Live and let die – Arabidopsis nonhost resistance to powdery mildews

ARTICLE IN PRESS European Journal of Cell Biology 89 (2010) 194–199

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Live and let die – Arabidopsis nonhost resistance to powdery mildews Ulrike Lipka a,b, Rene Fuchs b,c, Christine Kuhns a,b,c, Elena Petutschnig b,c, Volker Lipka a,b,c,n a b c

ZMBP Plant Biochemistry, University T¨ ubingen, Auf der Morgenstelle 5, D-72076 T¨ ubingen, Germany The Sainsbury Laboratory, John Innes Centre, Colney, Norwich NR4 7UH, UK Georg-August-University G¨ ottingen, Albrecht-von-Haller-Institute for Plant Sciences, Department of Plant Cell Biology, Untere Karsp¨ ule 2, D-37073 G¨ ottingen, Germany

a r t i c l e in f o

Keywords: Nonhost resistance Plant innate immunity Powdery mildew Arabidopsis PEN genes Lipase-like genes Pre-invasion resistance Post-invasion resistance

a b s t r a c t The term ‘‘nonhost resistance’’ (NHR) describes the phenomenon that an entire plant species is resistant to all genetic variants of a non-adapted pathogen species. In nature, NHR represents the most robust form of plant immunity and is therefore of scientific as well as economic importance. Due to its highly complex nature, NHR has previously not been studied in detail. Recently, the establishment of model interaction systems utilizing Arabidopsis and non-adapted powdery mildews allowed the identification of several key components and conceptual conclusions. It is now generally accepted that NHR of Arabidopsis to powdery mildews comprises two distinct layers of defence: pre-invasion entry control at the cell periphery and post-invasion resistance based on cell death execution. The timely production and localised discharge of toxic compounds at sites of fungal attack appear to be pivotal for entry control. This process requires proteins involved in secretion and trans-membrane transport, synthesis and activation of indolic glucosinolates as well as gene regulation and post-translational protein modification. Post-invasion defence relies on lipase-like proteins and salicylic acid signalling. To what extent pathogen-associated molecular pattern- or effector-triggered immunity contribute to NHR remains to be investigated and is likely to depend on the model system studied. & 2009 Elsevier GmbH. All rights reserved.

Introduction Plants are constantly exposed to a plethora of potential pathogens with different infection strategies and life styles. Nevertheless, disease represents the exception in natural plant communities. The reason is that most plants are immune to the majority of would-be pathogens and susceptible to only a relatively small number of adapted microbes. The phenomenon that an entire plant species is resistant to all genetic variants of a non-adapted pathogen species (or bacterial pathovar [pv] or fungal forma specialis [f.sp.]) is termed nonhost resistance (NHR) and defines the most robust form of plant immunity (ThordalChristensen, 2003; Mysore and Ryu, 2004; Nuernberger and Lipka, 2005). Thus, NHR delimits the host range of phytopathogenic microorganisms and impinges on pathogen radiation and speciation. The abilities to overcome NHR, to colonise a plant species and to reproduce represent the basic requirements for pathogen host plant adaptation and establishment of basic compatibility. It is conceivable that adapted pathogens must have evolved mechanisms to evade or suppress the basal defence machinery

n ¨ Corresponding author at: Georg-August-University Gottingen, Albrecht-vonHaller-Institute for Plant Sciences, Department of Plant Cell Biology, ¨ ¨ Untere Karspule 2, D-37073 Gottingen, Germany. Tel.: + 49 551 391 3581; fax: + 49 551 391 0406 E-mail address: [email protected] (V. Lipka).

