Diversity and Function of Appressoria

Abstract

Endophytic, saprobic, and pathogenic fungi have evolved elaborate strategies to obtain nutrients from plants. Among the diverse plant-fungi interactions, the most crucial event is the attachment and penetration of the plant surface. Appressoria, specialized infection structures, have been evolved to facilitate this purpose. In this review, we describe the diversity of these appressoria and classify them into two main groups: single-celled appressoria (proto-appressoria, hyaline appressoria, melanized (dark) appressoria) and compound appressoria. The ultrastructure of appressoria, their initiation, their formation, and their function in fungi are discussed. We reviewed the molecular mechanisms regulating the formation and function of appressoria, their strategies to evade host defenses, and the related genomics and transcriptomics. The current review provides a foundation for comprehensive studies regarding their evolution and diversity in different fungal groups.

Full text

Pathogens 2021, 10, 746. https://doi.org/10.3390/pathogens10060746 www.mdpi.com/journal/pathogens
Review
Diversity and Function of Appressoria
K. W. Thilini Chethana
1,2,3
, Ruvishika S. Jayawardena
2,3
, Yi-Jyun Chen
2,3
, Sirinapa Konta
2,3
,
Saowaluck Tibpromma
4
, Pranami D. Abeywickrama
2,3,5
, Deecksha Gomdola
2,3
, Abhaya Balasuriya
6,†
,
Jianping Xu
7
, Saisamorn Lumyong
1,8
and Kevin D. Hyde
1,2,3,4,8,
*
1
Innovative Institute of Plant Health, Zhongkai University of Agriculture and Engineering,
Guangzhou 510225, China; tchethi@yahoo.com (K.W.T.C.); scboi009@gmail.com (S.L.)
2
Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand;
ruvi.jaya@yahoo.com (R.S.J.); yui2134000@gmail.com (Y.-J.C.); sirinapakonta@gmail.com (S.K.);
pranamiabeywickrama@yahoo.com (P.D.A.); deeckshagomdola@gmail.com (D.G.)
3
School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand
4
CAS Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany,
Chinese Academy of Science, Kunming 650201, China; saowaluckfai@gmail.com
5
Beijing Key Laboratory of Environment Friendly Management on Diseases and Pests of North China Fruits,
Institute of Plant and Environment Protection, Beijing Academy of Agriculture and Forestry Sciences,
Beijing 100097, China
6
Department of Plant Sciences, Faculty of Agriculture, Rajarata University of Sri Lanka,
Mihintale 50300, Sri Lanka; abhayab2006@gmail.com
7
Department of Biology, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4K1, Canada;
jpxu@mcmaster.ca
8
Center of Excellence in Microbial Diversity and Sustainable Utilization, Faculty of Science, Chiang Mai
University, Chiang Mai 50200, Thailand
* Correspondence: kdhyde3@gmail.com
† Retired professor.
Abstract: Endophytic, saprobic, and pathogenic fungi have evolved elaborate strategies to obtain
nutrients from plants. Among the diverse plant-fungi interactions, the most crucial event is the at-
tachment and penetration of the plant surface. Appressoria, specialized infection structures, have
been evolved to facilitate this purpose. In this review, we describe the diversity of these appressoria
and classify them into two main groups: single-celled appressoria (proto-appressoria, hyaline ap-
pressoria, melanized (dark) appressoria) and compound appressoria. The ultrastructure of appres-
soria, their initiation, their formation, and their function in fungi are discussed. We reviewed the
molecular mechanisms regulating the formation and function of appressoria, their strategies to
evade host defenses, and the related genomics and transcriptomics. The current review provides a
foundation for comprehensive studies regarding their evolution and diversity in different fungal
groups.
Keywords: host-defense; host-recognition; hyaline appressoria; infection process; melanized ap-
pressoria; proto-appressoria
1. Introduction
Host-fungal interactions found in nature are described as commensalism, symbiosis,
or pathogenesis. For any successful fungal-host interaction, the most crucial steps are at-
tachment, lysis, and mechanical breaching of the host surface [1,2]. To overcome the bar-
riers present in the host, fungi have evolved diverse strategies such as the formation of
specialized infection structures and the secretion of cell-wall-degrading enzymes, toxins,
and effectors [1,3–6]. Infection structures which are believed to have evolved from non-
pathogenic hyphae [1] are variable and belong to many types such as appressoria, ex-
pressoria, infection cushions, infection hyphae, and penetration hyphae [1,7,8].
Citation: Chethana, K.W.T.;
J
ayawardena, R.S.; Chen, Y.J.; Konta,
S.; Tibpromma, S.; Abeywickrama,
P.D.; Gomdola, D.; Balasuriya, A.;
Xu, J.; Lumyong, S.; Hyde, K.D.
Diversity and function of appresso-
ria. Pathogens 2021, 10, 746.
https://doi.org/10.3390/
pathogens10060746
Academic Editor: Nemat O. Keyhani
Received: 29 April 2021
Accepted: 8 June 2021
Published: 12 June 2021
Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional
claims in published maps and institu-
tional affiliations.
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license
(http://creativecommons.org/licenses
/by/4.0/).

Pathogens 2021, 10, 746 2 of 23
Appressoria, discovered by Frank [9] from the pathogenic fungus Colletotrichum lin-
demuthanium as the ‘spore-like organ’ or the ‘adhesion organ’, represent the most studied
form of infection structure. Different types of appressoria and their mechanisms of pene-
tration have been studied extensively in plant pathogens, and also in endophytes, sap-
robes, other symbionts (including arbuscular mycorrhizal fungi and lichens), and ento-
mopathogenic fungi [3,4,6,8,10]. Highly specialized, melanized appressoria with ex-
tremely high turgor pressure, which is critical for cuticle penetration, have been identified
from Pyricularia grisea (Pyr.), Pyr. oryzae, and Colletotrichum (Col.) species [1,4,8]. Non- or
lightly-melanized appressoria have been identified from Blumeria graminis (Bl.), Ustilago
maydis, and Cochliobolus (Coc.) species [1,11]. Similarly, infection cushions have been iden-
tified from Fusarium graminearum, Rhizoctonia sp., Sclerotium sp., Sclerotinia sp. (Sc.), and
Botrytis cinerea (Bot.) [12–14]. Furthermore, different appressoria have been observed in
entomopathogenic Beauveria bassiana (Be.), mutualistic arbuscular mycorrhizae in Glom-
eromycota, and the oomycetes such as Phytophthora (Ph.), Pythium (Pyt.), and Peronospora
species [10,15]. For example, oomycete appressoria are non-melanized or non-pigmented
and smaller compared to fungal appressoria, and they are separated from germ tubes by
false septa, while in fungi a septum separates the appressorium from the spore [16].
Adherence of the fungal spore to the host plant is considered as a critical requirement
for spore germination, germ tube elongation, and appressorium formation during the
plant-fungal interactions [2]. These morphogenetic events from spore attachment to ap-
pressorium formation are stimulated by a variety of host plant signals and environmental
factors such as substratum hydrophobicity [17], topographic signals [18], cutin monomers
[19], and ethylene signals [2]. Several important fungal signaling pathways have been
heavily studied for their involvement in the formation and functioning of appressoria,
including G proteins, cAMP, MAP kinases, and calcium/calmodulin-mediated signaling
pathways [20,21]. Many comprehensive studies have been conducted on diverse fungal
invasion strategies, and the morphology of these processes are well-documented [22–27].
Genomics and their interconnected disciplines have provided a fertile platform to
address most of these issues and controversies related to the formation and functioning
of appressoria. Genomic data and novel techniques have contributed towards a better un-
derstanding of these fungal-host interactions from appressorium formation to host inva-
sion [28–32]. Advances in reverse genetic techniques have enabled cloning of diverse
genes predicted to be involved in these interactions, and many are characterized via var-
ious functional genomic approaches [17,21,28,33–42].
This study brings together diverse information on appressoria. The review explains
what appressoria are, the genera that produce appressoria, how appressoria are involved
in host colonization, the ultrastructure of appressorial colonization, and their latest mo-
lecular understandings. Further, recent discoveries from omics studies including ge-
nomics, transcriptomics, and proteomics related to appressorial formation and function
are discussed. This knowledge is important to understand the appressorial diversity and
evolution among different fungal life strategies.
2. What Are Appressoria?
Appressoria have been defined in a number of ways, often depending on the disci-
pline of the definer. The commonly used definitions are given in Table 1. Most definitions
refer to appressoria as being infection structures of pathogens. Fungal appressoria are,
however, also found in epiphytes, endophytes, and even saprobes. Many entomo-patho-
genic fungi also develop appressoria [10,41]. We, therefore, propose to define appressoria
in a more general sense.
General Definition
Appressoria are specialized cells or adhesion structures produced by fungi from
which a penetration peg emerges that pierces or enters the host tissues.

