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ANNUAL REVIEWS

Further

Fungal Effector Proteins


Ioannis Stergiopoulos1 and Pierre J.G.M. de Wit1,2
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Wageningen University and Research Center (http://www.php.wur.nl/uk), Laboratory of Phytopathology, 6709 PD Wageningen, The Netherlands; email: ioannis.stergiopoulos@wur.nl Centre for BioSystems Genomics, 6700 AB Wagengen, The Netherlands; email: pierre.dewit@wur.nl

Annu. Rev. Phytopathol. 2009. 47:23363 The Annual Review of Phytopathology is online at phyto.annualreviews.org This articles doi: 10.1146/annurev.phyto.112408.132637 Copyright c 2009 by Annual Reviews. All rights reserved 0066-4286/09/0908/0233$20.00

Key Words
avirulence, cysteine-rich proteins, diversifying selection, guard model, resistance, virulence

Abstract
It is accepted that most fungal avirulence genes encode virulence factors that are called effectors. Most fungal effectors are secreted, cysteinerich proteins, and a role in virulence has been shown for a few of them, including Avr2 and Avr4 of Cladosporium fulvum, which inhibit plant cysteine proteases and protect chitin in fungal cell walls against plant chitinases, respectively. In resistant plants, effectors are directly or indirectly recognized by cognate resistance proteins that reside either inside the plant cell or on plasma membranes. Several secreted effectors function inside the host cell, but the uptake mechanism is not yet known. Variation observed among fungal effectors shows two types of selection that appear to relate to whether they interact directly or indirectly with their cognate resistance proteins. Direct interactions seem to favor point mutations in effector genes, leading to amino acid substitutions, whereas indirect interactions seem to favor jettison of effector genes.

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INTRODUCTION
The gene-for-gene hypothesis states that for every dominant avirulence (Avr) gene in the pathogen there is a cognate resistance (R) gene in the host, and the interaction between the products of these genes leads to activation of host defense responses, such as the hypersensitive response (HR) that arrests the growth of biotrophic fungi (40). Many plant pathologists have been searching for molecular and biochemical evidence of the genefor-gene concept. The molecular cloning of the rst bacterial Avr gene was reported in 1984 (114), the rst fungal Avr gene in 1991 (141), and the rst oomycete Avr gene followed in 2004 (67, 124). Over the past two decades, numerous novel Avr genes and cognate R genes have been discovered, and our molecular understanding of the gene-for-gene relationship has increased considerably. It is now accepted that plants contain two lines of defense. The rst line provides basal defense against all potential pathogens and is based on recognition of conserved microbial features known as pathogen-associated molecular patterns (PAMPs) by so-called PAMP-recognition receptors (PRRs) that activate PAMP-triggered immunity (PTI) and prevent further colonization of the host (23, 61, 91). One of the bestknown microbial PAMPs is chitin, a major structural component of fungal cell walls, for which two LysM-type of receptor-like kinases involved in its perception have been characterized in rice and Arabidopsis, respectively (66, 90). Evidence is now accumulating that Avr genes encode effectors that suppress PTI, thus enabling a pathogen to infect its host plant and cause disease. Once the basal defense system of plants is overcome by pathogens, plants respond with the development of a more specialized recognition system based on effector perception by R proteins and subsequent activation of effector-triggered immunity (ETI) that leads to rapid and acute defense responses in plants, the hallmark of which is the HR. This triggers a second wave of coevolutionary arms race between pathogens and plants, during which
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pathogens respond by mutating or losing effectors, or by developing novel effectors that can avoid or suppress ETI, whereas plants develop novel R proteins mediating recognition of novel effectors (23, 61). Many reviews on bacterial, fungal, and oomycete Avr genes and their cognate R genes in plants have appeared in recent years (8, 24, 47, 67, 68). In this review, we will provide an update on Avr genes from extracellular fungi, including Cladosporium fulvum, Fusarium oxysporum f. sp. lycopersici, Leptosphaeria maculans, Magnaporthe oryzae, and Rhynchosporium secalis, and from obligate fungal pathogens that form haustoria, such as Melampsora lini and Blumeria graminis f. sp. hordei (Table 1). Their structure, intrinsic functions, localization, perception by R proteins, and evolution will be discussed.

EFFECTORS OF EXTRACELLULAR FUNGAL PATHOGENS Cladosporium fulvum


Cladosporium fulvum (syn. Passalora fulva) is an asexual extracellular fungal pathogen of tomato (25, 63, 123). To date, four avirulence (Avr) genes have been cloned from C. fulvum that all encode small cysteine-rich proteins that are secreted during infection. These are the Avr2, Avr4, Avr4E, and Avr9 effector proteins whose recognition in tomato is mediated by the cognate Cf (C. fulvum) R proteins Cf-2, Cf-4, Cf4E, and Cf-9, respectively (25, 63, 123). Four additional extracellular proteins (Ecps), namely Ecp1, Ecp2, Ecp4, and Ecp5 have been characterized from C. fulvum that invoke an HR in tomato lines that carry a cognate Cf-Ecp gene (22, 75), whereas Ecp6 and Ecp7 have also recently been identied, but for these two effectors no responding tomato lines have yet been reported (13). Although demonstrated in only some cases, all Avrs and Ecps are assumed to be virulence factors (13, 123, 139, 140). Avr2 effector. Avr2 encodes a preprotein of 78 amino acids (aa), which matures into a 58 aa

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protein with eight cysteine residues that induces HR in tomato plants that carry the cognate Cf-2 resistance gene (26, 82). During infection, Avr2 inhibits at least four tomato cysteine proteases including Rcr3, Pip1, aleurain, and TDI65, and as such, plays an offensive role in virulence by targeting and inhibiting host proteases that are important for host defense (74, 102, 107). Indeed, the role of Avr2 in virulence is demonstrated not only for C. fulvum but also for other fungal tomato pathogens, including Botrytis cinerea and Verticillium dahliae, as heterologous expression of Avr2 in Arabidopsis thaliana enhances susceptibility toward these pathogens (140). In the presence of Cf-2, Avr2 behaves as an avirulence factor, and its recognition is mediated by Rcr3pimp (required for C. fulvum resistance), a cysteine protease originating from Lycopersicon pimpinellifolium (74, 102). Despite the fact that the exact mechanism of Avr2 perception is not yet known, structural modication of Rcr3 by Avr2, rather than Rcr3 inhibition, is the most likely cause of triggering Cf-2-mediated defense signaling, as a natural variant of Rcr3 occurs in Lycopersicon esculentum (Rcr3esc ) that causes spontaneous HR in the presence of Cf-2 in an Avr2-independent manner. The Rcr3esc protein still shows protease activity and is likely to have a modied tertiary structure as compared with the Rcr3pimp protein (74). Circumvention of Avr2-triggered Cf2-mediated HR can be achieved by point mutations, deletions, or transposon insertions in the Avr2 gene (82). A recent survey of polymorphisms present in the Avr2 alleles of a worldwide collection of C. fulvum isolates revealed an excess of nonsynonymous polymorphisms compared with synonymous ones, suggesting positive diversifying selection. These were mainly insertions or deletions in the coding region of the gene that lead to the production of truncated Avr2 proteins (116). Avr4 effector. Avr4 encodes a 135 aa preprotein that is C- and N-terminally processed after secretion in the apoplast into an 86 aa mature protein with eight cysteine residues (62, 64). Based on its disulde-bond pattern, Avr4 shows

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structural similarity to proteins with an invertebrate chitin-binding domain, such as tachycitin (inv ChBD) (110). Indeed, binding of Avr4 to chitin has been experimentally conrmed, and it is further shown that Avr4 can protect chitinous fungi, such as Trichoderma viride and Fusarium solani, against basic plant chitinases. Therefore, a role for Avr4 in protection of C. fulvum against chitinases during infection has been proposed (130132). The virulence function of this protein is further supported by the fact that Avr4 is expressed only during infection of the host, when the fungus is exposed to chitinases, and that silencing of Avr4 in C. fulvum signicantly reduces virulence (139). In addition, tomato plants expressing Avr4 are more susceptible to C. fulvum and other chitinous fungal tomato pathogens (139). In the presence of Cf-4, Avr4 induces HR, but natural isoforms of this effector protein occur that no longer trigger Cf-4-mediated HR. Such isoforms show an excess of nonsynonymous substitutions compared with synonymous ones, suggesting that Avr4 is under positive diversifying selection (116), whereas the majority of the natural variation observed in Avr4 involves point mutations that mostly cause substitutions of cysteine residues. The resulting unstable Avr4 variants are more sensitive to proteases but are still able to bind chitin, thus preserving the virulence function of the protein but preventing accumulation of Avr4 in the apoplast and triggering of HR in Cf-4 plants (64, 132). Avr4E effector. Avr4E codes for a cysteinerich 101 aa protein that is secreted during infection and triggers Cf-4E-mediated HR (145). Various strains of C. fulvum have been identied that evade Cf-4E-mediated resistance that all show two identical point mutations in Avr4E that translate into stable Avr4E proteins with two aa substitutions (Avr4ELT ). Targeted mutagenesis showed that one of these mutations in Avr4E, namely the Phe62>Leu substitution, is sufcient for evasion of Avr4E-triggered Cf4E-mediated HR (145). Analysis of natural populations of the fungus also shows that a significant number of strains evade Cf-4E-mediated
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Table 1 Function/ Cysteines 8 Cf-2 (eLRR-TM) 20 Protease inhibitor Chitin-binding Apoplast In planta Apoplast In planta Inhibits Rcr3 and other proteases Protects against chitinases Unknown Unknown Disruption leads to reduced virulence Disruption leads to reduced virulence In planta In planta In planta Unknown Unknown Knock-down leads to reduced virulence Apoplast Probably in cytoplasm Unknown Probably in apoplast In planta In planta and in vitro In planta and in vitro Unknown Unknown Unknown Cf-4 (eLRR-TM) Hcr9-4E (eLRR-TM) Cf-9 (eLRR-TM) Cf-Ecp1 Not cloned Yes SignalP homology in plant Expression virulence (type) selection Localization Role in R-gene Positive

