* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download A cellular backline: specialization of host membranes for defence
Survey
Document related concepts
Cell encapsulation wikipedia , lookup
Magnesium transporter wikipedia , lookup
G protein–coupled receptor wikipedia , lookup
Organ-on-a-chip wikipedia , lookup
Membrane potential wikipedia , lookup
Lipid bilayer wikipedia , lookup
Theories of general anaesthetic action wikipedia , lookup
Model lipid bilayer wikipedia , lookup
Ethanol-induced non-lamellar phases in phospholipids wikipedia , lookup
SNARE (protein) wikipedia , lookup
Signal transduction wikipedia , lookup
Cytokinesis wikipedia , lookup
List of types of proteins wikipedia , lookup
Transcript
Journal of Experimental Botany, Vol. 66, No. 6 pp. 1565–1571, 2015 doi:10.1093/jxb/erv021 Advance Access publication 25 February 2015 Review Paper A cellular backline: specialization of host membranes for defence Christine Faulkner* John Innes Centre, Norwich Research Park, Colney Lane, Norwich NR4 7UH, UK * To whom correspondence should be addressed. E-mail: [email protected] Received 3 November 2014; Revised 11 December 2014; Accepted 19 December 2014 Abstract In plant–pathogen interactions, the host plasma membrane serves as a defence front for pathogens that invade from the extracellular environment. As such, the lipid bilayer acts as a scaffold that targets and delivers defence responses to the site of attack. During pathogen infection, numerous changes in plasma membrane composition, organization, and structure occur. There is increasing evidence that this facilitates the execution of a variety of responses, highlighting the regulatory role membranes play in cellular responses. Membrane microdomains such as lipid rafts are hypothesized to create signalling platforms for receptor signalling in response to pathogen perception and for callose synthesis. Further, the genesis of pathogen-associated structures such as papillae and the extra-haustorial membrane necessitates polarization of membranes and membrane trafficking pathways. Unlocking the mechanisms by which this occurs will enable greater understanding of how targeted defences, some of which result in resistance, are executed. This review will survey some of the changes that occur in host membranes during pathogen attack and how these are associated with the generation of defence responses. Key words: Callose, haustoria, lipid raft, papillae, pathogen, plasma membrane. Introduction Most plant pathogens infect tissues via an extracellular route. Thus, the cell wall and plasma membrane act as key defence fronts during an attempted pathogen invasion. Pattern recognition receptors (PRRs) that detect the presence of a pathogen threat via pathogen-associated molecular patterns (PAMPs) are anchored in the membrane and trigger signalling cascades that alter both the intracellular and extracellular environment, in some contexts by the transfer of signals and/or molecules across the lipid bilayer. Host membranes polarize in response to cellular penetration by filamentous pathogens, targeting defence machinery to the site of attack. They also remodel to accommodate subsequent invasive structures such as haustoria. There is evidence that the host plasma membrane changes in both composition and organization in order to mediate these interactions and to execute defence responses. This appears to have an association with dynamic membrane microdomains (possibly lipid rafts) that might recruit relevant proteins and thus activate receptor signalling and downstream responses. On a larger scale, the membranes that surround penetration events and haustoria are different in proteinaceous and lipid composition relative to the rest of the plasma membrane. Thus, it is clear that host cell membranes function as specific and dynamic scaffolds for the execution of many defence responses. Membrane-associated pathogen perception and signalling The recognition of pathogens by PRRs and their downstream activation of defence is one of the best explored elements of defence responses. PRRs are membrane-located receptors © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: [email protected] 1566 | Faulkner that, in exposing receptor domains at the extracellular face of the membrane, are able to bind PAMPs and thus recognize a pathogen threat (Macho and Zipfel, 2014). PRRs are receptor kinases or receptor proteins that span, or are tethered to, the plasma membrane. Consequently, PRR signalling activity, which occurs when a PRR binds its cognate PAMP ligand, is anchored in the membrane in which it resides (Figure 1). PRR activation triggers downstream signalling via several different pathways. Amongst these are at least three membrane-associated responses: the production of reactive oxygen species (ROS) by NADPH oxidases; the influx of calcium ions from the cell wall into the cytoplasm; and the production of callose by membrane-resident callose synthases (see below). The production of ROS (known as the oxidative burst) and the influx of Ca2+ ions from the apoplast are interdependent processes (Segonzac et al., 2011). The influx of Ca2+ from the apoplast is mediated by the