Fungal Effector Proteins Annurev - Phyto.112408

ANRV384-PY47-11 ARI 12 July 2009 7:25
ANNUAL
REVIEWS Further Fungal Effector Proteins
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Annu. Rev. Phytopathol. 2009. 47:233–63 Key Words

The Annual Review of Phytopathology is online at avirulence, cysteine-rich proteins, diversifying selection, guard model,
phyto.annualreviews.org
resistance, virulence
This article’s doi:
10.1146/annurev.phyto.112408.132637 Abstract
Copyright c 2009 by Annual Reviews. It is accepted that most fungal avirulence genes encode virulence factors
All rights reserved
that are called effectors. Most fungal effectors are secreted, cysteine-
0066-4286/09/0908/0233$20.00 rich 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 indi-
rectly recognized by cognate resistance proteins that reside either inside
the plant cell or on plasma membranes. Several secreted effectors func-
tion inside the host cell, but the uptake mechanism is not yet known.
Variation observed among fungal effectors shows two types of selec-
tion 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.
233
ANRV384-PY47-11 ARI 12 July 2009 7:25
INTRODUCTION pathogens respond by mutating or losing ef-

fectors, or by developing novel effectors that
The gene-for-gene hypothesis states that for
can avoid or suppress ETI, whereas plants de-
every dominant avirulence (Avr) gene in the
velop novel R proteins mediating recognition
pathogen there is a cognate resistance (R)
of novel effectors (23, 61).
gene in the host, and the interaction between
Many reviews on bacterial, fungal, and
the products of these genes leads to activa-
oomycete Avr genes and their cognate R genes
tion of host defense responses, such as the
in plants have appeared in recent years (8, 24,
hypersensitive response (HR) that arrests the
47, 67, 68). In this review, we will provide an
growth of biotrophic fungi (40). Many plant
update on Avr genes from extracellular fungi,
pathologists have been searching for molec-
including Cladosporium fulvum, Fusarium oxys-
ular and biochemical evidence of the gene-
porum f. sp. lycopersici, Leptosphaeria maculans,
for-gene concept. The molecular cloning of
Magnaporthe oryzae, and Rhynchosporium secalis,

the first bacterial Avr gene was reported in
and from obligate fungal pathogens that form

1984 (114), the first fungal Avr gene in 1991
haustoria, such as Melampsora lini and Blumeria
(141), and the first oomycete Avr gene fol-
graminis f. sp. hordei (Table 1). Their structure,
lowed in 2004 (67, 124). Over the past two
intrinsic functions, localization, perception by
decades, numerous novel Avr genes and cog-
R proteins, and evolution will be discussed.
nate R genes have been discovered, and our
molecular understanding of the gene-for-gene
relationship has increased considerably. It is EFFECTORS OF
now accepted that plants contain two lines of EXTRACELLULAR FUNGAL
defense. The first line provides basal defense PATHOGENS
against all potential pathogens and is based
on recognition of conserved microbial features Cladosporium fulvum
known as pathogen-associated molecular pat- Cladosporium fulvum (syn. Passalora fulva) is an
terns (PAMPs) by so-called PAMP-recognition asexual extracellular fungal pathogen of tomato
receptors (PRRs) that activate PAMP-triggered (25, 63, 123). To date, four avirulence (Avr)
immunity (PTI) and prevent further coloniza- genes have been cloned from C. fulvum that
tion of the host (23, 61, 91). One of the best- all encode small cysteine-rich proteins that are
known microbial PAMPs is chitin, a major secreted during infection. These are the Avr2,
structural component of fungal cell walls, for Avr4, Avr4E, and Avr9 effector proteins whose
which two LysM-type of receptor-like kinases recognition in tomato is mediated by the cog-
involved in its perception have been character- nate Cf (C. fulvum) R proteins Cf-2, Cf-4, Cf-
ized in rice and Arabidopsis, respectively (66, 90). 4E, and Cf-9, respectively (25, 63, 123). Four
Evidence is now accumulating that Avr genes additional extracellular proteins (Ecps), namely
encode effectors that suppress PTI, thus en- Ecp1, Ecp2, Ecp4, and Ecp5 have been char-
abling a pathogen to infect its host plant and acterized from C. fulvum that invoke an HR in
cause disease. Once the basal defense system tomato lines that carry a cognate Cf-Ecp gene
of plants is overcome by pathogens, plants re- (22, 75), whereas Ecp6 and Ecp7 have also re-
spond with the development of a more special- cently been identified, but for these two effec-
ized recognition system based on effector per- tors no responding tomato lines have yet been
ception by R proteins and subsequent activation reported (13). Although demonstrated in only
of effector-triggered immunity (ETI) that leads some cases, all Avrs and Ecps are assumed to be
to rapid and acute defense responses in plants, virulence factors (13, 123, 139, 140).
the hallmark of which is the HR. This trig-
gers a second wave of coevolutionary arms race Avr2 effector. Avr2 encodes a preprotein of
between pathogens and plants, during which 78 amino acids (aa), which matures into a 58 aa
234 Stergiopoulos · de Wit

ANRV384-PY47-11 ARI 12 July 2009 7:25
protein with eight cysteine residues that induces structural similarity to proteins with an inverte-
HR in tomato plants that carry the cognate brate chitin-binding domain, such as tachycitin
Cf-2 resistance gene (26, 82). During infec- (inv ChBD) (110). Indeed, binding of Avr4 to
tion, Avr2 inhibits at least four tomato cysteine chitin has been experimentally confirmed, and
proteases including Rcr3, Pip1, aleurain, and it is further shown that Avr4 can protect chiti-
TDI65, and as such, plays an offensive role in nous fungi, such as Trichoderma viride and Fusar-
virulence by targeting and inhibiting host pro- ium solani, against basic plant chitinases. There-
teases that are important for host defense (74, fore, a role for Avr4 in protection of C. fulvum
102, 107). Indeed, the role of Avr2 in viru- against chitinases during infection has been
lence is demonstrated not only for C. fulvum proposed (130–132). The virulence function of
but also for other fungal tomato pathogens, in- this protein is further supported by the fact that
cluding Botrytis cinerea and Verticillium dahliae, Avr4 is expressed only during infection of the
as heterologous expression of Avr2 in Arabidop- host, when the fungus is exposed to chitinases,
sis thaliana enhances susceptibility toward these and that silencing of Avr4 in C. fulvum sig-
pathogens (140). In the presence of Cf-2, Avr2 nificantly reduces virulence (139). In addition,
behaves as an avirulence factor, and its recogni- tomato plants expressing Avr4 are more sus-
tion is mediated by Rcr3pimp (required for C. ful- ceptible to C. fulvum and other chitinous fun-
vum resistance), a cysteine protease originating gal tomato pathogens (139). In the presence of
from Lycopersicon pimpinellifolium (74, 102). De- Cf-4, Avr4 induces HR, but natural isoforms of
spite the fact that the exact mechanism of Avr2 this effector protein occur that no longer trigger
perception is not yet known, structural modi- Cf-4-mediated HR. Such isoforms show an ex-
fication of Rcr3 by Avr2, rather than Rcr3 in- cess of nonsynonymous substitutions compared
hibition, is the most likely cause of triggering with synonymous ones, suggesting that Avr4
Cf-2-mediated defense signaling, as a natural is under positive diversifying selection (116),
variant of Rcr3 occurs in Lycopersicon esculen- whereas the majority of the natural variation
tum (Rcr3esc ) that causes spontaneous HR in the observed in Avr4 involves point mutations that
presence of Cf-2 in an Avr2-independent man- mostly cause substitutions of cysteine residues.
ner. The Rcr3esc protein still shows protease ac- The resulting unstable Avr4 variants are more
tivity and is likely to have a modified tertiary sensitive to proteases but are still able to bind
structure as compared with the Rcr3pimp pro- chitin, thus preserving the virulence function
tein (74). Circumvention of Avr2-triggered Cf- of the protein but preventing accumulation of
2-mediated HR can be achieved by point muta- Avr4 in the apoplast and triggering of HR in
tions, deletions, or transposon insertions in the Cf-4 plants (64, 132).
Avr2 gene (82). A recent survey of polymor-
phisms present in the Avr2 alleles of a world- Avr4E effector. Avr4E codes for a cysteine-
wide collection of C. fulvum isolates revealed an rich 101 aa protein that is secreted during in-
excess of nonsynonymous polymorphisms com- fection and triggers Cf-4E-mediated HR (145).
pared with synonymous ones, suggesting pos- Various strains of C. fulvum have been identi-
itive diversifying selection. These were mainly fied that evade Cf-4E-mediated resistance that
insertions or deletions in the coding region of all show two identical point mutations in Avr4E
the gene that lead to the production of trun- that translate into stable Avr4E proteins with
cated Avr2 proteins (116). two aa substitutions (Avr4ELT ). Targeted mu-
tagenesis showed that one of these mutations
Avr4 effector. Avr4 encodes a 135 aa prepro- in Avr4E, namely the Phe62>Leu substitution,
tein that is C- and N-terminally processed after is sufficient for evasion of Avr4E-triggered Cf-
secretion in the apoplast into an 86 aa mature 4E-mediated HR (145). Analysis of natural pop-
protein with eight cysteine residues (62, 64). ulations of the fungus also shows that a signif-
Based on its disulfide-bond pattern, Avr4 shows icant number of strains evade Cf-4E-mediated
www.annualreviews.org • Fungal Effectors 235

