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JIPB
Journal of Integrative
Plant Biology
From filaments to function: The role of the plant
actin cytoskeleton in pathogen perception,
signaling and immunity
Katie Porter1 and Brad Day1,2,3*
Graduate Program in Cell and Molecular Biology, Michigan State University, East Lansing, MI 48823, USA, 2Department of Plant, Soil and
Microbial Sciences, Michigan State University, East Lansing, MI 48823, USA, 3Graduate Program in Genetics, Michigan State University, East
Lansing, MI 48823, USA.
Brad Day
*Correspondence:
[email protected]
Abstract The eukaryotic actin cytoskeleton is required for
numerous cellular processes, including cell shape, development and movement, gene expression and signal transduction, and response to biotic and abiotic stress. In recent years,
research in both plants and animal systems have described a
function for actin as the ideal surveillance platform, linking the
function and activity of primary physiological processes to the
immune system. In this review, we will highlight recent
advances that have defined the regulation and breadth of
function of the actin cytoskeleton as a network required for
defense signaling following pathogen infection. Coupled with
The eukaryotic actin cytoskeleton is a dynamic network in
which activity is governed by tightly regulated spatial and
organizational changes in monomeric globular (G)- and
filamentous (F)-actin (Day et al. 2011). With more than 200
actin-binding proteins described in mammals, and nearly 75 in
plants, the actin cytoskeleton has been demonstrated to be
required for the function of a diverse suite of cellular
processes, including cell division and elongation (Barrero
et al. 2002), the establishment of cell polarity and movement
(Blanchoin et al. 2014), endocytosis and vesicle trafficking
(Robertson et al. 2009; Johnson et al. 2012; Mooren et al. 2012;
Wang and Hussey 2015), gene expression (Percipalle 2013) and
immunity (Tian et al. 2009; Day et al. 2011; Porter et al. 2012;
Henty-Ridilla et al. 2013; Li et al. 2015). At a fundamental level,
the sum expression and activity of the actin-binding protein
superfamily – key regulators of actin organization – not only
drive filament architecture, but also functionally and physically link actin to a diversity of cellular processes (Winder and
Ayscough 2005; Uribe and Jay 2009). In this regard, and as
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Keywords: Actin; cytoskeleton; immunity; pathogen; plant;
Pseudomonas syringae; surveillance
Citation: Porter K, Day B (2016) From filaments to function: The role of
the plant actin cytoskeleton in pathogen perception, signaling and
immunity. J Integr Plant Biol 58: 299–311 doi: 10.1111/jipb.12445
Edited by: Hailing Jin, University of California, Riverside, USA
Received Sept. 4, 2015; Accepted Oct. 28, 2015
Available online on Oct. 30, 2015 at www.wileyonlinelibrary.com/
journal/jipb
© 2015 Institute of Botany, Chinese Academy of Sciences
previously described (Staiger et al. 2009), actin’s ubiquity and
functional association with numerous signaling cascades
qualify it as the ideal cellular surveillance platform.
Here, we describe current research that defines the key
cellular processes in plants that link the activities of the actin
cytoskeleton and the host immune system. Additionally, we
will highlight current research demonstrating pathogen
targeting of actin and actin-associated processes, a relatively
new and understudied component of plant immunity. In this
regard, and in comparison to immune signaling in humans,
current evidence supports a role for the plant actin
cytoskeleton, not only as a key feature of the plant immune
system, but also as an important pathogen virulence target in
which subversion is tantamount to invasion and the elicitation
of disease. Indeed, recent work in this area has demonstrated
that plant pathogens specifically target the plant actin
cytoskeleton to block immune signaling processes, and
moreover, that or manipulation of critical steps in the actin
machinery result in a dampening of plant defense signaling.
Together, the identification of the actin cytoskeleton as a
critical component of the plant immune system, as well as a
April 2016 | Volume 58 | Issue 4 | 299–311
Free Access
INTRODUCTION
an overview of recent work demonstrating specific targeting
of the plant actin cytoskeleton by a diversity of pathogens,
including bacteria, fungi and viruses, we will highlight the
importance of actin as a key signaling hub in plants, one that
mediates surveillance of cellular homeostasis and the activation of specific signaling responses following pathogen
perception. B4ased on the studies highlighted herein, we
propose a working model that posits changes in actin filament
organization is in and of itself a highly specific signal, which
induces, regulates and physically directs stimulus-specific
signaling processes, most importantly, those associated with
response to pathogens.
Invited Expert Review
1
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Porter and Day
virulence target of plant pathogens, illustrate the importance
of actin as a key signaling hub in plants, one that mediates
surveillance of cellular homeostasis and the activation of
specific signaling responses following pathogen perception.
ASSEMBLY AND REGULATION OF THE
ACTIN CYTOSKELETON
The primary building block of the actin cytoskeleton is G-actin,
a 42-kDa adenosine triphosphate (ATP)-binding protein
capable of undergoing spontaneous self-assembly, a process
by which monomeric actin is added to the barbed ends of
existing F-actin filaments (Day et al. 2011; Figure 1). Actin
filament assembly is initiated via the formation of a homo/
hetero-trimer complex, a multi-step process referred to as
nucleation (Figure 1; reviewed in Campellone and Welch 2010),
a process influence by a number of factors, including: (i) the
availability of filament ends; (ii) the size of the cellular pool of
G-actin; (iii) the nucleotide-loaded state of the G-actin
monomers; and (iv) the spatial and temporal expression of
actin-binding proteins. In both plants and animals, each of
these four steps has been extensively characterized (Hussey
et al. 2006; Lee and Dominguez 2010; Mullins and Hansen
2013), and have been shown to be regulated by the activity of
a multi-protein complex referred to as the actin-related 2/3
(Arp2/3) complex (Mathur et al. 2003; Campellone and Welch
2010). Additional proteins required for actin nucleation
include formin (Chesarone et al. 2010), capping protein
(Huang et al. 2003) and gelsolin (Silacci et al. 2004). Once
nucleation is initiated, trimeric-actin seeds F-actin maturation
through filament elongation, a process that requires the
addition of ATPG-actin to the barbed plus end of either newly
nucleated actin-trimer or to a preformed severed F-actin
strand. As the filament matures, ATP hydrolysis, coupled to
the activity of actin depolymerizing factor (ADF) proteins,
drives the depolymerization of the filament at the pointed
ADP
F-actin end. This processes, referred to as “treadmilling”,
results in the remodeling of actin through the precise control
of balance and direction of F-actin formation. Herein, we
Figure 1. Schematic of actin remodeling in the plant cell
An illustration of the basic actin remodeling process, including the association and function of key actin binding proteins. Free
globular (G)-actin is initially sequestered by profilin in order to both prevent spontaneous nucleation and elongation, and to
incorporate G-actin into filamentous (F)-actin in a regulated manner. Nucleation of G-actin is aided by actin nucleators including:
Arp2/3, formins, and capping proteins (CPs). Elongation of F-actin occurs at the barbed end, and is achieved through the actions
of both formins and profilin. F-actin can then be bundled and/or branched through the activity of villin and the Arp2/3 complex.