balance of activity of membrane calcium channels and transporters (Frei dit Frey et al., 2012), while the initial ROS burst is mediated by the NADPH oxidase RESPIRATORY BURST OXIDASE D (RBOHD; Nuhse et al., 2007; Zhang et al., 2007), which, being located in the plasma membrane, produces ROS on the extracellular face of the membrane. It is thought that the production of ROS in the apoplast contributes to cell wall strengthening via cross-linking of glycoproteins (Bradley et al., 1992). The PRR FLAGELLIN SENSING2 (FLS2) binds flagellin and mediates the initiation of a suite of antibacterial defences including ROS burst, Ca2+ influx, and callose deposition. Flagellin binding occurs while FLS2 is resident in the plasma membrane, triggering accumulation of FLS2 in detergent-resistant membrane microdomains (Keinath et al., 2010). Following activation, and presumably any lateral rearrangement of FLS2 within the membrane, the receptor is internalized and enters the endocytic membrane trafficking pathway (Beck et al., 2012). It is unclear what FLS2 functions are associated with this change in localization, but this active rearrangement of FLS2-containing membranes must contribute to defence signalling, even in the simplest context in which internalization removes bound receptor from the plasma membrane to allow for newly synthesized receptor to take its place. FLS2 also mediates flagellin-triggered redistribution of the ABC transporter PENETRATION3 (PEN3; Underwood and Somerville, 2013). Following treatment of plant tissue with the flagellin derivative flg22, PEN3 localization shifts from uniform distribution in the plasma membrane to focal accumulations in what are assumed to be sites of detection (Underwood and Somerville, 2013). This rearrangement is actin dependent, but not dependent upon membrane secretion, suggesting the possibility of lateral rearrangement within the existing membrane. FLS2 signalling is dependent on the co-receptor BRI1-ASSOCIATED KINASE1 (BAK1) (Chinchilla et al., 2007), but, while PEN3 accumulation in response to flg22 is dependent on FLS2, it is not dependent on BAK1. Thus, it seems likely that PEN3 accumulation still requires FLS2 binding of flagellin, but that lateral membrane reorganization occurs via an unknown co-receptor or an entirely independent mechanism. Callose synthesis as a membraneanchored response The deposition of callose in the apoplast is triggered in a number of different contexts during plant–pathogen interactions. Callose is a β-1,3-glucan and has been hypothesized to fortify cell walls and tissues against an invading pathogen. Callose synthases (or glucan synthases) are located in membranes and synthesize callose towards the extracellular face to deposit it in the cell wall. Thus, while the callose deposition is detectable and measurable as an extracellular defence, the response is associated with the activity of proteins that are located in the plasma membrane. The perception of PAMPs such as chitin and flagellin causes what appears to be random deposition of callose in the apoplast. However, there are also examples of more targeted activity of callose synthases in response to pathogens. For example, papillae that form in response to attempted cellular penetration by filamentous pathogens are filled with callose, as are encasements that surround the haustoria of both fungal and oomycte pathogens (Figure 1). In these cases, callose synthesis is active at specific membrane domains. Similarly, callose is deposited at plasmodesmata in response to bacterial infection (Lee et al., 2011; Wang et al., 2013), again exemplifying targeted activity of callose synthases at a specialized membrane domain during infection. Callose deposition is well documented during papilla formation. Papillae are cell wall appositions that form at the site of attempted penetration of a filamentous pathogen such as the powdery mildews. While it was originally hypothesized that callose forms a crucial part of a physical or chemical barrier to penetration, recent data have cast some doubt on the simplicity of this model. According to the hypothesis that callose deposition increases structural resistance to penetration, it was reasonable to expect the mutants for the GLUCAN SYNTHASE-LIKE5 (GSL5; also known as POWDERY MILDEW RESISTANT4, PMR4) gene, that do not deposit callose in papillae during penetration, would show increased susceptibility to an adapted powdery mildew. However, such mutants exhibit increased resistance in this context (Jacobs et al., 2003; Nishimura et al., 2003; Ellinger et al., 2013). While increased resistance was co-incident with increased salicylic acid responses, the penetration success of the adapted pathogen Golovinomyces cichoracearum on pmr4 mutants was comparable with that observed in wild-type plants (Ellinger et al., 2013), indicating that callose deposition does not significantly contribute to any defensive function of papillae and that altered resistance in this interaction was the consequence of responses downstream of callose deposition in papillae. By contrast, the deposition of callose in papillae in response to attempted penetration by the non-adapted powdery