Table 1 Fungal effector proteins

ANRV384-PY47-11
Aa
ARI
Effector residues Function/ Localization Role in R-gene Positive

protein (mature) Cysteines SignalP homology in plant Expression virulence (type) selection References
Cladosporium fulvum (tomato):
236
Avr2 78 (58) 8 20 Protease Apoplast In planta Inhibits Rcr3 Cf-2 Yes (26)
inhibitor and other (eLRR-TM)
12 July 2009
proteases
Avr4 135 (86) 8 18 Chitin-binding Apoplast In planta Protects Cf-4 Yes (62)
Stergiopoulos
7:25
·
against (eLRR-TM)
chitinases
de Wit
Avr4E 121 (101) 6 10 Unknown Apoplast In planta Unknown Hcr9-4E Yes (145)
(eLRR-TM)
Avr9 63 (28) 6 23 Carboxypeptidase Apoplast In planta Unknown Cf-9 Yes (129)
inhibitor (eLRR-TM)
Ecp1 96 (65) 8 23 Tumor-necrosis Apoplast In planta Disruption Cf-Ecp1 No (129)
factor receptor leads to Not cloned
reduced
virulence
Ecp2 165 (143) 4 22 Unknown Apoplast In planta Disruption Cf-Ecp2 No (129)
leads to Not cloned
reduced
virulence
Ecp4 119 (101) 6 18 Unknown Apoplast In planta Unknown Cf-Ecp4 No (75)
Not cloned
Ecp5 115 (98) 6 17 Unknown Apoplast In planta Unknown Cf-Ecp5 No (75)
Not cloned
Ecp6 222 (199) 8 23 LysM-domains; Apoplast In planta Knock-down Unknown No (13)
chitin-binding leads to
reduced
virulence
Ecp7 ? (100) 6 ? Unknown Apoplast In planta Unknown Unknown Unknown (13)
Leptosphaeria maculans (oilseed rape):
AvrLm1 205 (183) 1 22 Unknown Probably in In planta and Unknown Rlm1 Yes (49)
cytoplasm in vitro Not cloned
AvrLm6 144 (124) 6 20 Unknown Probably in In planta and Unknown Rlm6 Unknown (44)
apoplast in vitro Not cloned
AvrLm4- 143 (122) 8 21 Unknown Probably in In planta Unknown Rlm4 and/or Yes (95)
ANRV384-PY47-11
7 apoplast (mainly) Rlm7

and in vitro Not cloned
ARI
Fusarium oxysporum f. sp. lycopersici (tomato):

Avr3 284 (189) 8 21 Unknown Xylem Stimulated Required for I-3 Unknown (99; M. Rep
(Six1) by living full virulence Not cloned pers.
cells comm.)
Six2 232 (172) 8 20 Unknown Xylem In planta Probably not Unknown Unknown (55; M. Rep
12 July 2009
required for pers.

virulence comm.)
7:25
Six3 163 (144) 3 (2) 19 Unknown Xylem In planta Required for I-2 (CC- Unknown (55; M. Rep
full virulence NBS-LRR) pers.
comm.)
Avr1 242 (184) 6 17 Unknown Xylem In planta Suppression of I or I-1 Unknown (55; M. Rep
(Six4) I-2 and I-3 Not cloned pers.
resistance comm.)
Magnaporthe oryzae (rice):
Avr-Pita 224 (176) 8 16 Homology to Cytoplasm In planta Not required Pi-ta (CC- Yes (93)
Metallopro- for virulence NBS-LRR)
teases on rice
Avr- 224 (?) 8 16 Homology to Probably in Unknown Probably not Pi-ta (CC- Yes (71)
Pita2 Metallopro- apoplast required for NBS-LRR)
teases virulence on
rice
Avr- 226 (?) 8 16 Homology to Probably in Unknown Probably not Unknown Yes (71)
Pita3 Metallopro- cytoplasm required for
teases virulence on
rice
Pwl1 147 (124) 2 23 Glycine-rich Probably in Unknown Unknown Unknown Unknown (70)
hydrophilic apoplast
protein
www.annualreviews.org • Fungal Effectors

Pwl2 145 (126) 1(1) 21 Glycine-rich Probably in Unknown Unknown Unknown Unknown (118)
237
hydrophilic apoplast
protein
Pwl3 137 (116) 0 21 Glycine-rich Probably in Unknown Non- Unknown Unknown (70)
hydrophilic apoplast functional
protein
(Continued )
ANRV384-PY47-11
ARI
238
12 July 2009
Table 1 (Continued )
Stergiopoulos
Aa
7:25
·
Effector residues Function/ Localization Role in R-gene Positive
protein (mature) Cysteines SignalP homology in plant Expression virulence (type) selection References
de Wit
Pwl4 138 (117) 0 21 Glycine-rich Probably in Unknown Non- Unknown Unknown (70)
hydrophilic apoplast functional
protein
Ace1 4035 43 - Hybrid Not secreted Expressed in Unknown Pi33 Unknown (11)
polyketide appressoria
synthase/
nonribosomal
peptide
synthetase
Avr1- Not - - Unknown Unknown Unknown Unknown Pi-CO39(t) Yes (37)
CO39 cloned
yet
Rhynchosporium secalis (barley):
Nip1 82 (60) 10 22 Non-specific Probably in Expressed in Not required Rrs-1 Not Yes (101)
toxin/induces apoplast vitro for virulence cloned
necrosis and
plasma-
membrane
H+ ATPase
Nip2 109 (?) 7 (6) 16 Non-specific Probably in Expressed in Not required Unknown Yes (101; W.
toxin/induces apoplast vitro for full Knogge
necrosis virulence pers.
comm.)
ANRV384-PY47-11
Nip3 115 (?) 9 (8) 17 Non-specific Probably in Expressed in Not required Unknown Yes (101; W.
ARI
toxin/induces apoplast vitro for full Knogge

necrosis virulence pers.
comm.)
Melampsora lini (flax):
12 July 2009
AvrL567 150 (127) 1 23 Unknown Cytoplasm Expressed in Unknown L5, L6 and L7 Yes (27)
(A, B haustoria (TIR-NBS-
and C) LRR)
7:25
AvrM 314 1 28 Unknown Cytoplasm Expressed in Unknown M (TIR- Yes (17)

haustoria NBS-LRR)
and in vitro
AvrP123 117 (94) 11 (10) 23 Kazal Ser Cytoplasm Expressed in Unknown P, P1, P2 Yes (17)
protease haustoria and/or P3
inhibitor (TIR-NBS-
LRR)
AvrP4 95 (67) 7 (6) 28 Cystine knotted Cytoplasm Expressed in Unknown P4 (TIR- Yes (17)
peptide haustoria NBS-LRR)
Blumeria graminis f. sp. hordei (barley):
Avra10 286 4 - >30 paralogues Probably in Unknown Unknown Mla10 (CC- Unknown (100)
in Bgh and cytoplasm NBS-LRR)
other f. sp.
Avrk1 177 3 - >30 paralogues Probably in Unknown Unknown Mlk1 (CC- Unknown (100)
in Bgh and cytoplasm NBS-LRR)
other f. sp.
www.annualreviews.org • Fungal Effectors