Fungal effector proteins

Aa References (26)

Effector

protein

residues (mature)

Cladosporium fulvum (tomato):

236

Avr2

78 (58)

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Avr4

135 (86)

18

Yes

(62)

6 6 8 23 Tumor-necrosis factor receptor Apoplast In planta 23 Carboxypeptidase Apoplast inhibitor In planta 10 Unknown Apoplast In planta 4 22 Unknown Apoplast In planta 6 6 8 23 LysM-domains; chitin-binding 17 Unknown Apoplast Apoplast 18 Unknown Apoplast 6 1 6 20 22 ? Unknown Unknown

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Avr4E

121 (101)

Yes Yes No

(145) (129) (129)

Avr9

63 (28)

Ecp1

96 (65)

Ecp2

165 (143)

Cf-Ecp2 Not cloned

No

(129)

Ecp4

119 (101)

Cf-Ecp4 Not cloned Cf-Ecp5 Not cloned Unknown

No No No

(75) (75) (13)

Ecp5

115 (98)

Ecp6

222 (199)

Ecp7

? (100)

Unknown Rlm1 Not cloned Rlm6 Not cloned

Unknown Yes Unknown

(13) (49) (44)

Leptosphaeria maculans (oilseed rape):

AvrLm1

205 (183)

AvrLm6

144 (124)

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AvrLm47

143 (122)

21

Unknown

Probably in apoplast

In planta (mainly) and in vitro Stimulated by living cells I-3 Not cloned Unknown In planta Probably not required for virulence Required for full virulence Suppression of I-2 and I-3 resistance Not required for virulence on rice Probably not required for virulence on rice Unknown Probably not required for virulence on rice Unknown Unknown I-2 (CCNBS-LRR) I or I-1 Not cloned Required for full virulence Unknown

Unknown

Rlm4 and/or Rlm7 Not cloned

Yes

(95)

Fusarium oxysporum f. sp. lycopersici (tomato): 8 21 Unknown Xylem (99; M. Rep pers. comm.) Unknown (55; M. Rep pers. comm.) Unknown (55; M. Rep pers. comm.) Unknown (55; M. Rep pers. comm.) Pi-ta (CCNBS-LRR) Pi-ta (CCNBS-LRR) Yes (93)

Avr3 (Six1) 8 20 Unknown Xylem

284 (189)

Six2

232 (172)

Six3

163 (144)

3 (2)

19

Unknown

Xylem

In planta

Avr1 (Six4)

242 (184)

17

Unknown

Xylem

In planta

Magnaporthe oryzae (rice): 8 16 Homology to Metalloproteases Homology to Metalloproteases Homology to Metalloproteases Glycine-rich hydrophilic protein Glycine-rich hydrophilic protein Glycine-rich hydrophilic protein Probably in apoplast Probably in cytoplasm Probably in apoplast Unknown Cytoplasm In planta

Avr-Pita

224 (176)

AvrPita2

224 (?)

16

Yes

(71)

AvrPita3

226 (?)

16

Unknown

Yes

(71)

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Pwl1

147 (124)

23

Unknown

Unknown

(70)

Pwl2

145 (126)

1(1)

21

Probably in apoplast Probably in apoplast

Unknown

Unknown

Unknown

Unknown

(118)

237

Pwl3

137 (116)

21

Unknown

Nonfunctional

Unknown

Unknown

(70)

(Continued )

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Stergiopoulos

Table 1 Function/ Cysteines 0 21 Glycine-rich hydrophilic protein Hybrid polyketide synthase/ nonribosomal peptide synthetase Unknown Unknown Unknown Not secreted Expressed in appressoria Unknown Probably in apoplast Unknown Nonfunctional SignalP homology in plant Expression virulence Localization Role in R-gene (type) Unknown

(Continued ) Positive selection Unknown References (70)

Aa

43 Pi33 Unknown 10 22 Non-specic toxin/induces necrosis and plasmamembrane H+ ATPase Non-specic toxin/induces necrosis Probably in apoplast Expressed in vitro Not required for virulence 7 (6) 16 Probably in apoplast Expressed in vitro Not required for full virulence

Effector

de Wit

protein

residues (mature)

Pwl4

138 (117)

Ace1

4035

Unknown

(11)

Avr1CO39

Not cloned yet

Pi-CO39(t)

Yes

(37)

Rhynchosporium secalis (barley): Rrs-1 Not cloned Yes (101)

Nip1

82 (60)

Nip2

109 (?)

Unknown

Yes

(101; W. Knogge pers. comm.)

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Nip3

115 (?)

9 (8)

17

Non-specic toxin/induces necrosis

Probably in apoplast

Expressed in vitro

Not required for full virulence

Unknown

Yes

(101; W. Knogge pers. comm.) (27)

Melampsora lini (ax): 1 23 Unknown Cytoplasm Expressed in haustoria Expressed in haustoria and in vitro Expressed in haustoria Unknown Unknown M (TIRNBS-LRR) P, P1, P2 and/or P3 (TIR-NBSLRR) P4 (TIRNBS-LRR) Unknown Mla10 (CCNBS-LRR) Unknown Mlk1 (CCNBS-LRR) Unknown L5, L6 and L7 (TIR-NBSLRR) Yes

AvrL567 (A, B and C) 1 28 Unknown Cytoplasm

150 (127)

AvrM

314

Yes

(17)

AvrP123

117 (94)

11 (10)

23

Kazal Ser protease inhibitor Cystine knotted peptide >30 paralogues in Bgh and other f. sp. >30 paralogues in Bgh and other f. sp. Probably in cytoplasm Probably in cytoplasm Unknown Cytoplasm Expressed in haustoria Unknown

Cytoplasm

Yes

(17)

AvrP4

95 (67)

7 (6)

28

Yes

(17)

Blumeria graminis f. sp. hordei (barley): 4 Unknown (100)

Avra10

286

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Avrk1

177

Unknown

Unknown

(100)

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HR by jettison of the Avr4E gene, indicating that the tness penalty associated with the loss of this gene is probably not very high (116). However, Avr4E-expressing tomato transformants are more susceptible to natural C. fulvum strains that lack Avr4E than control plants, suggesting that Avr4E is a virulence factor (H.P. van Esse and B.P.H.J. Thomma, personal communication). No homologs of Avr4E are present in public databases and its molecular function is still unknown (145).
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a major, but not an exclusive, positive regulator of Avr9 expression (96). Expression of all the other Avr and Ecp effector genes of C. fulvum that have been characterized so far is not induced under nitrogen-limiting conditions, and although they are all expressed in planta, their regulators have yet to be identied (12, 122). Ecp effectors. In addition to the Avrs, six more effector genes that code for Ecps have been cloned from C. fulvum, namely Ecp1, Ecp2, Ecp4, Ecp5, Ecp6, and Ecp7 (13, 75, 129). Ecps are abundantly secreted by all strains of C. fulvum during infection and possess an even number of cysteine residues that are most likely involved in intramolecular disulde bridges (80). Most Ecps share little or no homology with other proteins present in public databases, except for Ecp6 which, so far, is the only effector protein of C. fulvum with orthologs in many other fungal species. This is mainly due to the presence of LysM-domains in this protein, which in general are implicated in carbohydrate binding including chitin, suggesting that Ecp6 might be a functional homolog of Avr4 (13). Alternatively, Ecp6 might also be involved in scavenging of chitin fragments that are released from the fungal cell wall during infection by plant chitinases, thus preventing them from acting as PAMPs and eliciting basal defense responses (13). Ecp6, Ecp1, and Ecp2 are all virulence genes because silencing or disruption of these genes compromises virulence of C. fulvum on tomato (13, 77). For Ecp1, Ecp2, Ecp4, and Ecp5, tomato accessions have been identied that carry a single dominant Cf-Ecp gene and develop an HR upon inoculation with recombinant potato virus X (PVX) strains that express these Ecps, or after injection of the puried Ecp proteins (75, 76). However, although most Cf-Ecp genes have been genetically mapped, they still remain to be cloned (22, 113). In contrast to Cf-genes, Cf-Ecp genes have not yet been deployed in commercial tomato lines, which might explain why only a limited number of polymorphisms has been observed in Ecp genes of natural populations of C. fulvum (13, 116).