Pathogens 2021, 10, 746 3 of 23
Table 1. Different definitions for appressoria.
Definition
‘Peculiar spore-like organs produced by the germ tubes of spores of the bean anthracnose’ [43]
‘Localized swellings of the apices of germ tubes or older hyphae that develop in response to contact with the host’ [44]
‘The specialized cells, formed before the penetration of host tissue’ [45]
‘A swelling on a germ tube or hypha, especially for attachment in an early stage of infection’ [46]
‘Spore-like organs formed on germ tubes of Colletotrichum lindemuthianum, Polystigma rubrum and Fusicladium tremulae’ [9]
‘Hyphopodia are considered as attachment structures characteristic of a few families in three orders of fungi and appressoria as
attachment organs characteristic of germ tubes or the early stages of infection’ [47]
‘Hyphal ramification within the host may be much more extensive from appressoria than from hyphopodia, since from the latter, a
haustorium of only limited growth usually develops’ [47]
‘Appressoria can be defined as structures employed by fungal pathogens to press against and attach to the plant surface in prepa-
ration for infection’ [48]
‘Appressorium, a swelling on a germ-tube or hypha, especially for attachment in an early stage of infection, as in certain Puccin-
iales and in Colletotrichum; the expression of the genotype during the final phase of germination, whether or not morphologically
differentiated from vegetative hyphae, as long as the structure adheres to and penetrates the host’ [49]
‘The organ of attachment of a germ tube or hypha of certain parasitic fungi in early stages of infection’ [50]
‘A flattened and thickened apex of a hyphal branch, formed by some parasitic fungi, that facilitates penetration of the host plant’
[51]
The term ‘appressoria’ was first introduced by Frank when he studied diseases of
poplar trees and beans caused by Fusicladium sp. and Col. lindemuthianum, respectively [9].
He described them as spore-like organs, formed at the end of germ tubes. The term was
later coined as the ‘adhesion organ’ when he found that appressoria are tightly attached
to the host surface. Hasselbring confirmed Frank’s findings [43]. In Hasselbring’s experi-
ments, he placed the spores of Gloesporium fructigenum in a drop of water on a glass slide
to study appressoria formation. Within 12 to 18 hours, he found that the spores which
touched the surface of the glass generated short germ tubes; later, the apices of the germ
tubes became enlarged and formed thick-walled, brown spore-like bodies, which were
appressoria. Even after washing with water, the appressorium did not detach easily from
the glass surface. He obtained the same result when the experiment was carried out on an
apple surface instead of glass slide. He thus concluded that the spore must first come into
contact with the host surface and adhere tightly in order to generate appressoria [43]. Em-
mett and Parbery expanded the definition and described appressorium as a significant
penetration tool to break through the tough outer layer of plants, following adherence to
the host surface [14].
Even though they were first observed in plant pathogens, appressoria are present in
epiphytes, endophytes, saprobes, entomopathogens, and symbionts [4,14,41]. Appresso-
ria are therefore not organs specifically evolved for basic infection processes of plant path-
ogens. Hence, for the rest of this review, we will maintain that appressoria are, “special-
ized cells or adhesion structures from which a penetration peg emerges that pierces the
epidermal cell wall or enter the epidermis through stomatal apertures”.
3. Appressorial Categorization
Appressoria come in many different shapes and sizes and can be classified into two
major groups (Figure 1): single-celled and compound appressoria [14]. Single-celled ap-
pressoria are the most common type among many species and occur at the end of germ
tubes. This type of appressoria can be further classified into three groups: proto-, hyaline,
and dark/pigmented appressoria. These hyaline and dark/pigmented appressoria have
different shapes such as dome-shaped or lobed. Emmett and Parbery described proto-
appressorium as little more than slightly swollen, hyaline germ-tube apices, adhering to
the host surface with the formation of an infection-peg [14]. The proto-appressorium has
a short germ tube, with no septum separating the apex from the germ tube. Botrytis cinerea
elaborates a swollen cell at the apex of the germ tube in order to infect plants and was

Pathogens 2021, 10, 746 4 of 23
termed as a proto-appressorium [38]. Venturia (Ven.) and Pythium species too have formed
‘proto-appressoria’ when colonizing their hosts [14,52]. However, Venturia inaequalis has
a melanin-deposited ring structure in the appressorial cell wall surrounding the penetra-
tion pore [27]. The second group of single-celled appressoria termed hyaline or non-pig-
mented appressoria are swollen and generally separated from the germ tube by a septum.
Some genera in the orders of Erysiphales and Uredinales produce hyaline appressoria
[14,52]. These hyaline appressoria are of different shapes including hook-shaped in Bl.
graminis [53]; dome-shaped in Oxydothis species [54]; lobed in Erysiphe and Neoerysiphe
species [55]; elongated, nipple-shaped in Phyllactinia species [56]; and sickle-shaped in
Phythium species [57]. The third group is the dark or pigmented appressoria, usually thick-
walled relative to the spores, either sessile or subtended by a germ tube, from which they
can be separated by a septum. Take, for example, Colletotrichum, Pyricularia, Polystigma,
and Phyllosticta species, which produce melanized appressoria. These melanized appres-
soria are also formed in different shapes such as dome-shaped in Pyr. oryzae [58] and lobed
in Col. boninense [59]. The single-celled appressoria are formed not only terminally, such
as on germ tubes, but can also be found laterally or intercalary, when formed on hyphae
[14].
For all other multi-cellular appressoria, we grouped them into the compound appres-
soria category as in Emmett and Parbery [14] and Parbery and Emmett (1975) as cited in
[14]. These structures are formed in various forms and include infection cushions, infec-
tion plaques, and expressoria [7,13,60,61]. Infection cushions are complex, multi-cellular
appressoria with several penetration sites as in Bot. cinerea [13]; infection plaques are
formed when runner hyphae differentiate to discrete clusters of parenchyma-like cells
with multiple penetration sites as in Psuedocercosporella herpotrichoides [61]; expressoria are
intrinsecus appressoria that are formed at the tip of hyphae of endophytes such as Epichloe
festucae [7]. Most of the compound appressoria are hyaline [14]. Figure 2 exhibits different
appressorial types produced by different fungal taxa.
Figure 1. Classification of appressoria.