239
ANRV384-PY47-11 ARI 12 July 2009 7:25
HR by jettison of the Avr4E gene, indicating a major, but not an exclusive, positive regu-
that the fitness penalty associated with the loss lator of Avr9 expression (96). Expression of
of this gene is probably not very high (116). all the other Avr and Ecp effector genes of
However, Avr4E-expressing tomato transfor- C. fulvum that have been characterized so far
mants are more susceptible to natural C. fulvum is not induced under nitrogen-limiting condi-
strains that lack Avr4E than control plants, sug- tions, and although they are all expressed in
gesting that Avr4E is a virulence factor (H.P. van planta, their regulators have yet to be identified
Esse and B.P.H.J. Thomma, personal commu- (12, 122).
nication). No homologs of Avr4E are present
in public databases and its molecular function Ecp effectors. In addition to the Avrs, six more
is still unknown (145). effector genes that code for Ecps have been
cloned from C. fulvum, namely Ecp1, Ecp2, Ecp4,
Avr9 effector. Avr9 encodes a 63 aa prepro- Ecp5, Ecp6, and Ecp7 (13, 75, 129). Ecps are
tein that is C- and N-terminally processed by abundantly secreted by all strains of C. fulvum
fungal and plant proteases into a mature 28 aa during infection and possess an even number of
protein with six cysteine residues (129, 141). cysteine residues that are most likely involved
The 3-D structure of Avr9 resembles cystine- in intramolecular disulfide bridges (80). Most
knotted peptides (134, 143), which share struc- Ecps share little or no homology with other
tural, but little functional, homology (94). Ala- proteins present in public databases, except for
nine scanning of the Avr9 peptide revealed that Ecp6 which, so far, is the only effector protein
all six cysteine residues present in this pro- of C. fulvum with orthologs in many other fun-
tein are essential for its structure and necrosis- gal species. This is mainly due to the presence
inducing activity (73). Based on its overall of LysM-domains in this protein, which in gen-
tertiary structure, Avr9 shows structural but eral are implicated in carbohydrate binding in-
not functional homology to carboxypeptidase cluding chitin, suggesting that Ecp6 might be a
inhibitors (133, 134). Disruption of Avr9 in functional homolog of Avr4 (13). Alternatively,
C. fulvum by homologous recombination did Ecp6 might also be involved in scavenging of
not affect growth in vitro or virulence on chitin fragments that are released from the fun-
tomato plants, suggesting that Avr9 is not re- gal cell wall during infection by plant chitinases,
quired for full virulence (86). Indeed, all nat- thus preventing them from acting as PAMPs
ural strains that evade Cf-9-mediated HR lack and eliciting basal defense responses (13). Ecp6,
the Avr9 gene (116). However, Avr9-expressing Ecp1, and Ecp2 are all virulence genes because
tomato transformants are more susceptible to silencing or disruption of these genes compro-
natural C. fulvum strains that lack Avr9 than mises virulence of C. fulvum on tomato (13, 77).
control plants, suggesting that Avr9 may be For Ecp1, Ecp2, Ecp4, and Ecp5, tomato acces-
a redundant virulence factor (H.P. van Esse sions have been identified that carry a single
and B.P.H.J. Thomma, personal communica- dominant Cf-Ecp gene and develop an HR upon
tion). Expression of Avr9 in vitro is induced inoculation with recombinant potato virus X
under nitrogen-limiting conditions, suggesting (PVX) strains that express these Ecps, or af-
that the produced protein might be involved ter injection of the purified Ecp proteins (75,
in nitrogen metabolism (96, 122, 128). Fur- 76). However, although most Cf-Ecp genes have
thermore, an Nrf1 (nitrogen responsive factor) been genetically mapped, they still remain to be
gene has been identified in C. fulvum, and Nrf1 cloned (22, 113). In contrast to Cf-genes, Cf-Ecp
deletion mutants no longer express Avr9 under genes have not yet been deployed in commer-
nitrogen-limiting conditions and are compro- cial tomato lines, which might explain why only
mised in their virulence on Cf-0 tomato plants. a limited number of polymorphisms has been
However, these strains are still avirulent on observed in Ecp genes of natural populations of
Cf-9 tomato plants, suggesting that Nrf1 is C. fulvum (13, 116).

ANRV384-PY47-11 ARI 12 July 2009 7:25
Fusarium oxysporum f. sp. lycopersici well as Six2 could not be found outside the f. sp.
lycopersici lineage nor in nonpathogenic Fusar-
The vascular pathogen Fusarium oxysporum is
ium strains, suggesting that it represents a dis-
an asexual fungus with a broad host range and
pensable region that confers virulence specif-
causes wilt and root diseases. However, most
ically on tomato (135). In addition, the f. sp.
isolates of F. oxysporum can infect only a single
lycopersici lineage-specific effector gene Six3
or a few plant species and are therefore sub-
also resides on the same chromosome as Avr3
divided into different formae speciales (f. sp.)
and Six2, although not within the boundaries of
based on their host specificity (48). Thus, strains
the 8 kb region that harbors the latter two genes.
of F. oxysporum that cause tomato wilt are re-
This suggests that the ability to cause disease
ferred to as f. sp. lycopersici.
has likely arisen only once during evolution of
Fol by acquisition or emergence of the genomic
Six effectors. F. oxysporum f. sp. lycopersici region harboring the virulence genes neces-
(Fol) is an extracellular pathogen that colo- sary for infection of tomato. Subsequently, this
nizes the xylem vessels of tomato. Four small region might have spread to other clonal Fol
proteins, designated Six1 to Six4 (secreted in strains by horizontal gene transfer (135). Be-
xylem), have been identified in Fol and are cause the presence of Avr3 is required for full
produced during infection. Six1 (currently re- virulence on tomato, it raises the question how
named Avr3) is a small cysteine-rich protein Fol strains can overcome I-3-mediated resis-
of approximately 32 kDa that is N- and C- tance, as loss of Avr3 would pose a serious fit-
terminally processed by either fungal and/or ness penalty and mutants with only point mu-
plant proteases into a 12 kDa mature protein tations in Avr3 that avoid recognition by I-3
or an alternative form of 22 kDa, and which is but still remain virulent have not been reported.
also proven to be the active form of the pro- The recent functional characterization of Avr1
tein (98, 99). Avr3 is required for full virulence (previously known as Six4) might provide the
on tomato, but it also behaves as an Avr factor answer to this question. Avr1 is a small cysteine-
that triggers HR in the presence of the cog- rich secreted protein that confers avirulence to
nate I-3 resistance gene (56, 99). Interestingly, Fol strains on tomato lines carrying the I or I-1
expression of Avr3 has been detected only dur- resistance gene. However, unlike Avr3, Avr1 is
ing root colonization and infection of tomato not required for full virulence of Fol strains on
plants, but it is neither cultivar-specific nor de- tomato plants that lack the cognate R gene. In
pends on morphological features of the roots. contrast, Avr1 functions as a suppressor of I-
Instead, the presence of living plant tissue in 2- and I-3-mediated resistance (54). Indeed, al-
the vicinity of the fungus seems not only re- though Six3, which is required for full virulence
quired for Avr3 expression but might also trig- (M. Rep, personal communication), is present
ger a switch from a saprophytic to pathogenic in strains of Fol that are both virulent and avir-
lifestyle (136). In the genome of Fol, Avr3 re- ulent on I-3 tomato lines, Avr1 is only present
sides in an area of a small chromosome that in Fol strains virulent on I-3 lines. When strains
contains many transposable elements and thus of Fol that are avirulent on I-2 and/or I-3 lines
resembles pathogenicity islands. Pathogenic- were transformed with Avr1 they gained viru-
ity islands that harbor several virulence genes lence on these lines, indicating that Avr1 sup-
often reside on dispensable chromosomes and presses both I-2- and I-3-mediated resistance.
their existence has been described for other fun- The underlying molecular mechanism of sup-
gal pathogens as well, including Alternaria spp. pression is not yet known, but an evolutionary
(121), Nectria hematococca (79), L. maculans, B. model for this phenomenon has been proposed
graminis f. sp. hordei, M. oryzae, and possibly (54). In this model, it is suggested that the I-3
others (5, 70, 71, 111). Indeed, a large genomic resistance gene has evolved to recognize Avr3
area of approximately 8 kb that contains Avr3 as but because Avr3 is required for full virulence

ANRV384-PY47-11 ARI 12 July 2009 7:25
of Fol, evasion of I-3 recognition through loss other fungal Avr genes cloned so far. Both
of Avr3 would cause a serious fitness penalty. AvrLm1 and AvrLm6 are single copy genes, en-
Therefore, Avr1 might have been acquired by coding small secreted proteins of 205 aa and
Fol in order to avoid the fitness penalty as- 144 aa, respectively, that have no known ho-
sociated with the loss of Avr3 (and likely of mologs in public databases and lack any charac-
Six2) in overcoming I-2- and I-3-mediated re- teristic signatures that could point toward a pu-
sistance. This explains why all Fol strains an- tative intrinsic function. AvrLm1 and AvrLm6
alyzed so far contain Avr3, whereas Avr1 is are strongly induced in planta, particularly dur-
present only in strains that are virulent on I-2 ing the early stages of infection, and expres-
and/or I-3 lines. It is likely that the I-3 protein sion of the genes has also been observed in
operates in accordance with the guard model, vitro, although here expression of AvrLm1 is
where not the Avr3 protein, but the manipula- much higher than that of AvrLm6. Despite the
tion of its virulence target is recognized by I-3, clear avirulence properties of the AvrLm1 and
whereas Fol strains can (partially) regain viru- AvrLm6 genes, the cognate R genes Rlm1 and
lence toward I-3-containing lines by acquisition Rlm6 have not been cloned yet. Therefore, it
of Avr1. During evolution, tomato responded is currently unknown whether these effectors
to this adaptation with the development of the interact directly or indirectly with their cog-
I or the unlinked I-1 resistance gene that specif- nate R proteins and whether recognition oc-
ically recognizes and responds to Avr1 (54). curs outside or inside the host cells. In that re-
spect, AvrLm6 might be secreted and reside in
the apoplast as it contains six cysteine residues
Leptosphaeria maculans that could provide stability by forming disul-
At least nine distinct Avr genes, designated fide bridges. In contrast, AvrLm1 contains only
AvrLm1 to AvrLm9, have been genetically one cysteine residue, making it different from
identified in L. maculans, the causal agent of AvrLm6 and the apoplastic cysteine-rich effec-
stem canker on oilseed rape, that cause avir- tor proteins of C. fulvum and F. oxysporum f. sp.
ulence on plants carrying the cognate Rlm1 to lycopersici discussed above, and therefore, uptake
Rlm9 R genes, respectively. These Avr genes of AvrLm1 into the host cell seems more likely
have been mapped to at least four unlinked (49).
genomic regions, including the genetic clus- As has been observed for other fungi,
ters AvrLm1-AvrLm2-AvrLm6 and AvrLm3- AvrLm1 is absent in races that are virulent on
AvrLm4-AvrLm7-AvrLm9 (5). Rlm1 cultivars. Recently, a gain of virulence on
Rlm1 plants was observed in field populations
AvrLm1 and AvrLm6 effectors. AvrLm1 and of L. maculans in France that was due to a spe-
AvrLm6 are the first Avr genes that have been cific 260 kb deletion of a chromosomal segment
cloned from L. maculans using a map-based spanning AvrLm1. A similar deletion was ob-
cloning strategy (44, 49). The two Avrs are in served in more than 90% of 460 field isolates
relatively close proximity at a locus that also analyzed from a world population, indicating
harbors AvrLm2 (5). More specifically, AvrLm1 that jettison of the AvrLm1 is the main mech-
has been mapped on a 269 kb AT rich, gene- anism of adaptation to Rlm1 without causing a
poor heterochromatin-like region that consists significant fitness penalty for the fungus (6, 7,
of a number of degenerated, nested copies of 104).
four long-terminal repeat (LTR) retrotrans-
posons and is surrounded by GC-rich isochors. AvrLm4-7 effector. Using a map-based
Also, AvrLm6 maps within a 133 kb noncoding cloning strategy, a genetic locus of 238 kb
region that mainly contains LTR retrotrans- containing the AvrLm7 gene has been recently
posons. However, both AvrLm1 and AvrLm6 delineated in L. maculans that shows the
share a low GC content, in contrast to most same characteristics as the AvrLm1-2-6 locus,