Avr9 effector. Avr9 encodes a 63 aa preprotein that is C- and N-terminally processed by fungal and plant proteases into a mature 28 aa protein with six cysteine residues (129, 141). The 3-D structure of Avr9 resembles cystineknotted peptides (134, 143), which share structural, but little functional, homology (94). Alanine scanning of the Avr9 peptide revealed that all six cysteine residues present in this protein are essential for its structure and necrosisinducing activity (73). Based on its overall tertiary structure, Avr9 shows structural but not functional homology to carboxypeptidase inhibitors (133, 134). Disruption of Avr9 in C. fulvum by homologous recombination did not affect growth in vitro or virulence on tomato plants, suggesting that Avr9 is not required for full virulence (86). Indeed, all natural strains that evade Cf-9-mediated HR lack the Avr9 gene (116). However, Avr9-expressing tomato transformants are more susceptible to natural C. fulvum strains that lack Avr9 than control plants, suggesting that Avr9 may be a redundant virulence factor (H.P. van Esse and B.P.H.J. Thomma, personal communication). Expression of Avr9 in vitro is induced under nitrogen-limiting conditions, suggesting that the produced protein might be involved in nitrogen metabolism (96, 122, 128). Furthermore, an Nrf1 (nitrogen responsive factor) gene has been identied in C. fulvum, and Nrf1 deletion mutants no longer express Avr9 under nitrogen-limiting conditions and are compromised in their virulence on Cf-0 tomato plants. However, these strains are still avirulent on Cf-9 tomato plants, suggesting that Nrf1 is
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Fusarium oxysporum f. sp. lycopersici


The vascular pathogen Fusarium oxysporum is an asexual fungus with a broad host range and causes wilt and root diseases. However, most isolates of F. oxysporum can infect only a single or a few plant species and are therefore subdivided into different formae speciales (f. sp.) based on their host specicity (48). Thus, strains of F. oxysporum that cause tomato wilt are referred to as f. sp. lycopersici.
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Six effectors. F. oxysporum f. sp. lycopersici (Fol) is an extracellular pathogen that colonizes the xylem vessels of tomato. Four small proteins, designated Six1 to Six4 (secreted in xylem), have been identied in Fol and are produced during infection. Six1 (currently renamed Avr3) is a small cysteine-rich protein of approximately 32 kDa that is N- and Cterminally processed by either fungal and/or plant proteases into a 12 kDa mature protein or an alternative form of 22 kDa, and which is also proven to be the active form of the protein (98, 99). Avr3 is required for full virulence on tomato, but it also behaves as an Avr factor that triggers HR in the presence of the cognate I-3 resistance gene (56, 99). Interestingly, expression of Avr3 has been detected only during root colonization and infection of tomato plants, but it is neither cultivar-specic nor depends on morphological features of the roots. Instead, the presence of living plant tissue in the vicinity of the fungus seems not only required for Avr3 expression but might also trigger a switch from a saprophytic to pathogenic lifestyle (136). In the genome of Fol, Avr3 resides in an area of a small chromosome that contains many transposable elements and thus resembles pathogenicity islands. Pathogenicity islands that harbor several virulence genes often reside on dispensable chromosomes and their existence has been described for other fungal pathogens as well, including Alternaria spp. (121), Nectria hematococca (79), L. maculans, B. graminis f. sp. hordei, M. oryzae, and possibly others (5, 70, 71, 111). Indeed, a large genomic area of approximately 8 kb that contains Avr3 as

well as Six2 could not be found outside the f. sp. lycopersici lineage nor in nonpathogenic Fusarium strains, suggesting that it represents a dispensable region that confers virulence specifically on tomato (135). In addition, the f. sp. lycopersici lineage-specic effector gene Six3 also resides on the same chromosome as Avr3 and Six2, although not within the boundaries of the 8 kb region that harbors the latter two genes. This suggests that the ability to cause disease has likely arisen only once during evolution of Fol by acquisition or emergence of the genomic region harboring the virulence genes necessary for infection of tomato. Subsequently, this region might have spread to other clonal Fol strains by horizontal gene transfer (135). Because the presence of Avr3 is required for full virulence on tomato, it raises the question how Fol strains can overcome I-3-mediated resistance, as loss of Avr3 would pose a serious tness penalty and mutants with only point mutations in Avr3 that avoid recognition by I-3 but still remain virulent have not been reported. The recent functional characterization of Avr1 (previously known as Six4) might provide the answer to this question. Avr1 is a small cysteinerich secreted protein that confers avirulence to Fol strains on tomato lines carrying the I or I-1 resistance gene. However, unlike Avr3, Avr1 is not required for full virulence of Fol strains on tomato plants that lack the cognate R gene. In contrast, Avr1 functions as a suppressor of I2- and I-3-mediated resistance (54). Indeed, although Six3, which is required for full virulence (M. Rep, personal communication), is present in strains of Fol that are both virulent and avirulent on I-3 tomato lines, Avr1 is only present in Fol strains virulent on I-3 lines. When strains of Fol that are avirulent on I-2 and/or I-3 lines were transformed with Avr1 they gained virulence on these lines, indicating that Avr1 suppresses both I-2- and I-3-mediated resistance. The underlying molecular mechanism of suppression is not yet known, but an evolutionary model for this phenomenon has been proposed (54). In this model, it is suggested that the I-3 resistance gene has evolved to recognize Avr3 but because Avr3 is required for full virulence
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of Fol, evasion of I-3 recognition through loss of Avr3 would cause a serious tness penalty. Therefore, Avr1 might have been acquired by Fol in order to avoid the tness penalty associated with the loss of Avr3 (and likely of Six2) in overcoming I-2- and I-3-mediated resistance. This explains why all Fol strains analyzed so far contain Avr3, whereas Avr1 is present only in strains that are virulent on I-2 and/or I-3 lines. It is likely that the I-3 protein operates in accordance with the guard model, where not the Avr3 protein, but the manipulation of its virulence target is recognized by I-3, whereas Fol strains can (partially) regain virulence toward I-3-containing lines by acquisition of Avr1. During evolution, tomato responded to this adaptation with the development of the I or the unlinked I-1 resistance gene that specifically recognizes and responds to Avr1 (54).

Leptosphaeria maculans
At least nine distinct Avr genes, designated AvrLm1 to AvrLm9, have been genetically identied in L. maculans, the causal agent of stem canker on oilseed rape, that cause avirulence on plants carrying the cognate Rlm1 to Rlm9 R genes, respectively. These Avr genes have been mapped to at least four unlinked genomic regions, including the genetic clusters AvrLm1-AvrLm2-AvrLm6 and AvrLm3AvrLm4-AvrLm7-AvrLm9 (5). AvrLm1 and AvrLm6 effectors. AvrLm1 and AvrLm6 are the rst Avr genes that have been cloned from L. maculans using a map-based cloning strategy (44, 49). The two Avrs are in relatively close proximity at a locus that also harbors AvrLm2 (5). More specically, AvrLm1 has been mapped on a 269 kb AT rich, genepoor heterochromatin-like region that consists of a number of degenerated, nested copies of four long-terminal repeat (LTR) retrotransposons and is surrounded by GC-rich isochors. Also, AvrLm6 maps within a 133 kb noncoding region that mainly contains LTR retrotransposons. However, both AvrLm1 and AvrLm6 share a low GC content, in contrast to most
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other fungal Avr genes cloned so far. Both AvrLm1 and AvrLm6 are single copy genes, encoding small secreted proteins of 205 aa and 144 aa, respectively, that have no known homologs in public databases and lack any characteristic signatures that could point toward a putative intrinsic function. AvrLm1 and AvrLm6 are strongly induced in planta, particularly during the early stages of infection, and expression of the genes has also been observed in vitro, although here expression of AvrLm1 is much higher than that of AvrLm6. Despite the clear avirulence properties of the AvrLm1 and AvrLm6 genes, the cognate R genes Rlm1 and Rlm6 have not been cloned yet. Therefore, it is currently unknown whether these effectors interact directly or indirectly with their cognate R proteins and whether recognition occurs outside or inside the host cells. In that respect, AvrLm6 might be secreted and reside in the apoplast as it contains six cysteine residues that could provide stability by forming disulde bridges. In contrast, AvrLm1 contains only one cysteine residue, making it different from AvrLm6 and the apoplastic cysteine-rich effector proteins of C. fulvum and F. oxysporum f. sp. lycopersici discussed above, and therefore, uptake of AvrLm1 into the host cell seems more likely (49). As has been observed for other fungi, AvrLm1 is absent in races that are virulent on Rlm1 cultivars. Recently, a gain of virulence on Rlm1 plants was observed in eld populations of L. maculans in France that was due to a specic 260 kb deletion of a chromosomal segment spanning AvrLm1. A similar deletion was observed in more than 90% of 460 eld isolates analyzed from a world population, indicating that jettison of the AvrLm1 is the main mechanism of adaptation to Rlm1 without causing a signicant tness penalty for the fungus (6, 7, 104). AvrLm4-7 effector. Using a map-based cloning strategy, a genetic locus of 238 kb containing the AvrLm7 gene has been recently delineated in L. maculans that shows the same characteristics as the AvrLm1-2-6 locus,

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including the presence of multiple LTR retrotransposons and an AT-rich isochoric region next to a GC-equilibrated one (95). In total, 40 genes were predicted on this locus, 35 of which are present in the GC-equilibrated region and only ve in the AT-rich isochors. A single gene (AvrLm7) was identied in the gene-poor 60 kb AT-rich region that confers avirulence on both Rlm7 and Rlm4 genotypes. This gene was consequently renamed AvrLm4-7 for the dual specicity of its encoded protein toward the Rlm4 and Rlm7 genes. AvrLm4-7 encodes a putatively secreted preprotein of 143 aa that is further processed into a 122 aa mature protein with eight cysteine residues and no homology to any other proteins currently present in public databases. Expression of AvrLm4-7 is considerably upregulated during primary leaf infection, reaching a maximum at seven days post inoculation, whereas only low levels of expression are observed during in vitro growth of the fungus. Analysis of 300 eld isolates of L. maculans showed that complete or partial deletion of the AvrLm4-7 gene is the main mechanism for gaining virulence on both Rlm4 and Rlm7 genotypes, whereas most isolates virulent on Rlm4 genotypes alone showed only a single point mutation in the produced AvrLm4-7 protein (Gly120>Arg). Such strains were less t than strains with the wild-type AvrLm4-7 allele, indicating that AvrLm4-7 is important for tness of the fungus. No strains virulent only on Rlm7 plants were observed (95).