0171-9335/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejcb.2009.11.011

of the plant and to manipulate plant cell functions for their own benefit. They do so by utilising a repertoire of effector molecules that target a variety of distinct plant mechanisms (reviewed in Speth et al., 2007). However, infrequent historical host range shifts are indicative of the generally robust and durable nature of NHR (Heath, 2000; Nuernberger and Lipka, 2005). Consequently, mechanistic insights into NHR are considered to provide valuable clues to develop novel and promising strategies for crop plant disease control (Ellis, 2006). Stability of NHR has been proposed to be the consequence of several successive layers of protective mechanisms that include both constitutive barriers as well as inducible reactions (Thordal-Christensen, 2003; Nuernberger and Lipka, 2005). Because of its presumed multifactorial and complex nature, genetic dissection of NHR has not been attempted in the past. Despite these challenges, the successful establishment of genetically tractable nonhost model systems recently shed first light on the mechanisms of NHR to fungal and bacterial microbes. These ground-breaking discoveries provide first answers to the following questions that we will discuss further below: How many layers of defence account for NHR to non-adapted pathogens? What are the executive mechanisms that cause pathogen growth arrest? Preformed physical and chemical barriers like a rigid cell wall and toxic phytoanticipins are well known to be critical for invasion success of some non-adapted parasites (Thordal-Christensen, 2003; Nuernberger and Lipka, 2005). If a potential pathogen manages to

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overcome these constitutive defensive layers it faces the risk of recognition and activation of inducible plant defence reactions. It is now generally accepted that inducible components of NHR and basal resistance (which defines the ability of the plant to reduce the severity of disease caused by adapted pathogens) both involve recognition of slowly evolving microbial- or pathogen-associated molecular patterns (MAMPS or PAMPs) by transmembrane pattern recognition receptors (PRRs) (Schwessinger and Zipfel, 2008). Downstream cell-autonomous responses of PAMP-triggered immunity (PTI) include MAP kinase signalling, production of reactive oxygen species (ROS), NO and ethylene, ion fluxes, the transcriptional induction of pathogenesis-related (PR) genes, protein phosphorylation and callose deposits (Bittel and Robatzek, 2007). In contrast to cultivar-specific effector-triggered host plant immunity (ETI), which is mediated by resistance (R) proteins, NHR often (but not always) terminates pathogenesis of invaders without a hypersensitive cell death response (HR) (Mysore and Ryu, 2004). Chisholm et al. (2006) and Jones and Dangl (2006) recently proposed a conceptual framework for plant immunity evolution which suggests a sequential interplay of slowly evolving PTI, effector-triggered susceptibility (ETS) and comparatively rapidly evolving ETI. Referring to this concept, NHR can be considered a consequence of ineffective microbial effectors, resulting in no suppression of PTI. Alternatively, (individual) effectors might not have been selected to evade recognition and could thus be recognised in nonhost plants, resulting in ETI. These basic considerations provoke a storm of questions addressing the nature of NHR in an evolutionary context: Do both PTI (PRRs) and ETI (R-proteins) contribute to NHR? Is the remarkable efficiency and stability of NHR the consequence of multiple signal inputs and synergistic defence outputs? Does microbe invasion also trigger mechanical stimuli that contribute to NHR? Is NHR an ancient, static and non-adaptive trait or subject to evolutionary selection? What role does absence of matching plant compatibility factors and/or effector targets play? Do universal and basic effector targets exist? How many effectors (or sufficiently manipulated effector targets) does it take to convert a non-adapted into a successful pathogen (or a nonhost into a host plant)? In this review, we summarise insights into the function of genetically defined NHR components recently identified in a nonhost model system utilising interactions between Arabidopsis and non-adapted powdery mildew fungi. Moreover, we discuss conceptual implications and try to answer some of the questions raised above.

‘‘Live and let die’’ or ‘‘Live and let live’’ – Arabidopsis NHR and compatibility to powdery mildews Comparative cytological analyses of Arabidopsis inoculations with non-adapted and adapted powdery mildew species recently provided some conceptual clues about the differences between nonhost and host interactions. Unlike compatible powdery mildews such as Golovinomyces orontii (Go), which readily invades Arabidopsis (approximately 70% entry rate within 24 h post inoculation (hpi)), the barley and pea powdery mildews Blumeria graminis f.sp. hordei (Bgh) and Erysiphe pisi (Ep) fail to penetrate the majority of attacked epidermal pavement cells (approximately 95 and 75%, respectively; Lipka et al., 2005). Entry failure correlates with timely and localised defence responses, such as dynamic cytoskeletal rearrangements, organelle transport, protein translocation, secretion processes and focal cell wall remodelling (i.e. formation of multi-layered, callose-containing papilla (Fig. 1)) at sites of attempted fungal ingress (Hueckelhoven, 2007; Hardham et al., 2007). These pre-invasion