ANRV384-PY47-11 ARI 12 July 2009 7:25
including the presence of multiple LTR retro- characterized from M. oryzae, including Avr-
transposons and an AT-rich isochoric region Pita (93) that was originally called Avr2-YAMO
next to a GC-equilibrated one (95). In total, 40 (125), Avr1-CO39 (38), Pwl2 (118, 125), Pwl1
genes were predicted on this locus, 35 of which (70), and Ace1 (11). Avr1-CO39 was isolated
are present in the GC-equilibrated region from an M. oryzae isolate pathogenic on weep-
and only five in the AT-rich isochors. A single ing lovegrass and controls avirulence on rice
gene (AvrLm7) was identified in the gene-poor plants carrying the cognate Pi-CO39(t) resis-
60 kb AT-rich region that confers avirulence tance gene (18, 38). Almost all M. oryzae isolates
on both Rlm7 and Rlm4 genotypes. This gene that are virulent on CO39-rice cultivars lack the
was consequently renamed AvrLm4-7 for the Avr1-CO39 gene (37). Avr-Pita and Ace1 were
dual specificity of its encoded protein toward cloned from M. oryzae isolates pathogenic on
the Rlm4 and Rlm7 genes. AvrLm4-7 encodes a rice, whereas Pwl2 was originally identified in
putatively secreted preprotein of 143 aa that is a genetic cross between two laboratory strains
further processed into a 122 aa mature protein that infected rice. However, unlike the other
with eight cysteine residues and no homology two genes, Pwl2 is a species-specific gene that
to any other proteins currently present in confers avirulence on weeping lovegrass but not
public databases. Expression of AvrLm4-7 is on any known rice cultivars (118).
considerably upregulated during primary leaf
infection, reaching a maximum at seven days Avr-Pita effector. Avr-Pita encodes a pre-
post inoculation, whereas only low levels of sumably secreted preprotein of 223 aa with ho-
expression are observed during in vitro growth mology to fungal zinc-dependent metallopro-
of the fungus. Analysis of 300 field isolates of teases. This protein is further processed into an
L. maculans showed that complete or partial active 176 aa mature protein (Avr-Pita176 ) that
deletion of the AvrLm4-7 gene is the main is dispensable for virulence on rice (59, 93). Avr-
mechanism for gaining virulence on both Rlm4 Pita176 interacts directly with the cognate Pi-ta
and Rlm7 genotypes, whereas most isolates resistance protein, a predicted 928 aa receptor-
virulent on Rlm4 genotypes alone showed like protein with a central nucleotide-binding
only a single point mutation in the produced site (NBS) and a C-terminal leucine-rich repeat
AvrLm4-7 protein (Gly120>Arg). Such strains (LRR) domain. Direct interaction between the
were less fit than strains with the wild-type two proteins has been demonstrated by yeast
AvrLm4-7 allele, indicating that AvrLm4-7 is two-hybrid assays and by in vitro binding as-
important for fitness of the fungus. No strains says that showed binding of the Avr-Pita176 to
virulent only on Rlm7 plants were observed the LRR domain of Pi-ta (59). Furthermore,
(95). coexpression of the two proteins in rice cells
elicited plant defense responses, suggesting that
physical interaction inside host cells is required
Magnaporthe oryzae for activation of Pi-ta-mediated defense. In the
Magnaporthe oryzae (formerly known as M. same experiments, it was also shown that the
grisea) (21) is the causal agent of rice blast. Re- recognition specificity for Avr-Pita is deter-
sistant rice lines have been used extensively over mined by a difference in one aa residue (Ala-
the past decades to battle the disease, but R 918) of the Pi-ta protein present in resistant vs
genes present in these lines have often been susceptible rice varieties. On the other hand,
overcome rather quickly by the emergence of many mutations have been described in Avr-
virulent races. The rice blast pathosystem com- Pita that result in gain of virulence on Pi-ta
plies with the gene-for-gene model, and to date rice cultivars. In the genome of M. oryzae, Avr-
more than 40 major R genes, designated Pi, Pita resides close to the telomere of chromo-
have been identified. So far, five cultivar- and some 3, and this might account for the observed
species-specific Avr genes have been cloned and molecular instability of the genomic region

ANRV384-PY47-11 ARI 12 July 2009 7:25
containing this gene. Indeed, an array of mu- generally found in rice pathogens. At least four
tations has been described on the Avr-Pita lo- members of this family, designated Pwl1 to
cus in virulent strains of the fungus, including Pwl4 are present in M. oryzae, and act as Avr
various size deletions, point mutations, and a genes conferring species-specific avirulence
transposon insertion (15, 58, 69, 93, 150). on weeping lovegrass and finger millet but
have no effect on rice (70, 118). Pwl2 encodes
Avr-Pita-related effectors. Recently, it was a secreted glycine-rich, hydrophilic protein
shown that Avr-Pita (currently renamed to Avr- of 145 aa that confers avirulence on weeping
Pita1) belongs to a gene family with at least two lovegrass (118). This gene is located on a
additional members, Avr-Pita2 and Avr-Pita3 highly unstable genetic locus, where frequent
(71). Avr-Pita2 acts as elicitor of defense re- genetic rearrangements associated with large
sponses mediated by Pi-ta, but Avr-Pita3 does deletions lead to the emergence of spontaneous
not. Members of the Avr-Pita family are widely mutants virulent on weeping lovegrass. Pwl1
distributed among strains of M. grisea isolated (75% aa identity with Pwl2) and the allelic
from diverse hosts, including isolates that are Pwl3 (51% aa identity with Pwl2) and Pwl4
not pathogenic on rice. However, although (57% aa identity to Pwl2) were identified
Avr-Pita1 and Avr-Pita2 are present in both based on homology to Pwl2, but only Pwl1
M. oryzae and M. grisea isolates, Avr-Pita3 is is a functional homolog of Pwl2, conferring
present only in M. oryzae isolates, suggesting avirulence on weeping lovegrass. However,
that Avr-Pita1 and Avr-Pita3 are derived from a Pwl4 could be made functional when expressed
gene duplication event that must have occurred under the control of the Pwl2 promoter, which
after separation of M. oryzae from M. grisea (71). was not the case for Pwl3 (70).
Ace1 effector. Ace1 encodes a putative 4035

aa cytoplasmic fusion polypeptide containing Rhynchosporium secalis
a polyketide synthase (PKS) and a nonriboso- Rhynchosporium secalis, the causal agent of leaf
mal peptide synthetase (NRPS), two distinct scald on barley, secretes three low molecular
classes of enzymes that are involved in the pro- weight peptides, designated Nip1 to Nip3, that
duction of microbial secondary metabolites (11, function as nonspecific toxins on barley and
20). Ace1 seems to mediate avirulence indirectly several other plants. Nip1 and Nip3 are pro-
in the presence of the Pi33 rice R gene, by its duced both in vitro and in planta, and their pres-
involvement in the biosynthesis of a secondary ence correlates with the formation of necrotic
metabolite that most likely activates Pi33. The lesions that are most likely caused by an in-
nature of the secondary metabolite is not known direct stimulation of H+ -ATPase activity in
yet, but mutations in the PKS domain of Ace1 plant plasma membranes (146, 147). In addi-
abolish activation of Pi33, indicating that at tion, Nip1 triggers specific (non-HR) defense
least Ace1-mediated biosynthetic activity is re- responses in barley cultivars carrying the Rrs1
quired for avirulence on Pi33 plants (11). Ace1 is resistance gene (51). Nip1 (also known as Avr-
exclusively expressed in appressoria, suggesting Rrs1) encodes a preprotein of 82 aa, which ma-
that the produced secondary metabolite might tures into a 60 aa protein after removal of the
have a role in virulence, although mutants in signal sequence. Ten cysteine residues (101) are
which Ace1 was deleted were not compromised present in the mature Nip1 protein that is in-
in virulence (11, 43). volved in five intramolecular disulfide bonds
(45). NMR spectrometry showed that Nip1
Pwl effectors. Members of the Pwl mainly consists of two structural domains, one
(pathogenicity toward weeping lovegrass) with two β-sheets and another with three an-
gene family encode rapidly evolving small tiparallel strands (127). Strains of R. secalis vir-
glycine-rich secreted proteins that are ulent on Rrs1 plants either lack Nip1 or carry