Pathogens 2021, 10, 746 5 of 23
Figure 2. Single-celled appressorial types produced by fungal taxa. (a–d) Single-celled, hyaline, ir-
regular-shaped appressoria in Oxydothis garethjonesii [54]; (e–k) Single-celled, light green, irregular-
shaped appressoria in O. metroxylonicola [54]; (l–s) Single-celled, hyaline to dark green, irregular-
shaped appressoria in O. metroxylonis [54]; (t–x) Single-celled, hyaline to brown, irregular-shaped
appressoria in O. palmicola [54]; (y–ab) Single-celled, hyaline, globose to subglobose appressoria in
Leptosporella arengae [62]; (ac–ag) Single-celled, hyaline, globose to subglobose appressoria in Neo-
linocarpon rachidis [63]. Scale bar: a, y, ac = 50 µm; e, t = 20 µm; b–d, f–s, u–x = 10 µm; z–ab, ad–ag =
5 µm.
4. Ultrastructural Studies of Appressoria
Knowledge gained from ultrastructural studies, combined with histological observa-
tions and physiological investigations on pathogenesis, enhances our understanding of
host-fungal interactions at tissue and cellular levels. These studies provide information on
how organelles and cytoskeletons reorganize during the appressorial formation and func-
tion. The information resulted from these studies is important as it provides notable tar-
gets for developing effective control measures for pathogens. For example, the application
of biocontrol agents with fungicidal properties such as Bacillus subtilis on Pyr. oryzae in-
hibits spore germination, germ tube length, and appressorial formation, finally leading to
reduced pathogenicity [64]. Appressorial formation has been studied at the ultrastructural

Pathogens 2021, 10, 746 6 of 23
level in Col. truncatum, Col. lindemuthianum, and Pyr. oryzae on sterilized dialysis mem-
branes. To study appressoria at an ultrastructural level, the germlings were mainly ana-
lyzed by freeze-substitution with electron-microscopy [65]. Spore adhesion is an initial
critical step in the establishment of fungal colonization, which is associated with hydro-
phobic interactions involving cell surface proteins [66]. This adhesion of Col. lindemuthi-
anum may persist after the release of glycoprotein exudates at the substrate interface be-
fore germination [66]. As the spore germinates, the Col. lindemuthianum germ tube grows,
and during its growth, the germling surface is surrounded by an extracellular matrix as
visualized by colloidal gold [67]. The adhesion strength and its constituents differ among
pathogenic fungi [68]. By using freeze-substitution, it was shown that non-germinated
and germinated conidia of Col. lindemuthianum and Col. truncatum were coated with an
electron-dense, finely fibrillar layer (Figure 3A) [67,69]. Negatively stained cells through
a transmission electron microscope showed that fimbriae are protruded from the surface
of germ tubes and appressoria as shown in Figure 3A [66].
Figure 3. Ultrastructures of appressoria and extracellular matrix. (A) Ultrastructure of extracellular
matrix from the germ tube (GT) of Colletotrichum lindemuthianum. Long fibers (fimbriae) projecting
from the surface of the germ tube; (B) Ultrastructure of appressoria-like structures formed by Pyricu-
laria oryzae. The melanin layer (arrows) is absent in the pore area (between arrowheads); (C) Close-
up view of the rectangular section marked on Figure B; (D) Fully developed appressorium of C.
truncatum. A: electron-dense extracellular matrix (arrowheads) coating on appressorium; DM: dial-
ysis membrane; ECM: extracellular matrix; GT: germ tube; ML: melanin layer; N: nucleus; PC: par-
ent conidium; PP: penetration peg; S: septum visible at the base of appressorium; V: vesicle. Scale
bars: A, B, D = 1 µm. Re-drawn from A: [67], B: [23], C, D: [69].
The Pyricularia oryzae extracellular matrix is electron-dense and consists of amor-
phous and fibrous components [23], which is similar to the animal extracellular matrix
[23,68]. Melanin is also one of the cell surface compounds of several plant pathogenic
fungi including Col. lindemuthianum, Col. truncatum, and Pyr. oryzae (Figures 3B and 3C).
Immunoelectron microscopy showed that electron-dense melanin layers were positioned
between cell walls and plasma membranes of appressoria [23]. The electron-dense mela-
nin layers are assumed to be involved in adhesion, turgor generation, cell wall rigidity,
and protection against fungal enzymatic hydrolysis [23,67,69]. A study conducted on Ven.
inaequalis disproved the importance of melanin for appressorial adhesion by demonstrat-
ing that appressorial adhesion to various surfaces is not affected by the melanin in appres-
sorial cell wall [70]. Similarly, studies have shown that melanin is not essential for turgor
generation but contributed to cell wall rigidity [71–73]. In addition, these functions are
discussed in detail under section 6. A study conducted on Col. graminicola demonstrated

Pathogens 2021, 10, 746 7 of 23
that melanized appressoria are protected against exogenous cell-wall-degrading enzymes
whereas the non-melanized appressoria become lysed, suggesting that melanin has a pri-
mary role as a barrier for fungal cell-wall-degrading enzymes [34].
The conidial cytoplasm is comprised of ribosomes, mitochondria, lipid bodies, mi-
crobodies, vacuoles, multivesicular bodies, golgi bodies, woronin bodies, vesicles, and
microtubules. When conidia are attached to substrates, a number of ultrastructural
changes become evident [69]. In Col. truncatum, cristae of mitochondria became clearer
because of the tremendously decreased electron density of the mitochondrial matrix. Mi-
crobodies also become prominent in germinating conidia, while their electron density in-
creases [69]. During germination, the germ tube cytoplasm is often very dense, and the
cytoplasmic components move from germinating conidia to the germ tube [69,74,75].
Eventually, the nucleus moves from the germinating cell to the germ tube to start mitosis.
During mitosis, one of the resulting daughter nuclei moves into the germ tube apex, and
the other one moves to the back of the germ tube [69,76]. The nuclear division is reported
as a prerequisite for conidial germination and appressorial differentiation for both ento-
mopathogens [77] as well as for plant pathogens [78]. Numerous microtubules concentrate
in the conidial cell near the germ tube [75]. Vesicles are also abundant in the germinating
spore and in the developing germ tube. Vesicles which condense near the germ tube apex
are referred to as apical vesicles [65,74]. The migration and positioning of apical vesicles
of germling apex are influenced by the interaction of vesicles and cytoskeletons [74,79].
Once the germ tube is attached to the substrate, it starts to differentiate into an ap-
pressorium [75]. Early in the appressorium development, the distribution of microtubules
changes from a parallel direction with the original longitudinal axis of the germling to the
parallel region of a cell exhibiting lateral growth [74,80]. The vesicles originally sent to-
wards the apex change their distributions around the cells and cause cellular ballooning
[80]. During appressorium formation, the germ tube cytoplasm migrates into the swollen
apex [74,76]. Eventually, a septum develops to separate the mature appressorium and the
germ tube [69,74–76].
Near the center of the fully developed appressorium (Figure 3D) which directly con-
tacts with the substrate, the cell wall becomes extremely thin. Apical vesicles along with
multivesicular bodies, filasomes, and a few microtubules become concentrated in the apex
of the appressorium [65,79]. Eventually, a small penetration peg grows from the thin-
walled region. As evident in Col. truncatum [69] and Pyr. oryzae [79], many small apical
vesicles are present in the apex of the penetration peg.
5. Appressorial Infection Process
In this section, we will review our current understanding of appressoria and their
common mechanisms in pathogenesis related to selected fungal taxa. Pathogenesis in-
volves a series of events that eventually can lead to disease development in a host. Here,
we examine the main stages in the disease cycle of fungal pathogens on plant hosts (Figure
4), beginning with the landing of spores, their attachment, and entry into the plant tissues
via appressoria formation and penetration. Appressoria are produced when fungi need to
access nutrients from their hosts [60]. The very first event in a disease cycle is the contact
of the fungus with a susceptible host plant [81].

