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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 www.jipb.net 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 1 300 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 www.jipb.net 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. www.jipb.net April 2016 | Volume 58 | Issue 4 | 299–311 302 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 www.jipb.net Actin-based immune signaling in plants 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, www.jipb.net 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. April 2016 | Volume 58 | Issue 4 | 299–311 304 Porter and Day 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 April 2016 | Volume 58 | Issue 4 | 299–311 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 www.jipb.net 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 www.jipb.net 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. April 2016 | Volume 58 | Issue 4 | 299–311 306 Porter and Day 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 www.jipb.net 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. www.jipb.net 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 April 2016 | Volume 58 | Issue 4 | 299–311 308 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 www.jipb.net 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 historical perspective. Arterioscler Thromb Vasc Biol 31: 1734–1738 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 etch virus P3 protein forms mobile inclusions via the early secretory pathway and traffics along actin microfilaments. Virol 397: 56–63 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 REFERENCES Dopie J, Skarp KP, Rajakyla EK, Tanhuanpaa K, Vartiainen MK (2012) Active maintenance of nuclear actin by importin 9 supports transcription. 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