characterized from M. oryzae, including AvrPita (93) that was originally called Avr2-YAMO (125), Avr1-CO39 (38), Pwl2 (118, 125), Pwl1 (70), and Ace1 (11). Avr1-CO39 was isolated from an M. oryzae isolate pathogenic on weeping lovegrass and controls avirulence on rice plants carrying the cognate Pi-CO39(t) resistance gene (18, 38). Almost all M. oryzae isolates that are virulent on CO39-rice cultivars lack the Avr1-CO39 gene (37). Avr-Pita and Ace1 were cloned from M. oryzae isolates pathogenic on rice, whereas Pwl2 was originally identied in a genetic cross between two laboratory strains that infected rice. However, unlike the other two genes, Pwl2 is a species-specic gene that confers avirulence on weeping lovegrass but not on any known rice cultivars (118). Avr-Pita effector. Avr-Pita encodes a presumably secreted preprotein of 223 aa with homology to fungal zinc-dependent metalloproteases. This protein is further processed into an active 176 aa mature protein (Avr-Pita176 ) that is dispensable for virulence on rice (59, 93). AvrPita176 interacts directly with the cognate Pi-ta resistance protein, a predicted 928 aa receptorlike protein with a central nucleotide-binding site (NBS) and a C-terminal leucine-rich repeat (LRR) domain. Direct interaction between the two proteins has been demonstrated by yeast two-hybrid assays and by in vitro binding assays that showed binding of the Avr-Pita176 to the LRR domain of Pi-ta (59). Furthermore, coexpression of the two proteins in rice cells elicited plant defense responses, suggesting that physical interaction inside host cells is required for activation of Pi-ta-mediated defense. In the same experiments, it was also shown that the recognition specicity for Avr-Pita is determined by a difference in one aa residue (Ala918) of the Pi-ta protein present in resistant vs susceptible rice varieties. On the other hand, many mutations have been described in AvrPita that result in gain of virulence on Pi-ta rice cultivars. In the genome of M. oryzae, AvrPita resides close to the telomere of chromosome 3, and this might account for the observed molecular instability of the genomic region
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Magnaporthe oryzae
Magnaporthe oryzae (formerly known as M. grisea) (21) is the causal agent of rice blast. Resistant rice lines have been used extensively over the past decades to battle the disease, but R genes present in these lines have often been overcome rather quickly by the emergence of virulent races. The rice blast pathosystem complies with the gene-for-gene model, and to date more than 40 major R genes, designated Pi, have been identied. So far, ve cultivar- and species-specic Avr genes have been cloned and

containing this gene. Indeed, an array of mutations has been described on the Avr-Pita locus in virulent strains of the fungus, including various size deletions, point mutations, and a transposon insertion (15, 58, 69, 93, 150). Avr-Pita-related effectors. Recently, it was shown that Avr-Pita (currently renamed to AvrPita1) belongs to a gene family with at least two additional members, Avr-Pita2 and Avr-Pita3 (71). Avr-Pita2 acts as elicitor of defense responses mediated by Pi-ta, but Avr-Pita3 does not. Members of the Avr-Pita family are widely distributed among strains of M. grisea isolated from diverse hosts, including isolates that are not pathogenic on rice. However, although Avr-Pita1 and Avr-Pita2 are present in both M. oryzae and M. grisea isolates, Avr-Pita3 is present only in M. oryzae isolates, suggesting that Avr-Pita1 and Avr-Pita3 are derived from a gene duplication event that must have occurred after separation of M. oryzae from M. grisea (71). Ace1 effector. Ace1 encodes a putative 4035 aa cytoplasmic fusion polypeptide containing a polyketide synthase (PKS) and a nonribosomal peptide synthetase (NRPS), two distinct classes of enzymes that are involved in the production of microbial secondary metabolites (11, 20). Ace1 seems to mediate avirulence indirectly in the presence of the Pi33 rice R gene, by its involvement in the biosynthesis of a secondary metabolite that most likely activates Pi33. The nature of the secondary metabolite is not known yet, but mutations in the PKS domain of Ace1 abolish activation of Pi33, indicating that at least Ace1-mediated biosynthetic activity is required for avirulence on Pi33 plants (11). Ace1 is exclusively expressed in appressoria, suggesting that the produced secondary metabolite might have a role in virulence, although mutants in which Ace1 was deleted were not compromised in virulence (11, 43). Pwl effectors. Members of the Pwl (pathogenicity toward weeping lovegrass) gene family encode rapidly evolving small glycine-rich secreted proteins that are
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generally found in rice pathogens. At least four members of this family, designated Pwl1 to Pwl4 are present in M. oryzae, and act as Avr genes conferring species-specic avirulence on weeping lovegrass and nger millet but have no effect on rice (70, 118). Pwl2 encodes a secreted glycine-rich, hydrophilic protein of 145 aa that confers avirulence on weeping lovegrass (118). This gene is located on a highly unstable genetic locus, where frequent genetic rearrangements associated with large deletions lead to the emergence of spontaneous mutants virulent on weeping lovegrass. Pwl1 (75% aa identity with Pwl2) and the allelic Pwl3 (51% aa identity with Pwl2) and Pwl4 (57% aa identity to Pwl2) were identied based on homology to Pwl2, but only Pwl1 is a functional homolog of Pwl2, conferring avirulence on weeping lovegrass. However, Pwl4 could be made functional when expressed under the control of the Pwl2 promoter, which was not the case for Pwl3 (70).

Rhynchosporium secalis
Rhynchosporium secalis, the causal agent of leaf scald on barley, secretes three low molecular weight peptides, designated Nip1 to Nip3, that function as nonspecic toxins on barley and several other plants. Nip1 and Nip3 are produced both in vitro and in planta, and their presence correlates with the formation of necrotic lesions that are most likely caused by an indirect stimulation of H+ -ATPase activity in plant plasma membranes (146, 147). In addition, Nip1 triggers specic (non-HR) defense responses in barley cultivars carrying the Rrs1 resistance gene (51). Nip1 (also known as AvrRrs1) encodes a preprotein of 82 aa, which matures into a 60 aa protein after removal of the signal sequence. Ten cysteine residues (101) are present in the mature Nip1 protein that is involved in ve intramolecular disulde bonds (45). NMR spectrometry showed that Nip1 mainly consists of two structural domains, one with two -sheets and another with three antiparallel strands (127). Strains of R. secalis virulent on Rrs1 plants either lack Nip1 or carry

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alleles with point mutations that translate into single aa substitutions (72, 101, 105). Two of these aa substitutions are correlated with gain of virulence, and lack of H+ -ATPase activity and necrosis-inducing activity, suggesting that both the necrosis induction and elicitor activity are mediated by a single receptor that is most likely different from the Rrs1 protein (39). Recently, it was found that Nip1 interacts with a single plasma membrane receptor that is involved in both mediating virulence and triggering defense, but the exact receptor has not yet been characterized (126). A recent study of eld populations of the pathogen showed clear evidence of positive diversifying selection operating on the Nip1 locus (105). In total, 14 Nip1 isoforms were identied, of which at least three were correlated with gain of virulence on Rrs1 plants, while a high deletion frequency of Nip1 was also observed that was much higher than that observed for Nip2 and Nip3. As single amino acid substitutions in Nip1 that correlated with gain of virulence on Rrs1 plants were observed in much lower frequencies than gene deletions, the tness cost associated with the loss of this gene is likely not high (105). Recently, the Nip2 and Nip3 genes have also been cloned from R. secalis (W. Knogge, personal communication). Nip2 encodes a 109 aa protein with a predicted signal peptide of 16 aa, whereas Nip3 encodes a 115 aa protein with a predicted signal peptide of 17 aa. Both proteins have one cysteine residue in their signal sequences, and the mature Nip2 and Nip3 carry six and eight cysteines, respectively. Nip2 and Nip3 are also further processed at their C termini, but as the cleavage sites are not yet known, the exact number of aa (including cysteine) residues present in the mature proteins remains to be determined (W. Knogge, personal communication).

epidermis (powdery mildews) or in mesophyll cells (rust fungi) of their hosts during infection. Haustoria are not only specialized feeding structures required for acquisition of nutrients, but they also induce structural, cellular, and biochemical changes in the invaded host cells. In addition, they can facilitate delivery of effectors into the extrahaustorial matrix, several of which are subsequently translocated into host cells (16). Here, we discuss effectors produced by the ax rust fungus M. lini and the barley powdery mildew fungus B. graminis f. sp. hordei.

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Melampsora lini
The ax rust fungus M. lini is an obligate basidiomycete that infects ax (Linum usitatissimum) and other species of the genus Linum. At least 30 Avr genes corresponding to approximately 30 cognate ax R genes have been identied in genetic analyses (33). Flax R proteins are all members of the intracellular TIR-NBS-LRR class and are distributed among ve highly polymorphic loci designated K, L, M, N, and P (4, 29, 30, 32, 35, 78). To date, Avr genes have been cloned from four M. lini loci, namely AvrL567, AvrM, AvrP123, and AvrP4 that code for haustorially expressed secreted proteins (HESPs) and elicit HR in ax plants that carry the cognate R genes (17). Each of the characterized Avr loci consists of one to ve closely related homologous genes that code for small secreted proteins with no sequence similarity to other proteins present in public databases. Transient expression of the mature Avr proteins in ax plants carrying the cognate cytoplasmic R proteins induces an HR, indicating that they are translocated via the extrahaustorial matrix into host cells during infection (17, 27). The AvrL567A, AvrL567B, and AvrL567C genes cluster at the AvrL567 locus and trigger HR in ax lines that carry the L5, L6, and L7 resistance genes. They all encode 150 aa proteins, with a predicted 23 aa highly conserved N-terminal signal sequence resulting in a 127 aa mature protein. Specic expression of these genes in haustoria suggests that they might play a role in virulence. Approximately 25% of the aa residues within the mature protein retain
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EFFECTORS FROM HAUSTORIUM-FORMING FUNGAL PATHOGENS