Pathogens 2021, 10, 746 8 of 23
Figure 4. Events of a disease cycle.
When the microclimatic conditions are favorable and the nutrients are available,
spores or the primary inocula will start germinating [81]. Resting spores may wait until
the arrival of new roots in their vicinity, or else motile zoospores may be attracted to their
hosts. For example, Phytophthora cinnamomi is attracted to Persea americana, and Ph.
citrophthora is attracted to citrus plants [81].
Spreading spores land on aerial plant surfaces via wind, rain splash, or facilitation
by some animal activities. Some of these spores initiate the germination process immedi-
ately, when coming in contact with water or under humid conditions [81]. Some spores
require a host stimulus to initiate their germination. Spores of many facultative patho-
genic fungi germinate more rapidly in the presence of certain available nutrients. After
landing on the host plant surface, spore attachment, germ tube formation, and entry into
the host may follow to establish infection [72]. Fungal entry into the host occurs via natural
openings, via wounds, or through intact surfaces [81]. Most of the obligate pathogens en-
ter via stomata, and many facultative fungal pathogens enter directly by penetrating
through intact plant surfaces [60].
Upon recognition of physical clues on the host plant surface, such as surface hardness
and hydrophobicity, spores germinate and form germ tubes. Then, potentially different
appressoria are formed on the plant surface, depending on the location of the fungal en-
trance mentioned above (sometimes over the openings) [81]. These appressoria vary from
simple swollen cells to complex infection cushions (Figure 5), which anchor the fungus
before and during penetration [81]. Penetration pegs (a thin hypha) usually originate from
the center of the appressorium and extend towards underlying tissues assisted by cell-
wall-degrading enzymes and through physical force [71,81]. After penetration through
the outer cuticle, fungal growth and nutrient utilization vary significantly, depending on
the nature of the host-fungus interaction [81,82]. Different fungal species acquire nutrients
from their host plants in different ways, and their effects on the hosts differ accordingly.

Pathogens 2021, 10, 746 9 of 23
Figure 5. Different infection structures of phytopathogenic fungi. (A) An infection hypha of
Fusarium culmorum invading the epidermal cell wall of the lemma at 36 hours after inoculation (hai)
observed transmission electron microscope; (B) Transmission electron micrograph of Pyricularia
grisea appressorium formed on cellophane membrane with the penetration peg invading the sub-
stratum; (C,D) Light microscopy of compound appressoria development of Rhizoctonia solani on
cauliflower hypocotyls after trypan blue staining of hyphae. Bulbous foot structures branch from
runner hyphae on the plant (C). Lobate appressoria and infection cushions consist of agglomerated
and highly ramified hyphae (D). Scale bars: A, B = 1 µm; C, D = 100 µm. Pictures re-illustrated from:
A: [83], B: [3], C, D: reprinted with permission from Pannecoucque, J.; Höfte, M. Interactions be-
tween cauliflower and Rhizoctonia anastomosis groups with different levels of pathogenicity. BMC
Plant Biol. 2009, 9, 1–12 [84].
6. Molecular Mechanisms Involved in Appressorial Infection and Evasion of Host De-
fenses
Host-fungal interactions include pathogen’s offense, host defense, and pathogen’s
counter-attack. In each interaction, if the host plant can ward off the invading fungus,
disease resistance develops, but if the fungus can overcome host defense, then disease
develops. In the cases of endophytic and saprobic fungi, successful colonization occurs
when the fungus establishes a foothold on host plants. During their coevolution with
plants, fungi have developed counterproductive mechanisms to elude the recognition by
plant receptors or disturb the signaling cascades of the plant’s innate defenses [5,28].
The adhesion of fungal spores to the surface of a plant initiates disease establishment.
When a fungal spore lands on the cuticle or the surface wax layer, the fungi may sense the
molecules that inhibit its entrance into the plant. Upon receiving these topographic and
hydrophobic signals, fungal spores release adhesives specific to different fungi [28]. The
composition of these adhesives differs among different groups of fungi, depending on the
plant surface and environmental signals. The compositions of these adhesives include gly-
coproteins (Stagonospora nodorum (St.) [85]), hydrophobins [28,86,87], chitosan and chitin-
binding proteins [26,28], lipids, and polysaccharides [2]. Many studies have shown the
roles of these adhesives in attaching appressoria to plant surfaces [28]. In addition to their
role in adhesion, molecules such as hydrophobins shield fungal spores from being recog-
nized by plant immune cells, as well as from being recognized by the insect immune sys-
tems (Aspergillus fumigatus [86]). Furthermore, they contribute to the surface hydrophobi-
city of aerial hyphae to protect the aerial hyphae against waterlogging (e.g., in Pyr. oryzae
[87]). The importance of chitosan-synthesizing enzymes in the germling adhesion was
demonstrated in Pyr. oryzae [26]. However, the function of these adhesives is not con-
served in filamentous fungi. They vary depending on their infection strategies and the
mechanisms they employ to invade a host. Secretion of these adhesives is a passive pro-
cess for some fungi such as Pyr. grisea [60] which allows for rapid attachment. For other
fungal pathogens such as Col. graminicola, energy is required to complete the process. For
example, it takes more than 30 minutes for Col. graminicola to complete the attachment

Pathogens 2021, 10, 746 10 of 23
process [88]. Similarly, in entomopathogens such as Metarhizium anisopliae, the insect cu-
ticle influences the conidial adhesion, germination, and appressorial differentiation [89].
Entomopathogenic conidia most often attach to the bases of hair sockets or on the interseg-
mental membranes, where cuticle flexibility allows the conidial attachment [90]. Similar
to plant pathogens, these entomopathogenic fungi also secrete an adhesive mucus via co-
nidia prior to germination [90]. Upon germination, M. anisopliae produces a short germ
tube at the tip or the middle region of the conidia, which then proceeds to develop the
appressorium on the epicuticle. These appressoria vary in morphology such as cupped,
clavate, curly, and globose-shaped structures [90].
In addition to adhesives, spores and various fungal structures, including the infection
hyphae, appressoria, and haustoria, cells are often surrounded by an extracellular matrix
(ECM). This ECM is implemented during the fungal adhesion of many groups of fungi
[91]. However, the composition and the structure of ECM can differ among different
groups of fungi, depending on the surface and environmental signals; well-studied exam-
ples include Bipolaris species [92] and Cochliobolus species [37]. These ECMs consist of gly-
coproteins as in St. nodorum [85] and lytic enzymes such as esterases and cutinases as in
Col. graminicola [93]. Furthermore, the presence of these enzymes in the ECMs facilitates
breakdown of the cuticle, which allows the absorption of nutrients required for spore ger-
mination as seen in Bl. graminis [2]. In addition to the chemical responses stated above,
some fungi including Pyr. grisea, Col. graminicola, and Uromyces appendiculatus adhere
physically to the hydrophobic substrate upon recognizing the barriers [2].
After successful attachment, a series of developmental events occur, including spore
germination and germ tube elongation in response to plant and fungal signals [94]. Dif-
ferent plants secrete different signals, and such signals are perceived differently among
different fungi. Plant signals such as nutritional availability [48] and hydration as for Pyr.
grisea [2] stimulate spore germination. Chitin-binding proteins that are secreted by the
fungus mediate the perception of environmental cues such as hydrophobic surfaces to
facilitate spore adhesion and induce germ tube development as demonstrated in Pyr. ory-
zae [17]. After formation of a germ tube, it acts as the main site for plant-signal perception
[2]. During the germ-tube-elongation process, it receives plant signals such as surface hy-
drophobicity and hardness [17], topographic signals [18], cutin monomers [19], and eth-
ylene signals [2]. On receipt of these signals, germ tubes differentiate and form appresso-
ria [48]. This series of events determine the success of infection. Stimulation required for
spore germination differs according to the nutritional mode of the fungus. For example,
in rust fungi, recognition of the physical surface signals, such as ridges and the lips of
stomatal guard cells, induces appressorium differentiation [2]. Furthermore, past studies
have shown the role of calmodulin-dependent protein kinases in transducing surface sig-
nals and Ca2+ ions, which trigger appressorium differentiation from the germ tube not
only in plant pathogens such as Uromyces appendiculatus [95], but also in entomopathogens
such as Zoophthora radicans [96]. Hydrophobins secreted by fungi facilitate spore adhesion,
specifically class I hydrophobins involved in attaching the germ tube apex to the plant
surface, inducing cellular differentiation into an appressorium [87]. Some of the chemicals
secreted by plants during innate defenses are recognized by the fungus as inducers for
spore adhesion, germination, and appressorium formation. These inducers include fatty
acids, fatty alcohols, flavonoids, and aldehydes [97]. Fatty alcohols released during the
degradation of plant cutin of avocado hosts were shown to be the main inducer of appres-
sorium formation in Colletotrichum species [98]. Furthermore, alcohols and aldehydes
comprised in the cuticular wax promoted germ tube gemination, differentiation, and ap-
pressorial formation in Pyr. oryzae [30] and Bl. graminis [99], respectively. Similarly, plant
flavonoids produced by legume roots demonstrated their ability to induce spore germi-
nation in Nectria haematococca [100]. While fungal appressoria differentiate, the fungi are
required to evade host defenses simultaneously. A previous study identified a host-selec-
tive HC-toxin in Cochliobolus carbonum that is associated with appressorium differentia-
tion. This toxin has been confirmed to inhibit the histone deacetylase activity. Histone

