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MOLECULAR PLANT PATHOLOGY (2009) 10(4), 547–562 DOI: 10.1111/J.1364-3703.2009.00547.X Review The zig-zag-zig in oomycete–plant interactions INGO HEIN 1 , ELEANOR M. GILROY 2 , MILES R. ARMSTRONG 2 AND PAUL R. J. BIRCH 2,3, * 1 Genetics Programme, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK Plant Pathology Programme, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK 3 Division of Plant Science, College of Life Sciences, University of Dundee (at SCRI), Invergowrie, Dundee DD2 5DA, UK 2 SUMMARY In addition to a range of preformed barriers, plants defend themselves against microbial invasion by detecting conserved, secreted molecules, called pathogen-associated molecular patterns (PAMPs). PAMP-triggered immunity (PTI) is the first inducible layer of plant defence that microbial pathogens must navigate by the delivery of effector proteins that act to suppress or otherwise manipulate key components of resistance. Effectors may themselves be targeted by a further layer of defence, effector-triggered immunity (ETI), as their presence inside or outside host cells may be detected by resistance proteins. This ‘zig-zag-zig’ of tightly co-evolving molecular interactions determines the outcome of attempted infection. In this article, we consider the complex molecular interplay between plants and plant pathogenic oomycetes, drawing on recent literature to illustrate what is known about oomycete PAMPs and elicitors of defence responses, the effectors they utilize to suppress PTI, and the phenomenal molecular ‘battle’ between effector and resistance (R) genes that dictates the establishment or evasion of ETI. INTRODUCTION Above and below ground, plants face a constant barrage of invading microorganisms, including bacteria, fungi and oomycetes. Yet, only a small percentage of plant–pathogen interactions lead to successful disease development, as the majority of plant species are resistant to invasion by all isolates of any given microbial species. Pathogen host range is thought to be limited initially by the ability of a microbe to penetrate preformed barriers, such as the cuticle and cell wall, and to detoxify constitutively accumulating host antimicrobials (phytoanticipins) which vary between plant species (Ingle et al., 2006). A pathogen that overcomes these obstructions will encounter the plant non-self surveillance system that perceives attempted invasions, *Correspondence: E-mail: [email protected] © 2009 THE AUTHORS JOURNAL COMPILATION © 2009 BLACKWELL PUBLISHING LTD activating a diverse array of effective, broad-range defences (Ingle et al., 2006; Zipfel, 2008). Perception initially involves the detection of conserved molecules (microbe- or pathogenassociated molecular patterns, MAMPs or PAMPs) that are secreted or displayed on the surface of microorganisms. PAMPtriggered immunity (PTI) constitutes a front-line defence that must be overcome by microorganisms for successful colonization of plant tissues (Jones and Dangl, 2006; Zipfel, 2008). This is achieved by the secretion of virulence determinants, called effectors, which act either outside or inside the host cell to suppress or otherwise manipulate plant innate immunity. Effectortriggered susceptibility (ETS) includes the suppression of PTI, representing the first level at which molecular co-evolution between host and pathogen occurs, as pathogen effectors have increasingly been shown to suppress immunity via direct molecular interactions with host defence-associated proteins (Block et al., 2008; Chisholm et al., 2006; Grant et al., 2006; Jones and Dangl, 2006). Plants have a second line of defence, in the form of resistance (R) proteins, the products of which directly or indirectly detect effectors (termed avirulence proteins; AVRs), and confer immunity to pathogens that are successful in suppressing PTI. Effectortriggered immunity (ETI) represents a second level at which host–pathogen molecular co-evolution occurs, as effectors evolve to evade detection and R proteins evolve to establish or retain detection (Jones and Dangl, 2006). In this review, we consider the complex molecular interactions between plants and plant pathogenic oomycetes, drawing on recent literature to illustrate what is known about oomycete PAMPs and the effectors they utilize to suppress host immune systems. We also consider the remarkable molecular evolutionary ‘battle’ between effectors, their targets in the host and R proteins, which dictate whether ETI is activated or evaded (Fig. 1). Plant pathogenic oomycetes Oomycetes are a diverse group of organisms that morphologically resemble fungi, yet are members of the chromista, more closely related to key organisms in aquatic environments, such as brown algae (e.g. kelp), golden-brown algae and diatoms. The 547 548 I. HEIN et al. Fig. 1 The zig-zag-zig in oomycete–plant interactions (modified from Jones and Dangl, 2006). Shown are the characterized oomycete pathogen-associated molecular patterns (PAMPs) and other elicitors of PAMP-triggered immunity (PTI) and necrosis [represented by a dotted arrow extending PTI beyond the threshold for host programmed cell death (PCD)]; examples of oomycete effectors that contribute to effector triggered susceptibility (ETS); and examples of host resistance proteins that detect oomycete effectors to trigger immunity (ETI). The amplitude of defence is shown on the y axis, and the threshold for activation of host PCD is also indicated. CBEL, cellulose-binding elicitor lectin; CRN, crinkling and necrosis; NLP, Nep1-like protein; PRR, pattern recognition receptor; R, resistance; SCR, small cysteine-rich. oomycetes include a diverse range of free-living water moulds, as well as pathogens of plants, algae, insects, fish, crustaceans, mammals and various microbes, including fungi (Kamoun, 2003, 2006). Plant pathogenic oomycetes cause devastating diseases of crop, ornamental and native species and are arguably the most important pathogens of dicotyledenous plants. The most damaging groups comprise more than 80 species of Phytophthora (Blair et al., 2008), several genera of the downy mildews (the most important being Peronospora, Plasmopora and Bremia) (Göker et al., 2006) and more than 110 species of Pythium (Levesque and de Cock, 2004). There is great diversity in oomycete infection strategies, from largely opportunistic or weakly pathogenic necrotrophs with wide host ranges, typified by many soil-borne Pythium species, to the highly specialized, biotrophic, aerially disseminated, foliar downy mildews. The genus Phytophthora spans these two extremes of lifestyle, including species specialized in colonizing leaf litter to a range of highly specialized pathogenic hemibiotrophs. A common feature, however, is the absolute dependence on living plant tissue to complete their life cycle. Oomycetes undergo a series of developmental stages throughout a successful infection cycle, including the formation of sporangia, release of motile zoospores, their encystment and germination to form hyphae and appressoria, the production of primary and secondary infection hyphae, haustoria and, finally, sporangiophores (Birch and Cooke, 2004). These various developmental processes facilitate dispersal, recognition of the host, adhesion, penetration and colonization, encompassing biotrophic and/or necrotrophic phases of infection, finally leading once again to dispersal. Three plant pathogenic oomycetes, in particular, can be considered as models to study the molecular co-evolution with the host. The soybean pathogen, Phytophthora sojae, invades and colonizes roots, whereas the late blight pathogen of potato and tomato is an aerially disseminated, foliar pathogen. Both are hemi-biotrophs. In contrast, the downy mildew Hyaloperonospora arabidopsidis (formerly Peronospora parasitica) is an obligate biotrophic pathogen of the model plant Arabidopsis. The genomes of all three have been sequenced (Lamour et al., 2007; Tyler et al., 2006), which will help to reveal the full array and diversity of proteinaceous PAMPs and effectors that trigger and/or suppress the plant immune system. Examples will be drawn from each to illustrate the challenges faced by oomycetes in establishing infection, and in the molecular co-evolution that dictates the outcome of their interactions with plants. OOMYCETE PAMPS AND OTHER ELICITORS OF PLANT IMMUNITY All classes of microorganism possess characteristic structural and enzymatic proteins so essential for fitness that they are indispensable. Some act as PAMPs, recognized as non-self molecules by distinct pattern recognition receptors (PRRs) in plants in a manner analogous to the Toll- and Toll-like family of receptors of the mammalian innate immune system (Zipfel, 2008). The consequent PTI comprises a range of universal responses, including ion fluxes, mitogen-activated protein kinase (MAPK) cascades, production of reactive oxygen species (ROS), cell wall reinforcement and rapid induction of defence genes, largely regulated by WRKY transcription factors (Ingle et al., 2006). In order to infect a plant host, successful pathogens must evade recognition or evolve effectors, some capable of entering the © 2009 THE AUTHORS MOLECULAR PLANT PATHOLOGY (2009) 10(4), 547–562 JOURNAL COMPILATION © 2009 BLACKWELL PUBLISHING LTD Zig-zag-zig in oomycete–plant interactions 549 Table 1 Selected oomycete secreted proteins that trigger and/or suppress plant immune responses. Secreted oomycete protein Acts as a PAMP or elicitor of necrosis Elicits ETI Possible contribution to virulence GP42 (transglutaminase family) Pep-13 PAMP No None CBEL family Cellulose binding domain PAMP No Adhesion to the plant cell INF1 (member of elicitin family) Activates PTI and necrosis. INF1-dependent ROS production requires SERK3/BAK1 Triggers PTI and necrosis No None No PcF triggers necrosis and defence proteins Triggers necrosis No Unknown. Evidence of diversifying selection Unknown Unknown Facilitates switch from biotrophy to necrotrophy? Unknown No Unknown Inhibits host protease P69B No Unknown RXLR effector AVR3a No R3a-dependent RXLR effector Avr1b No RPS1b-dependent RXLR effector ATR1 No RPP1-dependent Inhibits host papain proteases PIP1 and RCR3 Suppresses INF1-mediated cell death Suppresses BAX-dependent cell death Suppresses PTI RXLR effector ATR13 No RPP13-dependent Suppresses PTI NLP family PcF, SCR74 and SCR91 Crinkling and necrosis (Crn) family Glucanase inhibitors GIP1 and GIP2 EPI1 (member of Kazal-like protease inhibitor family) EPIC2B (member of cystatin family) Unknown Inhibits host glucanases Selected references Nürnberger et al. (1994); Brunner et al. (2002) Gaulin et al. (2006); Dumas et al. (2008) Kamoun (2006); Heese et al. (2007) Fellbrich et al. (2002); Qutob et al. (2006). Orosomando et al. (2001); Liu et al. (2005) Torto et al. (2003) Rose et al. (2002); Damasceno et al. (2008) Tian et al. (2004) Tian et al. (2007); Song et al. (2009) Armstrong et al. (2005); Bos et al. (2006) Shan et al. (2004); Dou et al. (2008b) Rehmany et al. (2005); Sohn et al. (2007) Allen et al. (2004); Sohn et al. (2007) CBEL, cellulose-binding elicitor lectin; ETI, effector-triggered immunity; NLP, Nep1-like protein; PAMP, pathogen-associated molecular pattern; PIP1, Phytophthorainhibited protease 1; PTI, PAMP-triggered immunity; ROS, reactive oxygen species; SCR, small cysteine-rich; SERK3/BAK1, SOMATIC EMBRYOGENESIS RECEPTOR KINASE 3/brassinosteroid (BR)-associated kinase 1. host cell, to suppress key modulators of the downstream signalling cascade of PTI (Zipfel, 2008). A number of microbial