fungal effector proteins

ANRV384-PY47-11 ARI 12 July 2009 7:25

Fungal Effector ProteinsIoannis Stergiopoulos1 and Pierre J.G.M. de Wit1,2

1Wageningen University and Research Center (http://www.php.wur.nl/uk), Laboratory ofPhytopathology, 6709 PD Wageningen, The Netherlands;email: [email protected] for BioSystems Genomics, 6700 AB Wagengen, The Netherlands;email: [email protected]

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

The Annual Review of Phytopathology is online atphyto.annualreviews.org

This article’s doi:10.1146/annurev.phyto.112408.132637

Copyright c! 2009 by Annual Reviews.All rights reserved

0066-4286/09/0908/0233$20.00

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

AbstractIt is accepted that most fungal avirulence genes encode virulence factorsthat are called effectors. Most fungal effectors are secreted, cysteine-rich proteins, and a role in virulence has been shown for a few of them,including Avr2 and Avr4 of Cladosporium fulvum, which inhibit plantcysteine proteases and protect chitin in fungal cell walls against plantchitinases, respectively. In resistant plants, effectors are directly or indi-rectly recognized by cognate resistance proteins that reside either insidethe 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 indirectlywith their cognate resistance proteins. Direct interactions seem to favorpoint mutations in effector genes, leading to amino acid substitutions,whereas indirect interactions seem to favor jettison of effector genes.

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INTRODUCTIONThe gene-for-gene hypothesis states that forevery dominant avirulence (Avr) gene in thepathogen there is a cognate resistance (R)gene in the host, and the interaction betweenthe products of these genes leads to activa-tion of host defense responses, such as thehypersensitive response (HR) that arrests thegrowth of biotrophic fungi (40). Many plantpathologists have been searching for molec-ular and biochemical evidence of the gene-for-gene concept. The molecular cloning ofthe first bacterial Avr gene was reported in1984 (114), the first fungal Avr gene in 1991(141), and the first oomycete Avr gene fol-lowed in 2004 (67, 124). Over the past twodecades, numerous novel Avr genes and cog-nate R genes have been discovered, and ourmolecular understanding of the gene-for-generelationship has increased considerably. It isnow accepted that plants contain two lines ofdefense. The first line provides basal defenseagainst all potential pathogens and is basedon recognition of conserved microbial featuresknown as pathogen-associated molecular pat-terns (PAMPs) by so-called PAMP-recognitionreceptors (PRRs) that activate PAMP-triggeredimmunity (PTI) and prevent further coloniza-tion of the host (23, 61, 91). One of the best-known microbial PAMPs is chitin, a majorstructural component of fungal cell walls, forwhich two LysM-type of receptor-like kinasesinvolved in its perception have been character-ized in rice and Arabidopsis, respectively (66, 90).Evidence is now accumulating that Avr genesencode effectors that suppress PTI, thus en-abling a pathogen to infect its host plant andcause disease. Once the basal defense systemof plants is overcome by pathogens, plants re-spond with the development of a more special-ized recognition system based on effector per-ception by R proteins and subsequent activationof effector-triggered immunity (ETI) that leadsto rapid and acute defense responses in plants,the hallmark of which is the HR. This trig-gers a second wave of coevolutionary arms racebetween pathogens and plants, during which

pathogens respond by mutating or losing ef-fectors, or by developing novel effectors thatcan avoid or suppress ETI, whereas plants de-velop novel R proteins mediating recognitionof novel effectors (23, 61).

Many reviews on bacterial, fungal, andoomycete Avr genes and their cognate R genesin plants have appeared in recent years (8, 24,47, 67, 68). In this review, we will provide anupdate on Avr genes from extracellular fungi,including Cladosporium fulvum, Fusarium oxys-porum f. sp. lycopersici, Leptosphaeria maculans,Magnaporthe oryzae, and Rhynchosporium secalis,and from obligate fungal pathogens that formhaustoria, such as Melampsora lini and Blumeriagraminis f. sp. hordei (Table 1). Their structure,intrinsic functions, localization, perception byR proteins, and evolution will be discussed.

EFFECTORS OFEXTRACELLULAR FUNGALPATHOGENS

Cladosporium fulvum

Cladosporium fulvum (syn. Passalora fulva) is anasexual extracellular fungal pathogen of tomato(25, 63, 123). To date, four avirulence (Avr)genes have been cloned from C. fulvum thatall encode small cysteine-rich proteins that aresecreted during infection. These are the Avr2,Avr4, Avr4E, and Avr9 effector proteins whoserecognition in tomato is mediated by the cog-nate Cf (C. fulvum) R proteins Cf-2, Cf-4, Cf-4E, and Cf-9, respectively (25, 63, 123). Fouradditional extracellular proteins (Ecps), namelyEcp1, Ecp2, Ecp4, and Ecp5 have been char-acterized from C. fulvum that invoke an HR intomato lines that carry a cognate Cf-Ecp gene(22, 75), whereas Ecp6 and Ecp7 have also re-cently been identified, but for these two effec-tors no responding tomato lines have yet beenreported (13). Although demonstrated in onlysome cases, all Avrs and Ecps are assumed to bevirulence factors (13, 123, 139, 140).

Avr2 effector. Avr2 encodes a preprotein of78 amino acids (aa), which matures into a 58 aa

234 Stergiopoulos · de Wit

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protein with eight cysteine residues that inducesHR in tomato plants that carry the cognateCf-2 resistance gene (26, 82). During infec-tion, Avr2 inhibits at least four tomato cysteineproteases including Rcr3, Pip1, aleurain, andTDI65, and as such, plays an offensive role invirulence by targeting and inhibiting host pro-teases that are important for host defense (74,102, 107). Indeed, the role of Avr2 in viru-lence is demonstrated not only for C. fulvumbut also for other fungal tomato pathogens, in-cluding Botrytis cinerea and Verticillium dahliae,as heterologous expression of Avr2 in Arabidop-sis thaliana enhances susceptibility toward thesepathogens (140). In the presence of Cf-2, Avr2behaves as an avirulence factor, and its recogni-tion is mediated by Rcr3pimp (required for C. ful-vum resistance), a cysteine protease originatingfrom Lycopersicon pimpinellifolium (74, 102). De-spite the fact that the exact mechanism of Avr2perception is not yet known, structural modi-fication of Rcr3 by Avr2, rather than Rcr3 in-hibition, is the most likely cause of triggeringCf-2-mediated defense signaling, as a naturalvariant of Rcr3 occurs in Lycopersicon esculen-tum (Rcr3esc) that causes spontaneous HR in thepresence of Cf-2 in an Avr2-independent man-ner. The Rcr3esc protein still shows protease ac-tivity and is likely to have a modified tertiarystructure as compared with the Rcr3pimp pro-tein (74). Circumvention of Avr2-triggered Cf-2-mediated HR can be achieved by point muta-tions, deletions, or transposon insertions in theAvr2 gene (82). A recent survey of polymor-phisms present in the Avr2 alleles of a world-wide collection of C. fulvum isolates revealed anexcess of nonsynonymous polymorphisms com-pared with synonymous ones, suggesting pos-itive diversifying selection. These were mainlyinsertions or deletions in the coding region ofthe gene that lead to the production of trun-cated Avr2 proteins (116).

Avr4 effector. Avr4 encodes a 135 aa prepro-tein that is C- and N-terminally processed aftersecretion in the apoplast into an 86 aa matureprotein with eight cysteine residues (62, 64).Based on its disulfide-bond pattern, Avr4 shows

structural similarity to proteins with an inverte-brate chitin-binding domain, such as tachycitin(inv ChBD) (110). Indeed, binding of Avr4 tochitin has been experimentally confirmed, andit is further shown that Avr4 can protect chiti-nous fungi, such as Trichoderma viride and Fusar-ium solani, against basic plant chitinases. There-fore, a role for Avr4 in protection of C. fulvumagainst chitinases during infection has beenproposed (130–132). The virulence function ofthis protein is further supported by the fact thatAvr4 is expressed only during infection of thehost, when the fungus is exposed to chitinases,and that silencing of Avr4 in C. fulvum sig-nificantly reduces virulence (139). In addition,tomato plants expressing Avr4 are more sus-ceptible to C. fulvum and other chitinous fun-gal tomato pathogens (139). In the presence ofCf-4, Avr4 induces HR, but natural isoforms ofthis effector protein occur that no longer triggerCf-4-mediated HR. Such isoforms show an ex-cess of nonsynonymous substitutions comparedwith synonymous ones, suggesting that Avr4is under positive diversifying selection (116),whereas the majority of the natural variationobserved in Avr4 involves point mutations thatmostly cause substitutions of cysteine residues.The resulting unstable Avr4 variants are moresensitive to proteases but are still able to bindchitin, thus preserving the virulence functionof the protein but preventing accumulation ofAvr4 in the apoplast and triggering of HR inCf-4 plants (64, 132).