Pathogens 2021, 10, 746 11 of 23
deacetylase is involved with the defense of host plants and functions to prevent the acti-
vation of defense genes [101].
All the mechanisms from spore adhesion to the appressorial differentiation and host
invasion are dependent on the recognition and transduction of host or environmental sig-
nals to its downstream by signal transduction pathways, such as cyclic AMP, G proteins,
and mitogen-activated protein (MAP) kinase pathways [28,30,33,102,103]. For example,
appressorial formation in Pyr. oryzae is induced by a series of cAMP/PKA and DAG/PKC
signaling cascades triggered due to the presence of cutin monomers, resulting from the
cutinase-mediated degradation of the plant cuticle [30]. The highly conserved MAPK sig-
naling pathway plays a crucial role in appressorium formation and penetration. Many
MAPK proteins (Pmk1, MEKK-Mst11, Osm1 in Pyr. grisea) have been shown to play cru-
cial roles in forming appressoria [28,33]. For example, the targeted deletion of these MAPK
genes such as Pmk1 results in non-pathogenic mutants unable to produce appressoria
[102]. Targeted deletion of MAPK genes, Bbhog1 in Be. bassiana, resulted in reduced path-
ogenicity, demonstrating their importance for spore viability, adhesion to the insect cuti-
cle, and appressorium formation in entomopathogens [104]. Furthermore, Bbmpk1 in Be.
bassiana is another gene characterized to be involved in fungal adhesion to the cuticle,
appressorium formation, and fungal penetration [105]. Homologs of this gene have been
shown to be involved in similar functions in other plant pathogens such as Pyr. grisea
[106], Col. lagenarium [21]. In addition to the MAPK signaling pathway, both cAMP and
calcium signaling cascades regulate appressoria development and pathogenicity in plant
pathogens such as Pyr. oryzae [33] and Colletotrichum species [103], and entomopathogens
such as M. anisopliae [107]. A major cAMP-dependent protein kinase A (PKA) is related to
the production of functional appressoria (encoded by the cpkA gene). PKA regulates the
mobilization of lipids and carbohydrates to the appressorium. This was confirmed by
cpkA mutation studies in Pyr. grisea [72], Col. trifolii [108], Bl. graminis [22], and M. anisopliae
[107]. PKA mutations in these organisms resulted in appressoria without the capacity to
penetrate cuticles and cause disease [22,72,107,108]. Based on current knowledge, many
signaling cascade genes are involved in both appressorium formation and subsequent
processes during pathogenesis in many phytopathogenic fungi [28].
These intracellular signaling cascades regulate the activity of transcription factors
that are critical for activating gene expression in response to extracellular signals. Gene
characterization research provides ample evidence for transcription factors regulating
changes to gene expression during appressorium development [28,36,39,109]. These stud-
ies demonstrate the diverse roles played by these transcription factors during appresso-
rium morphogenesis and pathogenicity of Pyr. oryzae, Col. orbiculare, Sc. sclerotium, and
many other pathogens [28,36,39,109–113]. The transcription factors GcStuA in Glomerella
cingulata [109], and Mstu1 in Pyr. oryzae [110], regulate conidial reserves during the ap-
pressorial turgor generation; Moswi6 and MoMst12 in Pyr. oryzae are required for the pen-
etration peg formation [39]; Hox7 in Pyr. oryzae is vital for appressorial development [36];
SsNsd1 and Ssams2 in Sc. sclerotium are responsible for the infection cushion formation
and differentiation [111,112]; and CoHox3 in Col. orbiculare is responsible for appressorial
formation and maturation [113].
Appressoria facilitate fungal penetration into host tissues; however, the mechanism
behind this process has been controversial and subjected to comprehensive studies
[2,8,34,71–73,114]. During appressorium maturation, fungi synthesize and accumulate
both glycerol and melanin. They then form a thick melanin layer on the inner appressorial
wall, as a response to combat the plant’s innate defenses and induced immunity. During
appressorium development and maturation, various cellular pathways contribute to the
accumulation of glycerol in the appressorium by the use of dihydroxyacetone phosphate,
dihydroxyacetone, glyceraldehyde, or triacylglycerol as precursors [72]. The accumulated
glycerol draws water into the cell through osmosis and facilitates the generation of hydro-
static turgor pressure [2], and the appressorial wall remains in contact with the plant cu-
ticle. In pathogens such as Pyr. grisea and Col. lagenarium, a series of reactions involving