PAMPs known to elicit a defence response in plants have been characterized, including bacterial flagellin, cold-shock proteins, lipopolysaccharides (LPS), elongation factor Tu (EF-Tu), fungal chitin, b-glucans and ergosterol (Ingle et al., 2006; Nürnberger and Lipka, 2005; Nürnberger et al., 2004). Indeed, recognized patterns can be narrowed down to particular conserved motifs or domains within these molecules, such as the 22-amino-acid peptide flg22 of bacterial flagellin (Felix et al., 1999) and the RNA-binding motif RNP-1 of bacterial cold-shock proteins (Felix and Boller, 2003). However, not all PAMPs are recognized by all plant species; for example, only members of the Brassicaceae respond to EF-Tu (Felix and Boller, 2003). PAMPs have been defined as surface-exposed, abundant structures that are common to microbial sources, are not found in potential eukaryotic hosts and are indispensable for the microbial lifestyle (Medzhitov and Janeway, 1997). Additions to these definitions (Gijzen and Nürnberger, 2006) include that PAMPs act outside the plant plasma membrane and comprise activities that are not heat labile. The oomycetes form a phylogenetically distinct group of eukaryotic microorganisms and consequently possess unique, essential physiological and structural components that function as PAMPs. Thus far, many oomycete PAMPs and other elicitors are secreted proteins (Fig. 1). Consequently, this brings them into the vicinity of plant cell surface receptors that can trigger PTI. Some oomycete PAMPs share the characteristics of enzymes or protein toxins (Gijzen and Nürnberger, 2006). In addition to these proteinaceous PAMPs and elicitors, it has long been known that surface-exposed oomycete glucans also trigger defences in host plants (Sharp et al., 1984). Indeed, a glucan receptor in the plasma membrane of soybean root cells has been characterized (Umemoto et al., 1997). In this review, however, we focus on oomycete proteins that act as PAMPs and elicitors (Table 1), all of which activate defence responses in the host plants infected by these oomycetes. One of the first proteinaceous oomycete PAMPs to be identified is an abundant, calcium-dependent transglutaminase (TGase) named GP42 that functions in the cell wall of P. sojae (Brunner et al., 2002; Nürnberger et al., 1994). TGases are multifunctional enzymes vital for many physiological processes, such as cell differentiation and tissue regeneration (Langston et al., 2007). The acyl transferase activity of the enzyme performs various in vivo and in vitro protein cross-linking and modification functions (Brunner et al., 2002). The PAMP-like © 2009 THE AUTHORS JOURNAL COMPILATION © 2009 BLACKWELL PUBLISHING LTD MOLECULAR PLANT PATHOLOGY (2009) 10(4), 547–562 550 I. HEIN et al. activity of GP42 can be narrowed to Pep-13, a specific 13-amino-acid, high-affinity pattern that has been shown to be necessary and sufficient to stimulate PTI responses in parsley cells, including ion effluxes, ROS generation, defence-related gene activation and phytoalexin synthesis (Brunner et al., 2002; Nürnberger et al., 1994) (Table 1). TGases with highly conserved Pep-13 motifs have been identified in all Phytophthora species investigated (Brunner et al., 2002). Mutational analysis of Pep-13 revealed that the amino acids important for plant defence-eliciting activity are also essential for TGase activity, supporting the concept that plants have evolved PRRs that recognize genus-specific ‘epitope-like’ motifs that are essential for the function of pathogen-derived molecules (Brunner et al., 2002). A further oomycete-specific cell wall PAMP is the Phytophthora cellulose-binding elicitor lectin (CBEL) isolated from P. parasitica var. nicotianae and found to be a potent elicitor of necrosis and PTI (Mateos et al., 1997) (Table 1). Thus far, 42 CBEL-like domains have been identified in 28 putative proteins of P. infestans, P. sojae, P. ramorum and P. parasitica, suggesting that this domain is widely distributed in oomycetes (TortoAlalibo et al., 2005). Like other known PAMPS, an essential motif sufficient to stimulate defence responses in plants was pinpointed to two cellulose-binding domains (CBDs) within CBEL. CBDs belong to the carbohydrate-binding module 1 family, which is almost exclusively found in fungi (Gaulin et al., 2006). Unexpectedly, although CBEL contributes to adhesion, the virulence of transgenic strains of P. parasitica suppressed in CBEL expression was not seriously affected. In spite of this, the lack of CBEL correlated with the abnormal formation of papillae-like cell wall thickenings in Phytophthora, suggestive of a developmental function supporting its classification as a PAMP (Gaulin et al., 2002). Further studies mutating conserved aromatic residues in CBEL significantly reduced cellulose binding. Single mutations in either CBD abolished elicitor activity, whereas mutations in both CBDs were required to eliminate cell wall binding (Gaulin et al., 2006). The CBDs have no detectable hydrolytic activity and may be perceived by the plant via the local destabilization of cellulose caused by binding (Dumas et al., 2008). Supporting research using protoplasts has shown that the cell wall is necessary for CBEL to elicit a transient variation of cytosolic calcium levels in tobacco cells. Surprisingly, the cev Arabidopsis mutant, deficient in cellulose synthesis, was more resistant to cell wall-penetrating powdery mildew fungal pathogens. However, cev mutants constitutively express jasmonate- and ethylene-dependent defence pathways, suggestive of a surveillance mechanism functioning in the plant cell wall (Ellis and Turner, 2001). Insights into the nature of effectors likely to be important for zoospore attachment have come from studies such as the cloning of the PcVsv1 gene encoding the ventral vesicle adhesive in Phytophthora cinnamomi. PcVsv1 contains 47 copies of the thrombospondin type I repeat motif, a conserved 50-amino-acid sequence found in a range of extracellular adhesive molecules secreted by mammalian cells and malarial parasites (Robold and Hardham, 2005). Its occurrence in the soil-borne Phytophthora and Pythium species, as well as in representatives of the downy mildews and white rusts, suggests that it is widely conserved in the oomycetes. As was the case for CBEL (above), PcVsv1 is proposed to function in adhesion to plant cell walls (Hardham, 2007). It would be interesting to determine whether there are examples of plant species that have evolved to recognize PcVsv1 as a PAMP, as its cross-species conservation suggests it would be a likely candidate. A number of small cysteine-rich (SCR) proteins that elicit necrosis and defence responses in plants are secreted by oomycetes (Kamoun, 2006). In addition to elicitins (below), SCR proteins include PcF from Phytophthora cactorum, which induces necrosis and may act as a phytotoxin (Table 1) (Orosomando et al., 2001). SCR74 and SCR91 are two PcF-like secreted proteins from P. infestans. The Scr74 gene family is induced during infection, and has been subject to diversifying selection. It appears that scr74 has undergone gene duplication and recombination, followed by functional divergence that is reminiscent of molecular co-evolution with the host, suggesting that this family of proteins could be targets for detection by host surveillance systems (Liu et al., 2005). Elicitins are conserved 10-kDa proteins that are secreted by most species of Phytophthora and Pythium and that elicit a wide range of defence responses in plants, including the hypersensitive response (HR), a form of programmed cell death (PCD), in Nicotiana spp. (Kamoun, 2006) (Table 1). Class I elicitins can bind sterols, such as ergosterol, and function as sterol-carrier proteins (Vauthrin et al., 1999). This is of potential importance because Phytophthora spp. (and oomycetes in general) lack the squalene epoxidase and the 14a-demethylase enzymes required to convert sterol precursors. Consequently, they cannot synthesize sterols and must acquire them from external sources (Tyler et al., 2006). Sterol loading has been implicated in elicitin binding to a plasma membrane receptor in tobacco and in activating the HR (Osman et al., 2001). HR triggered by the elicitin INF1 from P. infestans has been shown to require the respiratory burst oxidase Nbrboh (Yoshioka et al., 2003), heat-shock proteins HSP70 and HSP90 (Kanzaki et al., 2003), the ubiquitin ligase-associated protein NbSGT1 (Peart et al., 2002) and a MAPK kinase (Takahashi et al., 2007). Recently, a lectin-like receptor kinase, NbLRK1, has been shown to interact with INF1. Virus-induced gene silencing of NbLRK1 delayed INF1-mediated HR, suggesting that it is a component of the protein complex responsible for INF1 perception and signal transduction (Kanzaki et al., 2008). Support for the view that INF1 elicitin can be regarded as a PAMP comes from the observation that ROS © 2009 THE AUTHORS MOLECULAR PLANT PATHOLOGY (2009) 10(4), 547–562 JOURNAL COMPILATION © 2009 BLACKWELL PUBLISHING LTD Zig-zag-zig in oomycete–plant interactions generation, following INF1 perception, is dependent on the leucine-rich repeat (LRR) receptor kinase, SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK)3/brassinosteroid (BR)associated kinase (BAK)1. SERK3/BAK1 has been implicated in the integration of diverse PAMP perception events into a general PTI response by forming complexes with PAMP receptors (Heese et al., 2007). It would thus be interesting to determine whether the Nicotiana benthamiana SERK3/BAK1 protein complexes with NbLRK1. The cell wall-associated, necrosis-inducing, Phytophthora protein 1 (NPP1) of P. parasitica induces similar PTI responses to Pep-13, but also induces cell death (Fellbrich et al., 2002; Qutob et al., 2006) (Table 1). NPP1-mediated plant defences, including the oxidative burst, accumulation of pathogenesisrelated (PR) proteins, callose deposition and necrosis, did not utilize the Pep-13 receptor, suggestive of a converging PTI signalling cascade with other PAMP receptors (Fellbrich et al., 2002; Qutob et al., 2006). NPP1 is a member of a large class of proteins, known as Nep1-like proteins (NLPs), present in predominantly plant pathogenic oomycetes, fungi, Gram-negative and Gram-positive bacteria (Gijzen and Nürnberger, 2006). Nep1 is a 24-kDa protein that induces necrosis and ethylene production in plants, initially identified from the culture filtrate of the cereal pathogen Fusarium oxysporum (Bailey, 1995). Further analyses of NLP sequences have indicated that an ancient and fundamental bifurcation occurred during their evolution. Type I NLP domains, with two conserved cysteines, are found in fungi, oomycetes and bacteria, whereas the Type II NLP domains, with four conserved cysteines, are not found in oomycetes (Gijzen and Nürnberger, 2006). Currently, there is debate about whether NLPs are PAMPs, as there are significant characteristic differences when compared with Pep-13 and flg22 responses (Gijzen and Nürnberger, 2006; Qutob et al., 2006). NLPs can be classed as PAMPs on the basis of the rapid triggering of plant defence responses associated with other PAMPs, and because they act outside of the plant cell membrane. However, unlike Pep-13 or flg22, no defined, conserved amino acid motif in NLPs has been shown to be required for PTI. Finally, the crinkling and necrosis (Crn) gene family has been shown to induce necrosis when expressed inside plant cells (Kamoun, 2007; Torto et al., 2003). Crn genes are numerous in oomycete genomes. They encode modular proteins with a highly conserved N-terminus, followed by highly variable combinations of domains (Win et al., 2006). The conserved N-terminal domain, following a signal peptide for secretion, contains a motif, LXLFLAK, which has been postulated to act in the translocation of these proteins to the inside of host cells (Kamoun, 2007). Future work will reveal whether CRN proteins act, like RXLR proteins (see next section), as cytoplasmic effectors that manipulate host defence or metabolic functions. 