Avr4E effector. Avr4E codes for a cysteine-rich 101 aa protein that is secreted during in-fection and triggers Cf-4E-mediated HR (145).Various strains of C. fulvum have been identi-fied that evade Cf-4E-mediated resistance thatall show two identical point mutations in Avr4Ethat translate into stable Avr4E proteins withtwo aa substitutions (Avr4ELT). Targeted mu-tagenesis showed that one of these mutationsin Avr4E, namely the Phe62>Leu substitution,is sufficient for evasion of Avr4E-triggered Cf-4E-mediated HR (145). Analysis of natural pop-ulations of the fungus also shows that a signif-icant number of strains evade Cf-4E-mediated

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Tab

le1

Fung

alef

fect

orpr

otei

ns

Effe

ctor

prot

ein

Aa

resi

dues

(mat

ure)

Cys

tein

esSi

gnal

PFu

ncti

on/

hom

olog

yL

ocal

izat

ion

inpl

ant

Exp

ress

ion

Rol

ein

viru

lenc

eR

-gen

e(t

ype)

Pos

itiv

ese

lect

ion

Ref

eren

ces

Cla

dosp

oriu

mfu

lvum

(tom

ato)

:Av

r278

(58)

820

Prot

ease

inhi

bito

rA

popl

ast

Inpl

anta

Inhi

bits

Rcr

3an

dot

her

prot

ease

s

Cf-

2(e

LR

R-T

M)

Yes

(26)

Avr4

135

(86)

818

Chi

tin-b

indi

ngA

popl

ast

Inpl

anta

Prot

ects

agai

nst

chiti

nase

s

Cf-

4(e

LR

R-T

M)

Yes

(62)

Avr4

E12

1(1

01)

610

Unk

now

nA

popl

ast

Inpl

anta

Unk

now

nH

cr9-

4E(e

LR

R-T

M)

Yes

(145

)

Avr9

63(2

8)6

23C

arbo

xype

ptid

ase

inhi

bito

rA

popl

ast

Inpl

anta

Unk

now

nC

f-9

(eL

RR

-TM

)Ye

s(1

29)

Ecp

196

(65)

823

Tum

or-n

ecro

sisfa

ctor

rece

ptor

Apo

plas

tIn

plan

taD

isrup

tion

lead

sto

redu

ced

viru

lenc

e

Cf-

Ecp1

Not

clon

edN

o(1

29)

Ecp

216

5(1

43)

422

Unk

now

nA

popl

ast

Inpl

anta

Disr

uptio

nle

adst

ore

duce

dvi

rule

nce

Cf-

Ecp2

Not

clon

edN

o(1

29)

Ecp

411

9(1

01)

618

Unk

now

nA

popl

ast

Inpl

anta

Unk

now

nC

f-Ec

p4N

otcl

oned

No

(75)

Ecp

511

5(9

8)6

17U

nkno

wn

Apo

plas

tIn

plan

taU

nkno

wn

Cf-

Ecp5

Not

clon

edN

o(7

5)

Ecp

622

2(1

99)

823

LysM

-dom

ains

;ch

itin-

bind

ing

Apo

plas

tIn

plan

taK

nock

-dow

nle

adst

ore

duce

dvi

rule

nce

Unk

now

nN

o(1

3)

Ecp

7?

(100

)6

?U

nkno

wn

Apo

plas

tIn

plan

taU

nkno

wn

Unk

now

nU

nkno

wn

(13)

Lept

osph

aeri

am

acul

ans(

oilse

edra

pe):

AvrL

m1

205

(183

)1

22U

nkno

wn

Prob

ably

incy

topl

asm

Inpl

anta

and

invi

tro

Unk

now

nRl

m1

Not

clon

edYe

s(4

9)

AvrL

m6

144

(124

)6

20U

nkno

wn

Prob

ably

inap

opla

stIn

plan

taan

din

vitr

oU

nkno

wn

Rlm

6N

otcl

oned

Unk

now

n(4

4)


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AvrL

m4-

714

3(1

22)

821

Unk

now

nPr

obab

lyin

apop

last

Inpl

anta

(mai

nly)

and

invi

tro

Unk

now

nRl

m4

and/

orRl

m7

Not

clon

ed

Yes

(95)

Fusa

rium

oxys

poru

mf.

sp.l

ycop

ersic

i(to

mat

o):

Avr3 (Six

1)28

4(1

89)

821

Unk

now

nX

ylem

Stim

ulat

edby

livin

gce

lls

Req

uire

dfo

rfu

llvi

rule

nce

I-3 Not

clon

edU

nkno

wn

(99;

M.R

eppe

rs.

com

m.)

Six2

232

(172

)8

20U

nkno

wn

Xyl

emIn

plan

taPr

obab

lyno

tre

quir

edfo

rvi

rule

nce

Unk

now

nU

nkno

wn

(55;

M.R

eppe

rs.

com

m.)

Six3

163

(144

)3

(2)

19U

nkno

wn

Xyl

emIn

plan

taR

equi

red

for

full

viru

lenc

eI-

2(C

C-

NBS

-LR

R)

Unk

now

n(5

5;M

.Rep

pers

.co

mm

.)Av

r1 (Six

4)24

2(1

84)

617

Unk

now

nX

ylem

Inpl

anta

Supp

ress

ion

ofI-

2an

dI-

3re

sista

nce

Ior

I-1

Not

clon

edU

nkno

wn

(55;

M.R

eppe

rs.

com

m.)

Mag

napo

rthe

oryz

ae(r

ice)

:Av

r-Pi

ta22

4(1

76)

816

Hom

olog

yto

Met

allo

pro-

teas

es

Cyt

opla

smIn

plan

taN

otre

quir

edfo

rvir

ulen

ceon

rice

Pi-t

a(C

C-

NBS

-LR

R)

Yes

(93)

Avr- Pita

222

4(?

)8

16H

omol

ogy

toM

etal

lopr

o-te

ases

Prob

ably

inap

opla

stU

nkno

wn

Prob

ably

not

requ

ired

for

viru

lenc

eon

rice

Pi-t

a(C

C-

NBS

-LR

R)

Yes

(71)

Avr- Pita

322

6(?

)8

16H

omol

ogy

toM

etal

lopr

o-te

ases

Prob

ably

incy

topl

asm

Unk

now

nPr

obab

lyno

tre

quir

edfo

rvi

rule

nce

onri

ce

Unk

now

nYe

s(7

1)

Pwl1

147

(124

)2

23G

lyci

ne-r

ich

hydr

ophi

licpr

otei

n

Prob

ably

inap

opla

stU

nkno

wn

Unk

now

nU

nkno

wn

Unk

now

n(7

0)

Pwl2

145

(126

)1(

1)21

Gly

cine

-ric

hhy

drop

hilic

prot

ein

Prob

ably

inap

opla

stU

nkno

wn

Unk

now

nU

nkno

wn

Unk

now

n(1

18)

Pwl3

137

(116

)0

21G

lyci

ne-r

ich

hydr

ophi

licpr

otei

n

Prob

ably

inap

opla

stU

nkno

wn

Non

-fu

nctio

nal

Unk

now

nU

nkno

wn

(70)

(Con

tinue

d)


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Tab

le1

(Con

tinue

d)

Effe

ctor

prot

ein

Aa

resi

dues

(mat

ure)

Cys

tein

esSi

gnal

PFu

ncti

on/

hom

olog

yL

ocal

izat

ion

inpl

ant

Exp

ress

ion

Rol

ein

viru

lenc

eR

-gen

e(t

ype)

Pos

itiv

ese

lect

ion

Ref

eren

ces

Pwl4

138

(117

)0

21G

lyci

ne-r

ich

hydr

ophi

licpr

otei

n

Prob

ably

inap

opla

stU

nkno

wn

Non

-fu

nctio

nal

Unk

now

nU

nkno

wn

(70)

Ace

140

3543

-H

ybri

dpo

lyke

tide

synt

hase

/no

nrib

osom

alpe

ptid

esy

nthe

tase

Not

secr

eted

Exp

ress

edin

appr

esso

ria

Unk

now

nPi

33U

nkno

wn

(11)

Avr1

-C

O39

Not

clon

edye

t

--

Unk

now

nU

nkno

wn

Unk

now

nU

nkno

wn

Pi-C

O39

(t)Ye

s(3

7)

Rhyn

chos

pori

umse

calis

(bar

ley)

:N

ip1

82(6

0)10

22N

on-s

peci

ficto

xin/

indu

ces

necr

osis

and

plas

ma-

mem

bran

eH

+AT

Pase

Prob

ably

inap

opla

stE

xpre

ssed

invi

tro

Not

requ

ired

forv

irul

ence

Rrs-

1N

otcl

oned

Yes

(101

)

Nip

210

9(?

)7

(6)

16N

on-s

peci

ficto

xin/

indu

ces

necr

osis

Prob

ably

inap

opla

stE

xpre

ssed

invi

tro

Not

requ

ired

forf

ull

viru

lenc

e

Unk

now

nYe

s(1

01;W

.K

nogg

epe

rs.

com

m.)


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Nip

311

5(?

)9

(8)

17N

on-s

peci

ficto

xin/

indu

ces

necr

osis

Prob

ably

inap

opla

stE

xpre

ssed

invi

tro

Not

requ

ired

forf

ull

viru

lenc

e

Unk

now

nYe

s(1

01;W

.K

nogg

epe

rs.

com

m.)

Mela

mps

ora

lini(

flax)

:Av

rL56

7(A

,Ban

dC

)

150

(127

)1

23U

nkno

wn

Cyt

opla

smE

xpre

ssed

inha

usto

ria

Unk

now

nL5

,L6

and

L7(T

IR-N

BS-

LR

R)

Yes

(27)

AvrM

314

128

Unk

now

nC

ytop

lasm

Exp

ress

edin

haus

tori

aan

din

vitr

o

Unk

now

nM

(TIR

-N

BS-L

RR

)Ye

s(1

7)

AvrP

123

117

(94)

11(1

0)23

Kaz

alSe

rpr

otea

sein

hibi

tor

Cyt

opla

smE

xpre

ssed

inha

usto

ria

Unk

now

nP,

P1,P

2an

d/or

P3(T

IR-N

BS-

LR

R)

Yes

(17)

AvrP

495

(67)

7(6

)28

Cys

tine

knot

ted

pept

ide

Cyt

opla

smE

xpre

ssed

inha

usto

ria

Unk

now

nP4

(TIR

-N

BS-L

RR

)Ye

s(1

7)

Blum

eria

gram

inis

f.sp

.hor

dei(

barl

ey):

Avra

1028

64

->

30pa

ralo

gues

inBg

han

dot

herf

.sp.

Prob

ably

incy

topl

asm

Unk

now

nU

nkno

wn

Mla

10(C

C-

NBS

-LR

R)

Unk

now

n(1

00)

Avrk

117

73

->

30pa

ralo

gues

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HR by jettison of the Avr4E gene, indicatingthat the fitness penalty associated with the lossof this gene is probably not very high (116).However, Avr4E-expressing tomato transfor-mants are more susceptible to natural C. fulvumstrains that lack Avr4E than control plants, sug-gesting that Avr4E is a virulence factor (H.P. vanEsse and B.P.H.J. Thomma, personal commu-nication). No homologs of Avr4E are presentin public databases and its molecular functionis still unknown (145).