For disassembly of F-actin filaments, the aging pointed end of F-actin is severed and/or depolymerized by actin depolymerizing
factor (ADF), allowing for recharging of adenosine diphosphate to adenosine triphosphate by cyclase-associated protein (CAP)
for eventual re-incorporation of G-actin into F-actin filaments.
April 2016 | Volume 58 | Issue 4 | 299–311
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Actin-based immune signaling in plants
highlight the function and activity of three actin-binding
proteins, formins, profilins and ADFs, which have been
demonstrated to have roles that potentially position them
at the interface of host defense and pathogen targeting of
immunity.
PREFORMED LINKAGES: FUNCTIONS,
BARRIERS AND TARGETS OF
PATHOGENESIS
How does the actin cytoskeleton mediate intercellular-toextracellular connectivity and communication? Plants have
evolved robust mechanisms to cope with stress, including the
ability to sense and specifically respond to potential threats. If
one considers the rapid nature of actin filament assembly and
turnover, as well as its intimate association with numerous
cellular processes, it is reasonable to hypothesize that actin
possesses all of the fundamental properties necessary to fulfill
a role in not only intercellular connectivity, but also in
indirectly bridging the extracellular matrix to a multitude of
intercellular processes. For example, and as a first point in
fulfillment of this role, numerous examples linking the activity
of actin with cell membrane-associated processes, including
301
receptor activation and attenuation (Beck et al. 2012) and the
regulated delivery and secretion of signals (e.g., antimicrobial
compounds, phosphoinositides) within and from the cell have
been described (Staiger et al. 2009; Lee and Dominguez 2010;
Day et al. 2011; Smethurst et al. 2013). In this regard, we posit
that actin’s most important role – as it relates to biotic stress
perception – may be in its function as an interface between
the cell and the processes that mediate inter-, intra- and extracellular communication.
Plasma membrane-cell wall connectivity
What role does the plant actin cytoskeleton play in linking
intercellular processes to the extracellular environment, and
how is this function associated with pathogen perception and
immune activation? There is a growing body of evidence that
at least two actin-binding proteins – formins and profilins –
play a role in mediating connectivity between the plasma
membrane and the plant cell wall ((Cvrckova 2013; van
Gisbergen and Bezanilla 2013; Fan et al. 2015); Figure 2). In
humans, actin connectivity to the extracellular environment is
mediated by a family of proteins known as integrins, a family
of highly conserved integral plasma membranes proteins
(Hynes 2002). In short, integrins play important roles in not
only sensing changes in extracellular homeostasis in response
Figure 2. Examples of preformed cellular functions of the actin cytoskeleton utilized in defense signaling and targeted by
pathogens
(A) Actin-dependent intracellular movement of the Tomato spotted virus wilt tospovirus (TSVW) N-protein. N-protein of TSVW
forms inclusion bodies that then associate with the endoplasmic reticulum (ER) and are trafficked through the endomembrane
system in an actin and myosin dependent manner. (B) Involvement of the actin cytoskeleton in the formation of the cell wall
apposition, a defense-related formation of anti-fungal compounds at the site of fungal penetration. Fungal penetration also
signals the recruitment of actin filaments toward the penetration site. (C) The actin cytoskeleton and myosin play key roles in the
clathrin-mediated endocytosis (CME) of pattern recognition receptors including flagellin sensing 2 (FLS2), which recognizes
bacterial flagellin. Inhibition of either myosin or the actin cytoskeleton results in improper internalization of and endomembrane
trafficking of FLS2.
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Porter and Day
to stress, but also serve as a key mechanism to survey the
extracellular environment for potential threats, including
pathogen invasion (Zhang and Wang 2012). Conversely, in
plants, while integrin-like proteins have been identified
(Monshausen and Gilroy 2009; Knepper et al. 2011; Sardesai
et al. 2013), functionally similar bridges between the inter- and
extracellular space remain undefined.
As an alternative to an integrin-based mechanism linking
the plant actin cytoskeleton to the extracellular space, one of
the best candidates described to date is that of the actinbinding protein formin, a regulator of actin filament
organization and polymerization at the barbed end of F-actin
(Lee et al. 2008; Cvrckova 2013; van Gisbergen and Bezanilla
2013). In support of this hypothesis, several key features of
plant formins satisfy a minimum set of criteria required for
such a role. First, formins possess many of the biochemical
features necessary to mediate the cytoskeletal-plasma
membrane continuum, possessing a signal peptide, a
predicted trans-membrane domain and a proline-rich peptide
that is hypothesized to interact with proteins within the cell
wall (Cvrckova 2013). Second, several members of the formin
family of proteins possess a phosphate and tensin homolog
(PTEN)-like domain that catalyzes and binds plasma membrane-localized phosphoinisotides (van Gisbergen et al. 2012),
while an even smaller and less conserved class of formins
interacts with the plasma membrane indirectly via contacts
with Rho-guanosine triphophatases (GTPases) (Bechtold et al.