Rusts and powdery mildews are obligate pathogenic fungi that produce haustoria in the

one or more polymorphisms, and several of these have been shown to affect a transition to virulence on plants carrying the cognate R proteins, suggesting that the Avr567 locus is under positive diversifying selection. Indeed, in a survey involving just six ax rust strains, twelve AvrL567 sequence variants (known as AvrL567A to AvrL567L) were identied, six of which (A, B, D, F, J, and L) were avirulent variants that triggered HR in ax lines carrying the L5, L6, or L7 genes, and ve (C, G, H, I, and K) were virulent variants that no longer triggered HR on these lines (28). These virulent variants exhibited substitutions in aa residues that are exposed to the surface of the protein and interact directly with the cognate R proteins, as conrmed in yeast two-hybrid assays (34, 144). Therefore, evading host recognition is most likely the source of the diversifying selection acting on the Avr567 locus. Other characterized HESPs from ax rust include AvrM that is recognized by the M resistance protein, AvrP4 that is recognized by P4, and the complex AvrP123 proteins that are variously recognized by P, P1, P2, and/or P3 resistance proteins (17). As for AvrL567, diversifying selection also operates at the AvrM, AvrP4, and AvrP123 loci with virulent ax strains carrying mutated alleles that encode Avrs that are no longer recognized by the cognate R proteins (17). At least ve different paralogs (AvrMA to AvrME) have been detected at the AvrM locus of an avirulent strain, although one paralog encodes an effector that is not recognized by any known ax R protein. The six AvrM proteins have no known homologs in the public databases and show signicant sequence and size variations caused by DNA insertions, deletions, or polymorphisms in the location of stop codons. AvrP123 proteins contain ten cysteine residues including the characteristic CX7CX6YX3CX2-3C signature present in the Kazal family of serine protease inhibitors, suggesting that host proteases might be a target of these effectors. AvrP4 also encodes a protein with six cysteine residues at the 28 aa C-terminal part of the mature protein that show a spacing (CX37CX4-6CX0-5CX1-4CX4-10C) typical for
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cystine-knotted peptides (94). Both AvrM and AvrP4 are expressed in planta, whereas AvrM is also expressed in vitro. Transient intracellular expression of AvrM and AvrP4 in ax plants carrying the cognate R genes triggers an HR, suggesting that effector translocation into the host cells occurs during infection, which is consistent with the predicted cytoplasmic location of M and P resistance proteins (4, 78). However, both effectors also induce an HR in ax when targeted into the apoplast, suggesting their reentry from the apoplast into host cells after secretion (17, 27, 31). A role in virulence for the Avr genes of M. lini has not been shown yet.

Blumeria graminis f. sp. hordei


Powdery mildews are a large group of ascomycete obligate biotrophic fungi that show a high degree of host specialization and, like rusts, produce haustoria in their host plants (46). Blumeria graminis f. sp. hordei (Bgh) causes powdery mildew on barley and interacts with its host in a gene-for-gene manner (149). At least 85 dominant or semidominant mildew R genes (Ml) with different recognition specicities have been characterized in barley, including Mlk genes and 28 highly homologous genes that all map to the Mla (mildew A) locus of barley chromosome 5 (57, 65). Six of the genes present at this locus, namely Mla1, Mla6, Mla7, Mla10, Mla12, and Mla13, have been cloned, and they all encode highly related intracellular CC-NBS-LRR type of R proteins. However, despite the high sequence similarity (>90% identity), they all recognize isolatespecic effectors of Bgh (52, 53, 109). Two Avr genes residing within a proximity of 30 kb in the genome have been cloned from Bgh (100). They are designated Avrk1 and Avra10 and induce defense responses in barley varieties containing the cognate Mlk1 and Mla10 R proteins, respectively. Both genes belong to a large multigene family of more than 30 paralogs in Bgh, whereas homologs are present in formae speciales that are pathogenic on other grasses. Of all plant pathogens studied so far, Bgh has the highest number of Avr genes, and

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most of them map to single loci, although genomic regions that contain clusters of several recombining Avr genes have also been reported (14). The predicted 286 and 177 aa proteins encoded by Avra10 and Avrk1, respectively, share approximately 60% similarity with each other, but both lack an N-terminal signal sequence or a signature for uptake by host cells, such as the RXLR motif present in oomycete effectors (67). This suggests an alternative but yet unidentied route of delivery of these effectors into host cells, where they exert their putative virulence function and induce defense responses mediated by the cognate R proteins in resistant plants (100). Recently, it was shown by uorescence microscopy that the majority of the Mla10 protein is localized in the cytoplasm and approximately 5% in the nucleus (9, 108). Perturbation of nucleo-cytoplasmic Mla10 partitioning by expression of an Mla10 fusion protein containing a nuclear export signal (NES) that enhances nuclear export over import, decreased Mla10-specied disease resistance (108). In the nucleus, Mla10 showed an Avr10-dependent physical association with two WRKY transcription factors (HvWRKY1 and HvWRKY2 TFs), suggesting that these TFs serve as immediate downstream targets of the activated receptor. The effector-dependent association between barley Mla10 and WRKY TFs contributes to Mla10-mediated resistance and host cell death at attempted fungal infection sites (108). The WRKY TFs interacting with Mla10 presumably act as repressors of PTI and might have a role in keeping basal defense below a certain threshold.

FUTURE HUNTS FOR FUNGAL EFFECTORS BY COMPARATIVE GENOMICS


Genetic and biochemical approaches based on map-based cloning and analyses of fungal secretomes during infection have been the two most popular strategies to identify effector genes from the pathogens discussed above. Other, but less widely applied, methods for discovery of effector genes

include a functional high-throughput screening for identication of HR-inducing cDNAs from plant pathogens based on Agrobacterium tumefaciens/PVX-mediated cDNA expression in host plants (119), a mutagenesis approach using restriction enzyme-mediated integration (REMI) (84), and screening of EST libraries for genes upregulated during infection, as was successfully applied for AvrL567 of M. lini (27). However overall these approaches have been very labor intensive, and their success rate was relatively low. Recently, whole genome sequencing of fungal pathogens has provided an enormous amount of data that can be analyzed for putatively secreted cysteine-rich proteins. Narrowing down the spectrum of additional effector candidates can be achieved by integration of genome transcriptome, proteome, and metabolome data, when available. Comparative secretome analysis and BLAST sequence similarity searches could also be used for the identication of potential effectors in sequenced genomes, but these methods have limitations due to low sequence similarities among fungal effector genes. However, this approach proved successful with the recent identication of the rst homologs of the C. fulvum effector genes Avr4, Ecp2, and Ecp6 in the genome of the banana pathogen Mycosphaerella jiensis (117) and the discovery of a functional homolog of the toxic peptide ToxA from Stagonospora nodorum in Pyrenophora titici-repentis (41, 42). Nevertheless, despite the great potential of using genome-wide searches for identifying candidate effector genes, their function still needs to be conrmed experimentally by overexpression, gene disruption or silencing in fungal isolates, and subsequent (a)virulence assays on host plants.

FUNCTIONS AND HOST TARGETS OF EFFECTORS Extracellular and Cytoplasmic Fungal Effectors
Fungal effector proteins can be roughly grouped into extracellular effectors that are
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secreted into the apoplast or xylem of their host plants and cytoplasmic effectors that are translocated into host cells (Table 1). Ace1 from M. grisea is the only known fungal effector protein that is not secreted by the fungus, but instead is suggested to be involved in the biosynthesis of a yet unknown secondary metabolite that is likely secreted (11). Despite the low degree of sequence conservation among fungal effectors, most of them code for small secreted proteins, of which some are translocated into host cells by a yet unknown mechanism. The only two members of putatively cytoplasmic effectors which lack a signal sequence are Avra10 and Avrk1 of B. graminis f. sp. hordei (100). Extracellular effectors are often further N- and sometimes C-terminally processed by plant and/or fungal proteases, but evidence for protein maturation is in some cases based only on in-silico predictions. In that respect, experimental evidence for secretion and processing of fungal effectors during infection is available only for the Avr and Ecp effectors of C. fulvum (24), the Six effectors of F. oxysporum f. sp. lycopersici (55), and the Nip effectors of R. secalis (146). Avr-Pita from M. grisea is also predicted to be a secreted processed protein, but this assumption is based on the fact that the 176 aa mature protein, but not the intact 223 aa protein, is the active form that interacts directly with the cognate cytoplasmic Pi-ta R protein (59). A second common feature of extracellular effectors, as well as some of the effectors that are active inside host cells, is the presence of multiple cysteine residues (Table 1). The cysteine residues might be involved in disuldebridge formation that provides protein stability in the harsh protease-rich environment of the host apoplast. Indeed, disulde bonds between cysteine residues have been reported to be required for stability and activity of at least Avr4 and Avr9 of C. fulvum (132, 134). However, mutational analysis of cysteine residues present in the Ecps from the same fungus suggested that not all cysteine residues are involved in disulde bridges or are crucial for induction of HR on plants carrying the cognate Cf-Ecp proteins
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(80). Finally, effectors active inside the host cell like those of M. lini possibly need proper folding and disulde-bridge formation outside the host before being taken up.

Intrinsic Functions
As sequence homology between most fungal effectors and other proteins present in public databases is limited, assigning functions to most effectors based on putative orthology alone has been limited (Table 1). Exceptions are the AvrPita (a putative metalloprotease) (93) and Ace1 (a hybrid polyketide synthase/nonribosomal peptide synthetase) (11) effector proteins of M. grisea (130), as well as Ecp6 (LysM-domains) (13) of C. fulvum. Characterization of the threedimensional structure as well as the disulde bond pattern between cysteine residues in some cases provided additional clues with respect to the intrinsic functions of some fungal effectors. Indeed, the disulde bond pattern and cysteine spacing in Avr4 from C. fulvum revealed structural and functional homology with an invertebrate chitin-binding domain present in tachycitin (130, 131). A second effector protein with a known intrinsic function is Avr2 from C. fulvum that proved to be an inhibitor of the Rcr3, Pip1, aleurain, and TDI65 cysteine proteases (102, 107, 140), although this function was not inferred from homology with other known cysteine protease inhibitors. Avr2 and Avr4 are the only two fungal effectors for which both an intrinsic and virulence function has been shown experimentally. Based on the spacing between the cysteine residues, AvrP123 of M. lini shows structural homology to members of the Kazal family of serine protease inhibitors, whereas AvrP4 shows homology to cystine-knotted proteins, as does Avr9 of C. fulvum (17, 134). However, despite the structural resemblance of the latter effectors to other enzymes or proteins, a functional homology has never been established. Finally, the 3-D structure of AvrL567 from M. lini has been resolved and binding of this protein to DNA has been demonstrated, but because not all AvrL567 variants bind DNA with the same

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efciency, the biological signicance of the observed activity is not yet clear (144).