ANRV384-PY47-11 ARI 12 July 2009 7:25
alleles with point mutations that translate into epidermis (powdery mildews) or in mesophyll
single aa substitutions (72, 101, 105). Two of cells (rust fungi) of their hosts during infec-
these aa substitutions are correlated with gain of tion. Haustoria are not only specialized feeding
virulence, and lack of H+ -ATPase activity and structures required for acquisition of nutrients,
necrosis-inducing activity, suggesting that both but they also induce structural, cellular, and bio-
the necrosis induction and elicitor activity are chemical changes in the invaded host cells. In
mediated by a single receptor that is most likely addition, they can facilitate delivery of effec-
different from the Rrs1 protein (39). Recently, tors into the extrahaustorial matrix, several of
it was found that Nip1 interacts with a sin- which are subsequently translocated into host
gle plasma membrane receptor that is involved cells (16). Here, we discuss effectors produced
in both mediating virulence and triggering de- by the flax rust fungus M. lini and the barley
fense, but the exact receptor has not yet been powdery mildew fungus B. graminis f. sp. hordei.
characterized (126). A recent study of field pop-

ulations of the pathogen showed clear evidence Melampsora lini

of positive diversifying selection operating on
The flax rust fungus M. lini is an obligate basid-
the Nip1 locus (105). In total, 14 Nip1 isoforms
iomycete that infects flax (Linum usitatissimum)
were identified, of which at least three were cor-
and other species of the genus Linum. At least
related with gain of virulence on Rrs1 plants,
30 Avr genes corresponding to approximately
while a high deletion frequency of Nip1 was
30 cognate flax R genes have been identified
also observed that was much higher than that
in genetic analyses (33). Flax R proteins are all
observed for Nip2 and Nip3. As single amino
members of the intracellular TIR-NBS-LRR
acid substitutions in Nip1 that correlated with
class and are distributed among five highly poly-
gain of virulence on Rrs1 plants were observed
morphic loci designated K, L, M, N, and P (4,
in much lower frequencies than gene deletions,
29, 30, 32, 35, 78). To date, Avr genes have been
the fitness cost associated with the loss of this
cloned from four M. lini loci, namely AvrL567,
gene is likely not high (105).
AvrM, AvrP123, and AvrP4 that code for haus-
Recently, the Nip2 and Nip3 genes have also
torially expressed secreted proteins (HESPs)
been cloned from R. secalis (W. Knogge, per-
and elicit HR in flax plants that carry the cog-
sonal communication). Nip2 encodes a 109 aa
nate R genes (17). Each of the characterized
protein with a predicted signal peptide of 16
Avr loci consists of one to five closely related
aa, whereas Nip3 encodes a 115 aa protein with
homologous genes that code for small secreted
a predicted signal peptide of 17 aa. Both pro-
proteins with no sequence similarity to other
teins have one cysteine residue in their sig-
proteins present in public databases. Tran-
nal sequences, and the mature Nip2 and Nip3
sient expression of the mature Avr proteins in
carry six and eight cysteines, respectively. Nip2
flax plants carrying the cognate cytoplasmic R
and Nip3 are also further processed at their C
proteins induces an HR, indicating that they
termini, but as the cleavage sites are not yet
are translocated via the extrahaustorial matrix
known, the exact number of aa (including cys-
into host cells during infection (17, 27). The
teine) residues present in the mature proteins
AvrL567A, AvrL567B, and AvrL567C genes
remains to be determined (W. Knogge, per-
cluster at the AvrL567 locus and trigger HR
sonal communication).
in flax lines that carry the L5, L6, and L7 re-
sistance genes. They all encode 150 aa pro-
teins, with a predicted 23 aa highly conserved
EFFECTORS FROM N-terminal signal sequence resulting in a 127
HAUSTORIUM-FORMING aa mature protein. Specific expression of these
FUNGAL PATHOGENS genes in haustoria suggests that they might play
Rusts and powdery mildews are obligate a role in virulence. Approximately 25% of the
pathogenic fungi that produce haustoria in the aa residues within the mature protein retain

ANRV384-PY47-11 ARI 12 July 2009 7:25
one or more polymorphisms, and several of cystine-knotted peptides (94). Both AvrM and
these have been shown to affect a transition AvrP4 are expressed in planta, whereas AvrM
to virulence on plants carrying the cognate R is also expressed in vitro. Transient intracellu-
proteins, suggesting that the Avr567 locus is lar expression of AvrM and AvrP4 in flax plants
under positive diversifying selection. Indeed, carrying the cognate R genes triggers an HR,
in a survey involving just six flax rust strains, suggesting that effector translocation into the
twelve AvrL567 sequence variants (known as host cells occurs during infection, which is con-
AvrL567A to AvrL567L) were identified, six of sistent with the predicted cytoplasmic location
which (A, B, D, F, J, and L) were avirulent vari- of M and P resistance proteins (4, 78). However,
ants that triggered HR in flax lines carrying the both effectors also induce an HR in flax when
L5, L6, or L7 genes, and five (C, G, H, I, and targeted into the apoplast, suggesting their re-
K) were virulent variants that no longer trig- entry from the apoplast into host cells after se-
gered HR on these lines (28). These virulent cretion (17, 27, 31). A role in virulence for the
variants exhibited substitutions in aa residues Avr genes of M. lini has not been shown yet.
that are exposed to the surface of the protein
and interact directly with the cognate R pro-
teins, as confirmed in yeast two-hybrid assays Blumeria graminis f. sp. hordei
(34, 144). Therefore, evading host recognition Powdery mildews are a large group of as-
is most likely the source of the diversifying se- comycete obligate biotrophic fungi that show
lection acting on the Avr567 locus. Other char- a high degree of host specialization and, like
acterized HESPs from flax rust include AvrM rusts, produce haustoria in their host plants
that is recognized by the M resistance protein, (46). Blumeria graminis f. sp. hordei (Bgh) causes
AvrP4 that is recognized by P4, and the complex powdery mildew on barley and interacts with
AvrP123 proteins that are variously recognized its host in a gene-for-gene manner (149). At
by P, P1, P2, and/or P3 resistance proteins (17). least 85 dominant or semidominant mildew R
As for AvrL567, diversifying selection also op- genes (Ml) with different recognition speci-
erates at the AvrM, AvrP4, and AvrP123 loci ficities have been characterized in barley, in-
with virulent flax strains carrying mutated alle- cluding Mlk genes and 28 highly homologous
les that encode Avrs that are no longer recog- genes that all map to the Mla (mildew A) lo-
nized by the cognate R proteins (17). At least cus of barley chromosome 5 (57, 65). Six of
five different paralogs (AvrMA to AvrME) have the genes present at this locus, namely Mla1,
been detected at the AvrM locus of an avirulent Mla6, Mla7, Mla10, Mla12, and Mla13, have
strain, although one paralog encodes an effec- been cloned, and they all encode highly related
tor that is not recognized by any known flax R intracellular CC-NBS-LRR type of R proteins.
protein. The six AvrM proteins have no known However, despite the high sequence similarity
homologs in the public databases and show sig- (>90% identity), they all recognize isolate-
nificant sequence and size variations caused by specific effectors of Bgh (52, 53, 109). Two
DNA insertions, deletions, or polymorphisms Avr genes residing within a proximity of 30
in the location of stop codons. AvrP123 pro- kb in the genome have been cloned from Bgh
teins contain ten cysteine residues including (100). They are designated Avrk1 and Avra10
the characteristic CX7CX6YX3CX2-3C sig- and induce defense responses in barley vari-
nature present in the Kazal family of serine eties containing the cognate Mlk1 and Mla10
protease inhibitors, suggesting that host pro- R proteins, respectively. Both genes belong to
teases might be a target of these effectors. a large multigene family of more than 30 par-
AvrP4 also encodes a protein with six cys- alogs in Bgh, whereas homologs are present in
teine residues at the 28 aa C-terminal part of formae speciales that are pathogenic on other
the mature protein that show a spacing (CX3- grasses. Of all plant pathogens studied so far,
7CX4-6CX0-5CX1-4CX4-10C) typical for Bgh has the highest number of Avr genes, and