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Fig. 1. Pre- and post-invasion defence mechanisms both contribute to Arabidopsis nonhost resistance to non-adapted powdery mildews. Aniline blue fluorochrome staining of Arabidopsis leaves inoculated with barley powdery mildew spores (sp) reveals focal callose accumulation at attempted fungal invasion sites (red arrowheads) and whole cell fluorescence (yellow arrowheads), indicative of epidermal cell death, in case of fungal entry. The sample was taken 48 h post inoculation. Fungal structures were stained with Coomassie Blue. Bar: 50 mm.

defence mechanisms are backed up by post-invasion resistance: at the few successful Bgh and Ep entry sites (approximately 5 and 25%, respectively) haustoria become encased in callose, and attacked epidermal cells undergo an HR-like cell death, which is accompanied by microscopically detectable whole-cell autofluorescence (Fig. 1). As powdery mildews are obligate biotrophic phytopathogens that exclusively feed on living epidermal cells, cell death ultimately terminates any further fungal development and prohibits colonisation. Consequently, efficient invasion and cell death suppression are the hallmarks of compatible and co-evolved biotrophic interactions. It is conceivable that adapted powdery mildews employ effector molecule transfer to interfere with both pre- and post-invasion defence mechanisms and to establish basic compatibility (reviewed by OConnell and Panstruga (2006)). Conversely, lack of co-evolution might result in production of effector variants that are either non-functional or subject to recognition, or, nonadapted isolates might even entirely lack the required effector repertoire or host-specific transport machinery. Which of these alternative scenarios actually accounts for NHR, is thus likely to depend on the extent of ecological co-evolution of the plant– microbe interaction partners under study.

Marshalling the troops – Powdery mildew entry control Genetic screens for chemically induced Arabidopsis mutants with altered nonhost interactions upon Bgh inoculation were recently used to molecularly dissect pre- and post-invasion defence mechanisms. These efforts allowed the identification of three genes PENETRATION1 (PEN1), PEN2 and PEN3 (Collins et al., 2003; Lipka et al., 2005; Stein et al., 2006), that limit entry success of non-adapted powdery mildews. Single mutants of these genes exhibit several-fold enhanced invasion frequencies of Bgh and Ep, but no increase in overall susceptibility due to concomitant HRlike cell death of invaded epidermal cells. Systematic gene interaction analyses suggest that PEN1 and PEN2 act in two distinct entry control mechanisms and that PEN2 co-operates

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with PEN3 (Lipka et al., 2005; Stein et al., 2006). These findings are further substantiated by the fact that pen1 mutations exclusively affect interactions with non-adapted powdery mildews (Bgh and Ep), whereas pen2 and pen3 mutants also show enhanced entry of the non-adapted hemibiotrophic oomycete Phytophthora infestans and impaired basal resistance to the adapted powdery mildews Go and Golovinomyces cichoracearum (Gc), and to the adapted necrotrophic ascomycete Plectosphaerella cucumerina (Lipka et al., 2005; Stein et al., 2006). PEN1 encodes a plasma membrane-anchored syntaxin with a SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) domain (Collins et al., 2003). SNARE domaincontaining proteins are key players in vesicle-associated membrane fusion and secretion processes, including exocytosis and endocytosis (reviewed in Lipka et al., 2007). The presence of a functional homologue (REQUIRED FOR MLO RESISTANCE 2 (ROR2)) in the distantly related monocot species barley supports the idea that PEN1-mediated defence represents an evolutionarily ancient mechanism (Collins et al., 2003). Cell biological analyses with functional GFP-PEN1 fusions demonstrated a pathogeninduced accumulation in lipid raft-like plasma membrane microdomains at sites of attempted ingress by powdery mildews (Assaad et al., 2004; Bhat et al., 2005) (Fig. 2) but not by adapted or non-adapted Colletotrichum species (Shimada et al., 2006). Moreover, these experiments suggested a role of the PEN1 gene product for timely secretion and cell wall remodelling processes (Assaad et al., 2004; Bhat et al., 2005). In-depth cell biological analyses further expanded that model and revealed a potential involvement of multivesicular bodies and exosomes in PEN1mediated secretion (Meyer et al., 2009). Moreover, genetic evidence for direct or indirect PEN1 repressor activity of salicylic acid (SA)-dependent defence responses recently added another level of complexity to PEN1 function (Zhang et al., 2007). Kwon et al. (2008a) could show that PEN1 forms a pathogeninduced ternary complex with the adaptor SNARE protein SNAP33 and two endomembrane compartment-associated SNARE proteins, VESICLE-ASSOCIATED MEMBRANE PROTEIN (VAMP) 721 and VAMP722. Moreover, transgenic expression of functional GFP-VAMP722 fusions driven by endogenous promoter sequences revealed an inducible cell autonomous expression in epidermal pavement cells that are under powdery mildew attack. In addition, the GFP signals tag endomembrane compartments that move along defined routes to sites of attempted fungal penetration. These experiments provide the