Role in Virulence
As with intrinsic functions, the contribution of individual fungal effectors to virulence has also proven difcult to establish. Functional analysis indicates that in some cases effector proteins constitute genuine virulence factors that are required for full virulence of the pathogens on their host plants (Table 1). Some effector genes are also reported to be clustered on pathogenicity islands, the presence of which is correlated with virulence (or avirulence) on specic hosts. Examples are the Six1 (Avr3), Six2, and Six3 genes of F. oxysporum f. sp. lycopersici that cluster in a genomic region that confers virulence on tomato plants (135), and the Pwl gene family of M. oryzae that is required for specicity on weeping lovegrass (70, 118). Also many effectors seem to work in concert, and their individual contribution to virulence is often minor, undetectable, or redundant, as their deletion has no apparent effect on tness or virulence (Table 1). This fact is also supported by studies on diversifying selection operating on a number of effector genes showing that mixed (sub)populations of some pathogens exist that consist of individuals that either carry or lack particular effector genes, indicating that the tness cost for the loss is not very high (105, 116). Some effectors might also provide overlapping activities as is possibly the case for the Avr4 and Ecp6 effectors of C. fulvum that both bind to chitin and thus could potentially protect the fungus against plant chitinases and/or suppress PTI by scavenging chitin fragments released from fungal cell walls in the apoplast during infection (13, 130). Finally, some effectors might play a more general role in virulence, as is, for example, the case for the Nip proteins of R. secalis (146, 147), whereas other effectors that lack a clear role in virulence seem to interfere with defense signaling pathways that are induced by other effectors/Avr factors with an important role in virulence. This seems to be the case for Avr1 (Six4) of F. oxysporum f. sp.

lycopersici, which is not a virulence factor but rather suppresses I-3-mediated resistance triggered by Avr3 (Six1), an effector that is required for full virulence of this pathogen (54).

Translocation of Fungal Effectors


Plant and animal pathogenic bacteria contain six secretory systems of which the type three secretion system (TTSS) is crucial for virulence as it is required for translocation of effectors into the host cell (1, 2, 36). Many TTSSinjected effectors suppress PTI, and for several of them the mechanism of suppression has been elucidated (1). Most oomycete secreted effectors contain an RXLR (10) motif that is required for their uptake into host cells, as has been shown for effectors of the Arabidopsis downy mildew pathogen Hyaloperonospora parasitica (97) and the potato pathogen Phytophthora infestans (148). Bioinformatics analysis has identied 425 genes potentially encoding secreted RXLR proteins in the P. infestans genome (60). As with bacteria and oomycete effectors, effectors of some fungal plant pathogens (e.g. rusts, powdery mildews, M. oryzae, and F. oxysporum f. sp. lycopersici) are putatively translocated into the host cell where they interact with cytoplasmic or nuclear R proteins (16, 27, 28, 34, 59, 108). However, so far in all fungal effectors no clear consensus signature has been identied that would point to a function in translocation and uptake into the plant cell. The host-selective protein toxin ToxA of P. tritici-repentis (19) is required for full virulence on wheat, and its solvent-exposed ArgGly-Asp (RGD) motif that interacts with the host plasma membrane is likely required for its internalization (85). A similar RGD motif mediating interaction with the plasma membrane is also present in the IpiO effector proteins of the oomycete P. infestans (106).

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EFFECTOR RECOGNITION BY R PROTEINS


To date, many R genes have been cloned from different plant species and the products of these
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genes show great diversity in their structural properties (24, 87, 120, 142). The vast majority of cytoplasmic R proteins consist of an NBS connected to a region of LRRs (NBS-LRRs), whereas the second major class of R genes encodes proteins with an extracellular LRR (eLRR) domain and a short transmembrane (TM) domain. The NBS-LRR class can be further subdivided into subclasses based on the structure of their N-terminal domains that contain either a coiled coil (CC) or Tol/interleukin1 receptor (TIR) motif (Figure 1). However, despite the fact that an increasing number of plant R proteins and their cognate effectors have been characterized, in most cases we still know little about the molecular mechanisms associated with effector perception by R proteins and subsequent R-mediated downstream plant defense responses (Figures 2 and 3). The simplest perception model proposed so far is based on a direct interaction between the cognate R protein and effector. This model is referred to as the receptor-ligand model, and indeed, a few examples of direct interactions between a fungal effector and plant R protein have been described. Yeast two-hybrid and in vitro binding assays demonstrate a physical interaction between Avr-Pita (Avr-Pita176 ) from M. oryzae and the cognate Pi-ta resistance protein from rice (59). Pi-ta codes for an intracellular NBS-LRR protein and mutational analysis showed that single aa substitutions in the LRR domain of this protein or in Avr-Pita176 can disrupt the physical interaction and abolish Pita-mediated defense responses (59). Physical interaction between effectors of M. lini and their cognate R proteins in ax has also been reported (28, 34, 144). The polymorphic AvrL567 locus of M. lini consists of at least three genes (AvrL567A, AvrL567B, and Avr567C) whose products are recognized by the cognate L5, L6, and L7 proteins of ax. Yeast two-hybrid assays demonstrated direct interaction between AvrL567 and the cognate L5/L6 proteins, whereas a mutation in the LRR domain of L abolishes the interaction, indicating binding of AvrL567 to the LRR domain.
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One implication of the receptor-ligand model is that given the enormous diversity of effector molecules, plants must carry numerous R proteins that would enable them to recognize all individual effectors and their allelic variants. However, most plants have developed R proteins that monitor modications in the plant targets of fungal effectors. In this so-called guard model, R proteins do not interact directly with an effector but guard its host target and respond to alterations in this target caused by the effector. Therefore, in contrast to the receptorligand model, which allows recognition of only a limited set of structurally related effectors, the guard model enables detection of multiple unrelated effectors that interact with the same host target guarded by a single R protein. A classical example of the guard model is represented by the indirect interaction between Avr2 and Cf-2, mediated by Rcr3 in the C. fulvumtomato interaction as discussed above (102). In many other pathosystems, indirect interaction between R protein and cognate Avr has been reported, suggesting that the majority of interactions would t into the guard model (61). This also seems to be the case for the Avr9 effector of C. fulvum and the tomato Cf-9 protein, whose interaction is mediated by a high afnity binding site (73, 81) (Figures 2 and 3). Whether direct or indirect interaction mediates perception of effector molecules by R proteins, both systems could complement each other. Indirect recognition enables plants to monitor multiple effectors by a relatively small number of R proteins, whereas redundancy in guardees allows monitoring of one target by different R proteins, as seems to be the case for Rlm4 and Rlm7, which both seem to guard the same virulence target of AvrLm4-7 (95). This strategy, however, is effective only toward effectors with plant targets but not against effectors with defensive fungal targets, such as Avr4 from C. fulvum, which binds to chitin present in the fungal cell wall but apparently does not interfere with the physiology of the host. Furthermore, direct interaction between effector and R protein can be overcome more readily by mutations in effectors that abolish recognition,

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RLPs

LysM-RK
Ve2: ? HcrVf: ? LeEix2: Eix
LysM

Cfs: Avrs Ecps

CERK1: Chitin

eLRR

eLRR

eLRR

LysM LysM

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TM

TM

TM

TM

ECS ER-r

PEST ECS

Cytosol
ECS
Kinase

NB-LRRs

TIR

NBS

LRR

L5, L6, L7: AvrL567 M: AvrM Pi-ta: Avr-Pita I-2: Six3

LZ/CC

NBS

LRR

Figure 1 Structure of different types of resistance proteins. (a) From left to right: RLPs: tomato Cfs directly or indirectly recognize Cladosporium fulvum Avr and Ecp proteins, respectively; tomato Ve2 and apple HcrVf provide resistance to Verticillium sp. and Venturia inaequalis, respectively, but their cognate effectors are not known yet; tomato LeEix2 mediates recognition of Trichoderma sp. Eix; Arabidopsis LysM-RK is required for fungal chitin elicitor defense signaling. Figure is not drawn to scale. (b) NB-LRR proteins: TIR-NB-LRRs: L5, L6, and L7 are cytoplasmic resistance proteins mediating recognition of Melampsora lini effectors AvrL567, whereas cytoplasmic resistance protein M mediates recognition of Melampsora lini effector AvrM; rice LZ-CC-NB-LRRs: rice Pi-ta mediates recognition of Magnaporthe grisea effector Avr-Pita; tomato I-2 mediates recognition of Fusarium oxysporum f. sp. lycopersici effecor Six3. Figure is not drawn to scale. Abbreviations: Avr, avirulence; AvrL567 and AvrM, avirulence proteins of Melampsora lini; CC, coiled-coil domain; CERK1, chitin elicitor receptor kinase 1; Cf, Cladosporium fulvum resistance protein; Ecp, extracellular protein; ECS, endocytosis signature (YXX : Tyr-X-X- ); EIX, ethylene inducing xylanase; eLRR, extracellular leucine-rich repeat proteins; ER-r, endoplasmic reticulum retrieval signature (KKRY: Lys-Lys-Arg-Tyr); HcrVf, homologue of the C. fulvum resistance genes of the Vf region; I-2, Fusarium oxysporum resistance protein-2; L5, L6, L7, and M, ax rusts resistance proteins; LeEix2, Lycopersicon esculentum Eix-responding protein LeEix2; LysM, Lysin motif; LysM-RK, LysM-receptor kinase; NB-LRR, nucleotide binding leucine-rich repeat; PEST, ProGluSerThr signature; Pi-ta, rice blast resistance protein; Six3, secreted in xylem 3; LZ/CC-NB-LRR, leucine zipper-NB-LRR; TIR-NB-LRR, tollinterleukin-1 receptor kinase-NB-LRR; TM, transmembrane; Ve2, Verticillium wilt resistance protein 2.
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Cf-2 Cf-4/Cf-9
Chitin Avr4 Avr4 C1 2 C2 C3 D E ? Avr9 HABs C2 C3 D E F ? B Avr2 Rcr3 C1 Avr2 B Cysteine proteases

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F VAP27

Cytosol

Hsp90 ?

