fungal and oomycete effectors – strategies to subdue a host · can. j. plant pathol. (2011),...
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Can. J. Plant Pathol. (2011), 33(4): 425–446
Minireview/Minisynthèse
Fungal and oomycete effectors – strategies to subdue a host
SHAWKAT ALI1,2 AND GUUS BAKKEREN1
1Agriculture and Agri-Food Canada, Pacific Agri-Food Research Center, Summerland, BC V0H 1Z0, Canada2Department of Botany, University of British Columbia, Vancouver, BC V6T 1Z4, Canada; current address: Horticulture R&D Center,Agriculture and Agri-Food Canada, St-Jean-sur-Richelieu Québec J3B 3E6, Canada
(Accepted 4 september 2011)
Abstract: Molecular studies focusing on the interface between microbes and plant hosts have provided major insights into the basisunderlying pathogenesis, symbiosis and plant defence and resistance mechanisms. A more recent focus on microbes, facilitated by thegeneration of complete genome sequences, has uncovered the sheer number of protein effectors microbes deliver in this interface as well asinside host cells to manipulate the plant immune system. Although studies on the characterization and roles of bacterial effectors are furtheradvanced, in this review we focus on the current knowledge of fungal and oomycete effectors and their roles. Examples are given of effectorsdisarming plant defence enzymes, such as the apoplastic effector AVR2 from Cladosporium fulvum which inhibits the tomato defence cysteineprotease. Other effectors interfere with the perception by the host of microbes exposing molecular determinants such as Phytophthorainfestans INF1 protein. Many effectors alter gene expression induced by the host during defence, exemplified in fungi by Ustilago maydisPit2 suppressing maize defence genes. Effectors recognized by resistance gene products, either directly or indirectly, and eliciting defence,represent the classical avirulence genes and almost 50 have now been cloned from fungi and oomycetes. Evolutionary adaptations and armsraces have produced diversification in both pathogen and host, and in pathogens, are the cause of breaking crop resistance in agriculturalsettings. Molecular insight provides valuable information for applications. For example, some effectors are crucial for pathogenesis, therebyrevealing targets for disease control and others interact with host resistance gene products and could be used to screen germplasm for novelsources of disease resistance. Variation among effectors will likely yield diagnostic tools for pathogen race identification. The study of modelsystems is providing insight into avenues by which other, major plant diseases can potentially be controlled.
Keywords: avirulence genes, disease resistance, effector proteins, microbe–plant interactions
Résumé: Les études moléculaires ciblant l’interface entre les microorganismes et les plantes hôtes ont ouvert de nouvelles perspectives sur lesbases sous-jacentes de la pathogenèse, de la symbiose ainsi que des mécanismes de défenses et de résistance. Une nouvelle approche desmicroorganismes, facilitée par la génération de séquences entières de génomes, a permis de découvrir l’importance du nombre de protéineseffectrices que les microorganismes sécrètent dans cette interface ainsi que dans les cellules hôtes afin de modifier le système immunitaire dela plante. Bien que les études sur la caractérisation et les rôles des effecteurs microbiens soient avancées, dans cet exposé, nous mettonsl’accent sur les connaissances actuelles relatives aux effecteurs fongiques et à ceux des oomycètes, ainsi que sur leurs rôles. On y présente desexemples d’effecteurs neutralisant les enzymes de défense de plantes, comme l’effecteur apoplastique AVR2 de Cladosporium fulvum quiinhibe chez la tomate les mécanismes de défense dépendant de la protéase à cystéine. D’autres effecteurs modifient la perception que l’hôte ade microorganismes exposant des déterminants moléculaires comme la protéine INF1 de Phytophthora infestans. Un grand nombred’effecteurs altèrent l’expression du gène induite durant la phase de défense, phénomène illustré chez les champignons par la protéinePit-2 d’Ustilago maydis qui inactive les gènes de défense du maïs. Les effecteurs reconnus, directement ou indirectement, par les produitsgéniques de résistance suscitant la défense sont les gènes classiques d’avirulence et presque 50 ont à ce jour été clonés à partir dechampignons et d’oomycètes. Les adaptations évolutives et les « courses aux armements » ont induit la diversification tant chez l’hôte quechez les agents pathogènes et, chez ces derniers, elles sont la cause de l’effondrement de la résistance des cultures commerciales. Lesperspectives moléculaires fournissent des données précieuses en vue d’applications utiles. Par exemple, certains effecteurs sont essentiels à la
Correspondence to: G. Bakkeren. E-mail: [email protected]
ISSN: 0706-0661 print/ISSN 1715-2992 online © 2011 The Canadian Phytopathological Societyhttp://dx.doi.org/10.1080/07060661.2011.625448
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pathogenèse, déterminant de ce fait des cibles de lutte contre les maladies des plantes; d’autres interagissent avec les produits géniques de larésistance de l’hôte et pourraient être utilisés pour analyser les germoplasmes afin d’y déceler de nouvelles sources de résistance aux maladies.La variation observée chez les effecteurs contribuera de toute évidence au développement d’outils de diagnostic permettant l’identification deraces pathogènes. L’étude de modèles ouvre de nouvelles voies grâce auxquelles d’autres maladies importantes pourront être traitées.
Mots clés: gènes d’avirulence, interactions microorganismes plantes, protéines effectrices, résistance aux maladies
Introduction
Among the myriad of microbes larger organismsencounter, there are a few that have adapted to over-come the many barriers and defence mechanisms of theseorganisms to become pathogens. The more tools suchpathogens have at their disposal, the more virulent theyare on a given host. Over the last 10 years, tremendousprogress has been made in the field of plant–microbeinteractions and the discovery of many pathogen and hostgenes involved in these interactions has provided someinsight into the molecular mechanisms that decide the out-come of such encounters i.e. plant disease or host defenceand resistance. Pathogens flood their hosts with numer-ous proteins and compounds to counteract the defenceand ensure proliferation. The host, on its part, resists andthe uncovering of the increasingly astonishing ways eachinteracting organism tries to decide the outcome of thisdelicate balance in their favour, promises novel strategiesto help plants boost their defence responses. Although thefocus of this paper is on fungal and oomycete effectors,many recent discoveries into their roles have emergedfrom work on bacterial systems, some of which will behighlighted because they might direct research on fungaland oomycete effectors.
Strategies used by pathogens to infect host plants
Pathogens use different strategies to enter their hostplants. Fungi and oomycetes enter either through nat-ural openings or directly through the plant epidermalcells by mechanical and chemical means, or expand theirhyphae on the top of, within or between the plant cells(Jones & Dangl, 2006). Pathogens need to adhere to theplant before penetrating the plant cuticle. Fungal hyphaeand spores use mucilaginous and adhesive substances attheir tips, and the intermolecular forces between plantand pathogen are responsible for the close contact. Mostrust fungi enter plants through stomata by developingappressoria over the stomata to penetrate into the cavitybelow, while ascomycetes such as the rice blast fungusMagnaporthe grisea and the powdery mildews, such asBlumeria graminis f. sp. hordei (Bgh), penetrate the cuti-cle of plants directly by means of an appressorium. For
direct penetration of the cuticle, M. grisea uses turgorpressure in its melanized appressorium while Bgh pene-trates cell walls by the combined activity of cellulases andturgor pressure (Pryce-Jones et al., 1999; Talbot, 2003).Other pathogens secrete cell wall-degrading enzymes,such as cutinases, cellulases, pectinases and wax degrad-ing enzymes for penetration of their host. After gainingentrance to the host plants, pathogens require additional‘tools’ to neutralize the defence reaction of the host and togain access to nutrients.
Resistance in plants to pathogens
Resistance in plants to pathogens is often categorized inthree types; nonhost, race-nonspecific and race-specificresistance. Nonhost resistance is defined by the resistanceof an entire plant species against a specific non-adaptedpathogenic microbe and is the most common and durableform of disease resistance exhibited by plants. It is theresult of both preformed and inducible defence mecha-nisms and seems to be under complex genetic controlwhich can involve multiple defence factors that individ-ually may segregate within host species without com-promising overall resistance (Heath, 2002; Mysore &Ryu, 2004). Race-nonspecific resistance is known as gen-eral, quantitative or partial resistance of a plant against aspecies of an adapted pathogen. It is generally durable andis mostly controlled by genes with incremental/additiveeffects, such as combinations of wheat leaf rust resis-tance gene Lr34 and several ‘slow-rusting’ genes (Singh& Rajaram, 1992). Race-specific resistance is qualitative,usually controlled by dominant avirulence (Avr) genesin the pathogen and corresponding dominant resistance(R) genes in a specific plant genotype or cultivar of anotherwise susceptible host species. This resistance is ofthe ‘gene-for-gene interaction’ type and is often easilyovercome by the pathogen.
Preformed physical barriers can include waxy cuticu-lar surface layers of the leaves and thick and stable cellwalls that most microbes are not equipped to penetrate.Even after successful penetration, the pathogen needs toovercome the biochemical barriers that include low pHof the apoplast, broad-spectrum antimicrobial compoundsand ‘defence’ enzymes that degrade microbial cell walls
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Fungal and oomycete effectors 427
(Felle, 1998; Dangl & Jones, 2001; Huckelhoven, 2007).Cell wall components such as actin microfilaments playan important role in defence against fungal penetrationand whose disruption results in loss of nonhost resistanceagainst several nonhost fungi (Kobayashi et al., 1997;Heath, 2002; Mysore & Ryu, 2004). Biochemical barriersconsist of many peptides, proteins and non-proteinaceoussecondary antimicrobial metabolites which may deter-mine the host range of some pathogens (Broekaert et al.,1995; Morrissey & Osbourn, 1999), and fungitoxic sub-stances on the surface of leaves in sufficient concen-tration to inhibit the germination of spores (Agrios,2005). Phytoanticipins, preformed antimicrobial com-pounds such as tannins and fatty acid-like dienes, oftenpresent in high concentration in cells of young fruits,leaves or seeds, or saponins, are responsible for resis-tance to pathogens that lack the corresponding detoxifyingenzymes (Osbourn et al., 1996; Pedras & Ahiahonu,2005). Other classes of antimicrobial compounds areproteases, such as cysteine proteases secreted into theapoplast by tomato and potato (Lucas, 1998; Kruger et al.,2002) and protease inhibitors, such as a cysteine proteaseinhibitor produced by pearl millet (Joshi et al., 1999).