mildew Blumeria graminis f. sp. hordei (Bgh) does contribute significantly to the defensive function of the papilla. Ellinger et al. (2013) observed a small but significant increase in penetration success of Bgh in pmr4 mutants, exhibiting 20% success compared with the 8% success rate observed on wild-type plants. While these data show that callose deposition does positively contribute to penetration resistance, the small nature of the Membranes in defence | 1567 change indicates that even in this context callose deposition is a minor component. Studies in which callose deposition was enhanced by overexpression of PMR4 suggest that despite the minor role played by callose in an endogenous context, callose can indeed act as a barrier to pathogen penetration. Plants which express 35S::PMR4-GFP exhibit complete penetration resistance to both adapted and non-adapted powdery mildews (Ellinger et al., 2013; Eggert et al., 2014). These lines also enabled high resolution imaging of callose deposition at papillae and identified that while callose is deposited primarily at the membrane face of the cell wall, as might be expected from the membrane location of callose synthases, the callose polymers can penetrate the cellulose matrix. In PMR4 overexpression lines, callose polymers completely penetrate the cell wall and form a layer of callose at the external surface of the wall (Eggert et al., 2014), leading to the hypothesis that callose deposition protects the cell wall from hydrolysing enzymes produced by fungal pathogens. Indeed, cellulose digestion of inoculated leaves induced degradation of the cellulose–callose network at papillae which was not observed in 35S::PMR4-GFP plants. In wild-type plants, cellulase had a greater effect on callose than on cellulose at the papilla, suggesting that callose may act as a sacrificial protectant against cellulose hydrolysis. The observation that callose deposition is targeted to specific sites, such as at the papillae and haustoria, supports the notion that callose plays some role in physical or chemical defence against pathogen invasion. Further it requires a mechanism by which callose synthases are activated in, or targeted to, specific domains within the membrane. Vesicle trafficking machinery has been implicated in deposition of callose at the papilla in barley and Arabidopsis. ARF GTPases recruit coat proteins to membrane domains and mediate vesicle budding; in barley, the GTPase ARFA1b/1c was found to be required for callose deposition at the papilla (Bohlenius et al., 2010), but not for formation of the papilla structure. ARFA1b/1c also contributes to penetration resistance of Bgh and functions in the same membrane trafficking pathway as the REQUIRED FOR MLO RESISTANCE2 (ROR2)/PENETRATION1 (PEN1) syntaxin (Bohlenius et al., 2010). The co-localization of ARFA1b/1c with ARA7 led the authors to hypothesize that callose synthesis might occur in multivesicular bodies (MVBs) prior to trafficking of the membranes and cargoes to the penetration site (Figure 1). However, ARA7 is also located in early endosomes (Ueda et al., 2004; Beck et al., 2012) and it was reported that callose could not be detected in MVBs, despite their proximity to the penetration site (An et al., 2006), suggesting that further information is required to determine if callose is made prior to targeted deposition at the papilla in this system. Further implicating membrane trafficking pathways in targeted callose deposition, recent work on the Arabidopsis– Golovinomyces interaction identified that a Rab GTPase, RabA4c, interacts directly with PMR4 and that this interaction might recruit PMR4 to the penetration site (Ellinger et al. 2014a). Callose synthesis appeared to be linked to the functional state and location of RabA4c rather than changes in its expression in response to infection, suggesting that membrane trafficking and protein interactions control the activity and location of callose synthesis in response to pathogen penetration. Another scenario which has implicated membrane composition in callose deposition is the production of lipase by the pathogen Fusarium graminarum. Production of the lipase reduces callose deposition during pathogen infection of spikelets, and this was hypothesized to occur via lipasemediated production of free fatty acids that possibly disrupts the lipid composition of the membrane and consequently reduces callose synthase activity (Ellinger et al., 2014b). Thus it seems not only that membranes act as a scaffold for callose synthesis but that membrane trafficking and composition define the activity of callose synthases. Callose deposition is also highly localized in the encasement of pathogen haustoria. Haustorial encasement can occur in both fungal and oomycete interactions, such as Hyaloperonospora arabidopsidis (Hpa)–Arabidopsis and Uromyces fabae–wheat. Like at the papilla, pmr4 mutants show reduced callose deposition at the Hpa encasement but increased resistance (Vogel and Somerville, 2000), suggesting that callose plays a minor role in defence. However, recent data provide evidence that the deposition of callose in the encasement positively regulates defence. The