ANRV384-PY47-11 ARI 12 July 2009 7:25
most of them map to single loci, although ge- include a functional high-throughput screen-
nomic regions that contain clusters of several ing for identification of HR-inducing cDNAs
recombining Avr genes have also been reported from plant pathogens based on Agrobacterium
(14). The predicted 286 and 177 aa proteins en- tumefaciens/PVX-mediated cDNA expression
coded by Avra10 and Avrk1, respectively, share in host plants (119), a mutagenesis approach
approximately 60% similarity with each other, using restriction enzyme-mediated integration
but both lack an N-terminal signal sequence (REMI) (84), and screening of EST libraries
or a signature for uptake by host cells, such for genes upregulated during infection, as was
as the RXLR motif present in oomycete ef- successfully applied for AvrL567 of M. lini
fectors (67). This suggests an alternative but (27). However overall these approaches have
yet unidentified route of delivery of these ef- been very labor intensive, and their success rate
fectors into host cells, where they exert their was relatively low. Recently, whole genome se-
putative virulence function and induce defense quencing of fungal pathogens has provided an
responses mediated by the cognate R proteins enormous amount of data that can be analyzed
in resistant plants (100). Recently, it was shown for putatively secreted cysteine-rich proteins.
by fluorescence microscopy that the majority Narrowing down the spectrum of additional ef-
of the Mla10 protein is localized in the cyto- fector candidates can be achieved by integra-
plasm and approximately 5% in the nucleus tion of genome transcriptome, proteome, and
(9, 108). Perturbation of nucleo-cytoplasmic metabolome data, when available. Comparative
Mla10 partitioning by expression of an Mla10 secretome analysis and BLAST sequence simi-
fusion protein containing a nuclear export sig- larity searches could also be used for the iden-
nal (NES) that enhances nuclear export over tification of potential effectors in sequenced
import, decreased Mla10-specified disease re- genomes, but these methods have limitations
sistance (108). In the nucleus, Mla10 showed due to low sequence similarities among fungal
an Avr10-dependent physical association with effector genes. However, this approach proved
two WRKY transcription factors (HvWRKY1 successful with the recent identification of the
and HvWRKY2 TFs), suggesting that these first homologs of the C. fulvum effector genes
TFs serve as immediate downstream targets of Avr4, Ecp2, and Ecp6 in the genome of the
the activated receptor. The effector-dependent banana pathogen Mycosphaerella fijiensis (117)
association between barley Mla10 and WRKY and the discovery of a functional homolog of
TFs contributes to Mla10-mediated resistance the toxic peptide ToxA from Stagonospora nodo-
and host cell death at attempted fungal infec- rum in Pyrenophora titici-repentis (41, 42). Nev-
tion sites (108). The WRKY TFs interacting ertheless, despite the great potential of using
with Mla10 presumably act as repressors of PTI genome-wide searches for identifying candi-
and might have a role in keeping basal defense date effector genes, their function still needs
below a certain threshold. to be confirmed experimentally by overexpres-
sion, gene disruption or silencing in fungal iso-
lates, and subsequent (a)virulence assays on host
FUTURE HUNTS FOR FUNGAL plants.
EFFECTORS BY COMPARATIVE
GENOMICS
Genetic and biochemical approaches based FUNCTIONS AND HOST
on map-based cloning and analyses of fun- TARGETS OF EFFECTORS
gal secretomes during infection have been
the two most popular strategies to iden-
Extracellular and Cytoplasmic
tify effector genes from the pathogens dis-
Fungal Effectors
cussed above. Other, but less widely ap- Fungal effector proteins can be roughly
plied, methods for discovery of effector genes grouped into extracellular effectors that are

ANRV384-PY47-11 ARI 12 July 2009 7:25
secreted into the apoplast or xylem of their (80). Finally, effectors active inside the host cell
host plants and cytoplasmic effectors that are like those of M. lini possibly need proper fold-
translocated into host cells (Table 1). Ace1 ing and disulfide-bridge formation outside the
from M. grisea is the only known fungal ef- host before being taken up.
fector protein that is not secreted by the fun-
gus, but instead is suggested to be involved in
the biosynthesis of a yet unknown secondary Intrinsic Functions
metabolite that is likely secreted (11). Despite As sequence homology between most fungal
the low degree of sequence conservation among effectors and other proteins present in public
fungal effectors, most of them code for small se- databases is limited, assigning functions to most
creted proteins, of which some are translocated effectors based on putative orthology alone has
into host cells by a yet unknown mechanism. been limited (Table 1). Exceptions are the Avr-
The only two members of putatively cytoplas- Pita (a putative metalloprotease) (93) and Ace1
mic effectors which lack a signal sequence are (a hybrid polyketide synthase/nonribosomal
Avra10 and Avrk1 of B. graminis f. sp. hordei peptide synthetase) (11) effector proteins of M.
(100). Extracellular effectors are often further grisea (130), as well as Ecp6 (LysM-domains)
N- and sometimes C-terminally processed by (13) of C. fulvum. Characterization of the three-
plant and/or fungal proteases, but evidence for dimensional structure as well as the disulfide
protein maturation is in some cases based only bond pattern between cysteine residues in some
on in-silico predictions. In that respect, exper- cases provided additional clues with respect to
imental evidence for secretion and processing the intrinsic functions of some fungal effec-
of fungal effectors during infection is available tors. Indeed, the disulfide bond pattern and
only for the Avr and Ecp effectors of C. fulvum cysteine spacing in Avr4 from C. fulvum re-
(24), the Six effectors of F. oxysporum f. sp. ly- vealed structural and functional homology with
copersici (55), and the Nip effectors of R. secalis an invertebrate chitin-binding domain present
(146). Avr-Pita from M. grisea is also predicted in tachycitin (130, 131). A second effector pro-
to be a secreted processed protein, but this as- tein with a known intrinsic function is Avr2
sumption is based on the fact that the 176 aa from C. fulvum that proved to be an inhibitor
mature protein, but not the intact 223 aa pro- of the Rcr3, Pip1, aleurain, and TDI65 cys-
tein, is the active form that interacts directly teine proteases (102, 107, 140), although this
with the cognate cytoplasmic Pi-ta R protein function was not inferred from homology with
(59). other known cysteine protease inhibitors. Avr2
A second common feature of extracellular and Avr4 are the only two fungal effectors for
effectors, as well as some of the effectors that which both an intrinsic and virulence function
are active inside host cells, is the presence of has been shown experimentally. Based on the
multiple cysteine residues (Table 1). The cys- spacing between the cysteine residues, AvrP123
teine residues might be involved in disulfide- of M. lini shows structural homology to mem-
bridge formation that provides protein stability bers of the Kazal family of serine protease in-
in the harsh protease-rich environment of the hibitors, whereas AvrP4 shows homology to
host apoplast. Indeed, disulfide bonds between cystine-knotted proteins, as does Avr9 of C.
cysteine residues have been reported to be re- fulvum (17, 134). However, despite the struc-
quired for stability and activity of at least Avr4 tural resemblance of the latter effectors to other
and Avr9 of C. fulvum (132, 134). However, mu- enzymes or proteins, a functional homology
tational analysis of cysteine residues present in has never been established. Finally, the 3-D
the Ecps from the same fungus suggested that structure of AvrL567 from M. lini has been
not all cysteine residues are involved in disul- resolved and binding of this protein to DNA
fide bridges or are crucial for induction of HR has been demonstrated, but because not all
on plants carrying the cognate Cf-Ecp proteins AvrL567 variants bind DNA with the same

ANRV384-PY47-11 ARI 12 July 2009 7:25
efficiency, the biological significance of the ob- lycopersici, which is not a virulence factor but
served activity is not yet clear (144). rather suppresses I-3-mediated resistance trig-
gered by Avr3 (Six1), an effector that is required
for full virulence of this pathogen (54).
Role in Virulence
As with intrinsic functions, the contribution of
individual fungal effectors to virulence has also Translocation of Fungal Effectors
proven difficult to establish. Functional anal- Plant and animal pathogenic bacteria contain
ysis indicates that in some cases effector pro- six secretory systems of which the type three se-
teins constitute genuine virulence factors that cretion system (TTSS) is crucial for virulence
are required for full virulence of the pathogens as it is required for translocation of effectors
on their host plants (Table 1). Some effec- into the host cell (1, 2, 36). Many TTSS-
tor genes are also reported to be clustered on injected effectors suppress PTI, and for sev-
pathogenicity islands, the presence of which is eral of them the mechanism of suppression has
correlated with virulence (or avirulence) on spe- been elucidated (1). Most oomycete secreted
cific hosts. Examples are the Six1 (Avr3), Six2, effectors contain an RXLR (10) motif that is
and Six3 genes of F. oxysporum f. sp. lycoper- required for their uptake into host cells, as
sici that cluster in a genomic region that con- has been shown for effectors of the Arabidopsis
fers virulence on tomato plants (135), and the downy mildew pathogen Hyaloperonospora para-
Pwl gene family of M. oryzae that is required sitica (97) and the potato pathogen Phytophthora
for specificity on weeping lovegrass (70, 118). infestans (148). Bioinformatics analysis has iden-
Also many effectors seem to work in concert, tified 425 genes potentially encoding secreted
and their individual contribution to virulence RXLR proteins in the P. infestans genome (60).
is often minor, undetectable, or redundant, as As with bacteria and oomycete effectors, ef-
their deletion has no apparent effect on fitness fectors of some fungal plant pathogens (e.g.
or virulence (Table 1). This fact is also sup- rusts, powdery mildews, M. oryzae, and F. oxys-
ported by studies on diversifying selection op- porum f. sp. lycopersici) are putatively translo-
erating on a number of effector genes showing cated into the host cell where they interact with
that mixed (sub)populations of some pathogens cytoplasmic or nuclear R proteins (16, 27, 28,
exist that consist of individuals that either carry 34, 59, 108). However, so far in all fungal effec-
or lack particular effector genes, indicating that tors no clear consensus signature has been iden-
the fitness cost for the loss is not very high (105, tified that would point to a function in translo-
116). Some effectors might also provide over- cation and uptake into the plant cell.
lapping activities as is possibly the case for the The host-selective protein toxin ToxA of
Avr4 and Ecp6 effectors of C. fulvum that both P. tritici-repentis (19) is required for full viru-
bind to chitin and thus could potentially pro- lence on wheat, and its solvent-exposed Arg-
tect the fungus against plant chitinases and/or Gly-Asp (RGD) motif that interacts with the
suppress PTI by scavenging chitin fragments host plasma membrane is likely required for its
released from fungal cell walls in the apoplast internalization (85). A similar RGD motif me-
during infection (13, 130). Finally, some effec- diating interaction with the plasma membrane
tors might play a more general role in virulence, is also present in the IpiO effector proteins of
as is, for example, the case for the Nip proteins the oomycete P. infestans (106).
of R. secalis (146, 147), whereas other effectors
that lack a clear role in virulence seem to inter-
fere with defense signaling pathways that are EFFECTOR RECOGNITION
induced by other effectors/Avr factors with an BY R PROTEINS
important role in virulence. This seems to be To date, many R genes have been cloned from
the case for Avr1 (Six4) of F. oxysporum f. sp. different plant species and the products of these