Some of the above-mentioned defence systems arepreformed or could be kicked into higher gear, orrepresent inducible mechanisms only activated in thehost when attacked. Examples are induced physicaldefences, such as the formation of cell wall deposi-tions (papilla) directly under the penetration sites ofpathogens; this can stop up to 90% of the penetra-tion attempts in the Bgh/Arabidopsis nonhost interaction(Collins et al., 2003). The production of phytoalexinsis often induced upon attack (Hammerschmidt, 1999).Pathogenesis-related (PR) defence proteins such as chiti-nases and β-1,3 glucanase, (per-)oxidases, proteinaseinhibitors, thionins and lipid-transfer proteins, are inducedin response to pathogen attack and the accumulation ofthese proteins is usually associated with the acquisitionof systemic resistance in plants against a wide rangeof other pathogens. These are specific families of pro-teins, currently in 17 classes (van Loon et al., 2006)although a larger group of ‘‘inducible defence-related pro-teins’’, including phenylalanine ammonia lyase, one ofthe key enzymes involved in the synthesis of aromaticcompounds, like phytoalexins, is often included in the PRproteins.
Pathogen-associated molecular patterns
In addition to preformed and inducible physical and bio-chemical barriers, plants also have evolved a surveillancesystem to recognize various pathogen surface-exposed
and cytoplasmic molecules known as pathogen (microbe)associated molecular patterns: PAMPs or MAMPs (Shiu& Bleecker, 2003). These are highly conserved moleculesof microbes that are perceived by host receptors (PAMPrecognition receptors or PRRs) at an early stage of infec-tion. Recognition results in induction of PAMP-triggeredimmunity (PTI). Examples of surface-exposed PAMPsthat have been shown to be capable of triggering PTIare bacterial flagellin (Felix et al., 1999), lipopolysaccha-rides (LPS; Meyer et al., 2001; Erbs & Newman, 2003),lipooligosaccharides from Gram-negative bacteria, chitinfrom cell walls of higher fungi (Bartnicki-Garcia, 1968;Ren & West, 1992), invertase from Saccharomyces cere-visiae (Basse et al., 1992), and 1,3-1,6-hepta-β-glucosidefrom the cell walls of Phytophthora (Sharp et al., 1984a,1984b). Examples of cytoplasmic PAMPs that inducehost defence are cold shock protein (CSP) and elonga-tion factor Tu (Ef-Tu; Felix & Boller, 2003; Kunze et al.,2004).
Pathogen effectors and their delivery duringplant–microbe interactions
Pathogens secrete a wide range of so-called ‘effectors’,small molecules and proteins that can modify host cellstructures or functions. Effectors from several groups ofpathogens such as bacteria, oomycetes and fungi can enterplant cells (Huang et al., 2003; Chisholm et al., 2006;Kamoun, 2007). Bacteria use the Type II, Type IV andType III Secretion System (T3SS) to deliver 20 to hun-dreds of protein effectors into the host plant (Lindeberget al., 2006; Cunnac et al., 2009). Oomycetes and fungilikely secrete even more protein effectors; computationalanalysis of their genomes has revealed hundreds (Kamperet al., 2006; Tyler et al., 2006; Jiang et al., 2008; Muelleret al., 2008; Haas et al., 2009; Schirawski et al., 2010),even more than 1700 of potential effectors for rust fungi(Duplessis et al., 2011).
Predicted effectors from oomycetes, in addition to aN-terminal signal peptide (SP), carry a host targeting sig-nal (HTS) next to the SP that contains conserved RXLRand a dEER motifs that can target them into host cells inthe absence of the pathogen (Rehmany et al., 2005; Tyleret al., 2006; Whisson et al., 2007; Jiang et al., 2008; Douet al., 2008b; Haas et al., 2009; Kale et al., 2010), whileeffectors from fungi have an N-terminal SP and in somecases an RXLR-like motif (Dean et al., 2005; Kamperet al., 2006; Kale et al., 2010; Schirawski et al., 2010).Many examples exist where predicted effectors with aSP are shown to be delivered into the host cytoplasm(Kemen et al., 2005; Catanzariti et al., 2006; Mosqueraet al., 2009; Doehlemann et al., 2009; Khang et al.,
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2010) and some can do so through N-terminal sequenceswithout the help of the pathogen (Rafiqi et al., 2010; Kaleet al., 2010). Recently, Kale et al. (2010) showed that theconserved RXLR motif from oomycetes and the RXLR-like motif from other fungi bind specifically to phos-pholipids, in particular phosphatidylinositol-3-phosphate(PI3P) on the surface of the plasma membrane and likelyenter the cell through lipid raft-mediated endocytosis;results on lipid-binding assays have been controversial(Gan et al., 2010). In a search for possible equivalentmotifs in other fungi, Godfrey et al. (2010) showed thatsmall secreted proteins from haustoria-forming fungalpathogens share a conserved Y/F/WxC motif in additionto an N-terminal secretion signal although no function hasyet been ascribed to these motifs.
Roles of effectors in infection
Breaking the physical barrier
After landing on the surface of plants, pathogens eitherenter their host through natural openings (stomata andhydathodes) or penetrate the surface directly. Bacteriause natural openings or wounds to enter the apoplastfor colonization. Pathogenic bacteria sense compoundsreleased during photosynthesis from stomata and movetowards them. The PRR of the guard cells can sensePAMPs such as bacterial flagellin or lipopolysaccharidesor lipooligosaccharides, which induces the closure ofstomata (Gohre & Robatzek, 2008). Pathogenic bacte-ria, such as Pseudomonas syringae, produce coronatine,a phytotoxin that mimics jasmonic acid (JA) to interferewith salicylic acid (SA) and abscisic acid (ABA) sig-nalling for reopening stomata to get access to the hostapoplast (Melotto et al., 2006). It is not yet known whetherMAMP-associated defence pathways also close stomatato eukaryotic pathogens and whether these pathogens,such as cereal rusts, use effectors in a similar way to over-come this hurdle. Powdery mildew fungi, such as Bgh,penetrate the cuticle of plant cells directly by forming anappressorium. It has been shown that effectors Avra10 andAvrk1 increase the penetration efficiency of Bgh on sus-ceptible barley cultivars but the exact mechanism is notyet known (Ridout et al., 2006). After getting into theplant apoplast, the next physical barrier is the plant cellwall. To promote nutrient leakage from the cytosol into theapoplast, lytic enzymes that degrade the cell wall locallyare secreted. At the same time, pathogens secrete effectormolecules to suppress the host defence that is activated bydanger-associated molecular patterns (DAMPs) generatedfrom degrading cell wall molecules and signalling dam-age to the plant integrity and hence potential danger ofinfection (Jha et al., 2007).
Disarming plant defence enzymes
Plants produce antimicrobial enzymes such as proteases,hydrolases, glucanases and chitinases that can degrade thecell wall of invading pathogenic fungi in the apoplast,without detrimental effect to the plant itself (Lucas, 1998).This has a dual role in defence; the degradation of cellwalls can attenuate fungal growth while on the other handthe molecules released from degraded cell walls serveas elicitors for the induction of host defence. Pathogensuse effector molecules either to stop the delivery ofthese antimicrobial enzymes and compounds by prevent-ing their secretion, or by inhibiting their activity after theyare secreted (Bent & Mackey, 2007), and/or by inhibitingdownstream signalling. Bacteria secrete plant cell wall-degrading enzymes locally in order to construct the T3SSand use effectors such as HopP1 to suppress defenceinduced by DAMPs in the N. benthamiana apoplast (Gustet al., 2007; Oh et al., 2007). HopP1 either sequestersor processes the fragments that are produced during cellwall degradation so that they cannot function as DAMPs(Gohre & Robatzek, 2008).
Examples from fungi are represented by the apoplasticfungus Cladosporium fulvum which secretes effectorAVR2, a cysteine protease inhibitor, during infection thatbinds directly to RCR3, a tomato cysteine protease, to pro-tect the fungus from the deleterious effect of the enzyme.AVR2 also promotes virulence for other fungal pathogensthat cause diseases in tomato, such as Verticillium dahliaeand Botrytis cinerea, when expressed heterologously inArabidopsis (van Esse et al., 2008). Similarly, C. fulvumeffector AVR4 binds to chitin of fungal cell walls to pro-tect it from tomato host chitinases (van den Burg et al.,2006). AVR4 can also protect chitin against plant chiti-nases in the cell wall of other fungi, such as Trichodermaviride and Fusarium solani f. sp. phaseoli (van den Burget al., 2006). In this way, AVR4 not only protects thefungi from hydrolysis by plant chitinases but also keepschitin fragments from eliciting PTI (Libault et al., 2007).Effector ECP6 from C. fulvum has a Lys-M domain thatbinds to carbohydrates including chitin and protects thepathogen from plant chitinases or may be involved inscavenging of chitin fragments that are released duringcell wall degradation by plant chitinases, thus preventingthem from inducing PTI (Bolton et al., 2008).