first evidence for SNARE complex formation in plants and, in analogy to yeast and mammal model systems, corroborate a likely role of PEN1, SNAP33 and VAMP721/722 in pathogen-induced exocytotic secretion. Recent structure–function analyses using Nterminal phosphorylation variants of the PEN1 protein revealed the importance of particular amino acid residues for in vivo (but not in vitro) function of PEN1 (Pajonk et al., 2008). Whether this is the consequence of altered interactions with yet to be identified regulatory factors or altered kinetics of SNARE complex formation and/or disassembly remains to be shown. Pajonk et al. (2008) carried out expression analyses with chimeric domain swapping constructs in which 175 N-terminal PEN1 residues were substituted by corresponding sequences of its closest homolog SYNTAXIN OF PLANTS 122 (SYP122). These experiments suggested structural adaptations for functionality in disease resistance but also for developmental aspects of plant biology (Pajonk et al., 2008). Likewise, VAMP721 and VAMP722 have a second functionally redundant role in a default secretory pathway, suggesting that an ancient transport mechanism involving these VAMP proteins was co-opted to form part of a secretory plant immune response (Kwon et al., 2008a). Homo-/ heterozygous knockout mutant combinations of vamp721 and vamp722 result in haploinsufficient nonhost and basal resistance to Ep, Go and the adapted oomycete Hyaloperonospora arabidopsidis (Ha) (Kwon et al., 2008a). Intriguingly, VAMP721 appears to contribute considerably more to basal resistance towards Ha than VAMP722, whereas the opposite holds true for pre-invasion NHR to Ep (Kwon et al., 2008a). This demonstrates discrete potentials to promote disease resistance against different pathogen classes which could be the consequence of distinct tissue specificity of Ep and Ha infections (i.e. epidermis and mesophyll, respectively) and possibly corresponding differential VAMP721/722 expression levels. Alternatively (or additionally), VAMP721 and 722 could localise to distinct endomembrane compartments harbouring secretory cargo with divergent specificity. Ultimately, this conundrum can only be resolved by cell biological studies and comparative proteome/metabolome analyses with purified VAMP721/722-tagged secretory membrane compartments. PEN2 encodes one out of 48 predicted Arabidopsis family 1 glycoside hydrolases (F1GHs) (Lipka et al., 2005). F1GHs are required for the enzymatic activation of small molecules from inactive glycosidic precursor metabolites and play diverse roles in plant biology (Xu et al., 2004). Catalytic activity was shown to be

Fig. 2. PEN proteins mediate entry control at the cell periphery. Confocal imaging of functional GFP-fusion proteins expressed in transgenic Arabidopsis plants reveals that PEN gene products are subject to pathogen-induced cell polarisation. (A) Focal accumulation of GFP-labelled Qa-SNARE PEN1 in a plasma membrane microdomain at a contact site with barley powdery mildew 22 h post inoculation. Note the fluorescence signal depletion in rest of the attacked cell. (B) Peroxisome-associated PEN2-GFP fusions concentrate at an incipient fungal invasion site 16 h post inoculation. Fungal structures were stained with FM 4-64. Bars: 25 mm (A); 10 mm (B). sp = spore. ap= appressorium.