PLASMODESMATA LOCATED PROTEIN1 (PDLP1) localizes to the extra-haustorial membrane (EHM) prior to development of the encasement and regulates callose deposition in the encasement as it develops (Caillaud et al., 2014). Like PMR4, overexpression of PDLP1 enhances callose deposition, which in this interaction is co-incident with enhanced membrane convolution surrounding the haustorium. Significantly, overexpression of PDLP1 increases resistance of Arabidopsis to Hpa, and pdp1,2,3 mutants show both increased susceptibility and reduced callose deposition in the encasement. This demonstrates that callose deposition does contribute significantly to defence in this interaction. The body of data relating to the role of callose deposition during defence provides what appears to be conflicting data regarding the significance of the response. However, it is clear that in some contexts callose deposition positively contributes to defence responses (Ellinger et al., 2013; Caillaud et al., 2014). It is possible that the simple hypothesis that callose deposition fortifies the apoplast against invasion is in part correct, but that the ultimate contribution that this makes to defence is dependent on spatio-temporal factors and crosstalk with other defence responses. Membrane specialization via protein composition Haustoria are assumed to act as sites of molecular exchange between host and pathogen, and therefore many of these processes must be localized at the EHM (Figure 1). While the EHM is continuous with the plasma membrane of the host cell, plasma membrane-resident proteins are not always present in the EHM, indicating that there is specialization of the membrane (Roberts et al., 1993; Koh et al., 2005; Wang 1568 | Faulkner Fig. 1. Membrane specialization during (A) PRR signalling, (B) papillae formation, and (C) haustorial accommodation. Lipid rafts that are enriched in sphingolipids and sterols, and contain remorins, have been implicated in the definition of membrane domains that facilitate receptor signalling (A) and callose synthesis (B, C). (A) The PRR FLS2 binds bacterial flagellin and this induces complex formation with the co-receptor BAK1. This triggers a calcium ion influx, the generation of reactive oxygen species by RBOHD, and callose deposition, as well as multiple non-membrane-anchored responses. (B) Papillae are cell wall appositions that form in response to attempted penetration by filamentous pathogens, here illustrated by what is known for powdery mildew pathogens. The final composition of papillae-associated membranes has been speculated to result from VAMP721/722-associated membrane trafficking and deposition of materials, such as lipids and callose synthases, at the papillae-associated membrane. Multivesicular bodies have been proposed to target PEN1 to papilla as PEN1 has been observed in membranes in the papillary matrix. It is also possible that lateral redistribution of some proteins such as the ABC transporter PEN3 results in accumulation beneath the papilla. (C) The source of membrane for the genesis of the extrahaustorial membrane (EHM) is unknown, but VAMP721/722 vesicles have been implicated in the trafficking of RPW8 and PDLP1 to the EHM. The lipid raft protein remorin has also been associated with the EHM, suggesting that it might be further specialized into microdomains. Membranes associated with the encasement are sites of callose synthesis. et al., 2009; Lu et al., 2012). Electron micrographs have also determined that the EHM is different in appearance (Chou, 1970; Knauf et al., 1989; Mims et al., 2002) from the plasma membrane. In several interactions the EHM appears convoluted or invaginated, which increases the membrane surface in proximity to the pathogen (Chou, 1970; Mims et al., 2002; Micali et al., 2011). It is assumed that pathogen effectors are delivered across this membrane, as are nutrients from host to pathogen, and thus it is conceivable that the increased surface of the membrane is manipulated by, and to the benefit of, the pathogen. However, as defence responses are spatially anchored in the membrane, it is also likely that this membrane convolution enhances the capacity for defence responses to be targeted at the site of attack. The identification of PDLP1 at the EHM and its association with callose deposition indicates that membrane convolutions in the EHM are positively correlated with increased callose deposition (Caillaud et al., 2014), suggesting that this increased membrane scaffold enhances at least one defence response. The trade-off between host and pathogen processes can only be unravelled as molecular players, such as pathogen effectors of known translocation mechanism and activity or host EHM-associated proteins with a known mechanism of defence activity, are identified. With respect to the protein composition of the EHM, Lu et al. (2012) observed that in the Hpa–Arabidopsis interaction the calcium ATPase AUTOINHIBITED CA2+-ATPASE (ACA8) and the aquaporin PLASMA MEMBRANE INTRINSIC PROTEIN1;4 (PIP1;4) were excluded from the EHM but maintained normal association with the plasma membrane during infection. The authors noted that this specificity indicated