Pathogens 2021, 10, 746 12 of 23
polyketide synthesis occur, leading to the polymerization of 1, 8-dihydroxynaphthalene
(DHN). This results in the production of DHN-melanin [8]. A study conducted on Col.
graminicola showed that melanin is not required for solute accumulation and turgor gen-
eration, contrary to that seen in Pyr. oryzae, Pyr. grisea, and Col. lagenarium [34]. This mel-
anin that is deposited between the appressorial cell wall and the plasma membrane binds
to the plant surface to lower the porosity of the appressorial wall, blocking osmolytes
[8,71]. As a result of glycerol accumulation and cell wall melanization, an enormous hy-
drostatic turgor pressure is generated [8,71]. This extreme pressure is translated into a
physical force that provides the structural rigidity required for the penetration hypha to
penetrate through the cuticle and epidermal layers of the host forcibly [71,73]. Bastmeyer
et al. [73] visualized and quantified the exerted physical force of the penetration pegs on
an optical waveguide. Bechinger et al. [114] showed that the exerted force by matured
appressoria increases rapidly, suggesting that the exerted physical force is not due to the
sudden release of turgor but sustained application of the physical force by the penetration
peg. In entomopathogens, appressoria produce a narrow peg-like structure, which pene-
trates the epicuticle and reaches the endocuticle, where they differentiate into a lateral
penetrant structure. These structures lyse the endocuticle layer using cuticle degrading
enzymes [115] to enter into the host [90]. Unlike most plant pathogens, host penetration
of M. anisopliae is mainly due to the chemical dissolution of structures rather than the me-
chanical force [90].
Appressorial pore is the point of infection which facilitates the contact of the fungal
plasma membrane with the plant surface. At the appressorial pore, actin cytoskeletons
organize as a network at the base of the appressorium, scaffolded by septin [116,117]. Sep-
tin are small morphogenetic guanosine triphosphates (GTPases) that assemble as a large
ring surrounding the F-actin network at the appressorium pore to provide the cortical
rigidity [116]. These septin also act as diffusion barriers to confine domain proteins that
function in generating the membrane curvature and protrusion of the penetration peg,
which is required to rupture the host cuticle [116]. In addition, studies have shown that
the catalyzation of reactive oxygen species burst by NADPH oxidases (Nox2) are required
for the appressorium repolarization process [117]. This Nox2-NoxR complex is essential
to organize the septin ring and F-actin network at the appressorium pore [79,117,118].
Gene mutation studies involving septin and F-actin have resulted in the failure to differ-
entiate appressorial pores, as well as stunted penetration pegs that failed to elongate and
break the cuticle [79,116–118].
During penetration of the cuticle into the plant cell, fungi must overcome the barrier
of the plant cell wall, which is composed of many polymers such as cellulose, xylan, and
pectin [5]. For fungi which produce non-melanized appressoria or inconspicuous appres-
soria such as Bot. cinerea, it is assumed that cell-wall-degrading enzymes play a more
prominent role during penetration than the physical force [106]. Gene-expression-profil-
ing studies have shown that cell-wall-degrading enzymes secreted through the penetra-
tion peg perform enzymatic softening of the substratum and allow fungal hyphae to pen-
etrate plant cells. Fungi with highly melanized appressoria such as Col. gloeosporioides
show evidence of cell-wall-degrading-enzyme secretion during penetration [119]. Fungi
with weakly melanized appressoria and melanin-deficient mutants such as Cochliobolus
species exhibit a lot higher cell-wall-degrading-enzyme activity during the penetration of
hyphae [1,5,120–124]. During penetration, cellulases, cutinases, endo- and exo-polygalac-
turonases, pectinases, pectate lyases, polymethylgalacturonases, pectic methylesterases,
and rhamnogalacturonases act in plant pectin degradation. Examples include four func-
tional pectate lyases in Nectria haematococca, five endo-PGs in Bot. cinerea, and four xy-
lanases in Coc. carbonum and Pyr. grisea [5,120–124]. Targeted silencing of single genes is
difficult due to the redundancy of the cell-wall-degrading genes responsible for the plant
cell wall degradation, hence identifying their exact role in host penetration is challenging
[125]. However, Tonukari et al. [121] demonstrated that the inactivation of SNF1 in Coc.
carbonum resulted in reduced penetration of the fungus. Similarly, reduced or impaired

Pathogens 2021, 10, 746 13 of 23
penetration was observed in the knockout mutants of xylanases [122] and cellulases [123]
in Pyr. oryzae. Pyricularia grisea cutinase2 genes are required for the appressorial differen-
tiation and host penetration [124]. In all these studies, the host penetration is reduced or
impaired as a result of gene mutation, suggesting that the physical force exerted by the
penetration peg alone is not sufficient, and enzymatic degradation of the cell wall also
assists in the host penetration in some fungi. Similar to plant pathogens, entomopathogens
also secrete toxins and degrading enzymes to reduce the host defense, transporters which
provide protection against host defenses or components of signal transduction pathways
that are necessary to sense the host environment [41,105,107,126–128].
When a fungus initiates plant infection, pathogenic fungi and their proteins are rec-
ognized by plant pattern recognition receptors and induce pathogen-associated molecular
patterns-triggered immunity responses. As a response, fungal taxa deploy a repertoire of
effectors to suppress the plant immunity through a penetration peg [24,129,130]. Several
studies have shown evidence of specialized focal secretion of effectors at the penetration
peg and an extending primary infection hyphae in Col. higginsianum [25], Col. orbiculare
[24], and Pyr. oryzae [129]. Effector genes such as pep1 and pit2 have been extensively ana-
lyzed in the corn smut pathogen U. maydis [42]. Gene deletion studies on these effectors
have proven the successful induction of plant defense responses [42,131]. It was also re-
vealed that some effectors have the ability to target plants’ secondary metabolite path-
ways, preventing plants’ induced immune responses, such as lignification [131]. Unlike
the biotrophic fungal effectors, necrotrophic fungal effectors such as polyketide toxins,
nonribosomal peptide toxins, necrosis- and ethylene-inducing peptide 1 (Nep1), and
Nep1-like proteins can induce cell death [5]. Many of these effectors are well studied and
have shown to facilitate fungal colonization by compromising the host defenses and con-
tributing to the establishment of symbiotic and pathogenic relationships [5]. However,
plants have evolved mechanisms that recognize these effectors using R proteins and in-
duce effector-triggered immune responses. As a response to these effector-triggered im-
mune responses, fungi secrete effectors encoded by avirulence genes, which target the R
proteins [130]. Such effectors have been identified in many hemibiotrophs, such as Pyr.
oryzae [132], Col. higginsianum [31], Verticillium dahliae, and F. oxysporum [133].
Appressorium pores and penetration pegs act as key hubs for the signaling and se-
cretion of host immune suppressors during plant infections. Therefore, fungal appressoria
have evolved mechanisms to evade plant defense responses from the initial spore attach-
ment to when they completely colonizes the plant tissue, as discussed above.
7. Genomics, Proteomics, and Metabolomics of Appressoria
Since the unraveling of the first genome for Pyr. oryzae [134], many genomes have
been published for appressoria-producing fungi, which include plant pathogens, entomo-
pathogens, saprobes, and endophytes [31,135]. As new genome data emerge for different
groups of fungi, DNA sequences and probes become important tools for phylogenetic
analyses related to fungal identifications, and for the detection and manipulation of the
expression of genes involved in pathogenesis. Many pathogenicity-related genes have
been identified and characterized over the years, such as MaSte12 in M. acridum [35], tran-
scriptional regulators in Pyr. oryzae [36], CBP1 [17] and MPG1 [87] in Pyr. grisea, SsNsd1 in
Sc. sclerotiorum [29], and effector-encoding Pep1 in U. maydis [42]. Appressorium formation
and function are complex morphogenetic processes that are tightly linked to genetic reg-
ulation. They are intricately coordinated by an array of genes and signaling pathways.
Many such genes involved with appressorial formation and function have been identified
and characterized, including exocyst components and proteins in Pyr. oryzae necessary for
appressorial repolarization and host cell invasion [79], ATG8 in Pyr. oryzae related to ap-
pressorium maturation and infection [136], MoMps1 related to appressorial function in
Pyr. oryzae [39], MAC1 in Pyr. grisea [137], MoSfl1 in Pyr. oryzae [33] and MaSte12 in M.
acridum [35] involved in appressorium formation, BcPIs1 related to appressorial penetra-
tion in Bot. cinerea [38], CgPKS1 involved with appressorial melanization in Col. graminicola