The oomycete pathogen, P. infestans, is known tosecrete a suite of Cysteine and Kazal family proteaseinhibitors. The tomato papain-like protease, PIP1, whichis induced by SA, is blocked by the EPIC2B inhibitorof P. infestans (Tian & Kamoun, 2005; Tian et al.,2007; van Esse et al., 2008). PIP1 is related to RCR3,and AVR2 from C. fulvum apparently can also inhibit
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PIP1 and two other plant cysteine proteases, aleurainand TDI65 (Rooney et al., 2005; Shabab et al., 2008;van Esse et al., 2008). P. infestans EPIC1 and EPIC2Bcan also bind and inhibit RCR3, similar to AVR2, butunlike AVR2, these EPICs do not elicit an HR on Cf-2/Rcr3pimp tomato plants, suggesting that P. infestansevolved stealthy effectors that can inhibit tomato pro-teases without activating defence responses (Song et al.,2009). These findings show that effectors from differ-ent pathogens can target the same apoplastic enzymesto increase pathogen fitness in the host (Shabab et al.,2008). Recently, another apoplastic tomato and potatopapain-like cysteine protease, C14, was shown to be tar-geted by EPIC1 and EPIC2B (Kaschani et al., 2010).Other P. infestans effectors, EPI1 and EPI10, target thesubtilisin-like serine protease of tomato P69B, a PR pro-tein (Tian et al., 2004; Tian & Kamoun, 2005; Tianet al., 2007). AVRP123, a secreted protein from the flaxrust fungus M. lini, also shows similarity to Kazal serineprotease inhibitors (Catanzariti et al., 2006). The soybeanpathogen, P. sojae, secretes glucanase inhibitor proteins,GIP1 and GIP2, that target endo-β-1,3-glucanaseA of thehost plant to protect the pathogen during infection andalso to prevent PTI induced by oligoglucosides (Roseet al., 2002) as discussed next.
Suppression of receptor activation
The recognition of PAMPs by plant PRRs leads to the acti-vation of defence responses against the pathogen. It wasproposed in the beginning that resistance induced byPRRs is only basic and not as strong as that induced byresistance genes, but it is clear now, at least in the caseof FLS2 (the flagellin receptor), that the contribution ofPRRs towards overall resistance is huge (He et al., 2006;de Torres et al., 2006; Hann & Rathjen, 2007). It is there-fore not surprising that pathogens have evolved to targetwith their effectors these signalling pathways in order toreduce resistance responses. Insight into the molecularmechanisms involved has emerged over the last few years.For example, several effectors target the PRRs directlyto jam all the downstream resistance responses (Jamiret al., 2004; Jones & Dangl, 2006; Blocker et al., 2008)such as P. syringae AVRPto and AVRPtoB which canalso suppress nonhost HR in N. benthamiana induced byFLG22 or P. infestans INF1 (Hann & Rathjen, 2007).
Several oomycete RXLR effectors can suppress hostcell immunity in a similar manner. AVR3A fromP. infestans can block an HR induced by INF1 (Boset al., 2009) as can several random P. infestans effectors(Oh et al., 2009). Similarly, AVR1B from P. sojaealso suppresses programmed cell death triggered by
the mouse protein, BAX, in plants and yeast (Douet al., 2008a) and M. oryzae AvrPiz-t can also suppressmouse BAX protein-mediated programmed cell death intobacco leaves (Li et al., 2009). ATR1 and ATR13 fromHyaloperonospora arabidopsidis increased the virulenceof P. syringae DC3000 on susceptible Arabidopsis whenthese effectors were delivered by the P. syringae T3SS,and ATR13 seemed to target PTI by suppressing calloseaccumulation and ROS production (Sohn et al., 2007).In the genus Phytophthora, effectors such as P. infes-tans IpiO1 bind to a membrane-spanning lectin receptorkinase and seem to prevent downstream defence sig-nalling (Bouwmeester et al., 2011).
Suppression of R gene-triggered resistance
Pathogenic fungi such as Fusarium oxysporum f. sp.lycopersici (Fol) can avoid host defences by evolvingeffectors that can suppress R gene-triggered resistance(Houterman et al., 2008). AVR3 and AVR2 are requiredfor full virulence on tomato plants but they are also rec-ognized by tomato lines that have resistance genes I-3or I-2, respectively, triggering an HR and cause arrestof the pathogen (Huang & Lindhout, 1997; Rep et al.,2004, 2005). In contrast, AVR1 is a small cysteine-richsecreted protein that is recognized by resistance gene Ior I-1 but is not required for virulence. It seems gener-ally true that effectors that contribute to full virulence aremaintained in the species and tellingly, AVR3 is presentin all Fol strains analysed, while AVR1 is present onlyin Fol strains that are virulent on I-3 lines. Houtermanet al. (2008) showed that AVR1 actually suppresses theresistance triggered by I-2 and I-3, as the transformationof Avr1 to Fol strains that were avirulent on I-2 or I-3became virulent on these lines. It was proposed that Folstrains acquired Avr1 as a mechanism of partial functionalredundancy so that they can avoid the consequences oflosing Avr3 and probably Avr2 that are required for fullvirulence (Stergiopoulos & de Wit, 2009). In the flax rustfungus, M. lini, the interaction of AvrL567 in strain CH5-89 with the flax cultivar ‘Barnes’ that contains the L7gene is inhibited by an inhibitor gene and thus results in alower virulence reaction (Lawrence et al., 2010). P. infes-tans secreted effector SNE1 can suppress programmedcell death resulting from the interaction of several Avr-Rprotein interactions (Kelley et al., 2010).
Alteration of the plant defence transcriptome
Bacterial effectors have been identified many years beforefungal and oomycete effectors and it is therefore not sur-prising that a better understanding of the impact of such
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effectors on the host transcriptome during infection hascome from the study of bacteria–plant interactions. Forexample, the perception of PAMPs such as bacterial flag-ellin changes the expression of at least 1000 genes inArabidopsis (Zipfel et al., 2004). Microbes have evolvedto become pathogens in part because their effectors inhibitthe activation of defence genes. Effectors can have an indi-rect effect on transcription by dephosphorylating MAPKsignalling components in order to suppress the defenceresponse triggered by the recognition of PAMPs by PRRs(reviewed in Gohre & Robatzek, 2008). Another mech-anism by which effectors have been shown to affectthe plant transcriptome is by causing changes in RNAstability. For example, P. syringae HopU1, a mono-ADP-ribosyltransferase, acts on glycine-rich RNA-binding pro-teins such as Arabidopsis AtGRP7 and AtGRP8 which areRNA chaperones and thus, HopUI changes the plant tran-scriptome by reducing transcript levels and hence affectsthe production of defence response proteins (Fu et al.,2007). Some pathogen effectors act as transcription fac-tors and can induce the expression of host genes for thebenefit of the pathogen. For example, the AVRBs3 fam-ily of effectors from X. campestris pv. vesicatoria hasplant nuclear localization signals and binds to a ‘upa-box’(upregulated by AVRBs3) that is found in the promoter ofUpa20, a master regulator of cell size inducing hypertro-phy, and several other host genes, ensuring proper nutrientsupply for pathogen multiplication (Szurek et al., 2002;Gurlebeck et al., 2006; Kay et al., 2007); however, inresistant plant cultivars, the promoter of Bs3 also carriesa ‘upa-box’ and, thus, binding of the AVRBs3 effectorinduces transcription of Bs3 and cell death (Kay et al.,2007). This shows that under selection pressure, plantscan evolve to recognize effectors and use them for theirown defence.
Several studies in fungal and oomycete pathosys-tems have reported on changes in host transcriptomes:in compatible interactions, initial defence reactions sup-posedly triggered by recognition of PAMPs, are theresult of the upregulation of genes in defence pathways;at later infection stages, these are suppressed and thiseffect is proposed to be caused by suites of secretedpathogen effectors. Such a scenario has been demon-strated in the smut fungus Ustilago maydis–maize inter-action (Doehlemann et al., 2008). In this latter system,effectors likely even reprogram specific developmentalpathways in the host to create the best niche for theirown development, depending on the age and part ofthe plant infected (Skibbe et al., 2010). One particulareffector, Pit2, was deduced to suppress defence genes(Doehlemann et al., 2011). In the M. oryzae–rice inter-action, changes in the host transcriptome were noted
including upregulation of susceptibility genes and down-regulation of defence genes and these effects on genetranscription were attributed to the actions of secretedeffectors, although no particular effector could be iden-tified to cause specific effects (Mosquera et al., 2009).
Killing of host cells
Necrotrophic pathogens produce several phytotoxins inaddition to cell wall hydrolysing enzymes in order tokill the host tissue for colonization during infection.Some necrotrophic fungi produce proteinaceous effectors,also called host selective toxins (HST), that are requiredfor infection on susceptible host plants that have thecorresponding dominant receptor gene (Wolpert et al.,2002). This represents a situation opposite to the classi-cal gene-for-gene interaction in which a dominant gene isrequired for disease resistance rather than susceptibility.Two wheat snecrotrophs, Stagonospora nodurum (Sn) andPyrenophora tritici-repentis (Ptr), produce several host-specific peptide effectors, such as PtrTOXA, SnTOX1,SnTOX2 and SnTOX4, that, when recognized by theircorresponding dominant susceptibility genes in wheat(TsN1, Snn1, Snn2 and Snn4), cause disease (Liu et al.,2004, 2006; Friesen et al., 2007; Abeysekara et al., 2009;de Wit et al., 2009; Manning et al., 2009). PtrTOXA, thebest-studied effector, has an N-terminal secretion signal,followed by an RGD domain (Arg-Gly-Asp-containingloop) for host targeting and a C-terminal domain witheffector function (Sarma et al., 2005; Manning et al.,2007). After entering the host cell, PtrTOXA was reportedto enter the chloroplast and bind to ToxABP1, therebyinterfering with functions of the chloroplast and affectingphotosystem I and II function in a light-dependent man-ner (Manning et al., 2007). PtrTOXA is an ortholog toSnTOX1 and both effectors are recognized by the samewheat susceptibility gene, Tsn1 (Liu et al., 2006).