2014). Based on these features, it is interesting to hypothesize
a role for formins in pathogen perception and immune
signaling. Indeed, in addition to roles for phosphoinisotides
and Rho-GTPase in the modulation of actin cytoskeletal
dyanmics, a large body of work has shown that phosphoinisotides and GTPases play important roles in plant-pathogenassociated processes, including pathogen entry and immune
activation (e.g., Hung et al. 2014; Kawano et al. 2014).
Finally, in addition to formins, and a further illustration of
the connectivity of actin to a multitude of cellular processes,
profilin also possesses many of the fundamental properties
that that would allow for its direct and/or indirect interaction
with the plasma membrane (Sun et al. 2013). For example, in
addition to binding actin, profilins also interacts with most
formins, via a conserved formin homology-1 domain
€ m 2010), and the sum of this association facilitates
(Aspenstro
further interactions with plasma membrane-localized phosphoinisotides (Sun et al. 2013). Based on this, it is tempting to
speculate that one could expect to find profilin and formin
within a complex of associated proteins at the plasma
membrane, and through further association with actin, fulfill a
role as a link to the extracellular space.
Endomembrane transport
A recent review highlights the importance, and numerous
functions, of the interactions between the eukaryotic actin
cytoskeleton and the endomembrane system, describing the
role of actin in the crosstalk between the nucleus (discussed
below), the Golgi (Akkerman et al. 2011) and endoplasmic
reticulum (ER), and as presented above, the plasma
membrane (Wang and Hussey 2015). While our understanding
of the interplay between host endomembrane dynamics, actin
and pathogen invasion is limited, there are several recent
reports that demonstrate the necessity and regulation of this
April 2016 | Volume 58 | Issue 4 | 299–311
interaction during pathogen infection and the activation of
immunity. In this regard, and as an example of the virulence
targeting of this network, the actin-endomembrane systems
have been characterized through work demonstrating actindependent hijacking of the ER by the plant enveloped virus
Tomato spotted wilt tospovirus (TSVW; (Feng et al. 2013;
Ribeiro et al. 2013); Figure 2A; Table 1). In brief, the TSWV
membrane envelope is predominantly formed by two viral
glycoproteins, Gc and Gn, both of which interact with the viral
nucleocapsid protein, N (Ribeiro et al. 2013). In addition to the
membrane envelope, TSWV also synthesizes a spherical viral
particle consisting of ribonucleoproteins, where the singlestranded genomic RNA is found in tight association with N
(Feng et al. 2013). From this work, it was demonstrated that
the nucleoprotein forms cytoplasmic inclusion bodies that
associate with, and are trafficked along, the host endoplasmic
reticulum in an actin- and myosin-dependent manner.
Interestingly, it was determined that this intracellular
trafficking, while actin-dependent, functions independently
of microtubules. In a parallel study, Ribeiro et al. (2013) came
to a similar conclusion, demonstrating that nucleoprotein
trafficking is actin-dependent and microtubule-independent,
while further showing that actin was not required for the
assembly of the viral glycoproteins with the nucleoprotein.
Taken together, these studies clearly demonstrate a role for
actin in the cellular trafficking of viral proteins during
infection. This is noteworthy that while there are numerous
examples of enveloped viruses in the animal kingdom, few
have been identified to infect plants, thus providing evidence
that TSVW represents an exciting foundation, and case study,
for the further analysis of actin-endomembrane dynamics and
function during host-virus interactions.
As a final illustration of the dynamic function of the
endomembrane-actin interface, a recent study has shown that
the P3 protein of the Soybean mosaic virus interacts directly
with ADF2 of soybean (Lu et al. 2015). This study further
supports previous work that demonstrated movement of P3
in the early secretory pathways in an actin-dependent manner,
while presenting a direct interaction of P3 with ADF2 of
soybean and suggests a component of that actin cytoskeleton, ADF2, may be the target of P3 for movement through the
endomembrane system (Cui et al. 2010). It should be noted
that the above two examples of actin hijacking by viruses to
facilitate cellular trafficking are not the only examples of
this well-studied occurrence; herein, we have chosen to
focuses on P3 based on its association with the actin-binding
protein ADF2. Additional examples of the interactions
between plant endomembrane dynamics and the actin
cytoskeleton have also been described (Haupt et al. 2005;
Harries et al. 2009; Grangeon et al. 2012), and further support a
role for actin in the important cellular component of the host
immune system.
Pathogen perception and receptor dynamics
One of the earliest events in the initiation of immune signaling
is mediated by responses associated with receptor-ligand
interactions at, or adjacent to, the outer surface of the plasma
membrane. In large part, these signaling processes – as will be
described in detail below – are associated with the recognition
of pathogens by host membrane-localized receptor complexes the function(s) of which is to survey the host
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303
Table 1. Pathogen virulence factors that specifically target the host cytoskeleton, actin, and/or actin binding proteins
Pathogen/elicitor
Type of virulence
factor
Tomato spotted wilt
tospovirus
Pseudomonas syringae pv.
tomato DC3000
Agrobacterium tumefaciens
N-protein
Whole pathogen
Magnaporthe grisea
Whole pathogen
Flagellin/flg22
PAMP/peptide ligand
Chitin
PAMP
Ef-Tu/elf26
PAMP/peptide ligand
HopW1
Bacterial effector
AvrPphB
Bacterial effector
VD toxin
ToxA
Toxin
Toxin
Whole pathogen
Effect on host
Reference
Targets actin and myosin to alter
endomembrane trafficking.
Alters actin dynamics by
increasing actin density.
Alters actin dynamics by
increasing actin density.
Alters actin dynamics by
increasing actin density.
Alters actin dynamics by
increasing actin density.
Alters actin dynamics by
increasing actin density.
Alters actin dynamics by
increasing actin density. In the
absence of Arabidopsis ADF4
this increased density was not
observed.
Alters actin dynamics and disrupts
endocytosis and cellular
trafficking in an actin
dependent manner.
Reduces flg22/FLS2 related MAPK
signaling in the absence of
Arabidopsis ADF4.
Disrupts the actin cytoskeleton.
Causes cell death when
internalized, possibly through
actin dependent CME.