Avr9 effector. Avr9 encodes a 63 aa prepro-tein that is C- and N-terminally processed byfungal and plant proteases into a mature 28 aaprotein with six cysteine residues (129, 141).The 3-D structure of Avr9 resembles cystine-knotted peptides (134, 143), which share struc-tural, but little functional, homology (94). Ala-nine scanning of the Avr9 peptide revealed thatall six cysteine residues present in this pro-tein are essential for its structure and necrosis-inducing activity (73). Based on its overalltertiary structure, Avr9 shows structural butnot functional homology to carboxypeptidaseinhibitors (133, 134). Disruption of Avr9 inC. fulvum by homologous recombination didnot affect growth in vitro or virulence ontomato plants, suggesting that Avr9 is not re-quired for full virulence (86). Indeed, all nat-ural strains that evade Cf-9-mediated HR lackthe Avr9 gene (116). However, Avr9-expressingtomato transformants are more susceptible tonatural C. fulvum strains that lack Avr9 thancontrol plants, suggesting that Avr9 may bea redundant virulence factor (H.P. van Esseand B.P.H.J. Thomma, personal communica-tion). Expression of Avr9 in vitro is inducedunder nitrogen-limiting conditions, suggestingthat the produced protein might be involvedin nitrogen metabolism (96, 122, 128). Fur-thermore, an Nrf1 (nitrogen responsive factor)gene has been identified in C. fulvum, and Nrf1deletion mutants no longer express Avr9 undernitrogen-limiting conditions and are compro-mised in their virulence on Cf-0 tomato plants.However, these strains are still avirulent onCf-9 tomato plants, suggesting that Nrf1 is

a major, but not an exclusive, positive regu-lator of Avr9 expression (96). Expression ofall the other Avr and Ecp effector genes ofC. fulvum that have been characterized so faris not induced under nitrogen-limiting condi-tions, and although they are all expressed inplanta, their regulators have yet to be identified(12, 122).

Ecp effectors. In addition to the Avrs, six moreeffector genes that code for Ecps have beencloned from C. fulvum, namely Ecp1, Ecp2, Ecp4,Ecp5, Ecp6, and Ecp7 (13, 75, 129). Ecps areabundantly secreted by all strains of C. fulvumduring infection and possess an even number ofcysteine residues that are most likely involvedin intramolecular disulfide bridges (80). MostEcps share little or no homology with otherproteins present in public databases, except forEcp6 which, so far, is the only effector proteinof C. fulvum with orthologs in many other fun-gal species. This is mainly due to the presenceof LysM-domains in this protein, which in gen-eral are implicated in carbohydrate binding in-cluding chitin, suggesting that Ecp6 might be afunctional homolog of Avr4 (13). Alternatively,Ecp6 might also be involved in scavenging ofchitin fragments that are released from the fun-gal cell wall during infection by plant chitinases,thus preventing them from acting as PAMPsand eliciting basal defense responses (13). Ecp6,Ecp1, and Ecp2 are all virulence genes becausesilencing or disruption of these genes compro-mises virulence of C. fulvum on tomato (13, 77).For Ecp1, Ecp2, Ecp4, and Ecp5, tomato acces-sions have been identified that carry a singledominant Cf-Ecp gene and develop an HR uponinoculation with recombinant potato virus X(PVX) strains that express these Ecps, or af-ter injection of the purified Ecp proteins (75,76). However, although most Cf-Ecp genes havebeen genetically mapped, they still remain to becloned (22, 113). In contrast to Cf-genes, Cf-Ecpgenes have not yet been deployed in commer-cial tomato lines, which might explain why onlya limited number of polymorphisms has beenobserved in Ecp genes of natural populations ofC. fulvum (13, 116).


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Fusarium oxysporum f. sp. lycopersiciThe vascular pathogen Fusarium oxysporum isan asexual fungus with a broad host range andcauses wilt and root diseases. However, mostisolates of F. oxysporum can infect only a singleor a few plant species and are therefore sub-divided into different formae speciales (f. sp.)based on their host specificity (48). Thus, strainsof F. oxysporum that cause tomato wilt are re-ferred to as f. sp. lycopersici.

Six effectors. F. oxysporum f. sp. lycopersici(Fol) is an extracellular pathogen that colo-nizes the xylem vessels of tomato. Four smallproteins, designated Six1 to Six4 (secreted inxylem), have been identified in Fol and areproduced during infection. Six1 (currently re-named Avr3) is a small cysteine-rich proteinof approximately 32 kDa that is N- and C-terminally processed by either fungal and/orplant proteases into a 12 kDa mature proteinor an alternative form of 22 kDa, and which isalso proven to be the active form of the pro-tein (98, 99). Avr3 is required for full virulenceon tomato, but it also behaves as an Avr factorthat triggers HR in the presence of the cog-nate I-3 resistance gene (56, 99). Interestingly,expression of Avr3 has been detected only dur-ing root colonization and infection of tomatoplants, but it is neither cultivar-specific nor de-pends on morphological features of the roots.Instead, the presence of living plant tissue inthe vicinity of the fungus seems not only re-quired for Avr3 expression but might also trig-ger a switch from a saprophytic to pathogeniclifestyle (136). In the genome of Fol, Avr3 re-sides in an area of a small chromosome thatcontains many transposable elements and thusresembles pathogenicity islands. Pathogenic-ity islands that harbor several virulence genesoften reside on dispensable chromosomes andtheir existence has been described for other fun-gal pathogens as well, including Alternaria spp.(121), Nectria hematococca (79), L. maculans, B.graminis f. sp. hordei, M. oryzae, and possiblyothers (5, 70, 71, 111). Indeed, a large genomicarea of approximately 8 kb that contains Avr3 as

well as Six2 could not be found outside the f. sp.lycopersici lineage nor in nonpathogenic Fusar-ium strains, suggesting that it represents a dis-pensable region that confers virulence specif-ically on tomato (135). In addition, the f. sp.lycopersici lineage-specific effector gene Six3also resides on the same chromosome as Avr3and Six2, although not within the boundaries ofthe 8 kb region that harbors the latter two genes.This suggests that the ability to cause diseasehas likely arisen only once during evolution ofFol by acquisition or emergence of the genomicregion harboring the virulence genes neces-sary for infection of tomato. Subsequently, thisregion might have spread to other clonal Folstrains by horizontal gene transfer (135). Be-cause the presence of Avr3 is required for fullvirulence on tomato, it raises the question howFol strains can overcome I-3-mediated resis-tance, as loss of Avr3 would pose a serious fit-ness penalty and mutants with only point mu-tations in Avr3 that avoid recognition by I-3but still remain virulent have not been reported.The recent functional characterization of Avr1(previously known as Six4) might provide theanswer to this question. Avr1 is a small cysteine-rich secreted protein that confers avirulence toFol strains on tomato lines carrying the I or I-1resistance gene. However, unlike Avr3, Avr1 isnot required for full virulence of Fol strains ontomato plants that lack the cognate R gene. Incontrast, Avr1 functions as a suppressor of I-2- and I-3-mediated resistance (54). Indeed, al-though Six3, which is required for full virulence(M. Rep, personal communication), is presentin strains of Fol that are both virulent and avir-ulent on I-3 tomato lines, Avr1 is only presentin Fol strains virulent on I-3 lines. When strainsof Fol that are avirulent on I-2 and/or I-3 lineswere transformed with Avr1 they gained viru-lence on these lines, indicating that Avr1 sup-presses both I-2- and I-3-mediated resistance.The underlying molecular mechanism of sup-pression is not yet known, but an evolutionarymodel for this phenomenon has been proposed(54). In this model, it is suggested that the I-3resistance gene has evolved to recognize Avr3but because Avr3 is required for full virulence


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of Fol, evasion of I-3 recognition through lossof Avr3 would cause a serious fitness penalty.Therefore, Avr1 might have been acquired byFol in order to avoid the fitness penalty as-sociated with the loss of Avr3 (and likely ofSix2) in overcoming I-2- and I-3-mediated re-sistance. This explains why all Fol strains an-alyzed so far contain Avr3, whereas Avr1 ispresent only in strains that are virulent on I-2and/or I-3 lines. It is likely that the I-3 proteinoperates in accordance with the guard model,where not the Avr3 protein, but the manipula-tion of its virulence target is recognized by I-3,whereas Fol strains can (partially) regain viru-lence toward I-3-containing lines by acquisitionof Avr1. During evolution, tomato respondedto this adaptation with the development of theI or the unlinked I-1 resistance gene that specif-ically recognizes and responds to Avr1 (54).

Leptosphaeria maculansAt least nine distinct Avr genes, designatedAvrLm1 to AvrLm9, have been geneticallyidentified in L. maculans, the causal agent ofstem canker on oilseed rape, that cause avir-ulence on plants carrying the cognate Rlm1 toRlm9 R genes, respectively. These Avr geneshave been mapped to at least four unlinkedgenomic regions, including the genetic clus-ters AvrLm1-AvrLm2-AvrLm6 and AvrLm3-AvrLm4-AvrLm7-AvrLm9 (5).