551 OOMYCETE ETS Plant pathogenic oomycetes must be capable of overcoming preformed and inducible defences in at least one host plant species. Physical barriers, such as the cell wall, cuticle and epicular waxes, successfully thwart infection by non-pathogenic oomycetes (Attard et al., 2008). Plant pathogenic oomycetes have evolved specialized infection strategies that facilitate the invasion of plant cells, including the development of appressoria for host cell penetration, and the secretion of pectinases and glucanases that weaken the cell wall, together allowing physical barriers to be overcome (Latijnhouwers et al., 2003). Oomycetes must invade plant tissues in such a way that they acquire sufficient nutrients to sustain themselves in the dynamic and potentially hostile environment of the host. Some effectors may thus serve structural roles, for example, in the extrahaustorial matrix (Schulze-Lefert and Panstruga, 2003). Others may promote pathogen dispersal or nutrient leakage, perhaps by modulating the transition from biotrophy to necrotrophy in the case of hemibiotrophs, such as P. infestans. Indeed, some of the PAMPs and elicitors referred to above have been proposed to play roles important for the infection process, other than to suppress host defences. Thus, CBEL has been implicated in host cell adhesion, elicitins in the assimilation of sterols, and NLPs, inducing necrosis in resistant and susceptible plants, may be important in facilitating the transition to the necrotrophic phase of plant colonization (Qutob et al., 2002). A role for NLPs in virulence is supported by the finding that a knock-out of the NLP1 homologue in the soft-rot enterobacterial pathogen Pectobacterium atrosepticum results in reduced virulence on potato (Pemberton et al., 2005). Recent research has provided the first examples of oomycete effectors that suppress PTI and thus overcome the initial hurdle in establishing a successful infection (Fig. 1). Two broad classes of effectors can be identified: those that act extracellularly to suppress secreted defence-related proteins, and those that act intracellularly, presumably to suppress the defence-related signal transduction, regulation and trafficking that lead to PTI or ETI. Extracellular oomycete effectors Several plant PR proteins, such as glucanases, chitinases and proteases, are secreted hydrolytic enzymes. From a host viewpoint, such enzymatic activities represent potentially important defence mechanisms through both direct degradation of the pathogen mycelial wall and, in the case of glucanases, indirectly via the release of b-1,3-glucan oligosaccharide elicitors (van Loon et al., 2006). Fungal and bacterial plant pathogens have evolved diverse mechanisms for protection against the activities of these PR proteins (Abramovitch and Martin, 2004; Chisholm © 2009 THE AUTHORS JOURNAL COMPILATION © 2009 BLACKWELL PUBLISHING LTD MOLECULAR PLANT PATHOLOGY (2009) 10(4), 547–562 552 I. HEIN et al. et al., 2006). Similarly, plant pathogenic oomycetes, such as Phytophthora, have also evolved mechanisms to escape the enzymatic activity of secreted hydrolases. Oomycetes contain little chitin in their cell wall and are therefore resistant to plant chitinases (Bartinicki-Garcia and Wang, 1983). In addition, they have evolved active counter-defence mechanisms by secreting inhibitory proteins that target host glucanases and proteases (Table 1). The glucanase inhibitor proteins GIP1 and GIP2, secreted by P. sojae, inhibit the soybean endo-b-1,3-glucanase EgaseA (Rose et al., 2002). Four genes with similarity to P. sojae GIPs have been identified in the P. infestans genome. Analyses of P. infestans-inoculated tomato leaves showed that P. infestans GIPs and tomato EGases are present in the apoplast and form stable complexes in planta. Codon evolution analyses of GIP homologues identified several positively selected peptide sites, and structural modelling has revealed that they are in close proximity to rapidly evolving EGase residues, suggesting that the interaction between GIPs and EGases has the hallmarks of tight molecular co-evolution (Damasceno et al., 2008), the principles of which are described in more detail below for the molecular co-evolution between effector and R genes. In addition, 18 extracellular protease inhibitor genes have been identified in P. infestans that belong to two major structural classes: (i) Kazal-like serine protease inhibitors (called EPI1–14); and (ii) cystatin-like cysteine protease inhibitors (EPIC1–4). EPI1 and EPI10 interact with and inhibit a secreted subtilisin-like serine protease of tomato, the PR protein P69B (Tian et al., 2004, 2005). EPIC1 and EPIC2B have been shown to be unstable in tomato apoplastic fluids and to be degraded by tomato P69B. However, EPI1 protects both proteins from degradation. Affinity-purified P69B was sufficient to degrade EPIC1 and EPIC2B, but not EPI1, suggesting selectivity in degradation by P69B. Co-immunoprecipitation experiments revealed that EPIC2 interacts with a novel papain-like extracellular cysteine protease, termed Phytophthora-inhibited protease 1 (PIP1). The interaction was further confirmed by co-immunoprecipitation using in planta-expressed PIP1 protein. Together, these findings suggest that a cascade of inhibition of host proteases initiated by EPI1 occurs in the tomato apoplast during infection by P. infestans. In addition, this study provides biochemical evidence for a direct contribution of the PR protein P69B subtilase to defence through the degradation of proteins from invading pathogens (Tian et al., 2007). Like GIPs, PIP1 has recently been shown to be under strong diversifying selection. In addition, it is a target of the Cladosporium fulvum avirulence protein AVR2, which acts as a protease inhibitor (Shabab et al., 2008). More recently, RCR3, a tomato protease targeted by the C. fulvum AVR2 protein, has also been shown to be targeted by EPIC1 and EPIC2B from P. infestans, demonstrating that unrelated pathogens manipulate the same defence targets in the host to establish infection (Song et al., 2009). The secre- tion of extracellular serine protease inhibitors seems likely to be part of a common infection strategy for Phytophthora spp., because P. parasitica expresses a gene similar to epi1 (Panabieres et al., 2005) and secreted Kazal-like proteins form a diverse family of at least 35 members from five plant pathogenic oomycetes, including the downy mildew Plasmopara halstedi (Kamoun, 2006). Intracellular oomycete effectors The biochemical activities of intracellular oomycete effectors are poorly understood. However, we might expect some common features with the biochemical functions of bacterial pathogen T3SS effectors that often mimic or inhibit eukaryotic cellular functions on delivery into the host cell (Block et al., 2008; Grant et al., 2006). In recent years, six oomycete cytoplasmic effectors have been discovered through their avirulence (AVR) activity, triggering an HR on host genotypes with corresponding disease resistance (R) genes (Allen et al., 2004; Armstrong et al., 2005; van Poppel et al., 2008; Rehmany et al., 2005; Shan et al., 2004; Vleeshouwers et al., 2008) (Table 2). All six AVR proteins contain, within the N-terminal 60 amino acids, a secretory signal peptide and a conserved domain featuring the motif RXLR, followed by a high frequency of acidic (D/E) residues, and a C-terminal domain(s) associated with virulence function (Birch et al., 2006, 2008; Win et al., 2007). The oomycete RXLR motif is similar in sequence and position to the host cell targeting signal (HT/Pexel motif) required for the translocation of proteins from malaria parasites (Plasmodium spp.) into host red blood cells (Hiller et al., 2004; Marti et al., 2004). Indeed, the oomycete RXLR signal has been demonstrated to function for effector delivery in the malarial system (Bhattacharjee et al., 2006). Whisson et al. (2007) and Dou et al. (2008a) have since shown that the RXLR motif is required for the translocation of the P. infestans effector AVR3a and the P. sojae effector Avr1b, respectively, inside host plant cells. Phytophthora infestans transformants expressing AVR3a, in which the RXLR and EER regions were replaced, singly or in combination, with alanine residues, or with alternative amino acids that conserve the physicochemical properties of the protein, failed to deliver AVR3a from the extrahaustorial matrix to the inside of the plant cell (Whisson et al., 2007). Translocation was confirmed using the Escherichia coli GusA gene fused to the signal peptide (SP)-RXLR-EERencoding domains of AVR3a. b-Glucuronidase (GUS) activity was observed in plant cells in contact with the haustoria of P. infestans transformants expressing the SP-RXLR-EER::GusA fusion, but not with transformants expressing a version in which the motifs were replaced with alanine residues (SP-AAAA-AAAGUS) (Whisson et al., 2007). Dou et al. (2008a) have demonstrated, using co-bombardment and a purified Avr1b::GFP fusion protein, that the RXLR © 2009 THE AUTHORS MOLECULAR PLANT PATHOLOGY (2009) 10(4), 547–562 JOURNAL COMPILATION © 2009 BLACKWELL PUBLISHING LTD Zig-zag-zig in oomycete–plant interactions 553 Table 2 Cloned resistance (R) genes and avirulence (Avr) genes from oomycete–plant interactions. Cloned R gene Source Reference Resistance to Cognate Avr gene Reference R1 R3a RB/Rpi-Blb1 Solanum demissum S. demissum S. bulbocastanum Phytophthora infestans P. infestans P. infestans Avr3a AVR-blb1(IpiO) Armstrong et al. (2005) Vleeshouwers et al. (2008) Rpi-sto1 Rpi-pta1 Rpi-Blb2 Unknown RPP1 (-WsC, -WsA, -WsB) RPP2 (RPP2A&B) RPP4 RPP5 RPP7 RPP8 RPP13 (-Nd, -RLD) Dm3 Unknown S. stoloniferum S. papita S. bulbocastanum S. demissum Arabidopsis thaliana Ballvora et al. (2002) Huang et al. (2005) Song et al. (2003); van der Vossen et al. (2003) Vleeshouwers et al. (2008) Wang et al. (2008) van der Vossen et al. (2005) Avr4 ATR1 van Poppel et al. (2008) Rehmany et al. (2005) ATR13 Allen et al. (2004) AVR1b-1 Shan et al. (2004) A. thaliana A. thaliana A. thaliana A. thaliana A. thaliana A. thaliana Lactuca sativa Glycine max Botella et al. (1998) Sinapidou et al. (2004) van der Biezen et al. (2002) Parker et al. (1997) Eulgem et al. (2007) McDowell et al. (1998) Bittner-Eddy et al. (2000) Shen et al. (2002) and EER motifs in Avr1b are required for translocation across the host plasma membrane, and that such translocation is independent of the pathogen. They showed that the amino acids R at position 1 and L at position 3 were critical for translocation by the RXLR motif. Dou et al. (2008a) and Grouffaud et al. (2008) have both shown that the Plasmodium HT/Pexel motif will function in Phytophthora for effector delivery into plant cells. This indicates an ancient, conserved means of effector delivery into plant and animal cells that has been exploited, possibly through convergent evolution, by these distantly related pathogens. The secreted RXLR complement of an oomycete can be considered analogous to the T3SS repertoire of a bacterial plant pathogen, and the discovery of the effector functions