Some pathogenic bacteria, oomycetes and fungi pro-duce NEP1-like (necrosis-inducing protein) proteins(NLPs), effectors that are toxic to only dicotyledonousplants, possibly because of the different molecularcompositions of dicot and monocot cell membranes(Pemberton & Salmond, 2004; Gijzen & Nurnberger,2006; Ottmann et al., 2009). The common hepta-peptidemotif, GHRHDWE, and two conserved cysteine residuesmake NLPs structurally similar to actinoporins, cytolytictoxins from marine organisms. Hemibiotrophs, such as P.infestans and P. sojae, produce NLPs such as NPP1 andPiNPP1, in the late necrotrophic phase which couldcontribute to disease development with their cytolyticactivities (Qutob et al., 2002; Kanneganti et al., 2006).
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Fungal and oomycete effectors 431
Suppression of host defence by symbionts
Symbiotic microorganisms also secrete effectors into hostplants to suppress host defence responses, a conditionessential for their lifestyle. Many rhizobial strains usethe T3SS to deliver effectors into host cells to suppresshost defence responses in a similar way that pathogenicbacteria do (Deakin & Broughton, 2009). This couldindicate that this ancient delivery system is used for dif-ferent purposes or that symbiotic organisms have evolvedtheir effectors such as to overcome most host defenceresponses. In the fungal kingdom, the genome of Laccariabicolor, an ectomycorrhizal fungus, revealed more than3000 predicted secreted proteins of which 10% were smallsecreted proteins (SSPs) of the effector-type (Martin et al.,2008; Martin & Selosse, 2008). Some of these SSPs arehomologous to rust fungus ‘haustoria-expressed secretedproteins’ (HESPs) and are differentially expressed dur-ing infection. One effector, MiSSP7, was recently shownto localize to host cell nuclei upon cell entry where italtered host transcription and was shown to be necessaryfor promoting symbiosis and evading the host defence(Plett et al., 2011).
Gene-for-gene or R-Avr gene interaction
Adapted pathogens secrete effector molecules into thehost plant to surmount physical barriers, neutralizepreformed antimicrobial compounds, and overcome PTI.As a result, during co-evolution of plant host and adaptedpathogen, plants have developed sophisticated recognitionsystems, usually encoded by R genes to recognize theseeffectors or their action and trigger defence responses;this induced resistance has been called effector-triggeredimmunity (ETI) and leads to a rapid and enhanced defenceresponse in the host plant often including an HR (Jones& Dangl, 2006). These effectors represent avirulence(AVR) factors since they activate the plant defence sys-tem and make the pathogen unable to cause disease whenthe plant has the ‘recognizing’ R gene. This geneticallysuperimposed R and Avr interaction has been known formany years as ‘gene-for-gene’ resistance, or host/cultivar-level resistance, as particular cultivars of the host witha certain R gene product recognize an Avr gene productfrom a particular race of the pathogen. This concept wasformulated after genetic studies on two fungal pathosys-tems: the Melampsora lini–flax (Flor, 1942) and theUstilago tritici–wheat interactions (Oort, 1944). As men-tioned, some Avr genes encode effectors that suppress hostdefence or carry out other essential functions and, thus,act as virulence factors when the plant does not have the‘recognizing’ resistance gene. ETI represents the quali-tative, secondary layer of resistance and has led to an
evolutionary arms race between the pathogen and the plantin which the pathogen either mutates or discards effectorsto avoid recognition by the host or develops new effectorsto suppress ETI, while the plant develops new R genes torecognize the mutated or new effectors (Jones & Dangl,2006; de Wit, 2007; Bent & Mackey, 2007)
Avr (-triggering effector) genes
Because, until recently, the term avirulence gene/factorfeatured prominently in the literature, we will discussthese below but would prefer the term ‘Avr-triggeringeffector genes’. The first Avr gene was cloned from abacterium in 1984 (Staskawicz et al., 1984), which wasfollowed by the cloning and characterization of more than40 bacterial Avr genes in the following decades (van ’tSlot & Knogge, 2002; Mudgett, 2005). The cloning andcharacterization of Avr genes from fungi and oomyceteslagged behind because of these organisms’ larger genomesizes and sometimes inefficient transformation systems.The first Avr gene from a fungus was isolated from C. ful-vum in 1991 (van Kan et al., 1991) and the first oomyceteAvr gene was cloned from P. sojae and P. infestans in2004 (Shan et al., 2004). In the following years, mostfungal Avr genes were cloned from ascomycetes, althoughmore recently, a few have been reported from a basid-iomycete, the flax rust fungus, M. lini (Catanzariti et al.,2006; Table 1).
Avr genes isolated and characterized to date differamong one another both in sequence and function, andthose from different plant pathogens do not seem toshare many common features, other than the ones alreadymentioned. Exceptions are found among some familymembers, e.g. in Pseudomonas and Xanthomonas species(Lahaye & Bonas, 2001; Deslandes et al., 2003). And it istrue that among genomes from related species, sometimeseven among genera, homologous effectors are found, butthese, or not all of these, have proven avirulence functions.Most fungal and oomycete Avr genes characterized to dateencode small proteins (28–311 amino acids), except theACE1 of M. grisea (see below) and most have a secre-tion signal/protein transport motif at the N-terminus (Elliset al., 2006), although there are exceptions (see further).
Fungal Avr (-triggering effector) genes
Cladosporium fulvum
This fungus is an apoplastic pathogen of the ascomycetesubgroup that reproduces asexually and causes leaf moldof tomato (de Wit et al., 1997; Joosten & de Wit, 1999;Thomma et al., 2005). Four avirulence genes have beencloned: Avr2, Avr4, Avr4E and Avr9, which all encodesmall secreted cysteine-rich effector proteins that are
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S. Ali and G. Bakkeren 432
Tabl
e1.
Eff
ecto
rpr
otei
nsof
fung
alan
doo
myc
ete
plan
tpat
hoge
ns.
Prot
ein
Org
anis
m
Len
gth
aare
sidu
es(m
atur
e)N
umbe
rof
cyst
eine
sSP
leng
th(a
a)B
iolo
gica
lact
ivity
/ho
mol
ogy
Prot
ein
loca
lizat
ion
Rol
ein
viru
lenc
e/pa
thog
enic
ityC
orre
spon
-din
gR
gene
Ref
eren
ces
AV
R2
C.f
ulvu
m78
(58)
820
Indu
ces
HR
inth
epr
esen
ceof
Tom
ato
Rcr
3,Pr
otea
sein
hibi
tor
Apo
plas
tIn
hibi
tsR
cr3
and
othe
rpr
otea
ses
Cf-
2St
ergi
opou
los
etal
.,20
10A
VR
4C
.ful
vum
135
(86)
818
Indu
ces
HR
,Chi
tin-b
indi
ng,
orth
olog
sin
som
eot
her
fung
iA
popl
ast;
fung
alce
llw
allc
hitin
Prot
ects
agai
nst
chiti
nase
sC
f-4
Joos
ten
etal
.,19
94;
Ster
giop
oulo
set
al.,
2010
;van
den
Bur
get
al.,
2006
AV
R4E
C.f
ulvu
m12
1(1
01)
610
Indu
ces
HR
Apo
plas
tU
nkno
wn
Hcr
9-4E
Wes
teri
nket
al.,
2004
AV
R9
C.f
ulvu
m63
(28)
623
Indu
ces
HR
,Car
boxy
pept
idas
ein
hibi
tor
Apo
plas
tU
nkno
wn
Cf-
9va
nde
nA
cker
veke
net
al.,
1993
EC
P1C
.ful
vum
96(6
5)8
23In
duce
sH
R,T
umor
-nec
rosi
sfa
ctor
rece
ptor
Apo
plas
tD
isru
ptio
nle
ads
tore
duce
dvi
rule
nce
Cf-
Ecp
1va
nde
nA
cker
veke
net
al.,
1993
EC
P2C
.ful
vum
165
(143
)4
22in
duce
sH
R,
Apo
plas
tD
isru
ptio
nle
ads
tore
duce
dvi
rule
nce
Cf-
Ecp
2va
nde
nA
cker
veke
net
al.,
1993
EC
P4C
.ful
vum
119
(101
)6
18In
duce
sH
RA
popl
ast
Unk
now
nC
f-E
cp4
Bol
ton
etal
.,20
08E
CP5
C.f
ulvu
m11
5(9
8)6
17In
duce
necr
osis
,A
popl
ast
Unk
now
nC
f-E
cp5
Bol
ton
etal
.,20
08E
CP6
C.f
ulvu
m22
2(1
99)
823
LysM
-dom
ains
;chi
tin-b
indi
ng,
orth
olog
foun
din
diff
eren
tpa
thog
enan
dno
npat
hoge
nic
sp.
Apo
plas
tK
nock
-dow
nle
ads
tore
duce
dvi
rule
nce
Unk
now
nde
Jong
e&
Tho
mm
a,20
09
EC
P7C
.ful
vum
–(1
00)
6–
Unk
now
nA
popl
ast
Unk
now
nU
nkno
wn
Bol
ton
etal
.,20
08N
IP1
R.s
ecal
is82
(60)
1022
non-
spec
ific
toxi
n/in
duce
sne
cros
isan
dpl
asm
a-m
embr
ane
H+
AT
Pase
Prob
ably
inap
opla
stN
otre
quir
edfo
rvi
rule
nce
Rrs
-1R
ohe
etal
.,19
95
NIP
2R
.sec
alis
109
(?)
7(6
)16
Non
-spe
cific
toxi
n/in
duce
sne
cros
isin
seve
ralp
lant
spp.
Prob
ably
inap
opla
stN
otre
quir
edfo
rfu
llvi
rule
nce
Unk
now
nR
ohe
etal
.,19
95;
Ster
giop
oulo
s&
De
Wit,
2009
NIP
3R
.sec
alis
115
(?)
9(8
)17
Non
-spe
cific
toxi
n/in
duce
sne
cros
isin
seve
ralp
lant
ssp
ecie
sPr
obab
lyin
apop
last
Not
requ
ired
for
full
viru
lenc
eU
nkno
wn
Roh
eet
al.,
1995
;St
ergi
opou
los
&de
Wit,
2009
AV
Ra1
0B
.gra
min
is28
64
–>
30pa
ralo
gsin
Bgh
and
othe
rf.
sp.