ECS ER-r

G ACIK1 CITRX

Figure 2 Cf-4-, Cf-9-, and Cf-2-mediated perception of Avr4, Avr9, and Avr2, respectively. From left to right: Avr4 is a chitin-binding effector that is directly or indirectly recognized by RLP Cf-4, whereas Avr9 is a cystineknotted effector that is most likely indirectly recognized by the RLP Cf-9 through the tomato HABS; VAP27, Hsp90, ACIK1 and CITRX affect downstream defense signaling of both RLPs; Avr2 inhibits several tomato cysteine proteases including cysteine protease Rcr3 that, after binding to Avr2, triggers Cf-2-mediated defense signaling. Figure is not drawn to scale. Abbreviations: B, C1, C2, C3, D, E, F, and G, domains present RLPs Cf-4, Cf-9, and Cf-2; ACIK1, Avr9/Cf-9-induced kinase 1; Avr2, avirulence protein 2 (cysteine protease inhibitor); Avr4, avirulence protein 4 (chitin-binding protein); Avr9, avirulence protein 9 (cystine-knotted protein); Cf-2, Cf-4, and Cf-9, Cladosporium fulvum resistance proteins 2, 4, and 9; CITRX, Cf-9-interacting thioredoxin; HABS, high afnity binding site; Hsp90, heat shock protein 90; Rcr3, required for Cladosporium resistance (cysteine protease); VAP27, vesicle-associated protein 27.

whereas jettison of effectors seems to mediate evasion of R-mediated resistance in cases of indirect interactions (23, 61). The guard model is strongly dependent on guarded effector targets, and therefore, natural selection is expected to favor partners with improved interaction. Furthermore, the virulence function of effector proteins is based on modication of their host targets, and natural selection in the host would favor the development of targets that are less or no longer recognized by effectors. As a result of these two opposing forces acting on effector targets, it was recently proposed that plants have likely developed
252 Stergiopoulos

decoy molecules that mimic the effector targets. Such decoys would trap effector proteins, diverting them from their real virulence targets and alert monitoring R proteins. Decoys either could arise from a guardee duplication event or could evolve independently of the effector targets that they are mimicking. This variant of the original guard model is proposed as the decoy model, and a few cases in support of this model have been suggested to operate in bacterial and fungal pathosystems (137). One of the proposed cases involves the interaction between Avr2 and Rcr3, where it is suggested that Rcr3 is the decoy molecule that diverts Avr2 away

de Wit

Cf-4

Cf-9
Avr-induced K+ K+ K+

Avr4 Oxidative burst

ROS
Avr9 HABs

H+ Ca2+ ATPase K+ ATPase ATPase

PR PR

Syntaxin PLC DGK VAP27


Hsp90 ?

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CITRX

PIP2

DAG

PA NADPH oxidase

ACIK1

PR

CITRX

?
EDS1 IP3

Ca2+ H+ H+ Ca2+ 2+ H+ Ca

CC

MAPKKK MAPKK

P P

CDPKs

NRC1
P
Hsp90 RAR1 ?

MAPK1-3

?
TFs

Cytosol

SGT1

Salicyclic acid

Vacuole
PR

ACS PR

TFs

Defense responses

Cell wall reinforcement Phytoalexins PR proteins Hypersensitive response


PR

Nucleus
Ca2+ Ca2+ 2+ Ca Ethylene

Figure 3 Avr4-triggered Cf-4-mediated, and Avr9-triggered Cf-9-mediated defense signaling. Left: EDS1 and NRC1 are required for Cf-4- and Cf-4-mediated resistance. PLC is activated leading to IP3 and DAG. DAG is converted by DGK into PA, which stimulates NADPH-oxidase causing the accumulation of ROS. IP3 releases Ca2+ from the vacuole. SGT1, RAR1 and Hsp90 are in a complex with NRC1 or act just downstream of it. The Hsp90 complex interacts with the MAPK cascade eventually phosphorylating TFs and ACS causing accumulation of ethylene. TFs induce various defense responses including SA and ethylene accumulation, cell wall enforcement, phosphorylation of syntaxin, accumulation of phytoalexins and PR proteins, and the hypersensitive response. Right: Avr9 interacts via the HABS with the Cf-9 protein stimulating phosphorylation of CDPKs that, in turn, stimulate the production of ethylene through ACS. Ethylene inhibits the MAPK cascade. ACIK1 interacts with the C terminus of Cf-9 and CITRX with both Cf-9 and Cf-4 and are required for the Cf-mediated hypersensitive response and resistance. Avr9 and Avr4 also trigger Cf-mediated opening of Ca2+ , K+ and H+ channels through Ca2+ -ATPase, K+ -ATPase and H+ -ATPase, respectively. Figure is not drawn to scale. Abbreviations: ACS, 1-aminocyclopropane-1-carboxylic acid synthase; CDPKs, calcium-dependent protein kinases; DAG, diacylglycerol; DGK, diacylglycerol kinase; EDS1, enhanced disease susceptibility-1; IP3, inositol triphosphate; MAPK1-3, mitogen-activated protein kinase1-3; MAPKK, MAPK kinase; MAPKKK, MAPKK kinase; NADPH-oxidae, nicotinamide adenine dinucleotide phosphateH-oxidase; NRC1: NB-LRR protein required for HR-associated cell death-1; PA, phosphatidic acid; PLC, phospolipase C; PIP2, phosphatidyl inositol disphosphate; PR, pathogenesis-related; RAR1, required for Mla12 resistance-1; ROS, reactive oxygen species; SGT1, suppressor of G-two allele of Skp-1; TFs, transcription factors. This gure is a slightly revised version of gure 1C from Reference 24.
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from its real target, the plant protease Pip1, and activates Cf-2-mediated Avr2-triggered resistance (107, 137). However, this model still is very speculative and not yet supported experimentally.

EFFECTOR AND RESISTANCE PROTEIN EVOLUTION


Hosts and pathogens are locked up in a perpetual arms race with only temporary winners and losers. Fungal effectors and plant R proteins are in the front line of this arms race, and rapid rates of effector evolution and maintenance of high allelic diversity at corresponding R loci in plants have been reported in different pathosystems (3, 28, 83, 112, 115). The high rate of molecular evolution observed in effectors as a result of mutation, selection, and reproduction has caused extensive sequence diversication, gene expansion, and other genetic rearrangements in effector genes that might explain the absence of homology among effector proteins and their different host specicities. Many effector genes are embedded in highly dynamic genome areas, such as the chromosome ends or amid multiple transposable elements that trigger frequent genomic rearrangements, thus enabling higher genetic exibility in rapidly overcoming R-mediated resistance, as already discussed above. In many of these cases, gain of virulence due to transposon insertions, gene deletions, and other genetic rearrangements has been frequently observed (37, 44, 49, 50, 71, 93, 95, 100, 118). Although sequence alterations in effector genes that lead to evasion of R-mediated resistance can be the result of point mutations, frameshifts, gene deletions, and transposon insertions, the specic nature of the interplay between effector and R proteins can affect the type of genetic adaptation occurring in both proteins. Surveys for allelic variation in effector genes suggest that direct recognition by R proteins favors sequence diversication in effectors that disrupts physical association between the two, but without affecting the effectors presumed virulence function. Indeed, diversifying
254 Stergiopoulos

selection operating on the AvrL567 locus of M. lini generated protein variants that are no longer recognized by the cognate L protein in ax. Polymorphic aa residues in these sequence variants occurred on the proposed solventexposed surface of the effector protein that interacts with the LRR domain of the cognate R protein, indicating that selection favored variants that abolish recognition but still maintain a putative virulence function (144). In a similar way, almost all sequence variants described for the chitin-binding Avr4 effector protein of C. fulvum showed single aa substitutions that abolish recognition by the cognate Cf-4 protein but retain their ability to bind to chitin (62, 64, 116, 130). Recent analysis of Avr4expressing tomato lines has shown that Avr4 triggers hardly any transcriptional responses in the absence of the cognate Cf-4 protein, indicating that it is unlikely that Avr4 has targets in the host other than Cf-4 (139). In a similar way, sequence diversication of pathogen effectors is adaptively matched fairly quickly by diversication of the host R proteins that results in new recognition specicities (88). Good examples of a dynamic arms race between effectors and R proteins are provided by the M. lini Avrs and cognate ax R proteins (28) and the H. parasitica Atr13 and Rpp13 proteins of Arabidopsis (3, 103). In contrast to the emerging picture for direct recognition, indirect recognition of effectors by R proteins imposes selection against Avr effector function rather than structure, and such effectors are either present or absent in pathogen populations. Absence would imply a minimal tness penalty for the pathogen, but under strong selection pressure, the conditional benets of such a loss could be higher than the costs of the loss. Alternatively, the effector function could be redundant and compensated for by other effectors. Effector gene deletions have been reported as the main mechanism for gain of virulence for the fungal effectors genes Avr9 and Avr4E of C. fulvum (116, 141, 145), AvrLm1 and AvrLm4-7 of L. maculans (50, 95), Nip1 of R. secalis (105), and Avr-Pita and Avr1CO39 of M. grisea (37, 93, 150). Loss of the