of the large number of secreted RXLR-containing proteins found in oomycete genome sequences will provide the focus for much future research in the field of oomycete plant pathology. Already, some insights into RXLR effector virulence functions have been published (Table 1). The P. infestans RXLR effector Avr3a was identified by association genetics, and it was shown that avirulent and virulent alleles differ at only two amino acid positions, K80E and I103M, respectively, in the C-terminal domain (Armstrong et al., 2005). AVR3aK80I103, in addition to being recognized by R3a, is able to suppress hypersensitive cell death induced by the P. infestans elicitin, INF1, pointing to a possible virulence function. The AVR3aE80M103 form, in contrast, is not recognized by R3a or able to suppress INF-mediated cell death (Bos et al., 2006). Cell death suppression activity is a common feature of bacterial effectors, and the observation that RXLRs have this ability in plants is entirely consistent with expected models of effector function. In addition to AVR3a, the P. sojae RXLR effector Avr1b can suppress PCD triggered by the mouse BAX protein in yeast, soybean (Glycine max) and N. benthamiana cells (Dou P. infestans P. infestans Hyaloperonospora arabidopsidis H. arabidopsidis H. arabidopsidis H. arabidopsidis H. arabidopsidis H. arabidopsidis H. arabidopsidis Bremia lactucae P. sojae et al., 2008b). Computational work has identified conserved motifs within the C-terminal domains of Avr1b, and many other effectors, that have been termed W, Y, K and L, and that can occur in multiple copies (Dou et al., 2008b; Jiang et al., 2008). PCD suppression by Avr1b is dependent on the W and Y motifs. Two additional effectors from P. sojae (PsAvh331 and PsAvh163) and one from H. arabidopsidis (HpRxL96), all encoding proteins containing multiple W and Y motifs, are also able to suppress PCD (Dou et al., 2008b). Two additional RXLR proteins have also been assigned virulence functions (Table 1). Alleles of ATR1 and ATR13 from H. arabidopsidis confer enhanced virulence to Pseudomonas syringae pathovar tomato DC3000 on susceptible Arabidopsis accessions, suggesting that ATR1 and ATR13 positively contribute to pathogen virulence inside host cells. In addition, ATR13 alleles have been shown to suppress bacterial PAMP-triggered callose deposition in susceptible Arabidopsis. Furthermore, expression of another allele of ATR13 in plant cells suppressed PAMPtriggered ROS production in addition to callose deposition (Sohn et al., 2007). ETI AND BEYOND Effectors deployed by invading pathogens to, for example, gain access to host cell nutrients and/or suppress host defences (ETS), simultaneously provide the plant with new targets for detection. In the next layer of defence, plants utilize R proteins in a surveillance system to reveal effector (AVR) proteins and activate ETI (Jones and Dangl, 2006) (Fig. 1). This response involves the HR, which has been associated with almost all forms of resistance towards oomycetes, including non-host resistance (Kamoun et al., 1999). © 2009 THE AUTHORS JOURNAL COMPILATION © 2009 BLACKWELL PUBLISHING LTD MOLECULAR PLANT PATHOLOGY (2009) 10(4), 547–562 554 I. HEIN et al. The traditional view of R gene perception of an Avr gene follows the gene-for-gene concept that suggests a direct interaction between both protein products, similar to the receptor– ligand relationship of antibodies and antigens (Flor, 1971). In the majority of cases, however, recognition of an AVR protein by a cognate R protein may be indirect, involving activation of the R protein on recognition of effector activity on virulence targets, also know as ‘pathogen-induced modified self-molecular patterns’, previously described in the ‘Guard’ hypothesis (van der Biezen and Jones, 1998; Dangl and Jones, 2001). With the ability to initiate PCD, R protein activation is tightly regulated. The largest class of R proteins comprises nucleotide-binding site (NBS) and LRR domains, and current models postulate that many form multiprotein ‘trigger’ complexes that, by intra- and intermolecular interactions, maintain the R proteins in an inactive form until effectors are detected (Belkhadir et al., 2004; Friedman and Baker, 2007). The LRR domain might play a dual role, acting both as a recognition specificity determinant and as a repressor that prevents inappropriate NBS activation (Belkhadir et al., 2004). Evolutionary analysis of R genes and their structure has demonstrated selection pressure on the LRR-encoding regions, and also on other domains within R genes which, in light of the dual function, may point towards the co-evolution of domains to establish or maintain the guarding function on the virulence target(s) whilst maintaining the inhibitory function (Belkhadir et al., 2004; McDowell and Simon, 2006). This scenario sets the scene for a very close evolutionary contest on a molecular level between pathogen effectors to evade detection and plant R proteins and virulence targets to retain recognition. It has been speculated that plant R proteins may guard key, general pathogen targets, new pathogen-specific virulence targets, and deploy decoys to maximize the chance of pathogen recognition, all of which could then be subject to evolutionary pressure (Friedman and Baker, 2007; van der Hoorn and Kamoun, 2008; McDowell and Simon, 2006). Analysis of the fully sequenced model plant Arabidopsis thaliana ecotype Columbia (Col-0) has revealed approximately 149 genes that encode NBS–LRR-containing R proteins (Meyers et al., 2003). This class can be further divided according to the N-terminal domain: either a Toll/interleukin-1 family of receptor (TIR) domain, or a coiled-coiled (CC) region (Meyers et al., 1999). Interestingly, about two-thirds of NBS–LRR genes are found in clusters (Meyers et al., 2003). This arrangement is thought to reflect the evolution of R genes by a process relying on duplication, subsequent diversification and adaptive selection, also known as birth-and-death (Lehmann, 2002; Michelmore and Meyers, 1998; Nei et al., 1997). R genes can be under either purifying selection, acting against gene variation to maintain R protein function, or diversifying selection, which provides a means for new R gene recognition specificities. The latter is characterized by a rate of non-synonymous nucleotide substitu- tions (KA), resulting in amino acid changes, that is greater than the rate of synonymous nucleotide substitutions (KS), which do not alter the amino acid (KA : KS ratio > 1). In contrast, purifying selection typically displays a KA : KS ratio < 1. Evolution of the RGC2 locus in cultivated lettuce and wild relatives has shown that different selective forces act on R genes. Multiple mechanisms, including recombination, point mutations and insertions/deletions, drive R gene evolution, and different genes within a cluster have the potential to evolve independently (Kuang et al., 2004, 2005). The RGC2 locus comprises a large cluster of R genes, and a gene for resistance towards root aphid has been mapped alongside at least 10 Dm genes that confer resistance towards the oomycete downy mildew pathogen Bremia lactucae (Meyers et al., 1998a, b). Within this one cluster, two types of RGC2 gene have been identified that reflect a heterogeneous rate of evolution, yielding fast-evolving ‘type I’ genes, characterized by frequent sequence exchanges associated with chimeric genes and haplotype diversity, including variable gene copy numbers, and more slowly evolving ‘type II’ genes, that infrequently exchange sequences and maintain a higher similarity between orthologues than paralogues. In line with type I genes, the RGC2 copy number within seven genotypes from three species ranged from 12 in cv. Calmar to 32 in cv. Diana (Kuang et al., 2004). Furthermore, gene variation studies focusing on the LRR region of three Arabidopsis genes have shown that some, including the hypervariable RPP13 gene effective against the oomycete H. arabidopsidis, exhibit levels of diversity within populations that are similar to the diversity observed between populations, and could thus provide a means to maintain a high level of polymorphism (Ding et al., 2007). Regardless of the mechanism of R gene diversification, in terms of plant–pathogen co-evolution, two extreme outcomes are conceivable: (i) balancing selection, resulting in long-lived and high allelic diversity of R genes by maintaining stable polymorphisms (Bergelson et al., 2001), previously described as ‘trench warfare’ (Stahl et al., 1999); and (ii) selective sweeps, resulting in young R genes and monomorphic R gene loci as a consequence of the transient maintenance of polymorphism and the replacement of old R genes by new variants. This has been described as an ‘arms race’ (Bergelson et al., 2001). Interestingly, a genome-wide survey of polymorphisms within the LRR region of 27 representative R genes in 96 Arabidopsis thaliana accessions has found that both extremes are not common, and that R gene co-evolution has resulted in a continuum of possible states between ‘trench warfare’ and an ‘arms race’ (Bakker et al., 2006). Cloned and characterized plant R genes conferring resistance to oomycetes, and oomycete Avr effectors, are shown in Table 2. To date, all oomycete AVR proteins are RXLR effectors, and computational analysis of the genome sequences of P. sojae, P. ramorum, P. infestans and H. arabidopsidis has predicted hun- © 2009 THE AUTHORS MOLECULAR PLANT PATHOLOGY (2009) 10(4), 547–562 JOURNAL COMPILATION © 2009 BLACKWELL PUBLISHING LTD Zig-zag-zig in oomycete–plant interactions dreds of candidate RXLR genes in each oomycete (Birch et al., 2008; Jiang et al., 2008; Jones and Dangl, 2006; Whisson et al., 2007; Win et al., 2007), which is in stark contrast with the small repertoire of approximately 30 T3SS effectors required for bacterial infection (Jones and Dangl, 2006). Furthermore, although only few RXLR-containing effectors appear to be sufficiently conserved between sequenced oomycetes to have originated from a common ancestor, it is apparent that many effectors within species contain paralogous family members (Win et al., 2007). Taken together, these observations suggest an extraordinary potential for virulence function redundancy that may be governed by family members and/or evolutionarily unrelated effector sequences (Birch et al., 2008). In the light of this, it is of little surprise that the oomycetes comprise such formidable pathogens. Win et al. (2007) determined the ratio between nonsynonymous and synonymous nucleotide substitutions among RXLR gene family members in P. sojae, P. ramorum and H. arabidopsidis, and demonstrated that a great proportion of gene families are under positive selection. Furthermore, the C-terminal regions of effectors, mainly associated with virulence function, displayed a significantly higher rate of positive selection than did the N-terminal regions, which contain domains implicated in effector secretion and host targeting (Win et al., 2007). Reminiscent of the genomic organization of R genes, many RXLR effectors under positive selection were found in clusters, which has been postulated to be an important mechanism of gene duplication and diversification, and is consistent with their roles as modulators and targets of host resistance (Win et al., 2007). Pathogen responses to effector detection can vary greatly and may be dependent on the level of functional redundancy within the pathogen. So far, and discussed below, evidence indicates that oomycetes can (i) lose effectors without loss of