Pathogens 2021, 10, 746 14 of 23
[34], SsNsd1 responsible for infection cushion formation in Sc. sclerotiorum [29], and MAF1
and CBP1 involved in appressorial differentiation in Col. lagenarium [21] and Py. grisea,
respectively [17]. Regulation of these genes is needed to ensure a successful disease estab-
lishment, in the case of pathogens or successful host colonization, and in cases of endo-
phytes and saprobes [2,28,138].
The most comprehensively studied appressoria are the highly melanized ones of Pyr.
grisea [17,87], Pyr. oryzae [33,39], Col. higginsianum, and Col. graminicola [31]. A second well-
studied group includes the non- or lightly melanized appressoria of Bl. graminis and other
oomycetes [138], and the appressoria of many entomopathogenic fungi [35,41]. A plethora
of infection-related gene families involved in spore adhesion and germination, appresso-
rial formation, and host penetration have been revealed through comparative genomic
and transcriptomic studies in many fungi. These gene families include ATP-binding cas-
sette (ABC) type transporter, major facilitator superfamily and ion transporter families
involved in cellular transportation, cholesterol, phospholipid, phosphatidylcholine and
peroxisome biosynthesis gene families involved in lipid and fatty acid metabolism, carbo-
hydrate metabolism gene families, proteases, protein ligases and glutamate dehydrogen-
ase gene families related to protein and amino acid metabolism, secondary metabolism
gene families encoding melanin biosynthesis, polyketide synthesis, cytochrome P450s,
non-ribosomal peptide synthetases (NRPS), chitinases, glucanases, glucosidases, polysac-
charide dehydrogenases, cutinases, lignin peroxidase gene families involved in cell wall
degradation, gene families encoding transcription factors, pathogenicity-related gene
families involved in GAS homologs, hydrophobin and candidate secreted effector protein
(CSEP) synthesis, and gene families related to signal transduction such as MAPK, phos-
pholipases, adenylyl cyclases, and protein kinases [28,31,32,40,41,133–135,139,140]. Previ-
ous studies report that these gene families are involved in infection structure formation,
including producing secreted effectors, adhesives, pectin-degrading enzymes, chitin-de-
grading enzymes, secondary metabolic enzymes, transporters, proteases, and peptidases,
with many of these gene families expanded in pathogenic fungal genomes. For example,
most of the pathogenicity-related gene families encoding secreted effectors, pectin-de-
grading enzymes, secondary metabolism enzymes, transporters, and peptidases are ex-
panded in Col. higginsianum [31]. These expanded gene families principally help in the
increased production of their encoding proteins and also provide the material for specific
adaptations leading to the evolution of new functional systems [31,41,126,139,141]. For
instance, a comparative analysis identified expansions in cutinase, cytochrome P450, and
serine protease gene families in Pyr. grisea compared to non-pathogens, and suggested
that these expansions might be associated with the pathogenic evolution of Pyr. grisea
[134]. Encoded proteins by these gene families serve as the raw materials for behavioral
and physiological adaptations to overcome host defenses. For example, expansions of cel-
lulose-binding module (CBM) genes in Sc. sclerotiorum and Bot. cinerea demonstrate their
stronger preference for vegetative plant tissues, confirming that these gene family expan-
sions are involved in adaptations to specific ecological niches [135].
Furthermore, the transcriptome analyses of these fungi during spore germination
and appressorium formation have helped reveal the genes with significant changes in
their expression. In the Pyr. oryzae genome, approximately 21% of the genes (2,154 genes)
showed differential expressions by more than two-fold during infection. The majority of
these genes exhibited increased gene expression during spore germination and appresso-
rium formation [40]. Specifically, 357 genes were differentially expressed during appres-
sorium formation. Among them, 240 genes increased their expression, whereas 117 re-
duced their gene expression. The genes that showed a significant increase in expression
when the genes were involved in protein and amino acid degradation, lipid metabolism,
secondary metabolite synthesis (melanin biosynthesis), and cellular transportation. In
contrast, the genes with significantly decreased expression during appressorium induc-
tion were those that were involved in protein synthesis related to ribosome biogenesis.
Transcriptome analysis showed differential expression of Mgd1, GAS1, and GAS2 genes

Pathogens 2021, 10, 746 15 of 23
that are related to appressorial formation, and SPM1 involved with host penetration. The
functional characterization and differential expression of these genes solely or in combi-
nation with other genes establish their involvement in appressorium morphogenesis and
provide an understanding of protein degradation during appressorium functioning [40].
In a similar study conducted on transcriptional profiling of Pyr. oryzae, different genes
upregulated at different stages of appressorial formation and function were identified
[32]. During spore germination and early appressorium development, several genes were
significantly upregulated, such as cdc14, Mis14, sudA, and tinA, all four genes under the
transcriptional control of the Pmk1 MAP-kinase pathway. According to the transcrip-
tional profile, genes regulating acetyl-CoA synthesis responsible for appressorium mor-
phogenesis, several polyketide synthase genes involved in the melanin biosynthesis path-
way, genes involved in the beta-oxidation of fatty acids, and several genes encoding cell-
wall-degrading enzymes (cutinases, xylanases, polygalacturonases, cellulases) showed in-
creased expression at appressorial development and maturation stages. Furthermore, sev-
eral transporter genes such as sugar transporters, MFS transporters and multidrug and
toxin extrusion (MATE) family of transporters, and ABC transporters upregulated during
appressorium function, and the latter one also during host penetration [32]. Another study
identified increased expression of mas1/mas2 genes responsible for germination and ap-
pressorium formation, genes encoding tetraspanin involved in appressorium function,
and genes involved in MAP kinase and cAMP-dependent signal transduction pathways
in Sc. sclerotiorum and Bot. cinerea [135]. In addition to these plant pathogens, entomopath-
ogenic M. acridum also demonstrated the importance of tetraspanin-encoding MaPls1 for
conidial germination, appressorium formation, and turgor pressure generation [127], and
perilipin-encoding Mpl1 responsible for the turgor generation in M. anisopliae [128]. Simi-
lar to previous studies, O'Connell et al. [31] identified five gene categories (encoding tran-
scription factors, secondary metabolism enzymes, CSEPs, CAZymes, and transporters)
with increased expression patterns relevant to pathogenicity in Col. higginsianum and Col.
graminicola at the appressorial formation stage. For example, genes encoding CAZymes
essential for the host penetration such as cutinases, cellulases, hemicellulases, and pecti-
nases were identified. Prior to the penetration and during biotrophy stages, a significant
increase in gene expression was observed for 12 different secondary metabolism gene
clusters, including CSEP-encoding genes (ChEC6 being the most highly induced) for host
manipulation. Similar genomic and transcriptomic studies conducted on different plant
and entomopathogenic fungi such as Be. bassiana, Bl. graminis, Candida albicans, Cordyceps
militaris (Cor.), F. graminearum, Metarhizium anisopliae, M. acridum, Pyr. grisea, and Uromyces
fabae identified similar genes homologous to the above-mentioned gene categories in-
volved in surface attachment, appressorium formation, and host penetration. These dis-
coveries lay the foundation for future research and design of novel strategies for disease
control such as improving biocontrol efficacies of fungicides and fungal insecticides
[21,33–35,38–41,137].
Comparative proteomic studies facilitated the identification of proteins involved in
the formation of functional appressorium [29,141–143]. Franck et al. compared the prote-
ome of an appressorium-producing Py. oryzae with a mutant strain that produces a non-
functional appressorium to determine the proteins essential for the appressorium function
[141]. According to Franck et al. [141], 193 proteins specific to germination and appresso-
rium formation were identified in Pyr. oryzae. The same study discovered 59 proteins in-
volved in the cAMP signaling pathway during appressorium formation. From the total
proteins identified, 193 proteins (6% of the total proteins) were identified to be involved
in conidial germination and appressorial formation. Among them, 40 were specific to ap-
pressorium formation. A comparison of the appressoria-enriched cDNA library of the rust
pathogen Phakopsora pachyrhizi (Pha.) with existing expressed sequence tags (ESTs) re-
vealed 29 ESTs specific to the appressoria-enriched library. Among these, 35% of the ESTs
are important for autophagy, mitosis, and fungal metabolism (cholesterol biosynthesis