AvrLm1 and AvrLm6 effectors. AvrLm1 andAvrLm6 are the first Avr genes that have beencloned from L. maculans using a map-basedcloning strategy (44, 49). The two Avrs are inrelatively close proximity at a locus that alsoharbors AvrLm2 (5). More specifically, AvrLm1has been mapped on a 269 kb AT rich, gene-poor heterochromatin-like region that consistsof a number of degenerated, nested copies offour long-terminal repeat (LTR) retrotrans-posons and is surrounded by GC-rich isochors.Also, AvrLm6 maps within a 133 kb noncodingregion that mainly contains LTR retrotrans-posons. However, both AvrLm1 and AvrLm6share a low GC content, in contrast to most

other fungal Avr genes cloned so far. BothAvrLm1 and AvrLm6 are single copy genes, en-coding small secreted proteins of 205 aa and144 aa, respectively, that have no known ho-mologs in public databases and lack any charac-teristic signatures that could point toward a pu-tative intrinsic function. AvrLm1 and AvrLm6are strongly induced in planta, particularly dur-ing the early stages of infection, and expres-sion of the genes has also been observed invitro, although here expression of AvrLm1 ismuch higher than that of AvrLm6. Despite theclear avirulence properties of the AvrLm1 andAvrLm6 genes, the cognate R genes Rlm1 andRlm6 have not been cloned yet. Therefore, itis currently unknown whether these effectorsinteract directly or indirectly with their cog-nate R proteins and whether recognition oc-curs outside or inside the host cells. In that re-spect, AvrLm6 might be secreted and reside inthe apoplast as it contains six cysteine residuesthat could provide stability by forming disul-fide bridges. In contrast, AvrLm1 contains onlyone cysteine residue, making it different fromAvrLm6 and the apoplastic cysteine-rich effec-tor proteins of C. fulvum and F. oxysporum f. sp.lycopersici discussed above, and therefore, uptakeof AvrLm1 into the host cell seems more likely(49).

As has been observed for other fungi,AvrLm1 is absent in races that are virulent onRlm1 cultivars. Recently, a gain of virulence onRlm1 plants was observed in field populationsof L. maculans in France that was due to a spe-cific 260 kb deletion of a chromosomal segmentspanning AvrLm1. A similar deletion was ob-served in more than 90% of 460 field isolatesanalyzed from a world population, indicatingthat jettison of the AvrLm1 is the main mech-anism of adaptation to Rlm1 without causing asignificant fitness penalty for the fungus (6, 7,104).

AvrLm4-7 effector. Using a map-basedcloning strategy, a genetic locus of 238 kbcontaining the AvrLm7 gene has been recentlydelineated in L. maculans that shows thesame characteristics as the AvrLm1-2-6 locus,


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including the presence of multiple LTR retro-transposons and an AT-rich isochoric regionnext to a GC-equilibrated one (95). In total, 40genes were predicted on this locus, 35 of whichare present in the GC-equilibrated regionand only five in the AT-rich isochors. A singlegene (AvrLm7) was identified in the gene-poor60 kb AT-rich region that confers avirulenceon both Rlm7 and Rlm4 genotypes. This genewas consequently renamed AvrLm4-7 for thedual specificity of its encoded protein towardthe Rlm4 and Rlm7 genes. AvrLm4-7 encodes aputatively secreted preprotein of 143 aa that isfurther processed into a 122 aa mature proteinwith eight cysteine residues and no homologyto any other proteins currently present inpublic databases. Expression of AvrLm4-7 isconsiderably upregulated during primary leafinfection, reaching a maximum at seven dayspost inoculation, whereas only low levels ofexpression are observed during in vitro growthof the fungus. Analysis of 300 field isolates ofL. maculans showed that complete or partialdeletion of the AvrLm4-7 gene is the mainmechanism for gaining virulence on both Rlm4and Rlm7 genotypes, whereas most isolatesvirulent on Rlm4 genotypes alone showedonly a single point mutation in the producedAvrLm4-7 protein (Gly120>Arg). Such strainswere less fit than strains with the wild-typeAvrLm4-7 allele, indicating that AvrLm4-7 isimportant for fitness of the fungus. No strainsvirulent only on Rlm7 plants were observed(95).

Magnaporthe oryzaeMagnaporthe oryzae (formerly known as M.grisea) (21) is the causal agent of rice blast. Re-sistant rice lines have been used extensively overthe past decades to battle the disease, but Rgenes present in these lines have often beenovercome rather quickly by the emergence ofvirulent races. The rice blast pathosystem com-plies with the gene-for-gene model, and to datemore than 40 major R genes, designated Pi,have been identified. So far, five cultivar- andspecies-specific Avr genes have been cloned and

characterized from M. oryzae, including Avr-Pita (93) that was originally called Avr2-YAMO(125), Avr1-CO39 (38), Pwl2 (118, 125), Pwl1(70), and Ace1 (11). Avr1-CO39 was isolatedfrom an M. oryzae isolate pathogenic on weep-ing lovegrass and controls avirulence on riceplants carrying the cognate Pi-CO39(t) resis-tance gene (18, 38). Almost all M. oryzae isolatesthat are virulent on CO39-rice cultivars lack theAvr1-CO39 gene (37). Avr-Pita and Ace1 werecloned from M. oryzae isolates pathogenic onrice, whereas Pwl2 was originally identified ina genetic cross between two laboratory strainsthat infected rice. However, unlike the othertwo genes, Pwl2 is a species-specific gene thatconfers avirulence on weeping lovegrass but noton any known rice cultivars (118).

Avr-Pita effector. Avr-Pita encodes a pre-sumably secreted preprotein of 223 aa with ho-mology to fungal zinc-dependent metallopro-teases. This protein is further processed into anactive 176 aa mature protein (Avr-Pita176) thatis dispensable for virulence on rice (59, 93). Avr-Pita176 interacts directly with the cognate Pi-taresistance protein, a predicted 928 aa receptor-like protein with a central nucleotide-bindingsite (NBS) and a C-terminal leucine-rich repeat(LRR) domain. Direct interaction between thetwo proteins has been demonstrated by yeasttwo-hybrid assays and by in vitro binding as-says that showed binding of the Avr-Pita176 tothe LRR domain of Pi-ta (59). Furthermore,coexpression of the two proteins in rice cellselicited plant defense responses, suggesting thatphysical interaction inside host cells is requiredfor activation of Pi-ta-mediated defense. In thesame experiments, it was also shown that therecognition specificity for Avr-Pita is deter-mined by a difference in one aa residue (Ala-918) of the Pi-ta protein present in resistant vssusceptible rice varieties. On the other hand,many mutations have been described in Avr-Pita that result in gain of virulence on Pi-tarice cultivars. In the genome of M. oryzae, Avr-Pita resides close to the telomere of chromo-some 3, and this might account for the observedmolecular instability of the genomic region


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containing this gene. Indeed, an array of mu-tations has been described on the Avr-Pita lo-cus in virulent strains of the fungus, includingvarious size deletions, point mutations, and atransposon insertion (15, 58, 69, 93, 150).

Avr-Pita-related effectors. Recently, it wasshown that Avr-Pita (currently renamed to Avr-Pita1) belongs to a gene family with at least twoadditional members, Avr-Pita2 and Avr-Pita3(71). Avr-Pita2 acts as elicitor of defense re-sponses mediated by Pi-ta, but Avr-Pita3 doesnot. Members of the Avr-Pita family are widelydistributed among strains of M. grisea isolatedfrom diverse hosts, including isolates that arenot pathogenic on rice. However, althoughAvr-Pita1 and Avr-Pita2 are present in bothM. oryzae and M. grisea isolates, Avr-Pita3 ispresent only in M. oryzae isolates, suggestingthat Avr-Pita1 and Avr-Pita3 are derived from agene duplication event that must have occurredafter separation of M. oryzae from M. grisea (71).

Ace1 effector. Ace1 encodes a putative 4035aa cytoplasmic fusion polypeptide containinga polyketide synthase (PKS) and a nonriboso-mal peptide synthetase (NRPS), two distinctclasses of enzymes that are involved in the pro-duction of microbial secondary metabolites (11,20). Ace1 seems to mediate avirulence indirectlyin the presence of the Pi33 rice R gene, by itsinvolvement in the biosynthesis of a secondarymetabolite that most likely activates Pi33. Thenature of the secondary metabolite is not knownyet, but mutations in the PKS domain of Ace1abolish activation of Pi33, indicating that atleast Ace1-mediated biosynthetic activity is re-quired for avirulence on Pi33 plants (11). Ace1 isexclusively expressed in appressoria, suggestingthat the produced secondary metabolite mighthave a role in virulence, although mutants inwhich Ace1 was deleted were not compromisedin virulence (11, 43).

Pwl effectors. Members of the Pwl(pathogenicity toward weeping lovegrass)gene family encode rapidly evolving smallglycine-rich secreted proteins that are

generally found in rice pathogens. At least fourmembers of this family, designated Pwl1 toPwl4 are present in M. oryzae, and act as Avrgenes conferring species-specific avirulenceon weeping lovegrass and finger millet buthave no effect on rice (70, 118). Pwl2 encodesa secreted glycine-rich, hydrophilic proteinof 145 aa that confers avirulence on weepinglovegrass (118). This gene is located on ahighly unstable genetic locus, where frequentgenetic rearrangements associated with largedeletions lead to the emergence of spontaneousmutants virulent on weeping lovegrass. Pwl1(75% aa identity with Pwl2) and the allelicPwl3 (51% aa identity with Pwl2) and Pwl4(57% aa identity to Pwl2) were identifiedbased on homology to Pwl2, but only Pwl1is a functional homolog of Pwl2, conferringavirulence on weeping lovegrass. However,Pwl4 could be made functional when expressedunder the control of the Pwl2 promoter, whichwas not the case for Pwl3 (70).

Rhynchosporium secalisRhynchosporium secalis, the causal agent of leafscald on barley, secretes three low molecularweight peptides, designated Nip1 to Nip3, thatfunction as nonspecific toxins on barley andseveral other plants. Nip1 and Nip3 are pro-duced both in vitro and in planta, and their pres-ence correlates with the formation of necroticlesions that are most likely caused by an in-direct stimulation of H+-ATPase activity inplant plasma membranes (146, 147). In addi-tion, Nip1 triggers specific (non-HR) defenseresponses in barley cultivars carrying the Rrs1resistance gene (51). Nip1 (also known as Avr-Rrs1) encodes a preprotein of 82 aa, which ma-tures into a 60 aa protein after removal of thesignal sequence. Ten cysteine residues (101) arepresent in the mature Nip1 protein that is in-volved in five intramolecular disulfide bonds(45). NMR spectrometry showed that Nip1mainly consists of two structural domains, onewith two !-sheets and another with three an-tiparallel strands (127). Strains of R. secalis vir-ulent on Rrs1 plants either lack Nip1 or carry


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alleles with point mutations that translate intosingle aa substitutions (72, 101, 105). Two ofthese aa substitutions are correlated with gain ofvirulence, and lack of H+-ATPase activity andnecrosis-inducing activity, suggesting that boththe necrosis induction and elicitor activity aremediated by a single receptor that is most likelydifferent from the Rrs1 protein (39). Recently,it was found that Nip1 interacts with a sin-gle plasma membrane receptor that is involvedin both mediating virulence and triggering de-fense, but the exact receptor has not yet beencharacterized (126). A recent study of field pop-ulations of the pathogen showed clear evidenceof positive diversifying selection operating onthe Nip1 locus (105). In total, 14 Nip1 isoformswere identified, of which at least three were cor-related with gain of virulence on Rrs1 plants,while a high deletion frequency of Nip1 wasalso observed that was much higher than thatobserved for Nip2 and Nip3. As single aminoacid substitutions in Nip1 that correlated withgain of virulence on Rrs1 plants were observedin much lower frequencies than gene deletions,the fitness cost associated with the loss of thisgene is likely not high (105).