virulence, pointing towards redundancy in virulence function; (ii) diversify effectors to maintain virulence function and simultaneously evade recognition; and (iii) utilize so far unidentified inhibitors that act downstream of the recognition of avirulence genes to prohibit resistance responses. The effector genes Avr4 from P. infestans and Avr1b from P. sojae provide evidence for functional redundancy by the approaches used to evade recognition. Avr4 is recognized by the uncharacterized potato R4 gene. Phytophthora infestans isolates that are virulent on R4-containing plants possess truncated and non-functional avr4 alleles, indicating that the loss of an effector has no detrimental effect on overall pathogen virulence (van Poppel et al., 2008). Avr1b, recognized as an Avr gene in soybean plants carrying the cognate Rps1b resistance gene, was identified by map-based cloning. All analysed P. sojae isolates virulent on Rps1b plants contain Avr1b, but either with numerous amino acid substitutions or, in other isolates, fail to accumulate Avr1b mRNA and thus avoid recognition without obvious consequences to pathogen fitness (Shan et al., 2004). Neverthe- 555 less, P. sojae transformants over-expressing Avr1b displayed increased virulence compared with control transformants (Dou et al., 2008b). Interestingly, Avr1b and the paralogous Avh1b protein exhibit sequence similarity to AVR3a from P. infestans, which triggers R3a-dependent PCD (Armstrong et al., 2005; Win et al., 2007). Indeed, Avh1b, but not Avr1b, is weakly recognized by R3a. Comparison with an Avr1b variant, Avr1bAAM20939, that also evades recognition by R3a, highlighted nine amino acids under positive selection that differ between the two Avr1b variants and Avh1b. Mutagenesis of an aromatic threonine from Avh1b into a basic lysine, as found in Avr1b-1, abolished the R3a-dependent HR eliciting activity (Win et al., 2007). Future work might determine whether Avh1b and Avr3a have similar functions and seek out similar virulence targets, or whether the elusive Rps1b gene and R3a guard similar or different virulence targets. In addition to the virulent or avirulent allele of Avr3a, the Avr3a locus contains two paralogues, referred to as PEX147-2 and PEX147-3. Phylogenetically, the two alleles display a closer relationship compared with the paralogues, which suggests that duplication and divergence under positive selection are the driving mechanisms to avoid detection in this locus (Armstrong et al., 2005). The R3a gene from Solanum demissum, located within the R3 locus on chromosome 11, was cloned by comparative genomics and has revealed homology and synteny to the I2 locus in tomato that mediates resistance towards the fungus F. oxysporum f. sp. lycopersici (Huang et al., 2005). This locus appears to be an R gene hotspot in the Solanaceae and contains, in addition to R3a, other P. infestans resistance genes, such as the closely linked R3b (Huang et al., 2004) and, potentially, R5-R11 (Huang, 2005), as well as quantitative resistance towards the cyst nematode Globodera rostochiensis in potato, R genes effective towards Stemphylium spp. and to yellow leaf curl virus in tomato, and resistance towards tobacco mosaic virus in pepper (Huang et al., 2005). Sequence analysis at both loci suggests that these R genes share a common ancestral gene, but has also shown that, in potato, the locus has not only expanded significantly in size, but also in the number of R genes. Within the R3a cluster, four paralogues were identified, all of which are constitutively expressed and show hallmarks of diversifying selection. R3a haplotype comparison between other S. demissum, as well as evidence of sequence exchange between paralogues, is consistent with a type I class of R gene that evolves rapidly (Huang et al., 2005). The more durable Rpi-blb2 resistance gene from Solanum bulbocastanum has been identified within a cluster of R genes on chromosome 6 and is similar to the Mi-1 gene from tomato that mediates resistance towards root-knot nematodes, aphids and white flies (van der Vossen et al., 2005). Overall, two clusters have been identified at both loci and, similar to the I2 locus, haplotype analysis of the Mi-1 loci in Solanum peruvianum and © 2009 THE AUTHORS JOURNAL COMPILATION © 2009 BLACKWELL PUBLISHING LTD MOLECULAR PLANT PATHOLOGY (2009) 10(4), 547–562 556 I. HEIN et al. S. lycopersicum has shown a comparable structure and organization indicative of a conserved and slow evolution. In contrast, the same clusters have expanded significantly in S. bulbocastanum and the numbers of homologues in both clusters has increased, reminiscent of the expansion of the R3 locus (van der Vossen et al., 2005). It is tempting to speculate that the contrasting evolutionary fates of the slowly evolving I2 and Mi-1 loci in tomato, and the faster evolving R3a and Rpi-blb2 clusters in potato, reflect the converse evolutionary potential of the interacting pathogen and/or the cognate effectors, with slower evolving F. oxysporum and root-knot nematodes on the one side and rapidly changing P. infestans on the other. A second, also potentially more durable R gene isolated from chromosome 8 in S. bulbocastanum, RB/Rpi-blb1, has been classified as a slow-evolving type II gene (Song et al., 2003; van der Vossen et al., 2003). A high level of synonymous nucleotide substitutions is apparent among paralogues of the Rpi-blb1 haplotype, which indicates synonymous divergence and suggests that these genes are relatively old and could be subject to balancing selection (van der Vossen et al., 2003). The cognate P. infestans Avr gene ipiO is highly expressed at the tips of invading hyphae (van West et al., 1998), and at least two closely linked genes ipiO1 and ipiO2 exist as part of the PexRD6 family of effectors (Pieterse et al., 1994; Vleeshouwers et al., 2008). Using a candidate gene approach, Vleeshouwers et al. (2008) identified and cloned functional Rpi-blb1 orthologues from Solanum stoloniferum (Rpi-sto1) and Solanum papita (Rpipta1), and their function could be demonstrated by transient co-expression with ipiO in N. benthamiana, yielding, in all cases, an HR similar to the co-expression of Rpi-blb1 with ipiO. In a similar study, Rpi-blb1 orthologues were successfully amplified, not only in S. stoloniferum and S. papita, but also in Solanum polytrichon, whereas amplification of orthologous genes from the unrelated Rpi-blb2 gene failed to yield homologous genes (Wang et al., 2008). Considering the R gene diversity and recognition specificity observed between and within populations, it is of no surprise that effector recognition between Solanum species and within species accessions varies significantly, as shown by Vleeshouwers et al. (2008). The lettuce gene Dm3, a fast-evolving type I RGC2 gene that confers resistance to B. lactucae races expressing the avirulence gene Avr3 (Kuang et al., 2004), was found at a very low frequency in wild lettuce accessions (Kuang et al., 2006). Indeed, within a comprehensive screen of 1033 accessions from 49 natural populations, only one contained Dm3, which could be interpreted as either a very recent evolutionary event that led to Dm3 specificity or deletion of this gene caused by unequal crossing over. The latter was further supported by a high conservation of the 5′ region of Dm3 in about 30% of accessions and the detection of smaller Dm3-specific sequence fragments in over 40% of accessions. This study also indicated a high level of diversity within the Dm LRR domains in these wild populations, which could indicate a high level of Dm gene variation, in line with at least eight resistance specificities to B. lactucae that have mapped to the RGC2 locus (Kuang et al., 2006). The R1 gene for potato resistance to late blight was isolated from an R gene hotspot on chromosome 5 that contains resistances towards potato virus X (X, Rx2 and Nb) and major quantitative trait loci for resistance to P. infestans, Globodera pallida (Gpa and Gpa5) and Globodera rostochiensis (Gpr1) (Ballvora et al., 2002; Kuang et al., 2005). Interestingly, the Rx2 gene is, similar to R1, a member of the CC-NBS-LRR family of R genes, but belongs to a different class, as both genes share only 32% sequence identity. A comparative study of the R1 locus in all three genomes (haplotypes) of the allohexaploid species S. demissum has revealed three different types of R gene with homology to R1, Prf and Bs4, indicative of a complex R gene cluster (Kuang et al., 2005). In agreement with previous findings, the evolutionary rates of these different R genes were unequal. Both Prf-like and Bs4-like potato homologues at the R1 cluster comprise slow-evolving type II genes, whereas R1 homologues form three distinct subgroups, display variation in copy numbers, contain chimeric sequences because of frequent sequence exchanges reminiscent of fast-evolving type I resistance genes, and show diversifying selection at the LRR region (Kuang et al., 2005). The interaction between Arabidopsis and the oomycete pathogen H. arabidopsidis provides a powerful model system to study the co-evolution of R genes and cognate Avr genes (Koch and Slusarenko, 1990). R genes in Arabidopsis that recognize effectors from the oomycete pathogen H. arabidopsidis are referred to as RPP genes and comprise both CC (e.g. RPP7, 8, 13) and TIR (e.g. RPP1, 2a, 5) types of R gene (Holub, 2001) (Table 2). Hyaloperonospora arabidopsidis Avr genes are referred to as ATR (Arabidopsis thaliana recognized) genes. ATR1 and ATR13, both encoding RXLR effectors, have been well studied (Allen et al., 2004; Rehmany et al., 2005). A number of RPP genes have been identified in R gene hotspots and cloned from various ecotypes (reviewed by Slusarenko and Schlaich, 2003). This review focuses on two genes, RPP1 and RPP13, as the cognate ATR1 and ATR13 genes from H. arabidopsidis are known. One clear example for balancing selection has been found for the single-copy RPP13 gene, the most polymorphic R gene analysed in Arabidopsis, which exhibits an extreme variability and diversifying selection within the LRR domain (Rose et al., 2004). A collection of diverse and functionally distinct RPP13 alleles has been maintained in populations and is mirrored by a balancing selection of the ATR13 gene, which, in turn, displays high accumulation of non-synonymous polymorphism that is not limited to alleles that avoid recognition by certain RPP13 alleles (Allen et al., 2004; Rose et al., 2004). A study of naturally occurring variation of ATR13 alleles has identified specific amino acid © 2009 THE AUTHORS MOLECULAR PLANT PATHOLOGY (2009) 10(4), 547–562 JOURNAL COMPILATION © 2009 BLACKWELL PUBLISHING LTD Zig-zag-zig in oomycete–plant interactions positions within a C-terminal domain that are associated with recognition by RPP13Nd (Niederzenz allele). The study showed that the N-terminal signal peptide and RXLR regions exhibit sequence conservation. Indeed, deletion of the N-terminal domains has no effect on recognition, reminiscent of the recognition of AVR3aK80I103 by R3a (Allen et al., 2008). Mutations in a conserved leucine/isoleucine repeat within ATR13 alter recognition specificity. However, these changes do not occur in natural populations, leading the authors to postulate that this domain is required for virulence (Allen et al., 2008). Domain swap experiments between Rpp13Nd (Niederzenz allele), which recognizes ATR13Emco5, and RPP13Col, which fails to recognize ATR13Emco5, have shown that the LRR domain is required for the recognition and subsequent HR (Rentel et al., 