No
N-t
erm
inal
SP,i
nduc
esH
RPr
obab
lyin
cyto
plas
mU
nkno
wn
Mla
10R
idou
teta
l.,20
06
AV
Rk1
B.g
ram
inis
177
3–
>30
para
logs
inB
ghan
dot
her
f.sp
.N
oN
-ter
min
alSP
,ind
uces
HR
Prob
ably
incy
topl
asm
Unk
now
nM
lk1
Rid
oute
tal.,
2006
AV
RL
567
(A,B
and
C)
M.l
ini
150
(127
)1
23U
nkno
wn,
indu
ces
HR
,fun
ctio
nal
RX
LR
like
mot
ifC
ytop
lasm
Unk
now
nL
5,L
6an
dL
7D
odds
etal
.,20
04;
Kal
eet
al.,
2010
;R
afiqi
etal
.,20
10
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Fungal and oomycete effectors 433
AV
RM
M.l
ini
314
(286
)1
28U
nkno
wn,
indu
ces
HR
,RX
LR
like
mot
ifC
ytop
lasm
Unk
now
nM
Cat
anza
riti
etal
.,20
06;K
ale
etal
.,20
10;R
afiqi
etal
.,20
10A
VR
P123
M.l
ini
117
(94)
1123
Indu
ces
HR
,Kaz
alSe
rine
prot
ease
inhi
bito
rPr
obab
lyin
cyto
plas
mU
nkno
wn
P,P
1,P
2an
d/or
P3
Cat
anza
riti
etal
.,20
06A
VR
P4M
.lin
i95
(67)
728
Indu
ces
HR
,cys
tein
e-kn
otte
dpe
ptid
ePr
obab
lyin
cyto
plas
mU
nkno
wn
P4
Cat
anza
riti
etal
.,20
06A
vr-P
ita1
M.o
ryza
e22
3(1
76)
816
Hom
olog
yto
met
allo
-pro
teas
es,R
XL
Rlik
em
otif
Cyt
opla
smN
otre
quir
edfo
rvi
rule
nce
onri
ceP
i-ta
Orb
ach
etal
.,20
00;
Kal
eet
al.,
2010
;K
hang
etal
.,20
10A
VR
-Pita
2M
.ory
zae
224
(?)
816
Hom
olog
yto
met
allo
-pro
teas
esPr
obab
lyin
apop
last
Prob
ably
not
requ
ired
for
viru
lenc
eon
rice
Pi-
taK
hang
etal
.,20
08
AV
R-P
ita3
M.o
ryza
e22
6(?
)8
16H
omol
ogy
tom
etal
lo-p
rote
ases
Prob
ably
incy
topl
asm
Prob
ably
not
requ
ired
for
viru
lenc
eon
rice
Unk
now
nK
hang
etal
.,20
08
PWL
1M
.ory
zae
147
(124
)2
23G
lyci
ne-r
ich
hydr
ophi
licpr
otei
nB
iotr
ophi
cin
terf
acia
lco
mpl
ex
Unk
now
nU
nkno
wn
Kan
get
al.,
1995
;K
hang
etal
.,20
10PW
L2
M.o
ryza
e14
5(1
24)
221
Gly
cine
-ric
hhy
drop
hilic
prot
ein
Cyt
opla
smU
nkno
wn
Unk
now
nSw
eiga
rdet
al.,
1995
;Kha
nget
al.,
2010
PWL
3M
.ory
zae
137
(116
)0
21G
lyci
ne-r
ich
hydr
ophi
licpr
otei
nPr
obab
lyin
apop
last
Non
-fun
ctio
nal
Unk
now
nK
ang
etal
.,19
95
PWL
4M
.ory
zae
138
(117
)0
21G
lyci
ne-r
ich
hydr
ophi
licpr
otei
nPr
obab
lyin
apop
last
Non
-fun
ctio
nal
Unk
now
nK
ang
etal
.,19
95
AC
E1
M.o
ryza
e40
3543
–H
ybri
dpo
lyke
tide
synt
hase
/no
nrib
osom
alpe
ptid
esy
nthe
tase
Not
secr
eted
Unk
now
nP
i33
Boh
nert
etal
.,20
04
AV
R1-
CO
39M
.ory
zae
notc
lone
dye
t–
–U
nkno
wn
Unk
now
nU
nkno
wn
Pi-
CO
39(t
)Fa
rman
etal
.,20
02
AV
R-P
iaM
.ory
zae
85(6
6)2
19In
duce
sH
RPr
obab
lyin
cyto
plas
mU
nkno
wn
Pia
Yos
hida
etal
.,20
09
AV
R-P
iiM
.ory
zae
70(5
1)3
19In
duce
sH
RPr
obab
lyin
cyto
plas
mU
nkno
wn
Pii
Yos
hida
etal
.,20
09
AV
R-P
iiM
.ory
zae
70(5
1)3
19In
duce
sH
RPr
obab
lyin
cyto
plas
mU
nkno
wn
Pii
Yos
hida
etal
.,20
09
AV
R-
Pik/
km/kp
M.o
ryza
e11
3(9
2)3
21In
duce
sH
RPr
obab
lyin
cyto
plas
mU
nkno
wn
Pik
Yos
hida
etal
.,20
09
AV
RL
m1
L.m
acul
ans
205
(183
)1
22In
duce
sH
RPr
obab
lyin
cyto
plas
mR
equi
red
for
full
viru
lenc
eR
lm1
Gou
teta
l.,20
06
AV
RL
m6
L.m
acul
ans
144
(124
)6
20U
nkno
wn,
func
tiona
lRX
LR
like
mot
ifPr
obab
lyin
apop
last
Unk
now
nR
lm6
Fuda
leta
l.,20
07;
Kal
eet
al.,
2010
(Con
tinu
ed)
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S. Ali and G. Bakkeren 434Ta
ble
1.(C
ontin
ued.
)
Prot
ein
Org
anis
m
Len
gth
aare
sidu
es(m
atur
e)N
umbe
rof
cyst
eine
sSP
leng
th(a
a)B
iolo
gica
lact
ivity
/ho
mol
ogy
Prot
ein
loca
lizat
ion
Rol
ein
viru
lenc
e/pa
thog
enic
ityC
orre
spon
-din
gR
gene
Ref
eren
ces
AV
RL
m4-
7L
.mac
ulan
s14
3(1
22)
821
Unk
now
nPr
obab
lyin
apop
last
Req
uire
dfo
rfu
llvi
rule
nce
Rlm
4an
d/or
Rlm
7Pa
rlan
geet
al.,
2009
AV
R3
(SIX
1)F.
oxys
po-
rum
f.sp
.ly
cope
r-si
ci
284
(189
)6
or8
21U
nkno
wn
Xyl
emR
equi
red
for
full
viru
lenc
eI-
3R
epet
al.,
2004
;St
ergi
opou
los
&de
Wit,
2009
AV
R4
(SIX
2)F.
oxys
po-
rum
f.sp
.ly
cope
r-si
ci
232
(172
)8
20U
nkno
wn
Xyl
emPr
obab
lyno
tre
quir
edfo
rvi
rule
nce
Unk
now
nH
oute
rman
etal
.,20
07;
Ster
giop
oulo
s&
deW
it,20
09A
VR
2(S
IX3)
F.ox
yspo
-ru
mf.
sp.
lyco
per-
sici
163
(144
)3
(2)
19In
duce
sH
R,u
nkno
wn,
func
tiona
lR
XL
Rlik
em
otif
Xyl
emR
equi
red
for
full
viru
lenc
eI-
2H
oute
rman
etal
.,20
07;
Ster
giop
oulo
s&
deW
it,20
09;K
ale
etal
.,20
10A
VR
1(S
IX4)
F.ox
yspo
-ru
mf.
sp.
lyco
per-
sici
242
(184
)6
17U
nkno
wn
Xyl
emSu
ppre
ssio
nof
I-2
and
I-3
resi
stan
ce
Ior
I-1
Hou
term
anet
al.,
2007
;St
ergi
opou
los
&de
Wit,
2009
AT
R1N
dWsB
H. ar
abid
op-
sidi
s
311
(296
)–
15in
duce
sH
R,R
XL
Rdo
mai
nC
ytop
lasm
Supp
ress
host
defe
nse
RP
P1W
sBan
dR
PP
1Nd
Reh
man
yet
al.2
005;
Sohn
etal
.,20
07
AT
R13
H. ar
abid
op-
sidi
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recognized in tomato by Cf2, Cf4, Cf4E and Cf9 resis-tance proteins, respectively (Table 1; de Wit et al., 1997;Joosten & de Wit, 1999; Thomma et al., 2005). Thevirulence function of AVR2 and AVR4 has been dis-cussed earlier. AVR4E is a 101 amino acid (aa) proteinbut does not have a known virulence function (Westerinket al., 2004). Several field isolates that overcome Cf4E-mediated resistance reveal point mutations in AVR4E ora complete loss of the Avr4E gene, indicating that lossof this effector does not affect fitness of the pathogensignificantly (Stergiopoulos et al., 2007). Avr9 encodes a28 aa mature protein with six cysteine residues after it isprocessed by plant and fungal proteases at its C- and N-termini (van Kan et al., 1991; van den Ackerveken et al.,1993). Structurally, AVR9 is similar to carboxy-peptidaseinhibitor but no definitive function has been identified sofar (van Kan et al., 1991; van den Ackerveken et al., 1993;van den Hooven et al., 2001). All natural strains of C.fulvum that overcome Cf9 resistance lack Avr9, suggest-ing that it is not required for full virulence (Stergiopouloset al., 2007). C. fulvum deleted for Avr9 is fully viru-lent on tomato plants, but the heterologous expression ofAvr9 in tomato plants makes it more susceptible to C.fulvum strains lacking Avr9, indicating some (redundant)virulence function (Marmeisse et al., 1993; de Wit et al.,2009).