Recently, the Nip2 and Nip3 genes have alsobeen cloned from R. secalis (W. Knogge, per-sonal communication). Nip2 encodes a 109 aaprotein with a predicted signal peptide of 16aa, whereas Nip3 encodes a 115 aa protein witha predicted signal peptide of 17 aa. Both pro-teins have one cysteine residue in their sig-nal sequences, and the mature Nip2 and Nip3carry six and eight cysteines, respectively. Nip2and Nip3 are also further processed at their Ctermini, but as the cleavage sites are not yetknown, the exact number of aa (including cys-teine) residues present in the mature proteinsremains to be determined (W. Knogge, per-sonal communication).

EFFECTORS FROMHAUSTORIUM-FORMINGFUNGAL PATHOGENSRusts and powdery mildews are obligatepathogenic fungi that produce haustoria in the

epidermis (powdery mildews) or in mesophyllcells (rust fungi) of their hosts during infec-tion. Haustoria are not only specialized feedingstructures required for acquisition of nutrients,but they also induce structural, cellular, and bio-chemical changes in the invaded host cells. Inaddition, they can facilitate delivery of effec-tors into the extrahaustorial matrix, several ofwhich are subsequently translocated into hostcells (16). Here, we discuss effectors producedby the flax rust fungus M. lini and the barleypowdery mildew fungus B. graminis f. sp. hordei.

Melampsora liniThe flax rust fungus M. lini is an obligate basid-iomycete that infects flax (Linum usitatissimum)and other species of the genus Linum. At least30 Avr genes corresponding to approximately30 cognate flax R genes have been identifiedin genetic analyses (33). Flax R proteins are allmembers of the intracellular TIR-NBS-LRRclass and are distributed among five highly poly-morphic loci designated K, L, M, N, and P (4,29, 30, 32, 35, 78). To date, Avr genes have beencloned from four M. lini loci, namely AvrL567,AvrM, AvrP123, and AvrP4 that code for haus-torially expressed secreted proteins (HESPs)and elicit HR in flax plants that carry the cog-nate R genes (17). Each of the characterizedAvr loci consists of one to five closely relatedhomologous genes that code for small secretedproteins with no sequence similarity to otherproteins present in public databases. Tran-sient expression of the mature Avr proteins inflax plants carrying the cognate cytoplasmic Rproteins induces an HR, indicating that theyare translocated via the extrahaustorial matrixinto host cells during infection (17, 27). TheAvrL567A, AvrL567B, and AvrL567C genescluster at the AvrL567 locus and trigger HRin 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 conservedN-terminal signal sequence resulting in a 127aa mature protein. Specific expression of thesegenes in haustoria suggests that they might playa role in virulence. Approximately 25% of theaa residues within the mature protein retain


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one or more polymorphisms, and several ofthese have been shown to affect a transitionto virulence on plants carrying the cognate Rproteins, suggesting that the Avr567 locus isunder positive diversifying selection. Indeed,in a survey involving just six flax rust strains,twelve AvrL567 sequence variants (known asAvrL567A to AvrL567L) were identified, six ofwhich (A, B, D, F, J, and L) were avirulent vari-ants that triggered HR in flax lines carrying theL5, L6, or L7 genes, and five (C, G, H, I, andK) were virulent variants that no longer trig-gered HR on these lines (28). These virulentvariants exhibited substitutions in aa residuesthat are exposed to the surface of the proteinand interact directly with the cognate R pro-teins, as confirmed in yeast two-hybrid assays(34, 144). Therefore, evading host recognitionis most likely the source of the diversifying se-lection acting on the Avr567 locus. Other char-acterized HESPs from flax rust include AvrMthat is recognized by the M resistance protein,AvrP4 that is recognized by P4, and the complexAvrP123 proteins that are variously recognizedby P, P1, P2, and/or P3 resistance proteins (17).As for AvrL567, diversifying selection also op-erates at the AvrM, AvrP4, and AvrP123 lociwith virulent flax strains carrying mutated alle-les that encode Avrs that are no longer recog-nized by the cognate R proteins (17). At leastfive different paralogs (AvrMA to AvrME) havebeen detected at the AvrM locus of an avirulentstrain, although one paralog encodes an effec-tor that is not recognized by any known flax Rprotein. The six AvrM proteins have no knownhomologs in the public databases and show sig-nificant sequence and size variations caused byDNA insertions, deletions, or polymorphismsin the location of stop codons. AvrP123 pro-teins contain ten cysteine residues includingthe characteristic CX7CX6YX3CX2-3C sig-nature present in the Kazal family of serineprotease inhibitors, suggesting that host pro-teases might be a target of these effectors.AvrP4 also encodes a protein with six cys-teine residues at the 28 aa C-terminal part ofthe mature protein that show a spacing (CX3-7CX4-6CX0-5CX1-4CX4-10C) typical for

cystine-knotted peptides (94). Both AvrM andAvrP4 are expressed in planta, whereas AvrMis also expressed in vitro. Transient intracellu-lar expression of AvrM and AvrP4 in flax plantscarrying the cognate R genes triggers an HR,suggesting that effector translocation into thehost cells occurs during infection, which is con-sistent with the predicted cytoplasmic locationof M and P resistance proteins (4, 78). However,both effectors also induce an HR in flax whentargeted into the apoplast, suggesting their re-entry from the apoplast into host cells after se-cretion (17, 27, 31). A role in virulence for theAvr genes of M. lini has not been shown yet.

Blumeria graminis f. sp. hordeiPowdery mildews are a large group of as-comycete obligate biotrophic fungi that showa high degree of host specialization and, likerusts, produce haustoria in their host plants(46). Blumeria graminis f. sp. hordei (Bgh) causespowdery mildew on barley and interacts withits host in a gene-for-gene manner (149). Atleast 85 dominant or semidominant mildew Rgenes (Ml) with different recognition speci-ficities have been characterized in barley, in-cluding Mlk genes and 28 highly homologousgenes that all map to the Mla (mildew A) lo-cus of barley chromosome 5 (57, 65). Six ofthe genes present at this locus, namely Mla1,Mla6, Mla7, Mla10, Mla12, and Mla13, havebeen cloned, and they all encode highly relatedintracellular CC-NBS-LRR type of R proteins.However, despite the high sequence similarity(>90% identity), they all recognize isolate-specific effectors of Bgh (52, 53, 109). TwoAvr genes residing within a proximity of 30kb in the genome have been cloned from Bgh(100). They are designated Avrk1 and Avra10and induce defense responses in barley vari-eties containing the cognate Mlk1 and Mla10R proteins, respectively. Both genes belong toa large multigene family of more than 30 par-alogs in Bgh, whereas homologs are present informae speciales that are pathogenic on othergrasses. 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 ge-nomic regions that contain clusters of severalrecombining Avr genes have also been reported(14). The predicted 286 and 177 aa proteins en-coded by Avra10 and Avrk1, respectively, shareapproximately 60% similarity with each other,but both lack an N-terminal signal sequenceor a signature for uptake by host cells, suchas the RXLR motif present in oomycete ef-fectors (67). This suggests an alternative butyet unidentified route of delivery of these ef-fectors into host cells, where they exert theirputative virulence function and induce defenseresponses mediated by the cognate R proteinsin resistant plants (100). Recently, it was shownby fluorescence microscopy that the majorityof the Mla10 protein is localized in the cyto-plasm and approximately 5% in the nucleus(9, 108). Perturbation of nucleo-cytoplasmicMla10 partitioning by expression of an Mla10fusion protein containing a nuclear export sig-nal (NES) that enhances nuclear export overimport, decreased Mla10-specified disease re-sistance (108). In the nucleus, Mla10 showedan Avr10-dependent physical association withtwo WRKY transcription factors (HvWRKY1and HvWRKY2 TFs), suggesting that theseTFs serve as immediate downstream targets ofthe activated receptor. The effector-dependentassociation between barley Mla10 and WRKYTFs contributes to Mla10-mediated resistanceand host cell death at attempted fungal infec-tion sites (108). The WRKY TFs interactingwith Mla10 presumably act as repressors of PTIand might have a role in keeping basal defensebelow a certain threshold.