Pathogens 2021, 10, 746 16 of 23
and amino acid metabolism) involved in appressoria formation. In addition to those, ser-
ine/threonine protein kinases, P-type ATPases involved in appressorial morphogenesis,
subtilase-type proteinases involved in the appressorial development, isocitrate lyases re-
lated to lipid metabolism (synthesis of glycerol), and septin involved in urediniospore
germination and germ tube elongation were also identified to be in abundance [143]. Sim-
ilar proteins were identified in Aspergillus oryzae, Be. bassiana, Bot. cinerea, Cor. militaris, F.
oxysporum, Glomus intraradices, M. roberstii, Ophiocordyceps sinensis, Ph. infestans, Pyr. grisea,
and Sc. sclerotiorum [29,41,141–144]. In almost all of the studies, a higher expression is ev-
ident in proteins responsible for the degradation and recycling of the proteins, and cell
contents. This can be explained as the fungus-need energy to carry out functions such as
spore germination, germ tube elongation, and appressoria formation prior to host pene-
tration. Hence, this is achieved by breaking down spore content. Furthermore, the pro-
teins related to lipid, glycogen, and sugar metabolism are necessary for the synthesis of
glycerol in the appressorium which is considered as one of the factors contributing to the
turgor pressure during host penetration. The significance of the proteins involved in the
breakdown and recycling of cellular components has been demonstrated in Pyr. oryzae,
Pyr. grisea [136], and Col. orbiculare [122].
In another study, Liu et al. [138] performed an untargeted metabolomic analysis to
profile the metabolome of developing appressoria and identified significant changes in
six key metabolic pathways in Pyr. oryzae. According to the KEGG analysis, these path-
ways include degradation of lipids, degradation of carbohydrates, arginine synthesis,
sphingolipid synthesis, sterol synthesis, and phospholipid metabolism. As one of the early
intermediates of the sphingolipid biosynthesis pathway, ceramides were shown to be es-
sential for normal appressorial development, specifically for appressorial morphology
and penetration peg formation. It was believed to affect the anaphase of mitosis and deg-
radation of nuclei in conidia. They are shown to be accumulated during appressorial mor-
phogenesis and metabolized during maturation in Pyr. oryzae. Similar processes were
identified in the Asian soybean rust fungus, Pha. pachyrhizi [145].
Genomics, transcriptomics, proteomics, metabolomics, and comparative approaches
facilitated the identification and functional characterization of differentially expressed
genes, and revealed their functions related to appressorial formation and function. For
example, using the targeted gene deletion approach, several studies have revealed the
importance of hydrophobin genes (sc3, sc1, and sc4 in Schizophyllum commune; mpg1 in Pyr.
grisea; cu in Ophiostoma quercus; and crp in Cryphonectria parasitica) for spore attachment,
germ tube attachment, and blockage of plant recognition in different fungi [28,86,87]. Most
importantly, major genes involved in the signaling pathways that regulate the appresso-
rial development processes (mac1, sum1-99, pmk1, and cpkA in Pyr. grisea; chk1 in Coc. het-
erostrophus; MAF1 in Col. lagenarium; bmp1 in Bot. cinerea; fmk1 in F. oxysporum; ptk1 in Pyr-
enophora teres; pka1 in Sc. sclerotiorum and M. anisopliae; and many more) have been char-
acterized in many fungi [2,20,21,28,102,103,107]. For example, the importance of these sig-
nal pathways is evident when a gene mac1, encoding a cAMP signal is deleted in Pyr.
grisea. This resulted in the complete loss of appressorium formation in the mutant [137].
Furthermore, studies on the expression of genes, which control the turgor development
in the appressoria, have shown functionally the importance of each of the genes [2,106].
Similarly, the function of cell-wall-degrading enzymes in the penetration process [5] and
effectors and Avr genes for the suppression of host defense have been reviewed and
demonstrated in many studies [2,24,25,42,130–132].
With improvements in genomics and biotechnological techniques, further gene func-
tions related to appressorium formation and function will be revealed. Specifically, gene
silencing and modification technologies will facilitate discovery of new genes and the key
signaling molecules important for plant-fungal interactions. An in-depth understanding
of these processes will provide an unprecedented opportunity for plant pathology re-
search and to the improvement of agriculture practices.

Pathogens 2021, 10, 746 17 of 23
8. Conclusions
While most of the studies discuss appressoria formation as an independent event in
the infection process that exclusively occurs in pathogenic taxa, we discuss it as a part of
a sequence of events that occurs when a fungus colonizes a host. In this study, we provide
a comprehensive review of our current understanding of the diversity of appressoria in
fungal taxa. We described how appressoria are classified among fungal taxa and the
changes that occur in their ultrastructure during the host attachment and colonization.
The application of functional genomics techniques reveals signaling events between the
fungus and the host. Furthermore, these ‘omics’ studies identify genes encoding the func-
tion and formation of different morphogenesis stages in various fungi such as Col. gra-
minearum, Col. gloeosporioides, Pyr. grisea, and Bot. cinerea. This knowledge base is likely to
grow due to the exponential growth of genomic and transcriptomic studies, and the dis-
coveries from these disciplines are discussed together with the potential for further devel-
opments. The applications of transcriptomics, proteomics, secretomics, and metabolomics
offer significant opportunities to advance the understanding of appressorium formation
and function during host-fungal interactions. Combining these approaches in a single
study has many advantages over a single ‘omics’ study based on a single technique alone.
Knowledge of genetic mechanisms and appressorial morphogenesis is important for de-
signing control strategies against plant fungal diseases. An example of using this
knowledge is the use of anti-penetrant fungicides to control Pyr. grisea-related fungal dis-
ease. The fungicide interferes with the melanin biosynthesis of the fungus, hence, produc-
ing non-functional or less effective appressoria [48].
Author Contributions: Conceptualization, K.D.H.; Writing—Original Draft Preparation, K.W.T.C.,
R.S.J., Y.-J.C., S.K., S.T., P.D.A., and D.G.; Writing—Review and Editing, K.W.T.C., K.D.H., A.B.,
and J.X.; Funding Acquisition, K.D.H. and S.L. All authors have read and agreed to the published
version of the manuscript.
Funding: We thank the Thailand Research Fund entitled ‘Impact of climate change on fungal diver-
sity and biogeography in the Greater Mekong Sub region’, grant number RDG6130001. KDH thanks
Chiang Mai University for the award of Visiting Professor. ST would like to thank the International
Postdoctoral Exchange Fellowship Program, grant number Y9180822S1, CAS President’s Interna-
tional Fellowship Initiative, grant number 2020PC0009, the China Postdoctoral Science Foundation
and the Yunnan Human Resources, and the Social Security Department Foundation for funding her
postdoctoral research. RSJ would like to thank National Research Council of Thailand, grant for new
researcher NRCT5-TRG630010-01, entitled “Biodiversity, taxonomy, phylogeny and evolution of
Colletotrichum in northern Thailand”.
Acknowledgments: Authors would like to thank Sirinapa Konta for assistance in preparing some
of the figures. Pranami D. Abeywickrama would like to thank Prof. Monica Höfte (Department of
Plants and Crops, Faculty of Bioscience Engineering, Ghent University, Belgium) for her kind per-
mission to reprint figures from Pannecoucque and Höfte (2009). K.W.T. Chethana would like to
thank Dr Saranyaphat Boonmee (Center of Excellence in Fungal Research, Mae Fah Luang Univer-
sity, Thailand) for her kind permission to reprint figures from Konta et al. (2016, 2017) and Dr Ming-
kwan Doilom (Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Insti-
tute of Botany, Chinese Academy of Science, People’s Republic of China) for her kind permission to
reprint figures from Hyde et al. (2019).
Conflicts of Interest: The authors declare no conflict of interest.
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