Feng et al. 2013
Henty-Ridilla et al. 2013
Henty-Ridilla et al. 2013
Henty-Ridilla et al. 2013
Henty-Ridilla et al. 2013
Henty-Ridilla et al. 2013
Henty-Ridilla et al. 2014
Kang et al. 2014
Porter et al. 2012
Yuan et al. 2006
Manning and Ciuffetti 2005
PAMP, pathogen associated molecular pattern; ADF4, actin depolymerizing factor 4; MAPK, mitogen-activated protein kinase;
CME, clathrin mediated endocytosis.
extracellular matrix for potential threats. Based on the critical
requirement for pathogen recognition, and based on actin’s
role in intercellular signaling events during pathogen infection, this begs the question: Do pathogens perturb the initial
recognition events and activation of immunity by targeting
actin-mediated receptor dynamics?
As recently reviewed (Bigeard et al. 2015), among the
earliest events required for plant perception of pathogens are
the highly regulated processes underpinning the initiation of
dynamic movement of host resistance components to and
from the plasma membrane. One of these key processes,
clathrin-mediated endocytosis (Fan et al. 2015), is responsible
for the transport of, for example, anti-microbial compounds
to the site of infection (Kwon et al. 2008), the movement and
assembly of activated signaling complexes required for the
activation of immune signaling (Robatzek 2007), as well as the
attenuation of immune signaling once pathogen infection has
been abrogated. As a function of actin-based immunity,
clathrin-mediated endocytosis represents yet another example of the physical and functional link between cellular
membranes and the activity and organization of the host actin
cytoskeleton. Originally defined in yeast (Kaksonen et al.
2003), a growing body of literature from studies in both plants
and animals has identified several actin-binding proteins,
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including Arp2/3, capping protein and ADFs, as being
indispensible for endocytosis (Galletta et al. 2010). Indeed,
recent work using mammalian models showed that actin links
endocytic processes to the plasma membrane, and is
necessary for the generation of the mechanical forces
required for alternations in membrane shape, inducing
membrane curvature, an initial key step in clathrin-mediated
endocytosis ((Galletta et al. 2010); e.g., Figure 2C). In plants,
and as an example of immune signaling activation through the
actin cytoskeleton, it has been demonstrated that endocytosis of the immune-related pattern recognition receptor
flagellin sensing 2 (FLS2) requires the function of the actin
cytoskeleton (Beck et al. 2012). Interestingly, treatment with
the actin depolymerization inhibitor latrunculin-B (LatB) did
not inhibit internalization of FLS2, but instead, LatB impaired
the trafficking of the FLS2 endosome, while the myosin
inhibitor, 2,3-butanedione monoxime, inhibited FLS2 endocytosis. Furthermore, this study demonstrated that inhibition of
receptor-mediated endocytosis both reduced the motility of
FLS2 endosomes as well as stabilized actin filaments. Taken
together, this work suggests a synergistic function for myosin
and the actin cytoskeleton in the internalization and
endomembrane trafficking of FLS2 during immune signaling
and attenuation.
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Further linking the activity and organization of the actin
cytoskeleton to the extracellular environment, and specifically
as a function of biotic stress signaling, actin has been shown to
play a role in the formation of cell wall apposition, the
accumulation of anti-fungal compounds, and resistance to
penetration by plant pathogenic fungi (Hardham et al. 2007;
Hardham et al. 2008; Kobayashi and Kobayashi 2013). For
example, research by Kobayashi and Kobayashi (2013) showed
that when plants are mechanically wounded – mimicking the
penetration stress response induced during fungal penetration – they have increased resistance to the pathogenic fungi
Blumeria graminis. This study further showed that treatment
with the actin polymerization inhibitor cytochalasin-A abolished penetration resistance. Additional work revealed fungal
induced rearrangement of the actin cytoskeleton, including
dynamic (re)-localization of the actin-binding protein profilin
to the site of infection (Schutz et al. 2006). The observed
accumulation of profilin to the plasma membrane, coupled
with reorientation of the actin cytoskeleton during oomycete
infection, further supports the potential for profilin to connect
the actin cytoskeleton and plasma membrane. More recent
work has expanded upon these earlier observations to include
the actin motor protein myosin as a component of the
cytoskeletal-immune signaling network ((Yang et al. 2014);
e.g., Figure 2B), giving support to the hypothesis that
reorganization of actin, the movement of organelles, and
the deposition of compounds to the cell wall apposition of
attempted penetration by Blumeria graminis f. sp. hordei, are
all either directly or indirectly linked to the function and
activity of myosin.
Actin in the guard cell: Controlling entry to the apoplast
Once viewed as passive portals to the intercellular space of
plants, stomata are now regarded as primary lines of defense,
preventing, for example, bacterial phytopathogen entry
following pathogen perception. Indeed, numerous studies
have shown that plant stomata close upon recognition of
bacterial pathogen-associated molecular patterns (PAMPs:
e.g., flg22), and as is the case of infection by Pst DC3000, are
actively reopened through the activity of a bacterially
produced toxin coronatine, a jasmonic acid mimic (Melotto
et al. 2006; Melotto et al. 2008). As a likely link between
pathogen penetration, immunity and the function of the actin
cytoskeleton, plant actin has been implicated in having a role
in Arabidopsis guard cell architecture (Higaki et al. 2010), thus
giving rise to the hypothesis that pathogen manipulation of
actin within the stomata may play a key role in immune
subversion. As a foundation for further work in this area,
Higaki and colleagues established a baseline for the definition
of stomatal actin dynamics through the development and
utilization of confocal microscopy-based tools, coupling
hierarchical cluster analysis, to quantitatively analyze cytoskeletal orientation, actin filament bundling (skewness) and
percent occupancy (density) during diurnal cycles. Using this
comprehensive, quantitative approach, it was observed that
stomatal-localized actin filaments assume a radial orientation
when stomata are open, and that actin is dynamically, yet
transiently, bundled during the stomata opening process.
However, once fully opened, the bundled structures disassociate. During diurnal cycling, when stomata are open (i.e.,
photosynthetically active under light), actin persists in a
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largely filament-bundled conformation; thus, as developed by
Higaki and colleagues, this quantitative cell biology-based
method provides a tractable system to correlate actin
bundling, stomata movement, and potentially, induced
changes in these processes to events associated with
pathogen invasion of plants.