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Avr2 gene product of C. fulvum is caused by insertions, deletions, and other frameshift mutations that result in truncated nonfunctional alleles. In contrast to gene deletions, such mutated alleles are preserved in the genome of C. fulvum and may become available for reversions once selection pressure has been lifted (116). Frameshift mutations in particular are often reverted by the occurrence of a second complementary frameshift that could restore the function of the protein (92). However, except for Avr2, which indirectly interacts with Cf-2, for most of the other effector gene deletions described above, it is currently unknown whether the products of these genes interact directly or indirectly with their cognate R proteins. In fact, Avr-Pita of M. grisea, which directly interacts with Pi-ta, shows various types of sequence alterations that can result in gain of virulence, including also gene deletions (59, 69, 93, 150). Point mutations contributing to virulence have also been observed for Nip1, although less frequently than gene deletions (105), as well as for Avr4E (116, 145) and AvrLm4-7 (95). From the examples presented above, it is clear that the type of genetic adaptation of effectors to overcome R-mediated recognition is not only determined by whether the interaction between an effector and an R protein is direct or indirect. However, theoretical modeling suggests that jettison of effector proteins can lead to long and stable resistance, whereas sequence diversication is likely to result rather quickly in new virulence phenotypes (138). In contrast to the receptor-ligand model, the guard model does not impose any selective pressure for sequence diversication on R genes but instead enhances diversifying selection on plant targets of effectors (89). In all the cases described so far, gain of virulence was correlated with sequence diversi-

cation or jettison of effector genes. However, a different scenario has been observed for the effector Avr3 of F. oxysporum f. sp. lycopersici that is required for full virulence, where suppression of ETI is correlated with the acquisition of an additional protein (Avr1) that suppresses I3- and I-2-mediated defense signaling pathways (54).

FUTURE CHALLENGES
Comparative genomics of fungal pathogens will be useful in identication of new effector proteins and possibly in prediction of their virulence functions. However, this approach is limited because of the low level of homology among fungal effectors. Some effectors reside on pathogenicity islands, and it will be interesting to nd out by comparative genomics how these islands have arisen during evolution. For most fungal effectors, a function in virulence can be investigated experimentally by gene disruption, gene knock-down, or overexpression assays, and it is expected that for number of them a role in suppressing PTI induced by fungal PAMPs will be shown. In addition to effectors, discovery of more fungal PAMPs in addition to chitin fragments is also anticipated in the near future, which would enrich our understanding of the plant defense system and the interplay between PTI and ETI. Elucidating the mechanisms of translocation of fungal effectors into host cells is also a major challenge for the future. A considerable amount of effort is currently being placed on identifying interactors of effector proteins by in situ microscopic techniques and uorescently labeled effectors. Finally, it is hoped that effectors will be identied that are crucial for virulence, as their cognate resistance genes are anticipated to be durable and could be used in resistance breeding.

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DISCLOSURE STATEMENT
The authors are not aware of any afliations, memberships, funding, or nancial holdings that might be perceived as affecting the objectivity of this review.

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ACKNOWLEDGMENTS
We kindly acknowledge Peter N Dodds, Wolfgang Knogge, Martijn Rep, Christopher J Ridout, Thierry Rouxel, and Bart PHJ Thomma for critical reading of the manuscript. Ioannis Stergiopoulos is nancially supported by an ERA-PG grant (ERA-PG 31855.00030) and Pierre JGM de Wit by the Royal Netherlands Academy of Arts and Sciences. This project was carried out within the research programme of the Centre of BioSystems Genomics (CBSG) which is part of the Netherlands Genomics Initiative/Netherlands Organization for Scientic Research. LITERATURE CITED
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Contents
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Volume 47, 2009

Look Before You Leap: Memoirs of a Cell Biological Plant Pathologist Mich` le C. Heath p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1 e Plant Disease Diagnostic Capabilities and Networks Sally A. Miller, Fen D. Beed, and Carrie Lapaire Harmon p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p15 Diversity, Pathogenicity, and Management of Verticillium Species Steven J. Klosterman, Zahi K. Atallah, Gary E. Vallad, and Krishna V. Subbarao p p p p p p p39 Bacterial/Fungal Interactions: From Pathogens to Mutualistic Endosymbionts Donald Y. Kobayashi and Jo Anne Crouch p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p63 Community Ecology of Fungal Pathogens Causing Wheat Head Blight Xiangming Xu and Paul Nicholson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p83 The Biology of Viroid-Host Interactions Biao Ding p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 105 Recent Evolution of Bacterial Pathogens: The Gall-Forming Pantoea agglomerans Case Isaac Barash and Shulamit Manulis-Sasson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 133 Fatty AcidDerived Signals in Plant Defense Aardra Kachroo and Pradeep Kachroo p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 153 Salicylic Acid, a Multifaceted Hormone to Combat Disease A. Corina Vlot, DMaris Amick Dempsey, and Daniel F. Klessig p p p p p p p p p p p p p p p p p p p p p p p p 177 RNAi and Functional Genomics in Plant Parasitic Nematodes M.N. Rosso, J.T. Jones, and P. Abad p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 207 Fungal Effector Proteins Ioannis Stergiopoulos and Pierre J.G.M. de Wit p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 233 Durability of Resistance in Tomato and Pepper to Xanthomonads Causing Bacterial Spot Robert E. Stall, Jeffrey B. Jones, and Gerald V. Minsavage p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 265
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Seed Pathology Progress in Academia and Industry Gary P. Munkvold p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 285 Migratory Plant Endoparasitic Nematodes: A Group Rich in Contrasts and Divergence Maurice Moens and Roland N. Perry p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 313 The Genomes of Root-Knot Nematodes David McK. Bird, Valerie M. Williamson, Pierre Abad, James McCarter, Etienne G.J. Danchin, Philippe Castagnone-Sereno, and Charles H. Opperman p p p p p 333
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Viruses of Plant Pathogenic Fungi Said A. Ghabrial and Nobuhiro Suzuki p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 353 Hordeivirus Replication, Movement, and Pathogenesis Andrew O. Jackson, Hyoun-Sub Lim, Jennifer Bragg, Uma Ganesan, and Mi Yeon Lee p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 385 Ustilago maydis as a Pathogen Thomas Brefort, Gunther Doehlemann, Artemio Mendoza-Mendoza, Stefanie Reissmann, Armin Djamei, and Regine Kahmann p p p p p p p p p p p p p p p p p p p p p p p p p p p p 423 Errata An online log of corrections to Annual Review of Phytopathology articles may be found at http://phyto.annualreviews.org/

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From the Annual Review of Ecology, Evolution, and Systematics, Volume 39 (2008) Sanctions, Cooperation, and the Stability of Plant-Rhizosphere Mutualisms E. Toby Kiers and R. Ford Denison The Performance of the Endangered Species Act Mark W. Schwartz Pandoras Box Contained Bait: The Global Problem of Introduced Earthworms Paul F. Hendrix, Mac A. Callaham, Jr., John Drake, Ching-Yu Huang, Sam W. James, Bruce A. Snyder, and Weixin Zhang From the Annual Review of Entomology, Volume 54 (2009) Adaptation and Invasiveness of Western Corn Rootworm: Intensifying Research on a Worsening Pest Michael E. Gray, Thomas W. Sappington, Nicholas J. Miller, Joachim Moeser, and Martin O. Bohn Impacts of Plant Symbiotic Fungi on Insect Herbivores: Mutualism in a Multitrophic Context Sue E. Hartley and Alan C. Gange Cellular and Molecular Aspects of Rhabdovirus Interactions with Insect and Plant Hosts El-Desouky Ammar, Chi-Wei Tsai, Anna E. Whiteld, Margaret G. Redinbaugh, and Saskia A. Hogenhout From the Annual Review of Genetics, Volume 42 (2008) How Saccharomyces Responds to Nutrients Shadia Zaman, Soyeon Im Lippman, Xin Zhao, and James R. Broach The Organization of the Bacterial Genome Eduardo P.C. Rocha Genomic Insights into Marine Microalgae Micaela S. Parker, Thomas Mock, and E. Virginia Armbrust From the Annual Review of Genomics and Human Genetics, Volume 9 (2008) Phylogenetic Inference Using Whole Genomes Bruce Rannala and Ziheng Yang From the Annual Review of Microbiology, Volume 62 (2008) Evolution of Intracellular Pathogens Arturo Casadevall Chlamydiae as Symbionts in Eukaryotes Matthias Horn
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From the Annual Review of Pharmacology and Toxicology, Volume 49 (2009) Lipid Mediators in Health and Disease: Enzymes and Receptors as Therapeutic Targets for the Regulation of Immunity and Inammation Takao Shimizu From the Annual Review of Plant Biology, Volume 60 (2009) DNA Transfer from Organelles to the Nucleus: The Idiosyncratic Genetics of Endosymbiosis Tatjana Kleine, Uwe G. Maier, and Dario Leister
Annu. Rev. Phytopathol. 2009.47:233-263. Downloaded from arjournals.annualreviews.org by Universita degli Studi di Padova on 10/30/09. For personal use only.

Jasmonate Passes Muster: A Receptor and Targets for the Defense Hormone John Browse A Renaissance of Elicitors Perception of Microbe-Associated Molecular Patterns and Danger Signals by Pattern-Recognition Recepts Thomas Boller and Georg Felix Biosynthesis of Plant Isoprenoids: Perspectives for Microbial Engineering James Kirby and Jay D. Keasling Roles of Plant Small RNAs in Biotic Stress Responses Virginia Ruiz-Ferrer and Olivier Voinnet

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