Besides Avr effectors, four extracellular cysteine-richproteins (ECP), such as ECP1, ECP2, ECP4 and ECP5,have been cloned and characterized from C. fulvum thatinduce an HR in tomato lines containing the correspond-ing Cf-Ecp resistance genes (Lauge et al., 2000; deKock et al., 2005). Ecp6 and Ecp7 have been clonedbut the corresponding tomato lines that recognize thesegenes have not yet been identified (Bolton et al., 2008).ECPs are present in all strains of C. fulvum and aresecreted during infection. They contain an even num-ber of cysteine residues that are most likely involved indisulphide bridge formation and secondary structure for-mation to protect them from apoplastic proteases (Ludereret al., 2002). Three of the ECPs, ECP1, ECP2 andECP6, have virulence functions on host plants, basedon data showing that deleting or suppressing expressionof these genes reduced virulence (Lauge et al., 1997;Bolton et al., 2008). Orthologs for AVR4 and ECP6 havebeen identified in several fungal species because of thepresence of CMB14 and lysin motif (LysM, carbohydrate-binding) domains in these proteins (Bolton et al., 2008).Orthologs of AVR4 and ECP2 have been identified inMycosphaerella fijiensis that causes black Sigatoka dis-ease of banana (Stergiopoulos et al., 2010). The M.fijiensis ortholog of AVR4 induces an HR in tomatolines containing the corresponding Cf4 gene and binds
to chitin of fungal cell walls to protect against cell walldegradation, similar to C. fulvum AVR4 (Stergiopouloset al., 2010). Similarly, M. fijiensis ECP2 is a functionalortholog of C. fulvum ECP2 and is recognized by Cf2 oftomato to induce an HR, while in the absence of Cf2, itpromotes virulence on tomato plants (Stergiopoulos et al.,2010).
Rynchosporium secalis
The imperfect fungus R. secalis causes leaf scald diseaseon barley by secreting low molecular-weight toxic pro-teins. The genes for three of these effectors, designatedas necrosis-inducing proteins NIP1, NIP2 and NIP3, havebeen cloned (Table 1). They encode small secreted pro-teins with toxicity in a genotype non-specific manner onbarley and related cereal plant species (Hahn et al., 1993;Rohe et al., 1995; Steiner-Lange et al., 2003). MatureNIP1 is a 60 aa protein with 10 cysteine residues thatare involved in intramolecular disulphide bond formation.NIP1 triggers specific defence responses without an HRon barley cultivars that have the corresponding resistancegene, Rrs1 (Lehnackers & Knogge, 1990). The injectionof NIP1 into leaves of barley and other cereal plant speciescauses scald-like lesion formation (Wevelsiep et al., 1991;van’t Slot et al., 2007). A nip1 disruption mutant of R.secalis is slightly less virulent than the wild type on sus-ceptible plants, demonstrating a (minor) role in virulence(Knogge & Marie 1997). Virulent strains of R. secalisovercome Rrs1 resistance of barley either by a point muta-tion in the ORF that results in a single aa substitution orby jettison of the Nip1 gene (Rohe et al., 1995). It hasbeen shown that NIP1 interacts with a single plasma mem-brane receptor (different from Rrs1) that is involved bothin necrosis and defence induction (van’t Slot et al., 2007).A field population study of this pathogen showed a posi-tive diversifying selection on the Nip1 locus, as three outof the 14 isoforms gained virulence on Rrs1 barley linesand a high deletion frequency was observed in the Nip1locus compared with Nip2 and Nip3 (de Wit et al., 2009).The deletion frequency of Nip1 was higher than the occur-rence of the point mutation that gains virulence indicatinga reduced fitness penalty for the loss of the Nip1 gene.Nip2 encodes a 109 aa protein with a predicted secre-tion signal of 16 aa and a mature protein with 6 cysteineresidues, while Nip3 encodes a 115 aa protein with a pre-dicted secretion signal of 17 aa and a mature protein with8 cysteine residues (de Wit et al., 2009).
Magnaporthe oryzae
M. oryzae (formerly known as M. grisea) is a filamentousascomycete fungus that causes rice blast disease,
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destructive to rice production worldwide, but can alsocause disease in many other members of graminaceousplants (Kato et al., 2000; Couch & Kohn, 2002). Morethan 40 resistance genes have been identified in rice con-trolling the blast fungus and several of them have beenextensively used in resistant rice lines in the past fewdecades (Bryan et al., 2000; Chen et al., 2006). Theseresistant rice lines are overcome quickly in the field by theemergence of new races of the pathogen through variousmechanisms in the pathogen, such as deletion of Avr genesfrom the genome (Yoshida et al., 2009), changes in theirgene expression (Kang et al., 2001; Fudal et al., 2005) orpoint mutations in their genes (Orbach et al., 2000) result-ing in escaping recognition by R genes (Kolmer, 1989;Leach et al., 2001; McDonald & Linde, 2002). Eightcultivar- and species-specific Avr genes have been clonedand characterized from M. oryzae: Avr-Pita, Avr1-CO39,Ace1, Pwl1, Pwl2, AvrPiz-t, Avr-Pia, Avr-Pii and Avr-pik/km/kp (Table 1; Valent et al., 1991; Farman & Leong,1998; Orbach et al., 2000; Bohnert et al., 2004; Collemareet al., 2008; Li et al., 2009; Miki et al., 2009; Yoshidaet al., 2009).
The AVR-Pita effector shows similarity to fungal metal-loproteases of the deuterolysin family and is not requiredfor full virulence on rice plants (Jia et al., 2000; Orbachet al., 2000). Avr-Pita encodes a 223 aa protein thatis predicted to be secreted and needs to be processedinto a 176 aa active form (AVR-Pita 176) to interactdirectly with the LRR (leucine-rich repeat) domain ofthe PITA resistance protein and trigger PITA-mediateddefence responses, as assayed by yeast 2-hybrid assays, invitro binding analyses and direct expression in rice cells(Jia et al., 2000). In certain strains of M. oryzae, Avr-Pita undergoes spontaneous mutations in the laboratoryand also under field conditions, such as deletion, pointmutation, and the insertion of transposons, all resultingin overcoming Pita resistance in rice cultivars (Orbachet al., 2000; Kang et al., 2001; Zhou et al., 2007; Khanget al., 2008). Avr-Pita is located close to the telomereof chromosome 3 in the genome of M. oryzae and thismay be responsible for the genetic instability of thisgene. Avr-Pita was renamed Avr-Pita1 after identificationof Avr-Pita2 and Avr-Pita3 (Khang et al., 2008). Avr-Pita2 is recognized by the rice Pita gene and elicits thedefence response while Avr-Pita3 is not recognized byPita (Khang et al., 2008).
Avr1-Co39 was isolated from M. oryzae isolate 4091-5-8 pathogenic on weeping lovegrass and specifies avir-ulence on rice cultivar CO39 that contains the resistantgene, Pi-CO39(t) in a gene-for-gene manner (Valent et al.,1991; Chauhan et al., 2002). Isolates virulent on ricecultivar ‘CO-39’, lack Avr1-CO39 in most of the cases
(Farman et al., 2002). It has been shown that Avr1-CO39is a species-specific rather than a cultivar-specific typeof Avr gene (Zheng et al., 2011). The Pwl genes stopthis pathogen from causing disease on weeping lovegrassand finger millet in a species-specific manner, but theystill can infect rice (Kang et al., 1995; Sweigard et al.,1995). PWL effectors are small glycine-rich secreted pro-teins that are evolving fast and belong to a gene familydesignated as PWL1-PWL4. Pwl2 strains virulent onweeping lovegrass appear due to spontaneous mutations,predominantly by genetic rearrangement and large dele-tions (Kang et al., 1995; Sweigard et al., 1995). In thethree homologs of Pwl2, identified by homology searches,only Pwl1 is the functional homolog while Pwl3 and Pwl4are not functional; however, Pwl4 is functional only whenexpressed under the control of the Pwl1 or Pwl2 pro-moter, while Pwl3 is not functional in that case (Kanget al., 1995). Three new Avr genes, Avr-Pia, Avr-Pii andAvr-Pik/km/kp, have been isolated from M. oryzae byassociation genetics, i.e. by looking for polymorphismsamong 1032 predicted secreted proteins in field isolates(Yoshida et al., 2009).
An Avr gene from a different class, Ace1, encodes a4035 aa polyketide synthase (PKS) fused to a nonriboso-mal peptide synthetase (NRPS); these are two differentclasses of enzymes that are probably involved in theproduction of a secondary metabolite that triggers Pi33-mediated resistance in rice cultivars (Bohnert et al., 2004).M. grisea genome analysis revealed that Ace1 is present ina cluster of 15 genes of which 14 encode enzymes such asa second PKS-NRPS (Syn2), two enoyl reductases (Rap1and Rap2) and a putative Zn(II)(2)Cys(6) transcriptionfactor (BC2) which probably all play a role in secondarymetabolism (Collemare et al. 2008). Ace1 and all othergenes in the cluster are specifically expressed during pen-etration into the host plant, defining an infection-specificgene cluster, which suggests that Ace1 might have a rolein virulence; however, an Ace1 disruption mutant did notshow any reduction in virulence (Bohnert et al., 2004;Fudal et al., 2005).