IMMUNE SIGNALING AND THE ACTINPATHOGEN CONNECTION
As noted above, numerous regulatory and functional parallels
exist between the plant actin cytoskeleton and the mammalian actin cytoskeleton. Not surprisingly, similarities are also
found between immune signaling mechanisms in plants and
animals (Ausubel 2005), including the requirement for
homologous receptor-ligand interactions (Chisholm et al.
2006; Chtarbanova and Imler 2011), the initiation and
specificity of mitogen-activated protein kinase (MAPK)
cascades (Rodriguez et al. 2010; Whelan et al. 2011), and the
transcriptional reprogramming of cellular processes associated with defense signaling (Pandey and Somssich 2009). As
such, the immune system – whether from plants or animals – is
among the best-characterized examples of a biological
surveillance platform.
PTI of the actin cytoskeleton
Plant immune responses are broadly classified based on the
activity of two primary modes of pathogen recognition:
PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI; (Chisholm et al. 2006; Dangl and Jones 2006)). In the
case of PTI, perception and activation is mediated by
extracellular recognition of PAMPs (e.g., flagellin, lipopolysaccharide, chitin, elf26) by plasma membrane-localized
pattern recognition receptors. Binding of PAMPs by their
respective pattern recognition receptors initiates downstream signaling, including the activation of the MAPK, the
generation of reactive oxygen species, and transcriptional
reprogramming of pathogen-responsive genes ((Zhang and
Zhou 2010); e.g., Figure 3A). As a comparison between plants
and animals, PTI responses appear to be highly conserved,
both with regard to the mechanism of activation (e.g.,
receptor-ligand interactions), as well as with respect to
regulation (e.g., MAPK signaling) and attenuation (i.e.,
programmed cell death). Given these broad similarities, and
further based on the general concept of surveillance, it is not
surprising that linkages between the immune system and the
function and organization of the actin cytoskeleton exist.
Indeed, several recent studies have demonstrated the
importance of actin, and actin-binding proteins, as a
component of plant immunity (Tian et al. 2009; Day et al.
2011; Porter et al. 2012; Henty-Ridilla et al. 2013). However, the
question remains: Are pathogen-induced changes in cytoskeletal dynamics a function of plant-derived activation of
immunity, or the consequence of pathogen virulence?
To illustrate the complexity of this question, and
moreover, to begin to define the role of the eukaryotic actin
cytoskeleton during immune activation, several recent key
studies have begun to define the relationship between
changes in actin reorganization and host response to
pathogen infection. For example, and as an illustration of
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Actin-based immune signaling in plants
305
Figure 3. Direct targeting of the actin cytoskeleton by pathogens to enhance virulence
(A) Examples of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). Recognition of conserved PAMPs
results in a multitude of cellular signaling, including the generation of reactive oxygen species, mitogen-activated protein kinase
(MPK) stimulation, transcriptional reprograming, and (B) actin remodeling. PTI responses function in broad-based, basal
resistance to pathogen infection. (C) Actin-depolymerizing factor 4 (ADF4) from Arabidopsis has been demonstrated to play
a role in actin remodeling associated with the PTI response of the pattern recognition receptor EF-Tu Receptor (EFR).
(D) Pathogenic effectors are secreted into the host cell in order to target components of the PTI response and ultimately block
resistance signaling in the host. (E) The bacterial effector HopW1 specifically targets actin and alters the endomembrane
trafficking associated with resistance through the actions of both actin and myosin. (F) Arabidopsis ADF4 has also been
demonstrated to play a role in MPK activation by the ligand flg22 through stimulation of the pattern recognition receptor flagellin
sensing 2 (PRR FLS2) in the presence of AvrPphB.
the superimposition of actin filament assembly with the
initiation of the first node of immune signaling following
pathogen perception, a recent study by Henty-Ridilla and
colleagues (2014) showed that within 1 hour of pathogen
perception, a rapid increase in actin filament density occurs;
interestingly, this response was induced following infection
with a myriad of plant pathogens, including Pseudomonas
syringae pv. tomato DC3000 (Pst DC3000), P. syringae pv.
phaseolicola, Agrobacterium tumefaciens and Magnaporthe
grisea ((Henty-Ridilla et al. 2013); Figure 3B; Table 1). As a
function of defining the specificity of this response, it was
further determined that purified PAMPs (e.g., flg22 and
chitin) also induced similar changes in actin cytoskeletal
organization, suggesting that these responses are specific
elicitors of actin-immune-associated signaling. To block this
response, and thus provide further evidence supporting
the hypothesis that these changes are required for immunity,
co-inoculation with LatB – an actin-binding agent that
prevents filament polymerization – resulted in increased
susceptibility to the Pst DC3000. A second study further
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implicated the importance of the actin binding protein ADF4
in these responses (Henty-Ridilla et al. 2014; Figure 3C;
Table 1). In this study, dark grown hypocotyls of Arabidopsis
were used to examine the changes in actin dynamism in
the adf4 mutant as compared to wild-type plants. It was
determined that actin filament density increased following
elf26 elicitation, and that this increase was not observed in the
adf4 mutant (Henty-Ridilla et al. 2014; Figure 3C). Additionally,
an increase in actin filament length, filament lifetime and
a concomitant decrease in severing frequency, were also
observed in the following elf26 treatment. These observations
were phenocopied in the adf4 mutant and furthermore, no
change of these outputs was measured in the adf4 mutant
with elf26 treatment (Henty-Ridilla et al. 2014). In total, these
data demonstrate not only the importance of actin filament
organization during PTI, but also point to the involvement of
specific (i.e., ADF4) actin binding proteins in this process.
Moreover, they provide prima facie evidence that actin
binding proteins, such as ADF4, may be pathogen virulence
targets.