FUTURE HUNTS FOR FUNGALEFFECTORS BY COMPARATIVEGENOMICSGenetic and biochemical approaches basedon map-based cloning and analyses of fun-gal secretomes during infection have beenthe two most popular strategies to iden-tify effector genes from the pathogens dis-cussed above. Other, but less widely ap-plied, methods for discovery of effector genes

include a functional high-throughput screen-ing for identification of HR-inducing cDNAsfrom plant pathogens based on Agrobacteriumtumefaciens/PVX-mediated cDNA expressionin host plants (119), a mutagenesis approachusing restriction enzyme-mediated integration(REMI) (84), and screening of EST librariesfor genes upregulated during infection, as wassuccessfully applied for AvrL567 of M. lini(27). However overall these approaches havebeen very labor intensive, and their success ratewas relatively low. Recently, whole genome se-quencing of fungal pathogens has provided anenormous amount of data that can be analyzedfor putatively secreted cysteine-rich proteins.Narrowing down the spectrum of additional ef-fector candidates can be achieved by integra-tion of genome transcriptome, proteome, andmetabolome data, when available. Comparativesecretome analysis and BLAST sequence simi-larity searches could also be used for the iden-tification of potential effectors in sequencedgenomes, but these methods have limitationsdue to low sequence similarities among fungaleffector genes. However, this approach provedsuccessful with the recent identification of thefirst homologs of the C. fulvum effector genesAvr4, Ecp2, and Ecp6 in the genome of thebanana pathogen Mycosphaerella fijiensis (117)and the discovery of a functional homolog ofthe toxic peptide ToxA from Stagonospora nodo-rum in Pyrenophora titici-repentis (41, 42). Nev-ertheless, despite the great potential of usinggenome-wide searches for identifying candi-date effector genes, their function still needsto be confirmed experimentally by overexpres-sion, gene disruption or silencing in fungal iso-lates, and subsequent (a)virulence assays on hostplants.

FUNCTIONS AND HOSTTARGETS OF EFFECTORS

Extracellular and CytoplasmicFungal Effectors

Fungal effector proteins can be roughlygrouped into extracellular effectors that are


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secreted into the apoplast or xylem of theirhost plants and cytoplasmic effectors that aretranslocated into host cells (Table 1). Ace1from M. grisea is the only known fungal ef-fector protein that is not secreted by the fun-gus, but instead is suggested to be involved inthe biosynthesis of a yet unknown secondarymetabolite that is likely secreted (11). Despitethe low degree of sequence conservation amongfungal effectors, most of them code for small se-creted proteins, of which some are translocatedinto host cells by a yet unknown mechanism.The only two members of putatively cytoplas-mic effectors which lack a signal sequence areAvra10 and Avrk1 of B. graminis f. sp. hordei(100). Extracellular effectors are often furtherN- and sometimes C-terminally processed byplant and/or fungal proteases, but evidence forprotein maturation is in some cases based onlyon in-silico predictions. In that respect, exper-imental evidence for secretion and processingof fungal effectors during infection is availableonly for the Avr and Ecp effectors of C. fulvum(24), the Six effectors of F. oxysporum f. sp. ly-copersici (55), and the Nip effectors of R. secalis(146). Avr-Pita from M. grisea is also predictedto be a secreted processed protein, but this as-sumption is based on the fact that the 176 aamature protein, but not the intact 223 aa pro-tein, is the active form that interacts directlywith the cognate cytoplasmic Pi-ta R protein(59).

A second common feature of extracellulareffectors, as well as some of the effectors thatare active inside host cells, is the presence ofmultiple cysteine residues (Table 1). The cys-teine residues might be involved in disulfide-bridge formation that provides protein stabilityin the harsh protease-rich environment of thehost apoplast. Indeed, disulfide bonds betweencysteine residues have been reported to be re-quired for stability and activity of at least Avr4and Avr9 of C. fulvum (132, 134). However, mu-tational analysis of cysteine residues present inthe Ecps from the same fungus suggested thatnot all cysteine residues are involved in disul-fide bridges or are crucial for induction of HRon plants carrying the cognate Cf-Ecp proteins

(80). Finally, effectors active inside the host celllike those of M. lini possibly need proper fold-ing and disulfide-bridge formation outside thehost before being taken up.

Intrinsic FunctionsAs sequence homology between most fungaleffectors and other proteins present in publicdatabases is limited, assigning functions to mosteffectors based on putative orthology alone hasbeen limited (Table 1). Exceptions are the Avr-Pita (a putative metalloprotease) (93) and Ace1(a hybrid polyketide synthase/nonribosomalpeptide synthetase) (11) effector proteins of M.grisea (130), as well as Ecp6 (LysM-domains)(13) of C. fulvum. Characterization of the three-dimensional structure as well as the disulfidebond pattern between cysteine residues in somecases provided additional clues with respect tothe intrinsic functions of some fungal effec-tors. Indeed, the disulfide bond pattern andcysteine spacing in Avr4 from C. fulvum re-vealed structural and functional homology withan invertebrate chitin-binding domain presentin tachycitin (130, 131). A second effector pro-tein with a known intrinsic function is Avr2from C. fulvum that proved to be an inhibitorof the Rcr3, Pip1, aleurain, and TDI65 cys-teine proteases (102, 107, 140), although thisfunction was not inferred from homology withother known cysteine protease inhibitors. Avr2and Avr4 are the only two fungal effectors forwhich both an intrinsic and virulence functionhas been shown experimentally. Based on thespacing between the cysteine residues, AvrP123of M. lini shows structural homology to mem-bers of the Kazal family of serine protease in-hibitors, whereas AvrP4 shows homology tocystine-knotted proteins, as does Avr9 of C.fulvum (17, 134). However, despite the struc-tural resemblance of the latter effectors to otherenzymes or proteins, a functional homologyhas never been established. Finally, the 3-Dstructure of AvrL567 from M. lini has beenresolved and binding of this protein to DNAhas been demonstrated, but because not allAvrL567 variants bind DNA with the same


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efficiency, the biological significance of the ob-served activity is not yet clear (144).

Role in VirulenceAs with intrinsic functions, the contribution ofindividual fungal effectors to virulence has alsoproven difficult to establish. Functional anal-ysis indicates that in some cases effector pro-teins constitute genuine virulence factors thatare required for full virulence of the pathogenson their host plants (Table 1). Some effec-tor genes are also reported to be clustered onpathogenicity islands, the presence of which iscorrelated with virulence (or avirulence) on spe-cific hosts. Examples are the Six1 (Avr3), Six2,and Six3 genes of F. oxysporum f. sp. lycoper-sici that cluster in a genomic region that con-fers virulence on tomato plants (135), and thePwl gene family of M. oryzae that is requiredfor specificity on weeping lovegrass (70, 118).Also many effectors seem to work in concert,and their individual contribution to virulenceis often minor, undetectable, or redundant, astheir deletion has no apparent effect on fitnessor virulence (Table 1). This fact is also sup-ported by studies on diversifying selection op-erating on a number of effector genes showingthat mixed (sub)populations of some pathogensexist that consist of individuals that either carryor lack particular effector genes, indicating thatthe fitness cost for the loss is not very high (105,116). Some effectors might also provide over-lapping activities as is possibly the case for theAvr4 and Ecp6 effectors of C. fulvum that bothbind to chitin and thus could potentially pro-tect the fungus against plant chitinases and/orsuppress PTI by scavenging chitin fragmentsreleased from fungal cell walls in the apoplastduring infection (13, 130). Finally, some effec-tors might play a more general role in virulence,as is, for example, the case for the Nip proteinsof R. secalis (146, 147), whereas other effectorsthat lack a clear role in virulence seem to inter-fere with defense signaling pathways that areinduced by other effectors/Avr factors with animportant role in virulence. This seems to bethe case for Avr1 (Six4) of F. oxysporum f. sp.

lycopersici, which is not a virulence factor butrather suppresses I-3-mediated resistance trig-gered by Avr3 (Six1), an effector that is requiredfor full virulence of this pathogen (54).

Translocation of Fungal EffectorsPlant and animal pathogenic bacteria containsix secretory systems of which the type three se-cretion system (TTSS) is crucial for virulenceas it is required for translocation of effectorsinto the host cell (1, 2, 36). Many TTSS-injected effectors suppress PTI, and for sev-eral of them the mechanism of suppression hasbeen elucidated (1). Most oomycete secretedeffectors contain an RXLR (10) motif that isrequired for their uptake into host cells, ashas been shown for effectors of the Arabidopsisdowny mildew pathogen Hyaloperonospora para-sitica (97) and the potato pathogen Phytophthorainfestans (148). Bioinformatics analysis has iden-tified 425 genes potentially encoding secretedRXLR proteins in the P. infestans genome (60).

As with bacteria and oomycete effectors, ef-fectors of some fungal plant pathogens (e.g.rusts, powdery mildews, M. oryzae, and F. oxys-porum f. sp. lycopersici) are putatively translo-cated into the host cell where they interact withcytoplasmic or nuclear R proteins (16, 27, 28,34, 59, 108). However, so far in all fungal effec-tors no clear consensus signature has been iden-tified that would point to a function in translo-cation and uptake into the plant cell.

The host-selective protein toxin ToxA ofP. tritici-repentis (19) is required for full viru-lence on wheat, and its solvent-exposed Arg-Gly-Asp (RGD) motif that interacts with thehost plasma membrane is likely required for itsinternalization (85). A similar RGD motif me-diating interaction with the plasma membraneis also present in the IpiO effector proteins ofthe oomycete P. infestans (106).

EFFECTOR RECOGNITIONBY R PROTEINSTo date, many R genes have been cloned fromdifferent plant species and the products of these


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genes show great diversity in their structuralproperties (24, 87, 120, 142). The vast major-ity of cytoplasmic R proteins consist of an NBSconnected to a region of LRRs (NBS-LRRs),whereas the second major class of R genesencodes proteins with an extracellular LRR(eLRR) domain and a short transmembrane(TM) domain. The NBS-LRR class can be fur-ther subdivided into subclasses based on thestructure of their N-terminal domains that con-tain either a coiled coil (CC) or Tol/interleukin-1 receptor (TIR) motif (Figure 1). However,despite the fact that an increasing number ofplant R proteins and their cognate effectorshave been characterized, in most cases we stillknow little about the molecular mechanisms as-sociated with effector perception by R proteinsand subsequent R-mediated downstream plantdefense responses (Figures 2 and 3).