that the mechanism by which proteins were targeted to the EHM was unlikely to involve diffusion of proteins from the plasma membrane, or the simple redirection of general membrane trafficking to the plasma membrane. In this study the EHM did contain plasma membrane proteins such as the PRR FLS2 and the syntaxin PEN1, and proteins that mark endomembrane compartments indicated that these accumulate around the haustoria. This suggests that membrane trafficking plays a role in the genesis of the EHM, and possibly in the mechanism by which this membrane is specified. Independent studies have implicated the R-SNARE VESICLE ASSOCIATED MEMBRANE PROTEIN (VAMP) 721/722 in membrane trafficking to the EHM and papillae. These vesicle-associated proteins are necessary to target the resistance protein RPW8 to the haustoria of Golovinomyces orontii (Kim et al., 2014), and have been identified in PDLP1-containing membranes (Caillaud et al., 2014), suggesting that both proteins use the same membrane trafficking pathway to target haustoria of unrelated pathogens. Similarly, VAMP721/722 were found to be required for resistance to penetration of non-adapted Erysiphe Membranes in defence | 1569 pisi on Arabidopsis and to form a complex with PEN1 and the t-SNARE SOLUBLE N-ETHYLMALEIMIDESENSITIVE FACTOR ADAPTOR PROTEIN33 (SNAP33; Kwon et al., 2008). This implicates the same membrane trafficking pathway in polarization of membranes at papillae. It is likely that characterization of this pathway will identify factors that define the specificity of the membrane at the host–pathogen interface, leading to dissection of the mechanisms by which the host exploits this membrane as a targeted site of defence responses. PEN1 is present in the plasma membrane when the cell is unchallenged by a pathogen. Following attempted penetration, PEN1 accumulates at membranes beneath papillae triggered by powdery mildew pathogens (Collins et al., 2003; Meyer et al., 2009). This accumulation has been associated with membranes in the extracellular papillary matrix (Meyer et al., 2009; Nielsen et al., 2012) rather than continuous with the plasma membrane, raising the possibility that PEN1 is secreted by exosomes. Two studies have independently tested the accumulation of PEN1 following pharmacological treatment that disrupts elements of the acto-myosin system. While both cytochalasin E (which disrupts actin filaments) and 2,3-butanedione monoxime (BDM; a myosin inhibitor) induce a subtle change in the distribution of PEN1 beneath papillae to a more diffuse and slightly punctate pattern, an accumulation still occurs (Underwood and Somerville, 2013; Yang et al., 2014). This was interpreted differently in each study as a dependence (Yang et al., 2014) and an independence (Underwood and Somerville, 2013) of PEN1 targeting on the actin cytoskeleton. Underwood and Somerville (2013) showed that PEN1 accumulation is dependent upon secretion, but the subtle change in PEN1 arrangement beneath the papillae may point to a two-step mechanism for focal accumulation. The ABC transporter PEN3 is located in the plasma membrane and also accumulates at membranes beneath papillae following a pathogen challenge (Stein et al., 2006). Unlike PEN1, PEN3 accumulation depends upon the actin cytoskeleton but not on secretion (Underwood and Somerville, 2013), suggesting that targeting of different proteins to the site of pathogen invasion employs different membrane trafficking and/or re-organization mechanisms. Membrane specialization via lipid composition Lipids within the plasma membrane are not homogenous and the spatial concentration of different types of lipids within the membrane can define clusters of functional specialization within the membrane. This is of particular significance to receptor signalling in animal cells, with activation or formation of receptor complexes dependent upon the lipid environment (Simons and Gerl, 2010). Lipid rafts are characterized by an increased concentration of sphingolipids and sterols, and are often correlated with detergent-resistant membrane (DRM) extracts. As evidence of the role of lipid composition in the polarization of membranes, sphingolipids were found to be essential for the polarization of both PIN1 and AUX1, and sterols for polarization of PIN2, in Arabidopsis mutants for the respective lipid synthesis pathways (Men et al., 2008; Markham et al., 2011). Further, the Fusarium toxin fumonisin, which prevents ceramide synthase activity, also disrupted the polarity of PIN proteins without disrupting endocytic pathways (Aubert et al., 2011). As the polarization and specialization of membranes is also observed in the genesis of papillae-associated membranes and the EHM, it is likely that lipid composition also contributes to this phenomenon. Indeed, both of these membrane subdomains have been associated with lipid rafts. Papillae formed by attempted penetration of the Bgh pathogen on the non-host Arabidopsis were stained with the fluorochrome filipin, which is sequestered by sterols, suggesting that the host membrane at the penetration site is enriched by sterols and thus shares characteristics with lipid rafts (Bhat et al., 2005). Remorins