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Pathogen effectors and the plant actin cytoskeleton actin
Do plant pathogens actively target the host immune system
through disruption of cytoskeletal dynamics? This question
has long been the subject of intense speculation in the field of
plant pathology, and until recently, there have been no
reports describing an actin-specific virulence function of plant
pathogens. As noted above, pathogens utilize a suite of
secreted effector molecules to subvert the host PTI response
and the initiation of processes aimed at abrogating pathogen
proliferation (Figure 3D). To prevent this, hosts have evolved
mechanisms (i.e., ETI) to recognize and respond to the
presence and activity of these pathogen-secreted proteins. As
virulence factors, pathogen effectors evolved to target host
processes that function in immunity, and as a result of their
activity, induce a wide range of cellular changes in their
host(s). Not surprisingly, numerous virulence targets of
pathogen effectors identified thus far are components of
PTI signaling pathways – the hypothesis being that targeting
of PTI components can lead to increased growth of the
pathogen ((Zhang et al. 2010; Zhang and Zhou 2010);
Figure 3D). In this regard, given the diversity of host targets
identified, it is not surprising that plant pathogen effectors,
similar in function to those from human pathogens (Stuart
et al. 2013), target the host actin cytoskeleton for the purpose
of blocking processes required for host cell processes,
including immune signaling. Recently, an effector, HopW1,
from Pseudomonas syringae pv. maculicola, was shown to
disrupt the actin cytoskeleton, thereby enhancing pathogen
virulence, infection, and ultimately, disease ((Kang et al. 2014);
Figure 3E; Table 1). In this study, it was demonstrated that the
effector HopW1 directly interacts with actin both in vitro and
in vivo. First, in vitro, HopW1 was shown to bind to actin, and
through this, disrupt the normal remodeling processes
associated with filament organization. Second, in vivo, similar
observations were made to those using in vitro-based
methods, yet also including the key finding that this process
resulted in enhanced bacterial growth in planta when infected
with Pst DC3000 expressing HopW1 ((Kang et al. 2014);
Figure 3E). While a target actin binding protein was not
identified, it is noteworthy that results suggest a specific
targeting of endocytosis and trafficking to the vacuole
through HopW1 activity, thereby providing the first evidence
of an actin-associated virulence function for a phytopathogenic effector protein (e.g., Figure 3E; Table 1).
As a final demonstration of the connection between
pathogen effectors, the host actin cytoskeleton, and the
numerous homeostatic processes in plants that require the
activity of actin for their function, the work of Tian and
colleagues (Tian et al. 2009) best described the relationships that underpin the link(s) between cytoskeletal dynamics
and immune signaling. The functional analysis of ADF4 has not
only shown that actin depolymerization is important for
immunity, but through a series of complementary genetic
and cell biology-based approaches, has shown that compromised immune signaling in the adf4 mutant is the result of a
drastic reduction in the expression of the mRNA encoding
the resistance protein RPS5 (resistance to Pseudomonas
syringae-5). As a mechanism supporting this function, Porter
et al. (2012) defined that phospho-regulation of ADF4
influences association with actin as well as correlates with
the expression of RPS5. In addition to phospho-dependent
April 2016 | Volume 58 | Issue 4 | 299–311
regulation of the association of ADF4 with actin, this work also
demonstrated that the loss of RPS5 mRNA expression did not
fully explain the parallel reduction in MAPK signaling also
observed in the adf4 mutant. It was only in the presence of the
bacterial effector AvrPphB that a reduction in MAPK signaling
was observed ((Porter et al. 2012); Figure 3F; Table 1). Because
this loss was not observed in the rps5 mutant in the presence
of AvrPphB, these data support a role for ADF4 in the
activation of MAPK signaling. Taken together, these findings
offer a unique example of a multi-layered interaction of a
bacterial effector targeting both PTI and ETI in an actindependent manner. Recently, a similar observation was made,
showing that host susceptibility to the fungal pathogen
Puccinia striiformis f. sp. tritici was enhanced as a result of
silencing an ADF4 homolog in wheat (Triticum aestivum; Fu
et al. 2014).
Pathogen toxins and the actin cytoskeleton
One of the best-characterized virulence mechanisms of
pathogens of plants and animals is the production, delivery
and site of action of host-specific toxins (Strange 2007; Duke
and Dayan 2011). As a class of highly conserved diffusible
compounds, toxins serve many functions during infection,
including roles as long-range signaling molecules, extracellular
triggers of host cell lysis, and internalized inducers of
programed cell death (Strange 2007; Duke and Dayan 2011).
Pathogens of plants, particularly fungi, have been shown to
perturb the homeostatic function of the host actin cytoskeleton through the delivery of strain-specific elicitors and toxins,
presumably as a mechanism to alter defense signaling,
including host-derived secretion of anti-fungal compounds.
In most cases described thus far, these toxins (Table 1) have
been shown to either mimic the biochemical activities of
eukaryotic actin-binding proteins, or more broadly, disrupt the
structure/function of the microfilaments themselves. To date,
two well-established examples of toxin-specific targeting of
the host actin cytoskeleton by plant pathogens have been
described. In the first, Yuan et al. (2006) showed that
treatment of Arabidopsis suspension-cultured cells with the
toxin, VD toxin, from the soil-borne fungal pathogen
Verticillium dahlia, is capable of inducting dose-dependent
changes in the organization of the host actin cytoskeleton. For
example, at low toxin concentrations, actin filament structure
was disrupted, while microtubule organization was unaffected. Conversely, at high concentrations, both actin and
microtubule structures were disrupted, suggesting a point of
convergence in the activity of VD toxin, presumably as a
function of the virulence strategy of V. dahlia. This work
therefore implicates the actin cytoskeleton and the microtubule network as virulence targets of fungal pathogens,
providing a unique pathosystem to not only define the activity
and specific interaction points of the toxin, but also the
cellular function and activity of toxin delivery and uptake.
One of the best-characterized fungal toxins described to
date is that of ToxA, produced by the necrotrophic fungus
Pyrenophora tritici-repentis. ToxA has been shown to induced
cell death when expressed in mesophyll cells from both
sensitive and insensitive plants, yet is only actively translocated into the cytoplasm of the sensitive wheat cells
((Manning and Ciuffetti 2005); Table 1). One of the most
interesting features of this toxin is the presence of an RGD
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Actin-based immune signaling in plants
tripeptide sequence (i.e., Arginine-Glycine-Aspartic acid),
which has been shown to be required for its function
(Meinhardt et al. 2002). RGD motifs are most commonly
associated with the function of mammalian integrins (described above), required for their actin-dependent association
with the extracellular matrix. In this regard, it is interesting to
speculate that this toxin has evolved a function for the RGD
motif to associate with, and possibly subvert, the host
immune system through disruption of the actin cytoskeleton.