The simplest perception model proposed sofar is based on a direct interaction between thecognate R protein and effector. This model isreferred to as the receptor-ligand model, andindeed, a few examples of direct interactionsbetween a fungal effector and plant R pro-tein have been described. Yeast two-hybrid andin vitro binding assays demonstrate a physi-cal interaction between Avr-Pita (Avr-Pita176)from M. oryzae and the cognate Pi-ta resis-tance protein from rice (59). Pi-ta codes foran intracellular NBS-LRR protein and muta-tional analysis showed that single aa substitu-tions in the LRR domain of this protein orin Avr-Pita176 can disrupt the physical inter-action and abolish Pita-mediated defense re-sponses (59). Physical interaction between ef-fectors of M. lini and their cognate R proteinsin flax has also been reported (28, 34, 144). Thepolymorphic AvrL567 locus of M. lini consistsof at least three genes (AvrL567A, AvrL567B,and Avr567C) whose products are recognizedby the cognate L5, L6, and L7 proteins offlax. Yeast two-hybrid assays demonstrated di-rect interaction between AvrL567 and the cog-nate L5/L6 proteins, whereas a mutation inthe LRR domain of L abolishes the interac-tion, indicating binding of AvrL567 to the LRRdomain.

One implication of the receptor-ligandmodel is that given the enormous diversity ofeffector molecules, plants must carry numer-ous R proteins that would enable them to rec-ognize all individual effectors and their allelicvariants. However, most plants have developedR proteins that monitor modifications in theplant targets of fungal effectors. In this so-calledguard model, R proteins do not interact directlywith an effector but guard its host target and re-spond to alterations in this target caused by theeffector. Therefore, in contrast to the receptor-ligand model, which allows recognition of onlya limited set of structurally related effectors,the guard model enables detection of multipleunrelated effectors that interact with the samehost target guarded by a single R protein. Aclassical example of the guard model is repre-sented by the indirect interaction between Avr2and Cf-2, mediated by Rcr3 in the C. fulvum-tomato interaction as discussed above (102). Inmany other pathosystems, indirect interactionbetween R protein and cognate Avr has been re-ported, suggesting that the majority of interac-tions would fit into the guard model (61). Thisalso seems to be the case for the Avr9 effec-tor of C. fulvum and the tomato Cf-9 protein,whose interaction is mediated by a high affinitybinding site (73, 81) (Figures 2 and 3).

Whether direct or indirect interaction me-diates perception of effector molecules by Rproteins, both systems could complement eachother. Indirect recognition enables plants tomonitor multiple effectors by a relatively smallnumber of R proteins, whereas redundancy inguardees allows monitoring of one target by dif-ferent R proteins, as seems to be the case forRlm4 and Rlm7, which both seem to guard thesame virulence target of AvrLm4-7 (95). Thisstrategy, however, is effective only toward ef-fectors with plant targets but not against effec-tors with defensive fungal targets, such as Avr4from C. fulvum, which binds to chitin presentin the fungal cell wall but apparently does notinterfere with the physiology of the host. Fur-thermore, direct interaction between effectorand R protein can be overcome more readily bymutations in effectors that abolish recognition,


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RLPs

NB-LRRs

a

b

eLRR eLRR

LRRNBSTIR

LRRNBSLZ/CC

eLRR

ECS PEST

TM TMTMTM

ER-r ECS ECSKinase

LysM

LysM

LysM

LysM-RK

Cytosol

Cfs:AvrsEcps

Ve2: ?HcrVf: ?

L5, L6, L7: AvrL567M: AvrM

Pi-ta: Avr-PitaI-2: Six3

LeEix2:Eix

CERK1:Chitin

Figure 1Structure of different types of resistance proteins. (a) From left to right: RLPs: tomato Cfs directly orindirectly recognize Cladosporium fulvum Avr and Ecp proteins, respectively; tomato Ve2 and apple HcrVfprovide resistance to Verticillium sp. and Venturia inaequalis, respectively, but their cognate effectors are notknown yet; tomato LeEix2 mediates recognition of Trichoderma sp. Eix; Arabidopsis LysM-RK is required forfungal 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 effectorsAvrL567, 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-2mediates 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-coildomain; 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, Fusariumoxysporum resistance protein-2; L5, L6, L7, and M, flax rusts resistance proteins; LeEix2, Lycopersiconesculentum 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 resistanceprotein; 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.


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Cf-4/Cf-9

E

F

G

Cf-2

G

ECSER-r

?

F

E

C

C2C3D

Chitin

Avr4

Hsp90?

VAP27 ACIK1

CITRX

Avr4

B

B

1

2C3D

C1

C2

?

Avr2

Rcr3 Avr2

HABs

Avr9

Cysteineproteases

Cytosol

Figure 2Cf-4-, Cf-9-, and Cf-2-mediated perception of Avr4, Avr9, and Avr2, respectively. From left to right: Avr4 isa 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 severaltomato cysteine proteases including cysteine protease Rcr3 that, after binding to Avr2, triggersCf-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, requiredfor Cladosporium resistance (cysteine protease); VAP27, vesicle-associated protein 27.

whereas jettison of effectors seems to mediateevasion of R-mediated resistance in cases of in-direct interactions (23, 61).

The guard model is strongly dependent onguarded effector targets, and therefore, naturalselection is expected to favor partners with im-proved interaction. Furthermore, the virulencefunction of effector proteins is based on modi-fication of their host targets, and natural selec-tion in the host would favor the developmentof targets that are less or no longer recognizedby effectors. As a result of these two opposingforces acting on effector targets, it was recentlyproposed that plants have likely developed

decoy molecules that mimic the effector tar-gets. Such decoys would trap effector proteins,diverting them from their real virulence targetsand alert monitoring R proteins. Decoys eithercould arise from a guardee duplication event orcould evolve independently of the effector tar-gets that they are mimicking. This variant ofthe original guard model is proposed as the de-coy model, and a few cases in support of thismodel have been suggested to operate in bacte-rial and fungal pathosystems (137). One of theproposed cases involves the interaction betweenAvr2 and Rcr3, where it is suggested that Rcr3is the decoy molecule that diverts Avr2 away


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NRC1

Vacuole

Cf-4 Cf-9

P

Avr-induced

P PR

PRPR

PR

PR

Cytosol

P

Hypersensitive response

PR proteins

Phytoalexins

Cell wall reinforcement

Salicyclic acid

PRNucleus

P

PP

PP

K+K+

K+

Oxidativeburst

ROS

H+H+

H+Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Avr4

CITRX

EDS1

CCIP3

ACS

TFs

MAPKKK CDPKs

MAPKK

MAPK1-3

CITRX

PLC PIP2 DGK DAG PA VAP27

Syntaxin

Ca2+

ATPase

H+

ATPase K+

ATPase

ACIK1

NADPHoxidase

Avr9

HABs

RAR1

SGT1

Hsp90?

Hsp90?

Ethylene Def

ense

resp

onse

s

?

?

?

TFs

Figure 3Avr4-triggered Cf-4-mediated, and Avr9-triggered Cf-9-mediated defense signaling. Left: EDS1 and NRC1 are required forCf-4- and Cf-4-mediated resistance. PLC is activated leading to IP3 and DAG. DAG is converted by DGK into PA, which stimulatesNADPH-oxidase causing the accumulation of ROS. IP3 releases Ca2+ from the vacuole. SGT1, RAR1 and Hsp90 are in a complexwith NRC1 or act just downstream of it. The Hsp90 complex interacts with the MAPK cascade eventually phosphorylating TFsand ACS causing accumulation of ethylene. TFs induce various defense responses including SA and ethylene accumulation, cell wallenforcement, phosphorylation of syntaxin, accumulation of phytoalexins and PR proteins, and the hypersensitive response. Right: Avr9interacts via the HABS with the Cf-9 protein stimulating phosphorylation of CDPKs that, in turn, stimulate the production of ethylenethrough ACS. Ethylene inhibits the MAPK cascade. ACIK1 interacts with the C terminus of Cf-9 and CITRX with both Cf-9 and Cf-4and 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, phosphatidylinositol 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.


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from its real target, the plant protease Pip1,and activates Cf-2-mediated Avr2-triggered re-sistance (107, 137). However, this model still isvery speculative and not yet supported experi-mentally.

EFFECTOR AND RESISTANCEPROTEIN EVOLUTIONHosts and pathogens are locked up in a per-petual arms race with only temporary winnersand losers. Fungal effectors and plant R proteinsare in the front line of this arms race, and rapidrates of effector evolution and maintenance ofhigh allelic diversity at corresponding R loci inplants have been reported in different pathosys-tems (3, 28, 83, 112, 115). The high rate ofmolecular evolution observed in effectors as aresult of mutation, selection, and reproductionhas caused extensive sequence diversification,gene expansion, and other genetic rearrange-ments in effector genes that might explain theabsence of homology among effector proteinsand their different host specificities. Many ef-fector genes are embedded in highly dynamicgenome areas, such as the chromosome endsor amid multiple transposable elements thattrigger frequent genomic rearrangements, thusenabling higher genetic flexibility in rapidlyovercoming R-mediated resistance, as alreadydiscussed above. In many of these cases, gainof virulence due to transposon insertions, genedeletions, and other genetic rearrangements hasbeen frequently observed (37, 44, 49, 50, 71, 93,95, 100, 118).

Although sequence alterations in effectorgenes that lead to evasion of R-mediated re-sistance can be the result of point mutations,frameshifts, gene deletions, and transposon in-sertions, the specific nature of the interplay be-tween effector and R proteins can affect thetype of genetic adaptation occurring in bothproteins. Surveys for allelic variation in effectorgenes suggest that direct recognition by R pro-teins favors sequence diversification in effectorsthat disrupts physical association between thetwo, but without affecting the effector’s pre-sumed virulence function. Indeed, diversifying

selection operating on the AvrL567 locus ofM. lini generated protein variants that are nolonger recognized by the cognate L protein inflax. Polymorphic aa residues in these sequencevariants occurred on the proposed solvent-exposed surface of the effector protein that in-teracts with the LRR domain of the cognate Rprotein, indicating that selection favored vari-ants that abolish recognition but still maintaina putative virulence function (144). In a simi-lar way, almost all sequence variants describedfor the chitin-binding Avr4 effector protein ofC. fulvum showed single aa substitutions thatabolish recognition by the cognate Cf-4 pro-tein but retain their ability to bind to chitin(62, 64, 116, 130). Recent analysis of Avr4-expressing tomato lines has shown that Avr4triggers hardly any transcriptional responses inthe absence of the cognate Cf-4 protein, indi-cating that it is unlikely that Avr4 has targets inthe host other than Cf-4 (139). In a similar way,sequence diversification of pathogen effectorsis adaptively matched fairly quickly by diversi-fication of the host R proteins that results innew recognition specificities (88). Good exam-ples of a dynamic arms race between effectorsand R proteins are provided by the M. lini Avrsand cognate flax R proteins (28) and the H. par-asitica Atr13 and Rpp13 proteins of Arabidopsis(3, 103).