are plant-specific proteins that are located in steroland sphingolipid-enriched membranes (Raffaele et al., 2009). During infection of Nicotiana benthamiana by the oomycte Phytophthora infestans, transient expression of the potato remorin StRem1.3 showed localization to the EHM (Bozkurt et al., 2014), similarly implicating lipid rafts in the specialization of defence-associated membranes. Further supporting the hypothesis that both papillae-associated membranes and the EHM contain lipid rafts, several studies have identified that DRMs in different plant species have β-1,3-glucan synthase activity and are capable of producing callose (Him et al., 2001; Bessueille et al., 2009; Cifuentes et al., 2010; Srivastava et al., 2013). This activity correlates with the identification of callose synthases in DRMs (Srivastava et al., 2013) and indirectly suggests the possibility that the papillae-associated membrane and the EHM have characteristics of lipid rafts as they are sites of callose synthesis. The observation that PDLP1 localizes to both the plasmodesmal plasma membrane and the EHM in the Arabidopsis–Hpa interaction (Caillaud et al., 2014), and influences callose deposition in both locations, suggests that callose deposition utilizes the same regulatory mechanisms in both membranes. Remorins have also been observed to localize to plasmodesmal membranes (Raffaele et al., 2009; Gui et al., 2014), raising the possibility that callose deposition in plasmodesmata, and thus possibly in the EHM, has an association with lipid rafts (Gui et al., 2014). Close examination of the localization of both RPW8 and PDLP1 to the EHM surrounding powdery mildew (Berkey et al., 2012) and downy mildew (Hpa; Caillaud et al., 2014) haustoria, respectively, identifies uneven distribution of the proteins. Similarly, StRem1.3 localization to the EHM of Phytophthora haustoria is not homogeneous (Bozkurt et al., 2014) and does not overlap with the localization of another EHM-resident protein, SYNAPTOTAGMIN1. Each of these uneven distributions occurs in haustoria from different pathogens, but identify membrane domains within the EHM. Again, this is an indirect and speculative association between possible lipid rafts and defence-associated membranes, but examination of all markers in a single interaction would determine if these microdomains are indeed lipid rafts (ie. colocalize with remorin). 1570 | Faulkner Membrane microdomains have also been implicated in PRR activation and signalling. Several components of PRR signalling have been identified in DRMs following elicitation with pathogen-derived molecules, including RBOHD (Mongrand et al., 2004; Noirot et al., 2014) and the PRR FLS2 (Keinath et al., 2010). It is possible that these microdomains form signalling platforms and recruit necessary components, but further characterization of the spatio-temporal dynamics of protein–protein interactions during defence activation, as well as protein–lipid interactions are needed to understand fully how lateral membrane reorganization contributes to defence. Conclusions Host membranes act as scaffolds and signalling platforms for defence responses in several contexts. In anchoring PRRmediated pathogen perception, they act as one of the earliest defence fronts and may depend upon membrane domains for activity. Dynamic reorganization of membranes not only accommodates invasive structures but targets defences such as callose deposition to specific subcellular sites. The mechanisms by which membranes facilitate and/or regulate this activity will elucidate not only how a single cell responds to pathogen attack but also how cellular polarization can occur in fully expanded cells. It is clear that while lipid composition is a poorly explored facet of plant cell biology, it has unconsidered regulatory roles in cellular responses. Further analysis of the composition and function of membrane domains will detail how membranes respond to pathogens, and orchestrate and target specific responses. References An Q, Huckelhoven R, Kogel KH, van Bel AJ. 2006. Multivesicular bodies participate in a cell wall-associated defence response in barley leaves attacked by the pathogenic powdery mildew fungus. Cellular Microbiology 8, 1009–1019. Aubert A, Marion J, Boulogne C, Bourge M, Abreu S, Bellec Y, Faure JD, Satiat-Jeunemaitre B. 2011. Sphingolipids involvement in plant endomembrane differentiation: the BY2 case. The Plant Journal 65, 958–971. Beck M, Zhou J, Faulkner C, MacLean D, Robatzek S. 2012. Spatio-temporal cellular dynamics of the Arabidopsis flagellin receptor reveal activation status-dependent endosomal sorting. The Plant Cell 24, 4205–4219. Berkey R, Bendigeri D, Xiao S. 2012. Sphingolipids and plant defense/ disease: the ‘death’ connection and beyond. Frontiers in Plant Science 3, 68. Bessueille L, Sindt N, Guichardant M, Djerbi S, Teeri TT, Bulone V. 2009. Plasma membrane microdomains from hybrid aspen cells are involved in cell wall polysaccharide biosynthesis. Biochemical Journal 420, 93–103. Bhat RA, Miklis M, Schmelzer E, Schulze-Lefert P, Panstruga R. 2005. Recruitment and interaction dynamics of plant penetration resistance components in a plasma