In further support of a role for the RGD, and RGD-like, motif in
plant immune signaling, it was recently demonstrated that the
immune signaling regulator, non-race specific disease resistance-1 (NDR1), is an integrin-like protein that plays a role in
cell wall-plasma membrane adhesion through the function of
an NGD-like (i.e., Asparagine-Glycine-Aspartic acid) motif
(Knepper et al. 2011). As described above, the process
of clathrin-mediated endocytosis requires the host actin
cytoskeleton; thus, it is tempting to hypothesize that actin
plays a role in the internalization of the ToxA protein through
a yet to be identified extracellular receptor (e.g., integrin-like
protein). In total, this is a nice example of cellular mimicry,
whereby the structure-function activity of a fungal toxin can
mimic the endogenous behavior of a cell wall-plasma
membrane process, thereby driving changes in host actin
cytoskeletal dynamics for the purpose of promoting pathogen
infection.
ACTIN AND THE NUCLEUS: THE FINAL
FRONTIER?
Actin was first observed in isolated nuclear fractions from
Xenopus laevis in the late 1970s (Clark and Merriam 1977), and
since this time, the proposed function(s) of actin within the
nucleus has been a point of discussion (Bettinger et al. 2004;
Belin and Mullins 2013). It was initially assumed that actin was
present in nucleus-enriched cell isolations as the result of
contamination during sample preparation, or simply resident
within the nucleus as the result of non-specific, passive
diffusion (Grosse and Vartiainen 2013). Thus, a bona fide role
for actin within the nucleus was often dismissed, giving rise to
the long-held belief that actin did not possess any specific role
related to nuclear physiology or function. Following the
discovery of actin within the nucleus, a number of studies
sought to demonstrate, and define, a role for actin within the
nucleus. At a fundamental level, early work, using microinjection of actin antibodies directly into the nuclei of salamander
oocytes, showed a cessation of RNA synthesis from the
lampbrush chromosomes (Scheer et al. 1984). This work was
followed by an elegant series of studies that collectively
demonstrated a role for actin in the processes that support
transcriptional activity by all three of the RNA polymerases
(Olave et al. 2002; Obrdlik and Percipalle 2011; Kapoor and
Shen 2014), defining that the assembly of transcriptional
complexes required for RNA polymerase function and
chromatin remodeling require a functional actin cytoskeleton
(Figure 4B). Similar observations in plants were made soon
after by Kandasamy et al. (2010), who demonstrated that
in Arabidopsis, the localization of multiple vegetative class
actin variants within the plant nucleus are organized by
distinct localization patterns based on actin isotype.
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307
Actin movement in and out of the nucleus
As noted above, and recently reviewed by Wang and Hussey
(2015), actin plays a key role in mediating the connectivity of
the nucleus with the endomembrane system. As a function of
this connectivity, and moreover, as a missing link in the role
of actin in nuclear dynamics (i.e., chromatin positioning and
architecture, transcription), the precise mechanism(s) by
which actin enters the nucleus is still unclear. It has been
hypothesized that actin is shuttled into the nucleus by ADF/
Cofilin, one of a few members of the actin-binding protein
superfamily that contains a nuclear localization signal (Dopie
et al. 2012). In humans, the import/translocation of ADF/
Cofilin-actin into the nucleus has been shown to require the
function of importin-9, while export of actin is mediated by
profilin, through an association with exportin-6 ((Wada et al.
1998; Dopie et al. 2012); Figure 4A). In plants, the import/
export control of actin and actin binding proteins into and
out of the nucleus is unclear; however, it has been
demonstrated that similar to that in mammalian systems,
plant nuclei contain actin binding proteins, including ADF1-4
and profilin (Kandasamy et al. 2010; Porter et al. 2012),
supporting the hypothesis that a similar mechanism of
transport to that in mammals may also exists in plants.
In addition to the active nuclear import/export of actin,
actin-binding proteins themselves have been identified to
have direct interactions with genes as well as the nuclear
machinery (Miyamoto and Gurdon 2012; Percipalle 2013).
While direct interactions have been observed in mammalian
systems, research in plant systems has only identified indirect
alterations in gene expression due to either loss of actinbinding proteins or alterations in cytoskeletal dynamics
(Burgos-Rivera et al. 2008; Porter et al. 2012; Moes et al.
2013). For example, in Arabidopsis, it has been observed that
mutation of ADF9 results in reduced expression of flowering
locus C (FLC; (Burgos-Rivera et al. 2008); Figure 4B).
Additionally, through the use of chromatin immunoprecipitation, it was further demonstrated that this reduction in gene
expression is mediated by concomitant reductions in histone
H3 lysine 4 trimethylation and histone H3 lysine 9 and 14
acetylation of the FLC promoter. Similarly, in Nicotiana
tabacum, it was observed that the LIM protein, WLIM2,
which is predicted to be both nuclearly and cytoplasmically
localized, as well as binding actin, interacts with the
Arabidopsis histone H4A748 (Moes et al. 2013), and that
stimulation with LatB results in increased nuclear occupancy
of WLIM2 (Figure 4C). In total, these data further support the
hypothesis of a functional link between actin and the
regulation of transcription.
Do pathogens actively target host nuclear actin? A recent
review by Deslandes and Rivas (2011) notes that the plant
nucleus is the next major area of study in plant immunity
research. Given the movement of actin and actin-binding
proteins into and out of the nucleus, as well as the
involvement of these components in gene transcription, the
role of nuclear actin during immune activation and signaling
should not be overlooked. Indeed, recent work by Porter et al.