In contrast to the emerging picture for di-rect recognition, indirect recognition of effec-tors by R proteins imposes selection againstAvr effector function rather than structure, andsuch effectors are either present or absent inpathogen populations. Absence would imply aminimal fitness penalty for the pathogen, butunder strong selection pressure, the conditionalbenefits of such a loss could be higher thanthe costs of the loss. Alternatively, the effectorfunction could be redundant and compensatedfor by other effectors. Effector gene deletionshave been reported as the main mechanism forgain of virulence for the fungal effectors genesAvr9 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 Avr1-CO39 of M. grisea (37, 93, 150). Loss of the


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Avr2 gene product of C. fulvum is caused byinsertions, deletions, and other frameshift mu-tations that result in truncated nonfunctionalalleles. In contrast to gene deletions, such mu-tated alleles are preserved in the genome ofC. fulvum and may become available for re-versions once selection pressure has been lifted(116). Frameshift mutations in particular are of-ten reverted by the occurrence of a second com-plementary frameshift that could restore thefunction of the protein (92). However, exceptfor Avr2, which indirectly interacts with Cf-2,for most of the other effector gene deletions de-scribed above, it is currently unknown whetherthe products of these genes interact directly orindirectly with their cognate R proteins. In fact,Avr-Pita of M. grisea, which directly interactswith Pi-ta, shows various types of sequence al-terations that can result in gain of virulence,including also gene deletions (59, 69, 93, 150).Point mutations contributing to virulence havealso been observed for Nip1, although less fre-quently than gene deletions (105), as well as forAvr4E (116, 145) and AvrLm4-7 (95). From theexamples presented above, it is clear that thetype of genetic adaptation of effectors to over-come R-mediated recognition is not only de-termined by whether the interaction betweenan effector and an R protein is direct or indi-rect. However, theoretical modeling suggeststhat jettison of effector proteins can lead tolong and stable resistance, whereas sequencediversification is likely to result rather quicklyin new virulence phenotypes (138). In contrastto the receptor-ligand model, the guard modeldoes not impose any selective pressure for se-quence diversification on R genes but insteadenhances diversifying selection on plant targetsof effectors (89).

In all the cases described so far, gain of vir-ulence was correlated with sequence diversifi-

cation or jettison of effector genes. However, adifferent scenario has been observed for the ef-fector Avr3 of F. oxysporum f. sp. lycopersici thatis required for full virulence, where suppres-sion of ETI is correlated with the acquisition ofan additional protein (Avr1) that suppresses I-3- and I-2-mediated defense signaling pathways(54).

FUTURE CHALLENGESComparative genomics of fungal pathogens willbe useful in identification of new effector pro-teins and possibly in prediction of their vir-ulence functions. However, this approach islimited because of the low level of homologyamong fungal effectors. Some effectors resideon pathogenicity islands, and it will be interest-ing to find out by comparative genomics howthese islands have arisen during evolution. Formost fungal effectors, a function in virulencecan be investigated experimentally by gene dis-ruption, gene knock-down, or overexpressionassays, and it is expected that for number ofthem a role in suppressing PTI induced by fun-gal PAMPs will be shown. In addition to effec-tors, discovery of more fungal PAMPs in addi-tion to chitin fragments is also anticipated inthe near future, which would enrich our under-standing of the plant defense system and the in-terplay between PTI and ETI. Elucidating themechanisms of translocation of fungal effectorsinto host cells is also a major challenge for thefuture. A considerable amount of effort is cur-rently being placed on identifying interactorsof effector proteins by in situ microscopic tech-niques and fluorescently labeled effectors. Fi-nally, it is hoped that effectors will be identifiedthat are crucial for virulence, as their cognateresistance genes are anticipated to be durableand could be used in resistance breeding.

DISCLOSURE STATEMENTThe authors are not aware of any affiliations, memberships, funding, or financial holdings thatmight be perceived as affecting the objectivity of this review.


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ACKNOWLEDGMENTSWe 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 JGMde Wit by the Royal Netherlands Academy of Arts and Sciences. This project was carried outwithin the research programme of the Centre of BioSystems Genomics (CBSG) which is part ofthe Netherlands Genomics Initiative/Netherlands Organization for Scientific Research.

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Annual Review ofPhytopathology

Volume 47, 2009Contents

Look Before You Leap: Memoirs of a “Cell Biological” PlantPathologistMichele C. Heath ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 1

Plant Disease Diagnostic Capabilities and NetworksSally A. Miller, Fen D. Beed, and Carrie Lapaire Harmon ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !15

Diversity, Pathogenicity, and Management of Verticillium SpeciesSteven J. Klosterman, Zahi K. Atallah, Gary E. Vallad, and Krishna V. Subbarao ! ! ! ! ! ! !39

Bacterial/Fungal Interactions: From Pathogens to MutualisticEndosymbiontsDonald Y. Kobayashi and Jo Anne Crouch ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !63

Community Ecology of Fungal Pathogens Causing Wheat Head BlightXiangming Xu and Paul Nicholson ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! !83

The Biology of Viroid-Host InteractionsBiao Ding ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 105

Recent Evolution of Bacterial Pathogens: The Gall-FormingPantoea agglomerans CaseIsaac Barash and Shulamit Manulis-Sasson ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 133

Fatty Acid–Derived Signals in Plant DefenseAardra Kachroo and Pradeep Kachroo ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 153

Salicylic Acid, a Multifaceted Hormone to Combat DiseaseA. Corina Vlot, D’Maris Amick Dempsey, and Daniel F. Klessig ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 177

RNAi and Functional Genomics in Plant Parasitic NematodesM.N. Rosso, J.T. Jones, and P. Abad ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 207

Fungal Effector ProteinsIoannis Stergiopoulos and Pierre J.G.M. de Wit ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 233

Durability of Resistance in Tomato and Pepper to XanthomonadsCausing Bacterial SpotRobert E. Stall, Jeffrey B. Jones, and Gerald V. Minsavage ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 265

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AR384-FM ARI 14 July 2009 23:48

Seed Pathology Progress in Academia and IndustryGary P. Munkvold ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 285

Migratory Plant Endoparasitic Nematodes: A Group Rich in Contrastsand DivergenceMaurice Moens and Roland N. Perry ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 313

The Genomes of Root-Knot NematodesDavid McK. Bird, Valerie M. Williamson, Pierre Abad, James McCarter,

Etienne G.J. Danchin, Philippe Castagnone-Sereno, and Charles H. Opperman ! ! ! ! ! 333

Viruses of Plant Pathogenic FungiSaid A. Ghabrial and Nobuhiro Suzuki ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 353

Hordeivirus Replication, Movement, and PathogenesisAndrew O. Jackson, Hyoun-Sub Lim, Jennifer Bragg, Uma Ganesan,

and Mi Yeon Lee ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 385

Ustilago maydis as a PathogenThomas Brefort, Gunther Doehlemann, Artemio Mendoza-Mendoza,

Stefanie Reissmann, Armin Djamei, and Regine Kahmann ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! 423

Errata

An online log of corrections to Annual Review of Phytopathology articles may be found athttp://phyto.annualreviews.org/

vi Contents

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From the Annual Review of Ecology, Evolution, and Systematics, Volume 39 (2008)

Sanctions, Cooperation, and the Stability of Plant-Rhizosphere MutualismsE. Toby Kiers and R. Ford Denison

The Performance of the Endangered Species ActMark W. Schwartz

Pandora’s Box Contained Bait: The Global Problem of Introduced EarthwormsPaul 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 Researchon a Worsening PestMichael E. Gray, Thomas W. Sappington, Nicholas J. Miller, Joachim Moeser,

and Martin O. BohnImpacts of Plant Symbiotic Fungi on Insect Herbivores: Mutualism in a

Multitrophic ContextSue E. Hartley and Alan C. Gange

Cellular and Molecular Aspects of Rhabdovirus Interactions with Insectand Plant HostsEl-Desouky Ammar, Chi-Wei Tsai, Anna E. Whitfield, Margaret G. Redinbaugh,

and Saskia A. Hogenhout

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

How Saccharomyces Responds to NutrientsShadia Zaman, Soyeon Im Lippman, Xin Zhao, and James R. Broach

The Organization of the Bacterial GenomeEduardo P.C. Rocha

Genomic Insights into Marine MicroalgaeMicaela S. Parker, Thomas Mock, and E. Virginia Armbrust

From the Annual Review of Genomics and Human Genetics, Volume 9 (2008)

Phylogenetic Inference Using Whole GenomesBruce Rannala and Ziheng Yang

From the Annual Review of Microbiology, Volume 62 (2008)

Evolution of Intracellular PathogensArturo Casadevall

Chlamydiae as Symbionts in EukaryotesMatthias 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 TherapeuticTargets for the Regulation of Immunity and InflammationTakao Shimizu

From the Annual Review of Plant Biology, Volume 60 (2009)

DNA Transfer from Organelles to the Nucleus: The Idiosyncratic Geneticsof EndosymbiosisTatjana Kleine, Uwe G. Maier, and Dario Leister

Jasmonate Passes Muster: A Receptor and Targets for the Defense HormoneJohn Browse

A Renaissance of Elicitors Perception of Microbe-Associated Molecular Patternsand Danger Signals by Pattern-Recognition ReceptsThomas Boller and Georg Felix

Biosynthesis of Plant Isoprenoids: Perspectives for Microbial EngineeringJames Kirby and Jay D. Keasling

Roles of Plant Small RNAs in Biotic Stress ResponsesVirginia Ruiz-Ferrer and Olivier Voinnet

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