2008). Candidate R genes for the complex RPP1 loci were first identified from the Arabidopsis accession Wassilewskija, and functional analysis showed that three out of four tightly linked TIRNBS-LRR genes (RPP1-WsA, RPP1-WsB, and RPP1-WsC), with a distinctive but overlapping recognition specificity, explained the resistance spectrum previously associated with RPP1, RPP10 and RPP14 (Botella et al., 1998). The underlying evolutionary mechanism in the generation of this locus could be explained by at least two tandem duplications, followed by sequence divergence and positive selection, mainly within the LRR domain, in the form of non-synonymous amino acid substitutions and the loss of a complete LRR in RPP1-WsC. Cloning of six diverse ATR1 alleles from eight H. arabidopsidis isolates has played a crucial role in unravelling the recognition specificity of RPP1 alleles. Again, the C-terminal part of the effector determines the recognition and is under strong diversifying selection (Rehmany et al., 2005). Some R genes, such as RPP1Nd (Niederzenz allele), recognized a single allelic form of ATR1NdWsB, whereas RPP1-WsB recognized three additional highly variable allelic forms, and the RPP1-WsA cognate ATR gene remains elusive (Rehmany et al., 2005). ETS2 and beyond? To avoid recognition, pathogens may ‘shed’ Avr genes, as shown for the apparently redundant effectors Avr4 from P. infestans and Avr1b from P. sojae, or enter a tightly entwined co-evolutionary battle with R genes, as shown above, for example, for ATR13 and RPP13. However, additional mechanisms are also conceivable. Two independent reports have shown that, although effector and R gene co-expression yields an HR, some pathogen isolates that express these effectors manage to evade recognition on plants carrying the cognate R gene. This could be explained by additional effectors that are deployed by the pathogen to suppress recognition or downstream signalling components of ETI. The Arabidopsis R gene RPP1-WsB from the accession Wassilewskija (see above) initiates an HR if co-bombarded with the ATR1NdWsB gene from 557 H. arabidopsidis isolate Emco5. However, although ATR1NdWsB is expressed in Emco5, this isolate does not trigger an HR in the Arabidopsis accession Ws-0 (Rehmany et al., 2005). Similarly, bacterial T3SS delivery of the Emco5 form of ATR13 triggers an HR in Arabidopsis accession Ws-0 (Sohn et al., 2007). A possible explanation is that Emco5 possesses an additional effector(s) that suppresses either the recognition of ATR1 and ATR13, or the downstream signalling or regulation of PCD following recognition. CONCLUSIONS Every step in pathogen invasion provides plants with new opportunities for pathogen recognition or to stop vital virulence components from either entering the plant or leaving pathogen cells. Similarly, pathogens could potentially target and manipulate every step in the plant disease resistance response. Consequently, co-evolution may affect a much larger number of plant and pathogen molecules than is currently known. Recently, the potential for opposing selective forces for the host virulence target of pathogen effectors was highlighted (van der Hoorn and Kamoun, 2008). Indeed, depending on the presence or absence of guarding R genes, virulence targets could potentially evolve to enhance effector recognition or to avoid effector manipulation, whilst maintaining their in planta function. Moreover, virulence targets could represent decoys that are deployed solely to detect effectors and are thus free to co-evolve to maintain effector interactions. Furthermore, a study of the RPP5 loci has shown that R genes in this cluster can be regulated by RNA silencing and might thus be able to respond to pathogens that perturb RNA silencing (Yi and Richards, 2007). The genomics era is providing us with both oomycete and host genome sequences which act as blueprints for the effectors that are required to suppress or otherwise manipulate plant responses to a wide range of conserved or rapidly evolving molecules. The scene is set for an expansion in genome sequencing, revealing the sequence diversity in PAMP, elicitor and effector proteins within and between oomycete species that reveals the complex molecular co-evolutionary battles between pathogen and host to establish or prevent disease. REFERENCES Abramovitch, R.B. and Martin, G.B. (2004) Strategies used by bacterial pathogens to suppress plant defenses. Curr. Opin. Plant Biol. 7, 356– 364. Allen, R.L., Bittner-Eddy, P.D., Grenville-Briggs, L.J., Meitz, J.C., Rehmany, A.P., Rose, L.E. and Beynon, J.L. (2004) Host–parasite coevolutionary conflict between Arabidopsis and downy mildew. Science, 306, 1957–1960. Allen, R.L., Meitz, J.C., Baumber, R.E., Hall, S.A., Lee, S.C., Rose, L.E. and Beynon, J.L. (2008) Natural variation reveals key amino acids in a © 2009 THE AUTHORS JOURNAL COMPILATION © 2009 BLACKWELL PUBLISHING LTD MOLECULAR PLANT PATHOLOGY (2009) 10(4), 547–562 558 I. HEIN et al. downy mildew effector that alters recognition specificity by an Arabidopsis resistance gene. Mol. Plant Pathol. 9, 511–523. Armstrong, M.R., Whisson, S.C., Pritchard, L., Bos, J.I., Venter, E., Avrova, A.O., Rehmany, A.P., Böhme, U., Brooks, K., Cherevach, I., Hamlin, N., White, B., Fraser, A., Lord, A., Quail, M.A., Churcher, C., Hall, N., Berriman, M., Huang, S., Kamoun, S., Beynon, J.L. and Birch, P.R.J. (2005) An ancestral oomycete locus contains late blight avirulence gene Avr3a, encoding a protein that is recognized in the host cytoplasm. Proc. Natl. Acad. Sci. USA, 102, 7766–7771. Attard, A., Gourgues, M., Galiana, E., Panabières, F., Ponchet, M. and Keller, H. (2008) Strategies of attack and defense in plant–oomycete interactions, accentuated for Phytophthora parasitica Dastur (syn. P. Nicotianae Breda de Haan). J. Plant Physiol. 165, 83–94. Bailey, B.A. (1995) Purification of a protein from culture filtrates of Fusarium oxysporum that induces ethylene and necrosis in leaves of Erythroxylum coca. Phytopathology, 85, 1250–1255. Bakker, E.G., Toomajian, C., Kreitman, M. and Bergelson, J. (2006) A genome-wide survey of R gene polymorphisms in Arabidopsis. Plant Cell, 18, 1803–1818. Ballvora, A., Ercolano, M.R., Weiss, J., Meksem, K., Bormann, C.A., Oberhagemann, P., Salamini, F. and Gebhardt, C. (2002) The R1 gene for potato resistance to late blight (Phytophthora infestans) belongs to the leucine zipper/NBS/LRR class of plant resistance genes. Plant J. 30, 361–371. Bartinicki-Garcia, S. and Wang, M.C. (1983) Biochemical aspects of morphogenesis in Phytophthora. In: Phytophthora: Its Biology, Taxonomy, Ecology and Pathology (Bartinicki-Garcia S. and Tsao P.H., eds), pp. 121–137. St. Paul, MN: American Phytopathological Society. Belkhadir, Y., Subramaniam, R. and Dangl, J.L. (2004) Plant disease resistance protein signaling: NBS-LRR proteins and their partners. Curr. Opin. Plant Biol. 7, 391–399. Bergelson, J., Kreitman, M., Stahl, E.A. and Tian, D. (2001) Evolutionary dynamics of plant R-genes. Science, 292, 2281–2285. Bhattacharjee, S., Hiller, N.L., Liolios, K., Win, J., Kanneganti, T.D., Young, C., Kamoun, S. and Haldar, K. (2006) The malarial hosttargeting signal is conserved in the Irish potato famine pathogen. PLoS Pathog. 2, e50. van der Biezen, E.A., Freddie, C.T., Kahn, K., Parker, J.E. and Jones, J.D. (2002) Arabidopsis RPP4 is a member of the RPP5 multigene family of TIR-NB-LRR genes and confers downy mildew resistance through multiple signalling components. Plant J. 29, 439–451. van der Biezen, E.A. and Jones, J.D.G. (1998) Plant disease-resistance proteins and the gene-for-gene concept. Trends Biochem. Sci. 23, 454– 456. Birch, P.R.J., Boevink, P.C., Gilroy, E.M., Hein, I., Pritchard, L. and Whisson, S.C. (2008) Oomycete RXLR effectors: delivery, functional redundancy and durable disease resistance. Curr. Opin. Plant Biol. 11, 373–379. Birch, P.R.J. and Cooke, D.E.L. (2004) Mechanisms of infection— oomycetes. In: Encyclopedia of Plant and Crop Science (Goodman, R.M., ed.), pp. 697–700. New York, NY: Marcel Dekker Inc. Birch, P.R.J., Rehmany, A.P., Pritchard, L., Kamoun, S. and Beynon, J.L. (2006) Trafficking arms: oomycete effectors enter host plant cells. Trends Microbiol. 14, 8–11. Bittner-Eddy, P.D., Crute, I.R., Holub, E.B. and Beynon, J.L. (2000) RPP13 is a simple locus in Arabidopsis thaliana for alleles that specify downy mildew resistance to different avirulence determinants in Peronospora parasitica. Plant J. 21, 177–188. Blair, J.E., Coffey, M.D., Park, S-Y., Geiser, D.M. and Kang, S. (2008) A multi-locus phylogeny for Phytophthora utilizing markers from complete genome sequences. Fungal Genet. Biol. 45, 266–277. Block, A., Li, G., Fu, Z.Q. and Alfano, J.R. (2008) Phytopathogen type III effector weaponry and their plant targets. Curr. Opin. Plant Biol. 11, 396–403. Bos, J.I., Kanneganti, T.D., Young, C., Cakir, C., Huitema, E., Win, J., Armstrong, M.R., Birch, P.R. and Kamoun, S. (2006) The C-terminal half of Phytophthora infestans RXLR effector AVR3a is sufficient to trigger R3a-mediated hypersensitivity and suppress INF1-induced cell death in Nicotiana benthamiana. Plant J. 48, 165–176. Botella, M.A., Parker, J.E., Frost, L.N., Bittner-Eddy, P.D., Beynon, J.L., Daniels, M.J., Holub, E.B. and Jones, J.D.G. (1998) Three genes of the Arabidopsis RPP1 complex resistance locus recognize distinct Peronospora parasitica avirulence determinants. Plant Cell, 10, 1847– 1860. Brunner, F., Rosahl, S., Lee, J., Rudd, J.J., Geiler, C., Kauppinen, S., Rasmussen, G., Scheel, D. and Nürnberger, T. (2002) Pep-13, a plant defense-inducing pathogen-associated pattern from Phytophthora transglutaminases. EMBO J. 21, 6681–6688. Chisholm, S.T., Coaker, G., Day, B. and Staskawicz, B.J. (2006) Host– microbe interactions: shaping the evolution of the plant immune response. Cell, 124, 803–814. Damasceno, C.M.B., Bishop, J.G., Ripoll, D.R., Win, J., Kamoun, S. and Rose, J.K.C. (2008) Structure of the glucanase inhibitor protein (GIP) family from Phytophthora species suggests coevolution with plant endob-1,3-glucanases. Mol. Plant–Microbe Interact. 21, 820–830. Dangl, J.L. and Jones, J.D. (2001) Plant pathogens and integrated defence responses to infection. Nature, 411, 826–833. Ding, J., Zhang, W., Jing, Z., Chen, J.Q. and Tian, D. (2007) Unique pattern of R-gene variation within populations in Arabidopsis. Mol. Gen. Genom. 277, 619–629. Dou, D., Kale, S.D., Wang, X., Chen, Y., Wang, Q., Wang, X., Jiang, R.X.Y., Arredondo, F.D., Anderson, R.G., Thakur, P.B., McDowell, J.M., Wang, Y. and Tyler, B.M. (2008b) Conserved C-terminal motifs required for avirulence and suppression of cell death by Phytophthora sojae effector Avr1b. Plant Cell, 20, 1118–1133. Dou, D., Kale, S.D., Wang, X., Jiang, R.H.Y., Bruce, N.A., Arredondo, F.D., Zhang, X. and Tyler, B.M. (2008a) RXLR-mediated entry of Phytophthora sojae effector Avr1b into soybean cells does not require pathogen-encoded machinery. Plant Cell, 20, 1930–1947. Dumas, B., Bottin, A., Gaulin, E. and Esquerré-Tugayé, M.T. (2008) Cellulose-binding domains: cellulose associated-defensive sensing partners? Trends Plant Sci. 13, 160–164. Ellis, C. and Turner, J.G. (2001) The Arabidopsis mutant cev1 has constitutively active jasmonate and ethylene signal pathways and enhanced resistance to pathogens. Plant Cell, 13, 1025–1033. Eulgem, T., Tsuchiya, T., Wang, X.J., Beasley, B., Cuzick, A., Tor, M., Zhu, T., McDowell, J.M., Holub, E. and Dangl, J.L. (2007) EDM2 is required for RPP7-dependent disease resistance in Arabidopsis and affects RPP7 transcript levels. Plant J. 49, 829–839. Felix, G. and Boller, T. (2003) Molecular sensing of bacteria in plants. The highly conserved RNA-binding motif RNP-1 of bacterial cold shock proteins is recognized as an elicitor signal in tobacco. J. Biol. Chem. 278, 6201–6208. © 2009 THE AUTHORS MOLECULAR PLANT PATHOLOGY (2009) 10(4), 547–562 JOURNAL COMPILATION © 2009 BLACKWELL PUBLISHING LTD Zig-zag-zig in oomycete–plant interactions Felix, G., Duran, J.D., Volko, S. and Boller, T. (1999) Plants have a sensitive perception system for the most conserved domain of bacterial flagellin. Plant J. 18, 265–276. Fellbrich, G., Romanski, A., Varet, A., Blume, B., Brunner, F., Engelhardt, S., Felix, G., Kemmerling, B., Krzymowska, M. and Nürnberger, T. (2002) NPP1, a Phytophthora-associated trigger of plant defense in parsley and Arabidopsis. Plant J. 32, 375–390. Flor, H.H. (1971) Current status of the gene-for-gene concept. Ann. Rev. Phytopathol. 