(2012) demonstrated a requirement for Arabidopsis ADF4 for
the proper expression of the resistance gene RPS5, and
ultimately, resistance to Pst DC3000 expressing the cysteine
protease AvrPphB. From this work, it was determined that
ADF4, and by extension, the proper regulation and activity of
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Porter and Day
Figure 4. Nuclear involvement of the actin cytoskeleton in gene expression and its targeting by plant pathogens
(A) Proposed translocation of actin into and out of the nucleus by the actin-binding proteins actin depolymerizing factors (ADFs)
and profilin, as demonstrated in mammalian systems. (B) Sub-nuclear functions of monomeric globular (G)-actin, filamentous
(F)-actin and ADFs in gene transcription. G- and F-actin, as well as Cofilin1 have been determined to play a role in gene expression
in mammalian systems. Arabidopsis ADF9 has been demonstrated to be required for expression of the flowering locus C (FLC) in a
histone modification dependent manner. (C) The Nicotiana tabaccum LIM protein, WLIM2, associated with both actin and histone
H4A748. Additionally WLIM2 has subcellular localization patterns in the cytosol and nucleus. (D) Turnip vein clearing virus (TVCV)
movement protein (MPTVCV) posses a strong nuclear localization signal and interacts with F-actin. Visualization of MPTVCV
revealed an association with F-actin structures within the nucleus as well as co-localization of MPTVCV with histone H2B.
the actin cytoskeleton, was not only required for expression
of RPS5, but also that this processes was highly dependent
upon phosphorylation events associated with the regulation
of ADF4 activity. In total, these data provide insight into
the potential mechanisms by which expression of host R
genes may be regulated by actin-binding proteins in a posttranslational manner, thus providing clues as to how
pathogens may subvert nuclear immunity by altering
cytoskeletal dynamics. Additional recent work in this area
has further demonstrated a function for actin in gene
expression and the induction of immunity through the
identification of a direct interaction of rice ADF with a lectin
receptor-like kinase (Cheng et al. 2013); specifically, mutation
of either OsleRK or ADF resulted in reduced expression of the
resistance-associated genes PR1a and LOX, leading to
enhanced susceptibility to multiple pathogens, including the
bacterium Xanthomonas oryzae pv. oryzae and the fungi
Magnaporthe grisea ((Cheng et al. 2013); e.g., Figure 4B).
Additional supporting studies linking immunity, gene expression and actin have also been reported ((Levy et al. 2013); e.g.,
Figure 4D), demonstrating that the role of nuclear actin is
central to numerous host immune functions, including serving
as a key target for pathogen manipulation. For example, a
recent study examined the movement protein (MP) of the
tobamovirus Turnip vein clearing virus (TVCV: MPTVCV) and
found that in addition to its expected localization to the
endoplasmic reticulum and plasmodesmata, MPTVCV was
April 2016 | Volume 58 | Issue 4 | 299–311
located in the plant nucleus in association with F-actin (Levy
et al. 2013; Figure 2A). There, MPTVCV did not co-localize with
nucleoli or Cajal bodies, but instead co-localized with histone
H2B. Based on these data, it is plausible that that MPTVCV may
directly alter nuclear actin dynamics to alter the expression of
genes in order to enhance virulence.
FINAL THOUGHTS
The plant actin cytoskeleton is ubiquitous, dynamic and highly
regulated, requiring the activity of more than 75 actin-binding
proteins for its assembly and function. In addition to the
basic processes that regulate the filament architecture and
organization, actin cytoskeletal dynamics are intimately
governed by a suite of host processes that require its
function, including those associated with growth and
development, movement and organization, and response to
stimuli. In recent years, advances in genomics and cell biology
have further enhanced our understanding of the processes
governing, and governed by, the actin cytoskeleton. From
these collective studies, it is evident that we have only begun
to scratch the surface of our understanding of the “hows” and
“whys” regarding the extent of the role of the actin
cytoskeleton in plant biology. Of particular interest is the
role of actin as a surveillance mechanism, continually sensing
the cell for perturbations, including both chemical and
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Actin-based immune signaling in plants
physical changes in the intracellular and extracellular
environment. As a central component of actin’s role as a
surveillance platform, the localization, including changes in
the subcellular concentration of actin and various actinbinding proteins, is noteworthy. To begin to address this
knowledge gap, studies using plant-pathogen models have
demonstrated that changes in actin-binding protein localization within the cell serves not only as a stimulus for
reorientation of actin filament architecture, but also as a
trigger that initiates the induction of processes, including
changes in signal transduction pathways and gene expression.
To this end, the role of actin in the nucleus represents largely
unexplored areas of research, possibly holding the answers to
areas of biology beyond the dynamics of actin assembly, and
the realm of actin as a mediator of gene activation and cellular
homeostasis.
309
Cheng X, Wu Y, Guo J, Du B, Chen R, Zhu L, He G (2013) A rice lectin
receptor-like kinase that is involved in innate immune responses
also contributes to seed germination. Plant J 76: 687–698
Chesarone MA, DuPage AG, Goode BL (2010) Unleashing formins to
remodel the actin and microtubule cytoskeletons. Nat Rev Mol
Cell Biol 11: 62–74
Chisholm ST, Coaker G, Day B, Staskawicz BJ (2006) Host-microbe
interactions: Shaping the evolution of the plant immune
response. Cell 124: 803–814
Chtarbanova S, Imler JL (2011) Microbial sensing by toll receptors: A
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Clark TG, Merriam RW (1977) Diffusible and bound actin nuclei of
Xenopus laevis oocytes. Cell 12: 883–891
Cui X, Wei T, Chowda-Reddy RV, Sun G, Wang A (2010) The Tobacco
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ACKNOWLEDGEMENTS
Cvrckova F (2013) Formins and membranes: Anchoring cortical actin to
the cell wall and beyond. Front Plant Sci 4: 436
KP was supported in part by a Barnett Rosenberg Fellowship
in Biological Sciences from Michigan State University. Work in
the laboratory of BD is supported by the National Science
Foundation (IOS-1021044).
Day B, Henty JL, Porter KJ, Staiger CJ (2011) The pathogen-actin
connection: A platform for defense signaling in plants. Ann Rev
Phytopathol 49: 483–506
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