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required for PEN2 function in powdery mildew entry control, suggesting that the PEN2 product(s) may have direct or indirect antifungal activity (Lipka et al., 2005). Indeed, two recent papers report a novel glucosinolate metabolism pathway in which PEN2 myrosinase activity is required for activation of toxic tryptophanderived indolics (Bednarek et al., 2009; Clay et al., 2009). The Arabidopsis cytochrome P450 monooxygenase CYP81F2 is a central component of this pathway as it is required for pathogen-induced accumulation of the PEN2 substrate 4-methoxyindol-3-ylmethylglucosinolate. In addition, Clay et al. (2009) claim that PEN2-mediated glucosinolate hydrolysis is also required for flg22-elicited callose deposition in Arabidopsis seedlings, suggesting a signalling function of PEN2 hydrolysis products for callose synthase activation. However, mature pen2 knockout mutant leaves exhibit hyper-accumulation of callose at sites of attempted powdery mildew invasion (V. Lipka, unpublished results) with no obvious negative effect on invasion success (Lipka et al., 2005). Thus, callose deposition is neither abolished in pen2 mutants nor per se a potent entry control mechanism, which also is reflected in only marginally enhanced penetration rates of non-adapted powdery mildews on pmr4/gsl5 callose synthase mutants (Jacobs et al., 2003). Consequently, the observed lack of flg22-induced callose deposition in pen2 mutants is likely to be either seedlingspecific and/or restricted to applications of individual PAMPs. Fluorescence labelling revealed an association of the PEN2 protein with the periphery of peroxisomes, which are subject to pathogen-induced cell polarisation (Fig. 2). Oxidative stress, wounding and pathogen attack are known to induce gene expression required for peroxisome biogenesis (Lo´pez-Huertas et al., 2000), which suggests pathogen-responsive peroxisome proliferation or de novo biogenesis. In addition intracellular transport of plant peroxisomes is known to occur along actin filaments (Jedd and Chua, 2002; Collings et al., 2002). Genetic interference with actin dynamics has recently confirmed that pathogen-induced actin cytoskeleton reorganisation plays a major role in efficient pre-haustorial NHR to powdery mildews (Miklis et al., 2007). Thus, production of PEN2 substrate(s) in functionally specialised peroxisomes (the subcellular localisation of CYP81F2 has not been reported yet!), cytoskeleton-mediated delivery to fungal entry sites and PEN2-mediated glucosinolate hydrolysis represent a potential underlying mechanism to accomplish high local concentrations of toxic end products. Phylogenetic analyses suggest that the PEN2 gene represents an evolutionarily recent acquisition of the Arabidopsis genome (Consonni et al., 2006) and that its gene product is only present in close relatives of Arabidopsis thaliana (Christine Kuhns and Volker Lipka, unpublished results). These findings demonstrate that NHR is not an exclusively ancient, fixed and non-adaptive trait and might also explain why pen2 mutations affect a wide range of pathogen interactions. PEN3 encodes one of the 15 pleiotropic drug resistance (PDR) ATP-binding cassette (ABC) transporters present in the Arabidopsis genome (Stein et al., 2006). ABC transporters are ubiquitous transmembrane proteins that function in the ATP-dependent transport of a wide variety of substrates across extra- and intracellular membranes (Crouzet et al., 2006). Like PEN1, PEN3 resides in the plasma membrane and accumulates at sites of attempted fungal penetration (Stein et al., 2006). Interestingly, lack of PEN3 results in hyper-activation of SA-dependent defence signalling pathways which explains the initially surprising enhanced basal resistance of pen3 mutants to adapted powdery mildews such as Gc and Go. Intriguingly, this phenotype is significantly attenuated in pen2 pen3 double mutants. One possible explanation is that PEN2 and PEN3 act concertedly in the localised activation and energy-dependent transport of toxic compounds into the plant apoplast at interaction sites with