Leptosphaeria maculans
This ascomycete fungus causes blackleg (phoma stemcanker) disease on oilseed rape (Brassica napus). Geneticanalysis of the interaction has revealed at least nineavirulence genes designated as AvrLm1–AvrLm9 that arerecognized by corresponding resistance genes Rlm1–Rlm9of the host (Balesdent et al., 2002; Rouxel & Balesdent,2005; Yu et al., 2005; Fitt et al., 2006). Seven ofthese nine genes are present in two unlinked clusters(AvrLm1-2-6 and AvrLm 3-4-7-9) while the remaining two
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are individual genes (Balesdent et al., 2002). AvrLm1,AvrLm6 and AvrLm4-7 have been cloned by a map-basedstrategy and all encode small putative secreted proteinsthat have no similarity to sequences in public databases(Table 1; Gout et al., 2006; Fudal et al., 2007; Parlangeet al., 2009). Both AvrLm1 and AvrLm6 are located ina gene-poor, AT-rich, non-coding heterochromatin-likeregion as solo genes in stretches of 269 and 131 kb,respectively, which contain a number of degeneratednested copies of long terminal repeat (LTR) retrotrans-poson (Gout et al., 2006; Fudal et al., 2007). Also, bothAvrLm1 and AvrLm6 are single copy genes that encodesmall proteins of 205 and 144 aa, respectively, with anN-terminal secretion signal but no other conserved motifor any similarity to each other. The expression of bothgenes is strongly induced during early leaf infection butthey are also expressed in culture media at relatively lowlevels when not in contact with the host (Gout et al.,2006; Fudal et al., 2007). AvrLm1 has only one cysteineresidue and is likely taken up by the host cell (Gout et al.,2006), while AvrLm6 contains six cysteine residues thatmake disulphide bridges that could provide stability inthe apoplastic environment (Fudal et al., 2007). Strainsvirulent on Rlm1 oilseed rape cultivars lack the AvrLm1gene. Repeat-induced point (RIP) mutation, a commonmechanism to inactivate genes in ascomycetes, and dele-tion were also responsible for gain of virulence on Rlm1cultivars (Fudal et al., 2009). Similar to the AvrLm1 andAvrLm6 loci, the isolated AvrLm4-7 gene was located toa 238 kb genetic locus having multiple LTR retrotrans-posons; this gene induces a resistance response in plantshaving either Rlm7 or Rlm4. Within the locus, this singlegene is embedded in a gene-poor, AT-rich, recombination-deficient 60 kb isochor (Parlange et al., 2009). It encodesa small cysteine-rich secreted protein of 143 aa with a pre-dicted N-terminal secretion signal of 21 aa. Like othereffectors from this fungus, the expression of AvrLm4-7is induced in the plant but is also expressed at low lev-els in culture media (Parlange et al., 2009). The partial orcomplete loss of AvrLm4-7 in field isolates is responsiblefor gain of virulence on both Rlm4 and Rlm7 cultivars,while a point mutation that changes a glycine to an argi-nine residue, can overcome recognition by Rlm4 (Parlangeet al., 2009).
Fusarium oxysporum
F. oxysporum f. sp. lycopersici (Fol) infects tomato rootsvia wounds or by direct penetration after which it colo-nizes xylem vessels. Several small cysteine-rich proteinswere identified from xylem sap during Fol infection, andthree of them induced resistance in an R gene-specific
manner. They are coded for by avirulence gene SIX(secreted in xylem) 1 to 4 (Table 1; Rep et al., 2004;Houterman et al., 2007, 2008, 2009). Fusarium genomecomparisons revealed that all the SIX genes are locatedon the same chromosome that is absent from non-pathogenic isolates. The transfer of this chromosome tonon-pathogenic strains converted them to pathogens (Maet al., 2010). The function of three of these genes invirulence has been discussed in the previous section.
Blumeria graminis
Powdery mildews are biotrophic ascomycete fungi thatcause diseases on various mono- and dicotyledonous plantspecies, including food and feed crops and ornamentalplants (Bushnell, 2002). They are obligate biotrophs thatneed a living host for growth and reproduction and pro-duce intracellular feeding structures, the haustoria, in theepidermis of their host plants (Yarwood, 1957; Glawe,2008). Bgh causes powdery mildew on barley and isthe most thoroughly studied powdery mildew fungus.It interacts with its host in a ‘gene-for-gene’ manner(Both et al., 2005; Zhang et al., 2005). Genetic anal-ysis revealed that there are more than 85 dominant orsemi-dominant mildew (Ml) resistance genes that rec-ognize different Bgh races including 28 highly similargenes at the Mla locus on barley chromosome 5 (Jensenet al., 1980). Seven Mla genes at this locus have beencloned and they all encode closely related intracellularcoiled-coil, nucleotide-binding site, leucine-rich repeat(CC-NBS-LRR) type R proteins that recognize differ-ent Bgh avirulent effector proteins (Halterman et al.,2001; Halterman & Wise, 2004). Two of these effectors,AVRa10 and AVRk1, that are recognized by the bar-ley R proteins, Mla10 and Mlk1, respectively, inducingan HR, have been cloned; however, they are also vir-ulence factors in that they promote infection on sus-ceptible barley cultivars (Table 1; Ridout et al., 2006).Both Avr genes belong to multi-gene families that havemore than 30 paralogs and they also have orthologsin other forma speciales that are pathogenic on othergrasses (Ridout et al., 2006). The predicted AVRa10 andAVRk1 effectors lack an N-terminal secretion signal orhost targeting sequence, but it has been shown by flu-orescence microscopy that the Mla10 protein is presentintracellularly, both in the cytoplasm and the nucleus ofinvaded barley cells. Assuming a necessary interactionwith effectors, this means that AVRa10 is secreted fromthe fungus by non-endomembranous pathways and takenup by the cell by an as yet unknown mechanism (Bieriet al., 2004; Shen et al., 2007). Using a novel approachto gene silencing, Nowara et al. (2010) showed that by
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expressing double-stranded or antisense RNA sequencesin barley but targeted to the Bgh-specific Avra10 andAvrk1 effector genes, they could reduce fungal develop-ment in hosts lacking the corresponding Mla10 resistancegene, confirming their role in virulence. Interestingly, noclear release from R-gene recognition could be seen bysilencing the Avra10 gene using this system which theytermed ‘Host-Induced Gene Silencing’ or HIGS. BesidesAVRa10 and AVRk1, two other Avr genes, Avra22 andAvra12, have been mapped recently (Skamnioti et al.,2008).
Melampsora lini
Another obligate biotrophic fungus, but one that belongsto the basidiomycetes, is M. lini that causes flax rustdisease not only in flax but also in other species ofthe genus Linum. A number of flax R proteins havebeen analysed; these are highly polymorphic cytoplasmic(Toll/Interleukin1 Receptor) TIR-NBS-LRR proteins thatrecognize effector proteins that are delivered into flax cellsduring colonization. This interaction triggers an HR in a‘gene-for-gene’ manner, arresting growth of the fungus(Lawrence et al., 2007). The genetic analysis of M. liniwith its host plant flax (the basis of the original ‘gene-for-gene’ theory by Flor in 1942) revealed at least 30Avr genes and corresponding R genes (Ellis et al., 1997).Several Avr genes have been cloned from M. lini, mainlyfrom four different loci: AvrL567, AvrM, AvrP123 andAvrP4 (Table 1). These genes encode haustoria-expressedsecreted proteins (HESPs), suggesting that they have vir-ulence functions, but elicit defence responses in hosts thathave the corresponding R genes (Catanzariti et al., 2006).The AvrL567 locus has a cluster of three polymorphicAvr genes: AvrL567A, AvrL567B and AvrL567C. All threeencode 127 aa mature proteins after cleavage of a 23 aaSP and are recognized directly by the L5, L6 and L7 pro-teins inside the cell (Dodds et al., 2004). The matureAVR proteins are highly polymorphic with at least oneor more aa substitutions in the exposed surface of theproteins, suggesting functional interactions (Ellis et al.,2007; Wang et al., 2007). Some of the isolates harbouringthese AVR proteins became virulent by defeating match-ing plant resistance genes, indicating that genes in theAvrL567 locus are under positive diversifying selection(Dodds et al., 2006). The analysis of six flax rust iso-lates revealed 12 members of the AvrL567 effector genefamily, including the three previously isolated AvrL567A-C genes. Seven of these caused avirulence while fivebelonged to virulent strains and could no longer be rec-ognized by L5, L6 or L7 (Dodds et al., 2006). TheAvrM gene family is a small family that consists of five
avirulence paralogs, AvrMA to AvrME, and one virulentone, avrM, which is not recognized by any known flaxR protein (Catanzariti et al., 2006). These AvrM proteinsdo not have any known homologs and are highly variableboth in sequence and size due to deletions or insertions ofDNA, or to premature termination of the protein becauseof the location of stop codons (Catanzariti et al., 2006).
AvrP123 is a small cysteine-rich protein that containsthe characteristic CX7CX6YX3CX2-3C signature of theKazal serine protease inhibitor family, suggesting its roleas an inhibitor of host proteases. This is similar to thefunction of C. fulvum AVR2 that inhibits the Rcr3 cysteineprotease in the tomato apoplast (Catanzariti et al., 2006).AvrP4 also encodes a small cysteine-rich protein of 67 aaafter cleavage of a 28 aa SP. The 28 aa C-terminal part ofAvrP4 has 6 cysteine residues with a spacing consensus ofa typical ‘cysteine-knotted’ peptide, similar to the C. ful-vum AVR9 protein (van den Hooven et al., 2001). AvrP4is expressed only in planta while AvrM is expressed bothin planta and in vitro (Stergiopoulos & de Wit, 2009).Agroinfiltration of AvrP4 and AvrM genes in flax plantswith matching resistance genes results in a HR indicat-ing that the produced effectors are functional in host cellsand are, therefore, likely translocated into the host dur-ing infections. This is in agreement with the predictedcytoplasmic location of P and M resistance proteins inflax (Anderson et al., 1997). Using a yeast 2-hybrid sys-tem, the AvrM-A and -D effectors were shown to interactdirectly with the M resistance protein resulting in fastnecrosis, and that a globular C-terminal domain, variableamong alleles, is important for this interaction (Catanzaritiet al., 2010).