9, 275–296. Friedman, A.R. and Baker, B.J. (2007) The evolution of resistance genes in multi-protein plant resistance systems. Curr. Opin. Gen. Dev. 17, 493–499. Gaulin, E., Dramé, N., Lafitte, C., Torto-Alalibo, T., Martinez, Y., Ameline-Torregrosa, C., Khatib, M., Mazarguil, H., VillalbaMateos, F., Kamoun, S., Mazars, C., Dumas, B., Bottin, A., EsquerréTugayé, M.T. and Rickauer, M. (2006) Cellulose binding domains of a Phytophthora cell wall protein are novel pathogen-associated molecular patterns. Plant Cell, 18, 1766–1777. Gaulin, E., Jauneau, A., Villalba, F., Rickauer, M., Esquerré-Tugayé, M.T. and Bottin, A. (2002) The CBEL glycoprotein of Phytophthora parasitica var-nicotianae is involved in cell wall deposition and adhesion to cellulosic substrates. J. Cell Sci. 115, 4565–4575. Gijzen, M. and Nürnberger, T. (2006) Nep1-like proteins from plant pathogens: recruitment and diversification of the NPP1 domain across taxa. Phytochemistry, 67, 1800–1807. Göker, M., Voglmayer, H., Riethmuller, A. and Oberwinkler, F. (2006) How do obligate parasites evolve? A multi-gene phylogenetic analysis of downy mildews. Fungal Genet. Biol. 44, 105– 122. Grant, S.R., Fisher, E.J., Chang, J.H., Mole, B.M. and Dangl, J.L. (2006) Subterfuge and manipulation: type III effector proteins of phytopathogenic bacteria. Annu. Rev. Microbiol. 60, 425–449. Grouffaud, S., van West, P., Avrova, A.O., Birch, P.R.J. and Whisson, S.C. (2008) Plasmodium falciparum and Hyaloperonospora parasitica effector translocation motifs are functional in Phytophthora infestans. Microbiology, 154, 3743–3751. Hardham, A.R. (2007) Cell biology of plant–oomycete interactions. Cell. Microbiol. 9, 31–39. Heese, A., Hann, D.R., Gimenez-Ibanez, S., Jones, A.M., He, K., Li J., Schroeder, J.I., Peck, S.C. and Rathjen, J.P. (2007) The receptor-like kinase SERK3/BAK1 is a central regulator of innate immunity in plants. Proc. Natl. Acad. Sci. USA, 104, 12 217–12 222. Hiller, N.L., Bhattacharjee, S., van Ooij, C., Liolios, K., Harrison, T., Lopez-Estrano, C. and Haldar, K. (2004) A host-targeting signal in virulence proteins reveals a secretome in malarial infection. Science, 306, 1934–1937. Holub, E.B. (2001) The arms race is ancient history in Arabidopsis, the wildflower. Nat. Rev. Genet. 2, 516–527. van der Hoorn, R.A.L. and Kamoun, S. (2008) From guard to decoy: a new model for perception of plant pathogen effectors. Plant Cell, 20, 2009–2017. Huang, S. (2005) The discovery and characterization of the major late blight resistance complex in potato-genomic structure, functional diversity, and implications. PhD Thesis. Wageningen University, The Netherlands. Huang, S., Vleeshouwers, V.G., Werij, J.S., Hutten, R.C., van Eck, H.J., Visser, R.G. and Jacobsen, E. (2004) The R3 resistance to Phytoph- 559 thora infestans in potato is conferred by two closely linked R genes with distinct specificities. Mol. Plant–Microbe Interact. 17, 428–435. Huang, S., van der Vossen, E.A., Kuang, H., Vleeshouwers, V.G., Zhang, N., Borm, T.J., van Eck, H.J., Baker, B., Jacobsen, E. and Visser, R.G. (2005) Comparative genomics enabled the isolation of the R3a late blight resistance gene in potato. Plant J. 42, 251–261. Ingle, R.A., Carstens, M. and Denby, K.J. (2006) PAMP recognition and the plant–pathogen arms race. Bioessays, 28, 880–889. Jiang, R.H.Y., Tripathy, S., Govers, F. and Tyler, B.M. (2008) RXLR effector reservoir in two Phytophthora species is dominated by a single rapidly evolving superfamily with more than 700 members. Proc. Natl. Acad. Sci. USA, 105, 4874–4879. Jones, J.D. and Dangl, J.L. (2006) The plant immune system. Nature, 444, 323–329. Kamoun, S. (2003) Molecular genetics of phytopathogenic oomycetes. Eukaryotic Cell, 2, 191–199. Kamoun, S. (2006) A catalogue of the effector secretome of plant pathogenic oomycetes. Annu. Rev. Phytopathol. 44, 41–60. Kamoun, S. (2007) Groovy times: filamentous pathogen effectors revealed. Curr. Opin. Plant Biol. 10, 358–365. Kamoun, S., Huitema, E. and Vleeshouwers, V.G. (1999) Resistance to oomycetes: a general role for the hypersensitive response? Trends Plant Sci. 4, 196–200. Kanzaki, H., Saitoh, H., Ito, A., Fujisawa, S., Kamoun, S., Katou, S., Yoshioka, H. and Terauchi, R. (2003) Cytosolic HSP90 and HSP70 are essential components of INF1-mediated hypersensitive response and non-host resistance to Pseudomonas cichorii in Nicotiana benthamiana. Mol. Plant Pathol. 4, 383–391. Kanzaki, H., Saitoh, H., Takahashi, Y., Berberich, T., Ito, A., Kamoun, S. and Terauchi, R. (2008) NbLRK1, a lectin-like receptor kinase protein of Nicotiana benthamiana, interacts with Phytophthora infestans INF1 elicitin and mediates INF1-induced cell death. Planta, 228, 977–987. Koch, E. and Slusarenko, A. (1990) Arabidopsis is susceptible to infection by a downy mildew fungus. Plant Cell, 2, 437–445. Kuang, H., Ochoa, O.E., Nevo, E. and Michelmore, R.W. (2006) The disease resistance gene Dm3 is infrequent in natural populations of Lactuca serriola due to deletions and frequent gene conversions at the RGC2 locus. Plant J. 47, 38–48. Kuang, H., Wei, F., Marano, M.R., Wirtz, U., Wang, X., Liu, J., Shum, W.P., Zaborsky, J., Tallon, L.J., Rensink, W., Lobst, S., Zhang, P., Tornqvist, C.E., Tek, A., Bamberg, J., Helgeson, J., Fry, W., You, F., Luo, M.C., Jiang, J., Robin Buell, C. and Baker, B. (2005) The R1 resistance gene cluster contains three groups of independently evolving type I R1 homologues and shows substantial structural variation among haplotypes of Solanum demissum. Plant J. 44, 37–51. Kuang, H., Woo, S.S., Meyers, B.C., Nevo, E. and Michelmore, R.W. (2004) Multiple genetic processes result in heterogeneous rates of evolution within the major cluster disease resistance genes in lettuce. Plant Cell, 16, 2870–2894. Lamour, K.H., Win, J. and Kamoun, S. (2007) Oomycete genomics: new insights and future directions. FEMS Microbiol. Lett. 274, 1–8. Langston, J., Blinkovsky, A., Byun, T., Terribilini, M., Ransbarger, D. and Xu, F. (2007) Substrate specificity of Streptomyces transglutaminases. Appl. Biochem. Biotechnol. 136, 291–308. Latijnhouwers, M., de Wit, P.J. and Govers, F. (2003) Oomycetes and fungi: similar weaponry to attack plants. Trends Microbiol. 11, 462– 469. © 2009 THE AUTHORS JOURNAL COMPILATION © 2009 BLACKWELL PUBLISHING LTD MOLECULAR PLANT PATHOLOGY (2009) 10(4), 547–562 560 I. HEIN et al. Lehmann, P. (2002) Structure and evolution of plant disease resistance genes. J. Appl. Genet. 43, 403–414. Levesque, C.A. and de Cock, W.A.M. (2004) Molecular phylogeny and taxonomy of the genus Pythium. Mycol. Res. 108, 1363–1383. Liu, Z., Bos, J.I.B., Armstrong, M., Whisson, S.C., da Cunha, L., Torto, T., Win, J., Avrova, A.O., Wright, F., Birch, P.R.J. and Kamoun, S. (2005) Patterns of diversifying selection in the phytotoxin-like scr74 gene family of Phytophthora infestans. Mol. Biol. Evol. 22, 659–672. van Loon, L.C., Rep, M. and Pieterse, C.M.J. (2006) Significance of inducible defense-related proteins in infected plants. Annu. Rev. Phytopathol. 44, 135–162. Marti, M., Good, R.T., Rug, M., Knuepfer, E. and Cowman, A.F. (2004) Targeting malaria virulence and remodeling proteins to the host erythrocyte. Science, 306, 1930–1933. Mateos, F.V., Rickauer, M. and Esquerré-Tugayé, M.T. (1997) Cloning and characterization of a cDNA encoding an elicitor of Phytophthora parasitica var. nicotianae that shows cellulose-binding and lectin-like activities. Mol. Plant–Microbe Interact. 10, 1045–1053. McDowell, J.M., Dhandaydham, M., Long, T.A., Aarts, M.G.M., Goff, S., Holub, E.B. and Dangl, J.L. (1998) Intragenic recombination and diversifying selection contribute to the evolution of downy mildew resistance at the RPP8 locus of Arabidopsis. Plant Cell, 10, 1861–1874. McDowell, J.M. and Simon, S.A. (2006) Recent insights into R gene evolution. Mol. Plant Pathol. 7, 437–448. Medzhitov, R. and Janeway, C.A. (1997) Innate immunity: impact on the adaptive immune response. Curr. Opin. Immunol. 9, 4–9. Meyers, B.C., Chin, D.B., Shen, K.A., Sivaramakrishnan, S., Lavelle, D.O., Zhang, Z. and Michelmore, R.W. (1998b) The major resistance gene cluster in lettuce is highly duplicated and spans several megabases. Plant Cell, 10, 1817–1832. Meyers, B.C., Dickerman, A.W., Michelmore, R.W., Sivaramakrishnan, S., Sobral, B.W. and Young, N.D. (1999) Plant disease resistance genes encode members of an ancient and diverse protein family within the nucleotide-binding superfamily. Plant J. 20, 317–332. Meyers, B.C., Kozik, A., Griego, A., Kuang, H. and Michelmore, R.W. (2003) Genome-wide analysis of NBS-LRR-encoding genes in Arabidopsis. Plant Cell, 15, 809–834. Meyers, B.C., Shen, K.A., Rohani, P., Gaut, B.S. and Michelmore, R.W. (1998a) Receptor-like genes in the major resistance locus of lettuce are subject to divergent selection. Plant Cell, 10, 1833–1846. Michelmore, R.W. and Meyers, B.C. (1998) Clusters of resistance genes in plants evolve by divergent selection and a birth-and-death process. Genome Res. 8, 1113–1130. Nei, M., Gu, X. and Sitnikova, T. (1997) Evolution by the birth-and-death process in multigene families of the vertebrate immune system. Proc. Natl. Acad. Sci. USA, 94, 7799–7806. Nürnberger, T., Brunner, F., Kemmerling, B. and Piater, L. (2004) Innate immunity in plants and animals: striking similarities and obvious differences. Immunol. Rev. 198, 249–266. Nürnberger, T. and Lipka, V. (2005) Non-host resistance in plants: new insights into an old phenomenon. Mol. Plant Pathol. 6, 335–345. Nürnberger, T., Nennstiel, D., Jabs, T., Sacks, W.R., Hahlbrock, K. and Scheel, D. (1994) High affinity binding of a fungal oligopeptide elicitor to parsley plasma membranes triggers multiple defense responses. Cell, 78, 449–460. Orosomando, G., Lornzi, M., Raffaelli, N., Dalla Rizza, M., Mezzetti, B. and Ruggieri, S. (2001) Phytotoxic protein PcF: purification, characteri- sation and cDNA sequencing of a novel hydroxyproline-containing factor secreted by the strawberry pathogen Phytophthora cactorum. J. Biol. Chem. 276, 21 575–21 584. Osman, H., Vauthrin, S., Mikes, V., Milat, M.L., Panabières, F., Marais, A., Brunie, S., Maume, B., Ponchet, M. and Blein, J.P. (2001) Mediation of elicitin activity on tobacco is assumed by elicitin–sterol complexes. Mol. Biol. Cell, 12, 2825–2834. Panabieres, F., Amselem, J., Galiana, E. and Le Berre, J.Y. (2005) Gene identification in the oomycete pathogen Phytophthora parasitica during in vitro vegetative growth through expressed sequence tags. Fungal Genet. Biol. 42, 611–623. Parker, J.E., Coleman, M.J., Szabo, V., Frost, L.N., Schmidt, R., van der Biezen, E.A., Moores, T., Dean, C., Daniels, M.J. and Jones, J.D.G. (1997) The Arabidopsis downy mildew resistance gene RPP5 shares similarity to the toll and interleukin-1 receptors with N and L6. Plant Cell, 9, 879–894. Peart, J.R., Lu, R., Sadanandom, A., Malcuit, I., Moffett, P., Brice, D.C., Schauser, L., Jaggard, D.A., Xiao, S., Coleman, M.J., Dow, M., Jones, J.D., Shirasu, K. and Baulcombe, D.C. (2002) Ubiquitin ligase-associated protein SGT1 is required for host and nonhost disease resistance in plants. Proc. Natl. Acad. Sci. USA, 99, 10 865– 10 869. Pemberton, C.L., Whitehead, N.A., Sebaihia, M., Bell, K.S., Hyman, L.J., Harris, S.J., Matlin, A.J., Robson, N.D., Birch, P.R.J., Carr, J.P., Toth, I.K. and Salmond, G.P.S. (2005) Novel quorum-sensingcontrolled genes in Erwinia carotovora subsp. carotovora: identification of a fungal elicitor homologue in a soft-rotting bacterium. Mol. Plant– Microbe. Interact. 