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invading fungi. In the absence of PEN2, its toxic product would no longer accumulate inside pen3 mutant cells and thus not unintentionally activate SA-dependent hypersensitive responses. The two distinct pre-haustorial nonhost defence mechanisms (defined by PEN1/SNAP33/VAMP721/VAMP722 and PEN2/PEN3, respectively) have in common that they are both subject to pathogen-induced cell polarisation (Fig. 2). This highly organised battle array and the timely commitment of forces is strikingly reminiscent of the execution of immune responses at the immunological synapse in vertebrate T cells (reviewed in Kwon et al., 2008b). Thus, plant innate immunity and vertebrate adaptive immunity feature mechanistic parallels that are worth to be further explored. Interestingly, all PEN genes identified so far are transcriptionally induced by the bacterial PAMP flg22 (Zipfel et al., 2004). This supports the idea that PAMPs activate a general suite of executive NHR mechanisms irrespective of the potential intruder, and that perception of fungal PAMPs is likely to induce PEN gene transcription in powdery mildew interactions. Indeed, chitininduced transcriptional activation of the PEN genes is impaired in Arabidopsis knockout mutants of the LysM receptor-like kinase CERK1 (Cyril Zipfel, Elena Petutschnig, Volker Lipka, unpublished), which is required for perception of the fungal PAMP chitin and resistance to fungal pathogens (Miya et al., 2007; Wan et al., 2008), but also for basal resistance to bacterial pathogens (Gimenez-Ibanez et al., 2009). Additionally, attempted microbe invasion generates endogenous plant (self) signals or mechanical stimuli which could potentially contribute to the activation of pre-invasion defence mechanisms. NHR might therefore be the consequence of multiple signal inputs and a synergistic defence output. Broad-spectrum resistance to adapted powdery mildews is conferred by loss-of-function mutant alleles of MILDEW RESISTANCE LOCUS O (MLO) genes in both barley (HvMlo) and Arabidopsis (AtMlo2, AtMlo6, AtMlo12) (Consonni et al., 2006). Powdery mildew fungi are believed to require a subset of functional MLO proteins as compatibility factors (‘‘entry portals’’) for successful invasion of host epidermis cells. Lack of these likely powdery mildew effector targets results in efficient pre-invasion resistance which shares many characteristics with NHR to powdery mildews (reviewed in Humphry et al., 2006). Intriguingly, mlo resistance in Arabidopsis requires all PEN genes described so far (Consonni et al., 2006). Moreover, recent experiments showed that transient overexpression of constitutively active variants of a calcium-dependent protein kinase (CDPK) affects both mlo and NHR in barley (Freymark et al., 2007). Together, these findings imply that powdery mildew fungi utilise a common and evolutionarily old host cell entry mechanism (dependent on wild-type MLO) and that mlo resistance represents a re-establishment of pre-invasion NHR. Interestingly, expression of all PEN genes and of other genes with function in powdery mildew entry control, such as VAMP722, SNAP33, CYP81F2, MLO2, MLO6 and MLO12, is specifically enriched in a developmentally defined cellular cluster of the Arabidopsis root (i.e., LOCALISED EXPRESSION DOMAIN 6 (LED6); Birnbaum et al., 2003). This might either reflect that the corresponding root domain is the prime target for soil-borne pathogen invasion and/ or corroborate the earlier notion that PEN genes have a dual function in plant development and innate immunity. Other components that were recently described to contribute to Arabidopsis pre-invasion NHR against non-adapted powdery mildews include the S-nitrosoglutathione reductase GSNOR1 and the plant transcription factor ATAF1 (Feechan et al., 2005; Jensen et al., 2007). Although the entry success of non-adapted powdery mildews on ataf1-1 and atgsnor1-3 mutants was only moderately enhanced (from 9% to 14% and from 1% to 3%, respectively) these

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results suggest both transcriptional and post-transcriptional regulation of penetration resistance.