membrane microdomain. Proceedings of the National Academy of Sciences, USA 102, 3135–3140. Bohlenius H, Morch SM, Godfrey D, Nielsen ME, ThordalChristensen H. 2010. The multivesicular body-localized GTPase ARFA1b/1c is important for callose deposition and ROR2 syntaxindependent preinvasive basal defense in barley. The Plant Cell 22, 3831–3844. Bozkurt TO, Richardson A, Dagdas YF, Mongrand S, Kamoun S, Raffaele S. 2014. The plant membrane-associated REMORIN1.3 accumulates in discrete perihaustorial domains and enhances susceptibility to Phytophthora infestans. Plant Physiology 165, 1005–1018. Bradley DJ, Kjellbom P, Lamb CJ. 1992. Elicitor- and wound-induced oxidative cross-linking of a proline-rich plant cell wall protein: a novel, rapid defense response. Cell 70, 21–30. Caillaud MC, Wirthmueller L, Sklenar J, Findlay K, Piquerez SJ, Jones AM, Robatzek S, Jones JD, Faulkner C. 2014. The plasmodesmal protein PDLP1 localises to haustoria-associated membranes during downy mildew infection and regulates callose deposition. PLoS Pathogens 10, e1004496. Chinchilla D, Zipfel C, Robatzek S, Kemmerling B, Nurnberger T, Jones JD, Felix G, Boller T. 2007. A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence. Nature 448, 497–500. Chou CK. 1970. An electron-microscope study of host penetration and early stages of haustorium formation of Peronospora parasitica (Fr.) Tul. on cabbage cotyledons. Annals of Botany 34, 189–204. Cifuentes C, Bulone V, Emons AM. 2010. Biosynthesis of callose and cellulose by detergent extracts of tobacco cell membranes and quantification of the polymers synthesized in vitro. Journal of Integrative Plant Biology 52, 221–233. Collins NC, Thordal-Christensen H, Lipka V, et al. 2003. SNAREprotein-mediated disease resistance at the plant cell wall. Nature 425, 973–977. Eggert D, Naumann M, Reimer R, Voigt CA. 2014. Nanoscale glucan polymer network causes pathogen resistance. Scientific Reports 4, 4159. Ellinger D, Glockner A, Koch J, Naumann M, Sturtz V, Schutt K, Manisseri C, Somerville SC, Voigt CA. 2014a. Interaction of the Arabidopsis GTPase RabA4c with its effector PMR4 results in complete penetration resistance to powdery mildew. The Plant Cell 26, 3185–3200. Ellinger D, Naumann M, Falter C, Zwikowics C, Jamrow T, Manisseri C, Somerville SC, Voigt CA. 2013. Elevated early callose deposition results in complete penetration resistance to powdery mildew in Arabidopsis. Plant Physiology 161, 1433–1444. Ellinger D, Sode B, Falter C, Voigt CA. 2014b. Resistance of callose synthase activity to free fatty acid inhibition as an indicator of Fusarium head blight resistance in wheat. Plant Signaling and Behavior 9, (in press) Frei dit Frey N, Mbengue M, Kwaaitaal M, et al. 2012. Plasma membrane calcium ATPases are important components of receptormediated signaling in plant immune responses and development. Plant Physiology 159, 798–809. Gui J, Liu C, Shen J, Li L. 2014. Grain setting defect 1, encoding a remorin protein, affects the grain setting in rice through regulating plasmodesmatal conductance. Plant Physiology 166, 1463–1478. Him JL, Pelosi L, Chanzy H, Putaux JL, Bulone V. 2001. Biosynthesis of (1→3)-beta-d-glucan (callose) by detergent extracts of a microsomal fraction from Arabidopsis thaliana. European Journal of Biochemistry 268, 4628–4638. Jacobs AK, Lipka V, Burton RA, Panstruga R, Strizhov N, SchulzeLefert P, Fincher GB. 2003. An Arabidopsis callose aynthase, GSL5, is required for wound and papillary callose formation. The Plant Cell 15, 2503–2513. Keinath NF, Kierszniowska S, Lorek J, Bourdais G, Kessler SA, Shimosato-Asano H, Grossniklaus U, Schulze WX, Robatzek S, Panstruga R. 2010. PAMP (pathogen-associated molecular pattern)induced changes in plasma membrane compartmentalization reveal novel components of plant immunity. Journal of Biological Chemistry 285, 39140–39149. Kim H, O’Connell R, Maekawa-Yoshikawa M, Uemura T, Neumann U, Schulze-Lefert P. 2014. The powdery mildew resistance protein RPW8.2 is carried on VAMP721/722 vesicles to the extrahaustorial membrane of haustorial complexes. The Plant Journal 79, 835–847. Knauf GM, Welter K, Müller M, Mendgen K. 1989. The haustorial host– parasite interface in rust-infected bean leaves after high-pressure freezing. Physiological and Molecular Plant Pathology 34, 519–530. Koh S, Andre A, Edwards H, Ehrhardt D, Somerville S. 2005. Arabidopsis thaliana subcellular responses to compatible Erysiphe cichoracearum infections. The Plant Journal 44, 516–529. Membranes in defence | 1571 Kwon C, Neu C, Pajonk S, et al. 2008. Co-option of a default secretory pathway for plant immune responses. Nature 451, 835–840. Lee JY, Wang X, Cui W, et al. 2011. A plasmodesmata-localized protein mediates crosstalk between cell-to-cell communication and innate immunity in Arabidopsis. The Plant Cell 23, 3353–3373. Lu YJ, Schornack S, Spallek T, Geldner N, Chory J, Schellmann S, Schumacher K, Kamoun S, Robatzek S. 2012. Patterns of plant subcellular responses to successful oomycete infections reveal differences in host cell reprogramming and endocytic trafficking. Cellular Microbiology 14, 682–697. Macho AP, Zipfel C. 2014. Plant PRRs and the activation