18, 343–353. Pieterse, C.M., van West, P., Verbakel, H.M., Brasse, P.W., van den Berg-Velthuis, G.C. and Govers, F. (1994) Structure and genomic organization of the ipiB and ipiO gene clusters of Phytophthora infestans. Gene, 138, 67–77. van Poppel, P.M.J.A., Guo, J., van de Vondervoort, P.J.I., Jung, M.W.M., Birch, P.R.J., Whisson, S.C. and Govers, F. (2008) The Phytophthora infestans avirulence gene Avr4 encodes an RXLR-dEER effector. Mol. Plant–Microbe Interact. 21, 1460–1470. Qutob, D., Kamoun, S. and Gijzen, M. (2002) Expression of a Phytophthora sojae necrosis-inducing protein occurs during transition from biotrophy to necrotrophy. Plant J. 32, 361–373. Qutob, D., Kemmerling, B., Brunner, F., Küfner, I., Engelhardt, S., Gust, A.A., Luberacki, B., Seitz, H.U., Stahl, D., Rauhut, T., Glawischnig, E., Schween, G., Lacombe, B., Watanabe, N., Lam, E., Schlichting, R., Scheel, D., Nau, K., Dodt, G., Hubert, D., Gijzen, M. and Nürnberger, T. (2006) Phytotoxicity and innate immune responses induced by Nep1-like proteins. Plant Cell, 18, 3721–3744. Rehmany, A.P., Gordon, A., Rose, L.E., Allen, R.L., Armstrong, M.R., Whisson, S.C., Kamoun, S., Tyler, B.M., Birch, P.R. and Beynon, J.L. (2005) Differential recognition of highly divergent downy mildew avirulence gene alleles by RPP1 resistance genes from two Arabidopsis lines. Plant Cell, 17, 1839–1850. Rentel, M.C., Leonelli, L., Dahlbeck, D., Zhao, B. and Staskawicz, B.J. (2008) Recognition of the Hyaloperonospora parasitica effector ATR13 triggers resistance against oomycete, bacterial, and viral pathogens. Proc. Natl. Acad. Sci. USA, 105, 1091–1096. Robold, A.V. and Hardham, A.R. (2005) During attachment Phytophthora spores secrete proteins containing thrombospondin type 1 repeats. Curr. Genet. 47, 307–315. © 2009 THE AUTHORS MOLECULAR PLANT PATHOLOGY (2009) 10(4), 547–562 JOURNAL COMPILATION © 2009 BLACKWELL PUBLISHING LTD Zig-zag-zig in oomycete–plant interactions Rose, J.K., Ham, K.S., Darvill, A.G. and Albersheim, P. (2002) Molecular cloning and characterization of glucanase inhibitor proteins: co-evolution of a counter-defense mechanism by plant pathogens. Plant Cell, 14, 1329–1345. Rose, L.E., Bittner-Eddy, P.D., Langley, C.H., Holub, E.B., Michelmore, R.W. and Beynon, J.L. (2004) The maintenance of extreme amino acid diversity at the disease resistance gene, RPP13, in Arabidopsis thaliana. Genetics, 166, 1517–1527. Schulze-Lefert, P. and Panstruga, R. (2003) Establishment of biotrophy by parasitic fungi and reprogramming of host cells for disease resistance. Annu. Rev. Phytopathol. 41, 641–667. Shabab, M., Shindo, T., Gu, C., Kaschani, F., Pansuriya, T., Chintha, R., Harzen, A., Colby, T., Kamoun, S. and van der Hoorn, R.A.L. (2008) Fungal effector protein AVR2 targets diversifying defense-related cys proteases of tomato. Plant Cell, 20, 1169–1183. Shan, W., Cao, M., Leung, D. and Tyler, B.M. (2004) The Avr1b locus of Phytophthora sojae encodes an elicitor and a regulator required for avirulence on soybean plants carrying resistance gene Rps1b. Mol. Plant–Microbe Interact. 17, 394–403. Sharp, J.K., Valent, B. and Albersheim, P. (1984) Purification and partial characterisation of a beta-glucan fragment that elicits phytoalexin accumulation in soybean. J. Biol. Chem. 259, 11 312–11 320. Shen, K.A., Chin, D.B., Arroyo-Garcia, R., Ochoa, O.E., Lavelle, D.O., Wroblewski, T., Meyers, B.C. and Michelmore, R.W. (2002) Dm3 is one member of a large constitutively expressed family of nucleotide binding site–leucine-rich repeat encoding genes. Mol. Plant–Microbe Interact. 15, 251–261. Sinapidou, E., Williams, K., Nott, L., Bahkt, S., Tor, M., Crute, I., Bittner-Eddy, P. and Beynon, J. (2004) Two TIR:NB:LRR genes are required to specify resistance to Peronospora parasitica isolate Cala2 in Arabidopsis. Plant J. 38, 898–909. Slusarenko, A.J. and Schlaich, N.L. (2003) Downy mildew of Arabidopsis thaliana caused by Hyaloperonospora parasitica (formerly Peronospora parasitica). Mol. Plant Pathol. 4, 159–170. Sohn, K.H., Lei, R., Nemri, A. and Jones, D.G. (2007) The downy mildew effector proteins ATR1 and ATR13 promote disease susceptibility in Arabidopsis thaliana. Plant Cell, 19, 4077–4090. Song, J., Bradeen, J.M., Naess, S.K., Raasch, J.A., Wielgus, S.M., Haberlach, G.T., Liu, J., Kuang, H., Austin-Phillips, S., Buell, C.R., Helgeson, J.P. and Jiang, J. (2003) Gene RB cloned from Solanum bulbocastanum confers broad spectrum resistance to potato late blight. Proc. Natl. Acad. Sci. USA, 100, 9128–9133. Song, J., Win, J., Tian, M., Schornack, S., Kaschani, F., Ilyas, M., van der Hoorn, R. and Kamoun, S. (2009) Apoplastic effectors secreted by two unrelated eukaryotic plant pathogens target the tomato defence protease Rcr3. Proc. Natl. Acad. Sci. USA, 106, 1654–1659. Stahl, E.A., Dwyer, G., Mauricio, R., Kreitman, M. and Bergelson, J. (1999) Dynamics of disease resistance polymorphism at the Rpm1 locus of Arabidopsis. Nature, 400, 667–671. Takahashi, Y., Nasir, K.H., Ito, A., Kanzaki, H., Matsumura, H., Saitoh, H., Fujisawa, S., Kamoun, S. and Terauchi, R. (2007) A highthroughput screen of cell-death-inducing factors in Nicotiana benthamiana identifies a novel MAPKK that mediates INF1-induced cell death signaling and non-host resistance to Pseudomonas cichorii. Plant J. 49, 1030–1040. Tian, M., Benedetti, B. and Kamoun, S. (2005) A second Kazal-like protease inhibitor from Phytophthora infestans inhibits and interacts 561 with the apoplastic pathogenesis-related protease P69B of tomato. Plant Physiol. 138, 1785–1793. Tian, M., Huitema, E., da Cunha, L., Torto-Alalibo, T. and Kamoun, S. (2004) A Kazal-like extracellular serine protease inhibitor from Phytophthora infestans targets the tomato pathogenesis-related protease P69B. J. Biol. Chem. 279, 26 370–26 377. Tian, M., Win, J., Song, J., van der Hoorn, R., van der Knaap, E. and Kamoun, S. (2007) A Phytophthora infestans cystatin-like protein targets a novel tomato papain-like apoplastic protease. Plant Physiol. 143, 364–377. Torto, T.A., Li, S., Styer, A., Huitema, E., Testa, A., Gow, N.A., van West, P. and Kamoun, S. (2003) EST mining and functional expression assays identify extracellular effector proteins from the plant pathogen Phytophthora. Genome Res. 13, 1675–1685. Torto-Alalibo, T., Tian, M., Gajendran, K., Waugh, M.E., van West, P. and Kamoun, S. (2005) Expressed sequence tags from the oomycete fish pathogen Saprolegnia parasitica reveal putative virulence factors. BMC Microbiol. 5, 46. Tyler, B.M., Tripathy, S., Zhang, X., Dehal, P., Jiang, R.H., Aerts, A., Arredondo, F.D., Baxter, L., Bensasson, D., Beynon, J.L., Chapman, J., Damasceno, C.M., Dorrance, A.E., Dou, D., Dickerman, A.W., Dubchak, I.L., Garbelotto, M., Gijzen, M., Gordon, S.G., Govers, F., Grunwald, N.J., Huang, W., Ivors, K.L., Jones, R.W., Kamoun, S., Krampis, K., Lamour, K.H., Lee, M.K., McDonald, W.H., Medina, M., Meijer, H.J., Nordberg, E.K., Maclean, D.J., Ospina-Giraldo, M.D., Morris, P.F., Phuntumart, V., Putnam, N.H., Rash, S., Rose, J.K., Sakihama, Y., Salamov, A.A., Savidor, A., Scheuring, C.F., Smith, B.M., Sobral, B.W., Terry, A., Torto-Alalibo, T.A., Win, J., Xu, Z., Zhang, H., Grigoriev, I.V., Rokhsar, D.S. and Boore, J.L. (2006) Phytophthora genome sequences uncover evolutionary origins and mechanisms of pathogenesis. Science, 313, 1261–1266. Umemoto, N., Kakitani, M., Iwamatsu, A., Yoshikawa, M., Yamaoka, M. and Ishida, I. (1997) The structure and function of a soybean beta glucan-elicitor binding protein. Proc. Natl. Acad. Sci. USA, 94, 1029– 1034. Vauthrin, S., Mikes, V., Milat, M.L., Ponchet, M., Maume, B., Osman, H. and Blein, J.P. (1999) Elicitins trap and transfer sterols from micelles, liposomes and plant plasma membranes. Biochim. Biophys. Acta, 1419, 335–342. Vleeshouwers, V.G., Rietman, H., Krenek, P., Champouret, N., Young, C., Oh, S.K., Wang, M., Bouwmeester, K., Vosman, B., Visser, R.G., Jacobsen, E., Govers, F., Kamoun, S. and Van der Vossen, E.A. (2008) Effector genomics accelerates discovery and functional profiling of potato disease resistance and Phytophthora infestans avirulence genes. PLoS ONE 3, e2875. van der Vossen, E., Gros, J., Sikkema, A., Muskens, M., Wouters, D., Wolters, P., Pereira, A. and Allefs, S. (2005) The Rpi-blb2 gene from Solanum bulbocastanum is an Mi-1 gene homolog conferring broadspectrum late blight resistance in potato. Plant J. 44, 208–222. van der Vossen, E., Sikkema, A., Hekkert, B.L., Gros, J., Stevens, P., Muskens, M., Wouters, D., Pereira, A., Stiekema, W. and Allefs, S. (2003) An ancient R gene from the wild potato species Solanum bulbocastanum confers broad-spectrum resistance to Phytophthora infestans in cultivated potato and tomato. Plant J. 36, 867–882. Wang, M., Allefs, S., van den Berg, R.G., Vleeshouwers, V.G., van der Vossen, E.A.G. and Vosman, B. (2008) Allele mining in Solanum: © 2009 THE AUTHORS JOURNAL COMPILATION © 2009 BLACKWELL PUBLISHING LTD MOLECULAR PLANT PATHOLOGY (2009) 10(4), 547–562 562 I. HEIN et al. conserved homologues of Rpi-blb1 are identified in Solanum stoloniferum. Theor. Appl. Genet. 116, 933–943. van West, P., de Jong, A.J., Judelson, H.S., Emons, A.M. and Govers, F. (1998) The ipiO gene of Phytophthora infestans is highly expressed in invading hyphae during infection. Fungal Genet. Biol. 23, 126– 138. Whisson, S.C., Boevink, P.C., Moleleki, L., Avrova, A.O., Morales, J.G., Gilroy, E.M., Armstrong, M.R., Grouffaud, S., van West, P., Chapman, S., Hein, I., Toth, I.K., Pritchard, L. and Birch, P.R.J. (2007) A translocation signal for delivery of oomycete effector proteins into host plant cells. Nature, 450, 115–119. Win, J., Kanneganti, T.D., Torto-Alalibo, T. and Kamoun, S. (2006) Computational and comparative analyses of 150 full-length cDNA sequences from the oomycete plant pathogen Phytophthora infestans. Fungal Genet. Biol. 43, 20–23. Win, J., Morgan, W., Bos, J., Krasileva, K.V., Cano, L.M., ChaparroGarcia, A., Ammar, R., Staskawicz, B.J. and Kamoun, S. (2007) Adaptive evolution has targeted the C-terminal domain of the RXLR effectors of plant pathogenic oomycetes. Plant Cell, 19, 2349– 2369. Yi, H. and Richards, E.J. (2007) A cluster of disease resistance genes in Arabidopsis is coordinately regulated by transcriptional activation and RNA silencing. Plant Cell, 19, 2929–2939. Yoshioka, H., Numata, N., Nakajima, K., Katou, S., Kawakita, K., Rowland, O., Jones, J.D. and Doke, N. (2003) Nicotiana benthamiana gp91phox homologs NbrbohA and NbrbohB participate in H2O2 accumulation and resistance to Phytophthora infestans. Plant Cell, 15, 706– 718. Zipfel, C. (2008) Pattern-recognition receptors in plant innate immunity. Curr. Opin. Immun. 20, 10–16. © 2009 THE AUTHORS MOLECULAR PLANT PATHOLOGY (2009) 10(4), 547–562 JOURNAL COMPILATION © 2009 BLACKWELL PUBLISHING LTD