. Structural variations (duplications, black;
deletions, blue) were identified by CNVnator, using publicly available resequencing
data of multiple Z. tritici strains [29]. (D) The number of nucleotides (in Mb) of
the Z. tritici genome covered by different histone regions (defined by RSEG) for euchromatin
(green) and heterochromatin (orange, red) are shown by bar charts. (E) The number
of duplications and deletions overlapping with histone regions (see above) are shown
by bar charts.
Chromatin biology impacts the adaptive evolution of filamentous plant pathogens
Chromatin, a complex of nucleic acids and proteins, determines the physical shape
and organization of DNA within the nucleus [9]. In many eukaryotes, highly repetitive
regions are composed of tightly condensed chromatin, referred to as heterochromatin,
as opposed to open chromatin or euchromatin. Heterochromatin functions to silence
repetitive and neighboring DNA and to repress recombination in many eukaryotic genomes
[10]. However, these observations are inconsistent with data from fungal plant pathogens
since repeat-rich regions of the two-speed genome are enriched in structural variations
[7,11], and genes located in these regions often show highly coordinated expression
[4]. To address this conundrum, and to understand the formation, maintenance, and
transcriptional regulation of bipartite genomes, it is necessary to analyze genome
structure and organization through the study of chromatin biology.
Chromatin and genome organization
Chromatin features such as accessibility, histone modifications, and chromosome conformation
contribute to the organization of a genome. Recent modeling of mammalian and yeast
genomes suggests that genomic rearrangements can be accurately predicted based on
two chromatin features alone; chromatin openness and DNA contact in the nucleus (Fig
2A) [12]. Chromatin studies in most fungi remain scarce, but recent work on the fungal
wheat pathogen Z. tritici shows marked differences in histone modifications between
core and conditionally dispensable chromosomes (Fig 1C) [13]. Only half of the genome
is occupied by histones carrying modifications that are commonly associated with heterochromatic
regions, such as histone methylation of lysine residues on histone 3 at position 9
or 27 (H3K9me3 or H3K27me3). Notably, the majority of the detected structural variations
colocalize with these heterochromatic regions (Fig 1C–1E). This suggests a further
link between chromatin and genome structure, and despite the general thought that
heterochromatin suppresses genomic variation, heterochromatic regions appear highly
variable in the plant pathogenic fungi analyzed to date (see [6] for additional examples).
10.1371/journal.ppat.1005920.g002
Fig 2
Impact of chromatin organization on adaptive (genome) evolution.
(A) Genome rearrangements (red arrows) occur in open chromatin regions (euchromatin,
green background; heterochromatin, red background) that are in spatial proximity within
the nucleus. Chromosomes are shown as lines and genes as differentially shaped symbols.
Spatial organization of the nucleus is highlighted by nuclear regions that are occupied
by distinct chromosomes (different colors). (B) Chromatin influences gene expression,
as genes located in open chromatin are transcribed (arrows), while genes located in
heterochromatin are transcriptionally silent. (C) Interspecific genome hybridization
leads to genome restructuring, often accompanied by extensive gene loss, and changes
in transcriptional profiles, which can be influenced by differences in chromatin between
parental species (genes from two parental lineages are indicated by orange and blue,
respectively). (D) Host jumps, specialization towards a specific host, and evasion
of host immunity can be influenced by changes in chromatin that translate to alterations
in gene expression or DNA content.
A possible mechanism to explain the link between chromatin and genome structure is
that heterochromatic regions are more prone to DNA breaks during replication that,
when repaired, could result in structural variations [1,14]. In Neurospora crassa,
heterochromatin is highly enriched for a specific modification of histone 2, γH2A
(γH2A.X in mammals), which generally marks double-strand DNA breaks [15]. It is not
known why this histone modification marks these regions, but if it follows its canonical
role, these regions showing elevated rates of DNA breaks would be prone to erroneous
replication-based repair. Additionally, N. crassa chromosome conformation maps indicate
frequent and strong contact between heterochromatic regions [16]. Taken together,
the increased rates of genomic variation at heterochromatic regions may arise from
DNA damage during the replication of heterochromatic DNA, followed by error-prone
replication-based repair or template switching between contacting loci.