of innate immune signaling. Molecular Cell 54, 263–272. Markham JE, Molino D, Gissot L, Bellec Y, Hematy K, Marion J, Belcram K, Palauqui JC, Satiat-Jeunemaitre B, Faure JD. 2011. Sphingolipids containing very-long-chain fatty acids define a secretory pathway for specific polar plasma membrane protein targeting in Arabidopsis. The Plant Cell 23, 2362–2378. Men S, Boutte Y, Ikeda Y, Li X, Palme K, Stierhof Y-D, Hartmann M-A, Moritz T, Grebe M. 2008. Sterol-dependent endocytosis mediates post-cytokinetic acquisition of PIN2 auxin efflux carrier polarity. Nature Cell Biology 10, 237–244. Meyer D, Pajonk S, Micali C, O’Connell R, Schulze-Lefert P. 2009. Extracellular transport and integration of plant secretory proteins into pathogen-induced cell wall compartments. The Plant Journal 57, 986–999. Micali CO, Neumann U, Grunewald D, Panstruga R, O’Connell R. 2011. Biogenesis of a specialized plant–fungal interface during host cell internalization of Golovinomyces orontii haustoria. Cellular Microbiology 13, 210–226. Mims CW, Rodriguez-Lother C, Richardson EA. 2002. Ultrastructure of the host–pathogen interface in daylily leaves infected by the rust fungus Puccinia hemerocallidis. Protoplasma 219, 221–226. Mongrand S, Morel J, Laroche J, Claverol S, Carde JP, Hartmann MA, Bonneu M, Simon-Plas F, Lessire R, Bessoule JJ. 2004. Lipid rafts in higher plant cells: purification and characterization of Triton X-100insoluble microdomains from tobacco plasma membrane. Journal of Biological Chemistry 279, 36277–36286. Nielsen ME, Feechan A, Bohlenius H, Ueda T, Thordal-Christensen H. 2012. Arabidopsis ARF-GTP exchange factor, GNOM, mediates transport required for innate immunity and focal accumulation of syntaxin PEN1. Proceedings of the National Academy of Sciences, USA 109, 11443–11448. Nishimura MT, Stein M, Hou BH, Vogel JP, Edwards H, Somerville SC. 2003. Loss of a callose synthase results in salicylic acid-dependent disease resistance. Science 301, 969–972. Noirot E, Der C, Lherminier J, Robert F, Moricova P, Kieu K, Leborgne-Castel N, Simon-Plas F, Bouhidel K. 2014. Dynamic changes in subcellular distribution of the tobacco ROS-producing enzyme RBOHD in response to the oomycete elicitor cryptogein. Journal of Experimental Botany 65, 5011–5022. Nuhse TS, Bottrill AR, Jones AM, Peck SC. 2007. Quantitative phosphoproteomic analysis of plasma membrane proteins reveals regulatory mechanisms of plant innate immune responses. The Plant Journal 51, 931–940. Raffaele S, Bayer E, Lafarge D, et al. 2009. Remorin, a solanaceae protein resident in membrane rafts and plasmodesmata, impairs potato virus X movement. The Plant Cell 21, 1541–1555. Roberts AM, Mackie AJ, Hathaway V, Callow JA, Green JR. 1993. Molecular differentiation in the extrahaustorial membrane of pea powdery mildew haustoria at early and late stages of development. Physiological and Molecular Plant Pathology 43, 147–160. Segonzac C, Feike D, Gimenez-Ibanez S, Hann DR, Zipfel C, Rathjen JP. 2011. Hierarchy and roles of pathogen-associated molecular patterninduced responses in Nicotiana benthamiana. Plant Physiology 156, 687–699. Simons K, Gerl MJ. 2010. Revitalizing membrane rafts: new tools and insights. Nature Reviews. Molecular Cell Biology 11, 688–699. Srivastava V, Malm E, Sundqvist G, Bulone V. 2013. Quantitative proteomics reveals that plasma membrane microdomains from poplar cell suspension cultures are enriched in markers of signal transduction, molecular transport, and callose biosynthesis. Molecular and Cellular Proteomics 12, 3874–3885. Stein M, Dittgen J, Sanchez-Rodriguez C, Hou BH, Molina A, Schulze-Lefert P, Lipka V, Somerville S. 2006. Arabidopsis PEN3/ PDR8, an ATP binding cassette transporter, contributes to nonhost resistance to inappropriate pathogens that enter by direct penetration. The Plant Cell 18, 731–746. Ueda T, Uemura T, Sato MH, Nakano A. 2004. Functional differentiation of endosomes in Arabidopsis cells. The Plant Journal 40, 783–789. Underwood W, Somerville SC. 2013. Perception of conserved pathogen elicitors at the plasma membrane leads to relocalization of the Arabidopsis PEN3 transporter. Proceedings of the National Academy of Sciences, USA 110, 12492–12497. Vogel J, Somerville S. 2000. Isolation and characterization of powdery mildew-resistant Arabidopsis mutants. Proceedings of the National Academy of Sciences, USA 97, 1897–1902. Wang W, Wen Y, Berkey R, Xiao S. 2009. Specific targeting of the Arabidopsis resistance protein RPW8.2 to the interfacial membrane encasing the fungal haustorium renders broad-spectrum resistance to powdery mildew. The Plant Cell 21, 2898–2913. Wang X, Sager R, Cui W, Zhang C, Lu H, Lee JY. 2013. Salicylic acid regulates plasmodesmata closure during innate immune responses in Arabidopsis. The Plant Cell 25, 2315–2329. Yang L, Qin L, Liu G, Peremyslov VV, Dolja VV, Wei Y. 2014. Myosins XI modulate host cellular responses and penetration resistance to fungal pathogens. Proceedings of the National Academy of Sciences, USA 111, 13996–14001. Zhang J, Shao F, Li Y, et al. 2007. A Pseudomonas syringae effector inactivates MAPKs to suppress PAMP-induced immunity in plants. Cell Host and Microbe 1, 175–185.