To take one for the team – Post-invasion defence Arabidopsis mutants allowing enhanced entry of non-adapted powdery mildew fungi are however still nonhost plants for these pathogens due to effective post-entry cell death execution. Systematic mutant combination analyses revealed that posthaustorial NHR is controlled by ENHANCED SUSCEPTIBILITY 1 (EDS1), PHYTOALEXIN DEFICIENT 4 (PAD4) and SENESCENCE ASSOCIATED GENE 101 (SAG101) (Lipka et al., 2005; Stein et al., 2006). These lipase-like proteins constitute a regulatory node that is essential for basal defence against Ha, SA-mediated signalling and R-gene mediated resistance pathways (reviewed by Wiermer et al. (2005)). EDS1, PAD4 and SAG101 single mutants have no or only a minor effect on pre-invasion resistance to powdery mildews. However, at successful invasion sites, HR is less frequent and allows ectoparasitic secondary hyphal growth and microcolony formation of Bgh and Ep (Lipka et al., 2005; Stein et al., 2006). Intriguingly, NHR to Ep, but not to Bgh, is fully compromised on pen2 pad4 and pen3 eds1 double mutants, indicated by occasional conidiophore formation. On pen2 pad4 sag101 triple mutants, Bgh is also capable to complete its life cycle, whereas Ep growth and reproduction is now even macroscopically detectable and indistinguishable from infections with adapted powdery mildews. Thus, removal of only 3 genes is sufficient to make Arabidopsis a fully susceptible host plant for the non-adapted pea powdery mildew and to allow the monocot pathogen Bgh to establish basic compatibility. This is remarkable, as it suggests that both pathogens do not lack any principal adaptations to establish biotrophic interactions and to complete their life cycles, as soon as the plant basal defence machinery breaks down. The quantitative differences in virulence between Ep and Bgh presumably reflect the fact that Arabidopsis is more closely related to pea than to the monocot barley. Consequently, Ep is likely to harbour an effector complement that is more similar to powdery mildews infecting Arabidopsis, such as Gc, Go and Erysiphe cruciferarum. Comparative powdery mildew effector studies are urgently required to unequivocally resolve this issue. In summary, Arabidopsis NHR to non-adapted biotrophic powdery mildews is based upon two successive, multi-component and independently effective defence systems: PEN gene-mediated preinvasion resistance and EDS1/PAD4/SAG101 controlled postinvasion immunity. We already mentioned above that the EDS1/PAD4/SAG101 node is essential for a subset of R gene-mediated defence pathways. Thus, it was obvious to test whether or not R-genemediated ETI contributes to post-invasion resistance against Bgh and Ep. The proteins REQUIRED FOR MLA12 RESISTANCE (RAR1) and SUPPRESSOR OF G2 ALLELE OF SKP1 (SGT1) function as cofactors in HEAT SHOCK PROTEIN 90 (HSP90)-mediated stabilisation of R-protein complexes and represent a genetic convergence point for EDS1/PAD4/SAG101 and other R gene signalling pathways (Azevedo et al., 2006; Noel et al., 2007). Double and triple mutant combinations of either pen2 or pen3 with rar1 and sgt1b had a significantly weaker effect on colonisation success of Bgh or Ep than combinations with eds1, pad4 and sag101. Also, they did not allow fungal life cycle completion (unpublished results and (Stein et al., 2006)). These finding suggest that R genes play a minor if any role in post-haustorial Arabidopsis NHR to the tested non-adapted powdery mildews. Perhaps such a scenario is more likely in interactions where would-be pathogen and nonhost plants share more recent compatible ancestry (e.g. in barley/wheat interactions with the wheat/barley powdery mil-

dew; reviewed by Schweizer (2007)) or where a brisk intra- or interspecific transfer of effector molecules determines the host range of a pathogen. Horizontal gene transfer of individual genes has recently been described for fungal plant pathogens (Friesen et al., 2006; Richards et al., 2006). However, its role as a major driving force for host specialisation is generally better established and accepted for prokaryotic pathogens. Recent comparative genomics studies suggest that interspecific horizontal gene transfer-mediated acquisition of virulence-associated genes such as type III effectors controls host specificity of the approximately 50 different pathovars of Pseudomonas syringae (Sarkar et al., 2006). Analogous studies with a diverse set of powdery mildew species (together with comprehensive comparative genome analysis of their respective host and nonhost plants) are urgently required to understand how reciprocal selection shapes the interactions between plants and their powdery mildew pathogens.

Conclusions Entry control and post-invasion defence mechanisms appear to be pivotal components of NHR to non-adapted pathogens. However, the actual mechanisms by which a plant species detects and contains non-adapted would-be pathogens undoubtedly vary greatly with the model system and, most importantly, the evolutionary relationship of the interaction partners. Thus, NHR eludes modelling by a single overarching and universally valid concept. This is also illustrated by the fact that NHR can be monogenic, as recently demonstrated for homologs of the maize Hm1 gene which are exclusively conserved in the grass lineage and are suggested to have provided evolutionarily stable NHR to the hemibiotrophic fungal maize pathogen Cochliobolus carbonum (Sindhu et al., 2008). In the near future, insights from other nonhost model systems that are currently under investigation will help to further define the genetic and mechanistic requirements for plant NHR. They will also reveal to what extent molecular mechanisms and elements are shared among plant species and how virulent pathogens managed to overcome them. In the long run, transfer of knowledge obtained from these studies may allow engineering agronomically important crops with durable disease resistance.

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