ANRV384-PY47-11 ARI 12 July 2009 7:25
genes show great diversity in their structural One implication of the receptor-ligand
properties (24, 87, 120, 142). The vast major- model is that given the enormous diversity of
ity of cytoplasmic R proteins consist of an NBS effector molecules, plants must carry numer-
connected to a region of LRRs (NBS-LRRs), ous R proteins that would enable them to rec-
whereas the second major class of R genes ognize all individual effectors and their allelic
encodes proteins with an extracellular LRR variants. However, most plants have developed
(eLRR) domain and a short transmembrane R proteins that monitor modifications in the
(TM) domain. The NBS-LRR class can be fur- plant targets of fungal effectors. In this so-called
ther subdivided into subclasses based on the guard model, R proteins do not interact directly
structure of their N-terminal domains that con- with an effector but guard its host target and re-
tain either a coiled coil (CC) or Tol/interleukin- spond to alterations in this target caused by the
1 receptor (TIR) motif (Figure 1). However, effector. Therefore, in contrast to the receptor-
despite the fact that an increasing number of ligand model, which allows recognition of only
plant R proteins and their cognate effectors a limited set of structurally related effectors,
have been characterized, in most cases we still the guard model enables detection of multiple
know little about the molecular mechanisms as- unrelated effectors that interact with the same
sociated with effector perception by R proteins host target guarded by a single R protein. A
and subsequent R-mediated downstream plant classical example of the guard model is repre-
defense responses (Figures 2 and 3). sented by the indirect interaction between Avr2
The simplest perception model proposed so and Cf-2, mediated by Rcr3 in the C. fulvum-
far is based on a direct interaction between the tomato interaction as discussed above (102). In
cognate R protein and effector. This model is many other pathosystems, indirect interaction
referred to as the receptor-ligand model, and between R protein and cognate Avr has been re-
indeed, a few examples of direct interactions ported, suggesting that the majority of interac-
between a fungal effector and plant R pro- tions would fit into the guard model (61). This
tein have been described. Yeast two-hybrid and also seems to be the case for the Avr9 effec-
in vitro binding assays demonstrate a physi- tor of C. fulvum and the tomato Cf-9 protein,
cal interaction between Avr-Pita (Avr-Pita176 ) whose interaction is mediated by a high affinity
from M. oryzae and the cognate Pi-ta resis- binding site (73, 81) (Figures 2 and 3).
tance protein from rice (59). Pi-ta codes for Whether direct or indirect interaction me-
an intracellular NBS-LRR protein and muta- diates perception of effector molecules by R
tional analysis showed that single aa substitu- proteins, both systems could complement each
tions in the LRR domain of this protein or other. Indirect recognition enables plants to
in Avr-Pita176 can disrupt the physical inter- monitor multiple effectors by a relatively small
action and abolish Pita-mediated defense re- number of R proteins, whereas redundancy in
sponses (59). Physical interaction between ef- guardees allows monitoring of one target by dif-
fectors of M. lini and their cognate R proteins ferent R proteins, as seems to be the case for
in flax has also been reported (28, 34, 144). The Rlm4 and Rlm7, which both seem to guard the
polymorphic AvrL567 locus of M. lini consists same virulence target of AvrLm4-7 (95). This
of at least three genes (AvrL567A, AvrL567B, strategy, however, is effective only toward ef-
and Avr567C) whose products are recognized fectors with plant targets but not against effec-
by the cognate L5, L6, and L7 proteins of tors with defensive fungal targets, such as Avr4
flax. Yeast two-hybrid assays demonstrated di- from C. fulvum, which binds to chitin present
rect interaction between AvrL567 and the cog- in the fungal cell wall but apparently does not
nate L5/L6 proteins, whereas a mutation in interfere with the physiology of the host. Fur-
the LRR domain of L abolishes the interac- thermore, direct interaction between effector
tion, indicating binding of AvrL567 to the LRR and R protein can be overcome more readily by
domain. mutations in effectors that abolish recognition,

ANRV384-PY47-11 ARI 12 July 2009 7:25
RLPs
LysM-RK
a Cfs:
Avrs
Ve2: ?
HcrVf: ?
LeEix2:
Eix
CERK1:
Chitin
Ecps
LysM
eLRR eLRR eLRR
LysM
LysM
TM TM TM TM
Cytosol
ECS PEST Kinase
ER-r ECS ECS
NB-LRRs
b TIR NBS LRR

L5, L6, L7: AvrL567
M: AvrM
Pi-ta: Avr-Pita
LZ/CC NBS LRR I-2: Six3
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, flax 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, Pro–Glu–Ser–Thr signature; Pi-ta, rice blast resistance
protein; Six3, secreted in xylem 3; LZ/CC-NB-LRR, leucine zipper-NB-LRR; TIR-NB-LRR, toll-
interleukin-1 receptor kinase-NB-LRR; TM, transmembrane; Ve2, Verticillium wilt resistance protein 2.

ANRV384-PY47-11 ARI 12 July 2009 7:25
Cf-2
B
Cf-4/Cf-9
Cysteine
proteases
Chitin ? B
Avr2 C1
Avr4
Avr4 Rcr3 Avr2
C1
?
C22 Avr9 C2
C3 C3
D D
E HABs E
F F
VAP27
G ACIK1 G
Cytosol Hsp90
?
ECS CITRX
ER-r
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 cystine-
knotted 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 affinity 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 decoy molecules that mimic the effector tar-
evasion of R-mediated resistance in cases of in- gets. Such decoys would trap effector proteins,
direct interactions (23, 61). diverting them from their real virulence targets
The guard model is strongly dependent on and alert monitoring R proteins. Decoys either
guarded effector targets, and therefore, natural could arise from a guardee duplication event or
selection is expected to favor partners with im- could evolve independently of the effector tar-
proved interaction. Furthermore, the virulence gets that they are mimicking. This variant of
function of effector proteins is based on modi- the original guard model is proposed as the de-
fication of their host targets, and natural selec- coy model, and a few cases in support of this
tion in the host would favor the development model have been suggested to operate in bacte-
of targets that are less or no longer recognized rial and fungal pathosystems (137). One of the
by effectors. As a result of these two opposing proposed cases involves the interaction between
forces acting on effector targets, it was recently Avr2 and Rcr3, where it is suggested that Rcr3
proposed that plants have likely developed is the decoy molecule that diverts Avr2 away

ANRV384-PY47-11 ARI 12 July 2009 7:25
Cf-4 Cf-9
Avr-induced
K+
K+ K+
Avr4 H+
ROS
Ca2+ ATPase K+
ATPase ATPase
Oxidative Avr9 PR
burst
HABs PR
Syntaxin
CITRX PLC DGK ACIK1 VAP27 P PR