Oomycete Avr (-triggering effector) genes
Oomycetes belong to the kingdom Stramenopila and areevolutionarily related to algae and include some well-known plant pathogens. Many effector genes have beenisolated, some of which have proven avirulence functions(Table 1).
Phytophthora infestans
P. infestans causes late blight disease in potato andtomato and was responsible for the ‘Irish Famine’ in1840. Many effectors have been identified and isolatedfrom P. infestans such as INF1 ‘elicitin’, a secreted pro-tein that elicits hypersensitive cell death and is used inmany current functional assays (Kamoun et al., 1997;Kamoun, 2006; Haas et al., 2009). However, only onerace-specific avirulence gene, Avr3a has been cloned.Avr3a encodes a cytoplasmic RXLR effector and when
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transiently expressed lacking its SP, in potato cells, R3a-mediated cell death ensues illustrating its recognition inthe cytoplasm and avirulence function (Armstrong et al.,2005). The mature secreted protein is 147 aa in lengthand allelic polymorphic residues distinguishing function-ality were identified in various pathogen races: one allelicvariant Avr3AK80/I103 (abbreviated as Avr3A KI) causesavirulence on potato plants expressing R3a with the lysineresidue at position 80 being critical for recognition, whilethe virulent allele has two aa substitutions, glutamate atposition 80 and methionine at position 103 (designatedas Avr3AE80/M103 or Avr3AEM). In the absence of R3a,Avr3A KI strongly suppresses P. infestans INF1 elicitin-induced cell death in plants, thereby illustrating a majorvirulence function. The virulent allele Avr3a EM can-not induce an R3a-mediated HR but can still (weakly)suppress INF1-induced cell death suggesting that thiseffector has various parts with distinct functions thatlikely act through amino acid interactions with host pro-teins (Bos et al., 2006). These two distinct activitieshave been separated through saturated high-throughputmutant screens revealing important aa residues (Bos et al.,2009). Deletions or mutations in the C-terminal residueat tyrosine 147 maintained the R3A-mediated HR activ-ity while it caused the loss of the ability to suppressINF1-induced cell death. Molecular analysis of its celldeath-suppressing function showed that Avr3AKI interactsand stabilizes the host ubiquitin E3-ligase CMPG1 whichis required for INF1-induced cell death (Bos et al., 2010).The effector Avr3A likely targets CMPG1, interferingwith the ubiquitin proteasome system to block signaltransduction upon pathogen perception (Gilroy et al.,2011). P. infestans Avr3a is located in a region syn-tenic to a part in the genome of another oomycete,Hyaloperonospora arabidopsidis which harbours aviru-lence effector ATR1NdWsB (see below), suggesting thatthis locus is ancient in these oomycetes.
The P. infestans genome sequence revealed many pre-dicted secreted effectors and targeted searches for func-tion revealed novel Avr activities for some effectorsinteracting with specific resistance genes found in cer-tain potato species to induce hypersensitive cell death(Oh et al., 2009). Another secreted effector, SNE1, likelyis delivered into the plant host nucleus where it orches-trates the suppression of the effects of cell death-inducingeffectors secreted during the biotrophic phase (Kelleyet al., 2010).
Phytophthora sojae
P. sojae causes root and stem rot of soybean, resultingin huge damage to soybean production in North America
(Shan et al., 2004). Four avirulence genes, designatedAvr1b-1, Avr1a, Avr3a and Avr3c, have been cloned fromthis pathogen; they are recognized by soybean resis-tance genes Rps1b, Rps1a, Rps3a and Rps3c, respectively(Table 1; Shan et al., 2004; Dong et al., 2009; Qutob et al.,2009). Avr1b-1 was cloned by map-based cloning and ispredicted to encode a secreted protein of 138 aa witha RXLR motif. Interestingly, this gene requires anothergene, Avr1-b2, at the locus for accumulation of Avr1b-1 mRNA. Virulent P. sojae isolates such as P6497 andP9073, harbour a complete Avr1b-1 gene but cannot accu-mulate Avr1b-1 mRNA like avirulent strains (Shan et al.,2004). In addition to recognition by Rps1B, Avr1B-1 isalso recognized in plants having the Rpsk1 resistancegene resulting in limited cell death and showing that sucheffectors might have multiple targets (Kamoun, 2006).As R proteins from the Rps1 gene clusters are cytoplas-mic, it is assumed that Avr1-B is recognized inside thehost cytoplasm (Bhattacharyya et al., 1997; Shan et al.,2004).
P. sojae Avr1a, Avr3a and Avr3c were cloned througha combination of genetic mapping, transcript profiling,and functional analysis (Qutob et al., 2009). All P. sojaestrains, whether virulent or avirulent on Rps1 plants,contain the Avr1a gene which is present in four nearlyidentical copies of 5.2 kb. Virulence was attributed to dif-ferences in transcription of Avr1a in some strains whereasin other virulent strains, two of the multiple copies ofthe fragments containing Avr1a were additionally deleted(Qutob et al., 2009). Avr3a and four other predicted ORFswere also found in four duplicated copies, of a fragmentof 10.8 kb. Transcriptional silencing of these four Avr3acopies was found to be responsible for avoiding recogni-tion by Rps3 plants in some P. sojae virulent strains, whilein other virulent strains, three copies were deleted and thefourth one was transcriptionally silenced (Qutob et al.,2009). The Avr3c gene was located to a 33.7 kb fragmenttogether with eight other predicted ORFs and three near-identical copies of this fragment are present in a tandemarray in the P. sojae genome (Dong et al., 2009). Avr3cvirulent strains avoid recognition by Rps3A through spe-cific mutations in the effector and subsequent sequenceexchange between two copies of Avr3c in the arrays. Thisexample illustrates ways by which pathogens can evolvetheir effector repertoire as to avoid recognition by host Rgenes (Dong et al., 2009).
As for P. infestans, the release of the P. sojae genomesequence has allowed the large-scale analysis of candidateeffectors predicted to be secreted through their RXLR-dEER motif. Among 169 recently tested effectors, mostcould suppress cell death triggered by BAX, by othereffectors and/or the PAMP INF, while several caused
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cell death themselves and are hence possible avirulencefactors (Wang et al., 2011).
Hyaloperonospora arabidopsidis
H. arabidopsidis, formerly known as (Hyalo)peronosporaparasitica, is an obligate oomycete pathogen that causesdowny mildew disease on the model plant Arabidopsis.From this pathogen, two avirulence genes have beencloned and characterized: (Arabidopsis thaliana recog-nized 1), Atr1 and Atr13 (Table 1). The product ofthe Atr1 gene is recognized by Arabidopsis R pro-teins from two ecotypes, Niederzenz (RPP1Nd) andWassilewskija (RPP1WsB) and hence the effector alleleis called Atr1NdWsB. Transient expression of Atr1NdWsB inArabidopsis leaves by particle bombardment triggered celldeath. Extensive searches for allelic variants found thatRPP1Nd recognized the product of a single allele of Atr1,while RPP1WsB recognized the products of four differentdiverged alleles and provided resistance to a wide rangeof isolates (Rehmany et al., 2005). Atr1NdWsB encodesa 311 aa protein with a predicted secretion signal anda conserved RXLR motif. Atr1NdWsB is a highly poly-morphic protein and six different alleles that differ inabout one-third of all residues were identified in eightisolates; intense diversifying selection and recombinationplayed an important role in the evolution of this locus(Rehmany et al., 2005; Kamoun, 2006). Recent molec-ular studies have shown that binding of RPP1 occurs atthe LRR domain and is a prerequisite for HR induction,while the actual induction is facilitated by the TIR domain(Krasileva et al., 2010).
ATR13 is recognized by resistance protein RPP13 andtriggers a HR when transiently expressed in Arabidopsiscells using particle bombardment. The ATR13 geneencodes a 187 aa effector protein for which no relatedsequences have been found in public databases (Allenet al., 2004). ATR13 revealed apparent domain structures:in addition to the N-terminal SP and an RXLR motif,it has a heptad leucine-isoleucine repeat motif that isrequired for RPP13 recognition, followed by an imper-fect direct repeat of 4 × 11 aa which lies between aaresidues 93 and 136. All pathogen Atr13 effector alle-les as well as identified alleles of the interacting hostRPP13 resistance gene reveal a high level of aa polymor-phisms among their protein products and apparently havebeen under intense diversifying selection suggesting a co-evolutionary arms race at these loci (Allen et al., 2004).Both ATR1 and ATR13 suppress basal defence responsesof host plants when delivered by the P. syringae T3SS,revealing a virulence function (Sohn et al., 2007).
Concluding remarks
Over the last two decades, the study of different fungal,oomycete and bacterial pathogen effectors and their func-tion during the interaction with host plants has led toremarkable insights into the molecular basis underpin-ning plant diseases. These effectors facilitate a pathogen’sentry into the host by suppressing defence responses atmultiple levels. Examples include interference with recog-nition of PAMPs by PRRs and/or downstream signallingthereby evading basal immunity that otherwise wouldbe sufficient to stop infection. Effectors also have rolesin establishing feeding interactions and/or nutrient leak-age from the host to the benefit of the pathogens. Theseeffectors function at the interface of pathogen and hostplant or inside plant cells. Inadvertently, when recognizedby components of the host surveillance system, they cantrigger defence responses, revealing avirulence or elici-tor functions. The sequencing of complete genomes ofmany bacterial, fungal, and oomycete plant pathogens andsubsequent computational analyses have revealed a myr-iad of effectors, many of which are potential avirulencegenes that can trigger ETI. These then have the poten-tial of revealing novel resistance potential, in particularresistance genes in varied germplasm if suitable assayscan be designed. This has become an important goal ofmany research laboratories and has already led to the dis-covery of novel resistance genes (van der Hoorn et al.,2000; Torto et al., 2003; Stergiopoulos et al., 2010). Theidentification of complete sets of pathogen effectors, theirfunctions, and their host targets will advance the knowl-edge of molecular plant–pathogen interactions and sparkthe design of novel disease control strategies.
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