Chromatin and gene expression
The majority of chromatin studies using plant-pathogenic fungi have focused on their
transcriptional impacts (Fig 2B) [17–19]. Canonical repressive chromatin marks, such
as H3K9me2/3 or H3K27me2/3, are significantly enriched at regions known to harbor
genes important for pathogenicity [17,18]. Additionally, the canonical activating
mark H3K4me2/3 was shown to play a significant role in regulating gene expression
and promoting growth and virulence in Magnaporthe oryzae [19]. Collectively, these
studies indicate that deregulating chromatin significantly impacts fungal transcription,
growth, and development. To gain further understanding into the regulation of pathogenicity,
it is necessary to determine the key enzymes responsible for reading, writing, and
erasing these epigenetic modifications.
Chromatin and adaptation
Chromatin modifications can functionally diversify between species. For example, the
addition of methyl groups to histone 3 at lysine 36 (H3K36me3) functions in guiding
both DNA mismatch repair and messenger RNA (mRNA) splicing machinery in humans [20,21],
while in the yeast Saccharomyces cerevisiae, the same mark functions to suppress intragenic
transcripts [22]. Thus, although the overall localization of the mark at transcribed
genes is conserved, species evolved to utilize the mark differently. Along with functional
diversification between species, chromatin architecture can vary within a population
[23]. It has been proposed that environmental conditions can result in heritable epigenetic
variation and act as a substrate for so-called “Neo-Darwin” selection [24].
Given that chromatin regulation can evolve, is variable within populations, and can
be environmentally influenced, it can be anticipated that chromatin affects host–microbe
interactions. For example, several documented cases show that plant pathogens may
undergo, or are the result of, interspecific genome-hybridization events [25]. These
events are accompanied by complex gene expression changes, likely influenced by parental
chromatin structure, and by genome reorganization in the hybrids (Fig 2C). These changes
in expression and DNA content can lead to alterations in the hybrid’s ability to compete
for a particular niche and could facilitate a host jump (Fig 2D). Pathogens that are
well adapted to a specific host may also undergo chromatin-based regulatory changes
that alter their host interaction. For example, expression of the Avr3a effector in
Phytophthora sojae can change between generations, allowing for evasion of recognition
by the corresponding host immune receptor Rps3a in soybean [26]. The mechanism for
this switch has not been reported, but experimental evidence shows an enrichment of
small RNA (sRNA) at the locus in silenced versus Avr3a-expressing lines with no genetic
mutations reported, suggesting epigenetic regulation (Fig 2D). The prevalence of such
an epigenetic evasion strategy in other plant–microbe interactions remains unknown.
It is also interesting to speculate that variation in chromatin structure within a
pathogen population could influence a broad-host-range pathogen to form strains with
increased fitness on a particular host. Conceptually, this could be achieved through
alterations in the chromatin-based regulation for the optimal timing, rate, and order
of transcriptional events leading to successful infection. That is, alterations in
transcription to dampen or augment an infection strategy better suited for one host
versus another could influence the evolution of a plant–microbe relationship (Fig
2D). Future experiments to address these possibilities may uncover yet additional
layers influencing the evolution of plant–microbe interactions.
Outlook
Chromatin biology can impact filamentous pathogens across spatial and temporal scales,
from governing genome organization to controlling the expression of its individual
parts. We conceive that complex chromatin structures in pathogenic fungi will influence
not only coordinated effector expression in dynamic genomic regions but also structural
variations, thereby further linking genome and chromatin structure to genome evolution
and adaptation. Additionally, chromatin structure also plays crucial regulatory roles
in establishing symbiotic interaction between the fungus Epichloë festucae and its
plant host [27]. Thus, studying the impact of chromatin biology on genome organization
can broaden our knowledge and potentially provide mechanistic understanding of the
evolution of two-speed genomes in plant pathogens and, in general, of adaptive genome
evolution in plant–fungus interactions.