PIP2 DAG PA
P
CITRX Hsp90
NADPH ?
oxidase Ca2+ H+ H+
Ca2+ 2+ H+
? Ca
EDS1
IP3
CC MAPKKK P CDPKs
NRC1 MAPKK P
P Cytosol
?
Hsp90 RAR1 ? MAPK1-3
?
P TFs
SGT1
Salicyclic acid
Defense responses
TFs
Vacuole ACS
P P Cell wall reinforcement
PR
PR
Phytoalexins
Nucleus
PR proteins PR
Ca2+
Ca2+ 2+
Ca Ethylene Hypersensitive response
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 kinase-
1-3; 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 figure is a slightly revised version of figure 1C from Reference 24.
ANRV384-PY47-11 ARI 12 July 2009 7:25
from its real target, the plant protease Pip1, selection operating on the AvrL567 locus of
and activates Cf-2-mediated Avr2-triggered re- M. lini generated protein variants that are no
sistance (107, 137). However, this model still is longer recognized by the cognate L protein in
very speculative and not yet supported experi- flax. Polymorphic aa residues in these sequence
mentally. variants occurred on the proposed solvent-
exposed surface of the effector protein that in-
teracts with the LRR domain of the cognate R
EFFECTOR AND RESISTANCE protein, indicating that selection favored vari-
PROTEIN EVOLUTION ants that abolish recognition but still maintain
Hosts and pathogens are locked up in a per- a putative virulence function (144). In a simi-
petual arms race with only temporary winners lar way, almost all sequence variants described
and losers. Fungal effectors and plant R proteins for the chitin-binding Avr4 effector protein of
are in the front line of this arms race, and rapid C. fulvum showed single aa substitutions that
rates of effector evolution and maintenance of abolish recognition by the cognate Cf-4 pro-
high allelic diversity at corresponding R loci in tein but retain their ability to bind to chitin
plants have been reported in different pathosys- (62, 64, 116, 130). Recent analysis of Avr4-
tems (3, 28, 83, 112, 115). The high rate of expressing tomato lines has shown that Avr4
molecular evolution observed in effectors as a triggers hardly any transcriptional responses in
result of mutation, selection, and reproduction the absence of the cognate Cf-4 protein, indi-
has caused extensive sequence diversification, cating that it is unlikely that Avr4 has targets in
gene expansion, and other genetic rearrange- the host other than Cf-4 (139). In a similar way,
ments in effector genes that might explain the sequence diversification of pathogen effectors
absence of homology among effector proteins is adaptively matched fairly quickly by diversi-
and their different host specificities. Many ef- fication of the host R proteins that results in
fector genes are embedded in highly dynamic new recognition specificities (88). Good exam-
genome areas, such as the chromosome ends ples of a dynamic arms race between effectors
or amid multiple transposable elements that and R proteins are provided by the M. lini Avrs
trigger frequent genomic rearrangements, thus and cognate flax R proteins (28) and the H. par-
enabling higher genetic flexibility in rapidly asitica Atr13 and Rpp13 proteins of Arabidopsis
overcoming R-mediated resistance, as already (3, 103).
discussed above. In many of these cases, gain In contrast to the emerging picture for di-
of virulence due to transposon insertions, gene rect recognition, indirect recognition of effec-
deletions, and other genetic rearrangements has tors by R proteins imposes selection against
been frequently observed (37, 44, 49, 50, 71, 93, Avr effector function rather than structure, and
95, 100, 118). such effectors are either present or absent in
Although sequence alterations in effector pathogen populations. Absence would imply a
genes that lead to evasion of R-mediated re- minimal fitness penalty for the pathogen, but
sistance can be the result of point mutations, under strong selection pressure, the conditional
frameshifts, gene deletions, and transposon in- benefits of such a loss could be higher than
sertions, the specific nature of the interplay be- the costs of the loss. Alternatively, the effector
tween effector and R proteins can affect the function could be redundant and compensated
type of genetic adaptation occurring in both for by other effectors. Effector gene deletions
proteins. Surveys for allelic variation in effector have been reported as the main mechanism for
genes suggest that direct recognition by R pro- gain of virulence for the fungal effectors genes
teins favors sequence diversification in effectors Avr9 and Avr4E of C. fulvum (116, 141, 145),
that disrupts physical association between the AvrLm1 and AvrLm4-7 of L. maculans (50, 95),
two, but without affecting the effector’s pre- Nip1 of R. secalis (105), and Avr-Pita and Avr1-
sumed virulence function. Indeed, diversifying CO39 of M. grisea (37, 93, 150). Loss of the

ANRV384-PY47-11 ARI 12 July 2009 7:25
Avr2 gene product of C. fulvum is caused by cation or jettison of effector genes. However, a
insertions, deletions, and other frameshift mu- different scenario has been observed for the ef-
tations that result in truncated nonfunctional fector Avr3 of F. oxysporum f. sp. lycopersici that
alleles. In contrast to gene deletions, such mu- is required for full virulence, where suppres-
tated alleles are preserved in the genome of sion of ETI is correlated with the acquisition of
C. fulvum and may become available for re- an additional protein (Avr1) that suppresses I-
versions once selection pressure has been lifted 3- and I-2-mediated defense signaling pathways
(116). Frameshift mutations in particular are of- (54).
ten reverted by the occurrence of a second com-
plementary frameshift that could restore the
function of the protein (92). However, except FUTURE CHALLENGES
for Avr2, which indirectly interacts with Cf-2, Comparative genomics of fungal pathogens will
for most of the other effector gene deletions de- be useful in identification of new effector pro-
scribed above, it is currently unknown whether teins and possibly in prediction of their vir-
the products of these genes interact directly or ulence functions. However, this approach is
indirectly with their cognate R proteins. In fact, limited because of the low level of homology
Avr-Pita of M. grisea, which directly interacts among fungal effectors. Some effectors reside
with Pi-ta, shows various types of sequence al- on pathogenicity islands, and it will be interest-
terations that can result in gain of virulence, ing to find out by comparative genomics how
including also gene deletions (59, 69, 93, 150). these islands have arisen during evolution. For
Point mutations contributing to virulence have most fungal effectors, a function in virulence
also been observed for Nip1, although less fre- can be investigated experimentally by gene dis-
quently than gene deletions (105), as well as for ruption, gene knock-down, or overexpression
Avr4E (116, 145) and AvrLm4-7 (95). From the assays, and it is expected that for number of
examples presented above, it is clear that the them a role in suppressing PTI induced by fun-
type of genetic adaptation of effectors to over- gal PAMPs will be shown. In addition to effec-
come R-mediated recognition is not only de- tors, discovery of more fungal PAMPs in addi-
termined by whether the interaction between tion to chitin fragments is also anticipated in
an effector and an R protein is direct or indi- the near future, which would enrich our under-
rect. However, theoretical modeling suggests standing of the plant defense system and the in-
that jettison of effector proteins can lead to terplay between PTI and ETI. Elucidating the
long and stable resistance, whereas sequence mechanisms of translocation of fungal effectors
diversification is likely to result rather quickly into host cells is also a major challenge for the
in new virulence phenotypes (138). In contrast future. A considerable amount of effort is cur-
to the receptor-ligand model, the guard model rently being placed on identifying interactors
does not impose any selective pressure for se- of effector proteins by in situ microscopic tech-
quence diversification on R genes but instead niques and fluorescently labeled effectors. Fi-
enhances diversifying selection on plant targets nally, it is hoped that effectors will be identified
of effectors (89). that are crucial for virulence, as their cognate
In all the cases described so far, gain of vir- resistance genes are anticipated to be durable
ulence was correlated with sequence diversifi- and could be used in resistance breeding.
DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.

ANRV384-PY47-11 ARI 12 July 2009 7:25
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 Ster-
giopoulos is financially 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 Scientific Research.
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AR384-FM ARI 14 July 2009 23:48
Annual Review of
Phytopathology
Contents Volume 47, 2009
Look Before You Leap: Memoirs of a “Cell Biological” Plant

Pathologist
Michèle 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
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 Acid–Derived 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, D’Maris 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
v
AR384-FM ARI 14 July 2009 23:48
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
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/
vi Contents
AR384-FM ARI 14 July 2009 23:48
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From the Annual Review of Ecology, Evolution, and Systematics, Volume 39 (2008)
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ANRV384-PY47-11 ARI 12 July 2009 7:25

• Top downloaded articles Phytopathology, 6709 PD Wageningen, The Netherlands;

• Our comprehensive search email: ioannis.stergiopoulos@wur.nl

Annu. Rev. Phytopathol. 2009. 47:233–63 Key Words

INTRODUCTION pathogens respond by mutating or losing ef-

Magnaporthe oryzae, and Rhynchosporium secalis,

and from obligate fungal pathogens that form

234 Stergiopoulos · de Wit

www.annualreviews.org • Fungal Effectors 235

Table 1 Fungal effector proteins

Effector residues Function/ Localization Role in R-gene Positive

7 apoplast (mainly) Rlm7

Fusarium oxysporum f. sp. lycopersici (tomato):

required for pers.

www.annualreviews.org • Fungal Effectors

toxin/induces apoplast vitro for full Knogge

AvrM 314 1 28 Unknown Cytoplasm Expressed in Unknown M (TIR- Yes (17)

www.annualreviews.org • Fungal Effectors

240 Stergiopoulos · de Wit

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242 Stergiopoulos · de Wit

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Ace1 effector. Ace1 encodes a putative 4035

244 Stergiopoulos · de Wit

characterized (126). A recent study of ﬁeld pop-

ulations of the pathogen showed clear evidence Melampsora lini

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246 Stergiopoulos · de Wit

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248 Stergiopoulos · de Wit

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250 Stergiopoulos · de Wit

b TIR NBS LRR

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252 Stergiopoulos · de Wit

CITRX PLC DGK ACIK1 VAP27 P PR

254 Stergiopoulos · de Wit

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and plant defense. Annu. Rev. Phytopathol. 42:385–414

256 Stergiopoulos · de Wit

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Annu. Rev. Phytopathol. 46:189–215

258 Stergiopoulos · de Wit

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striking similarities and obvious differences. Immunol. Rev. 198:249–66

260 Stergiopoulos · de Wit

www.annualreviews.org • Fungal Effectors 261

Plant-Microbe Interact. 19:1420–30

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Contents Volume 47, 2009

Look Before You Leap: Memoirs of a “Cell Biological” Plant

Seed Pathology Progress in Academia and Industry

Hordeivirus Replication, Movement, and Pathogenesis

An online log of corrections to Annual Review of Phytopathology articles may be found at

From the Annual Review of Analytical Chemistry, Volume 2 (2009)

Nanoparticle PEBBLE Sensors in Live Cells and In Vivo

From the Annual Review of Biochemistry, Volume 78 (2009)

From the Annual Review of Biomedical Engineering, Volume 11 (2009)

From the Annual Review of Biophysics, Volume 38 (2009)

From the Annual Review of Entomology, Volume 54 (2009)

Adaptation and Invasiveness of Western Corn Rootworm: Intensifying Research

From the Annual Review of Genetics, Volume 42 (2008)