ARP_06

8/13/2019 ARP_06

1/20

A Catalogue of the EffectorSecretome of PlantPathogenic Oomycetes

Sophien Kamoun

Department of Plant Pathology, Ohio State University, Ohio Agricultural Researchand Development Center, Wooster, Ohio 44691; email: [email protected]

Annu. Rev. Phytopathol.2006. 44:4160

First published online as aReview in Advance on

January 30, 2006

TheAnnual Review ofPhytopathologyis online atphyto.annualreviews.org

doi: 10.1146/annurev.phyto.44.070505.143436

Copyright c2006 byAnnual Reviews. All rightsreserved

0066-4286/06/0908-0041$20.00

Key Words

virulence, pathogenesis, avirulence, defense suppression,Phytophthora, downy mildews

Abstract

The oomycetes form a phylogenetically distinct group of eukaryo

microorganisms that includes some of the most notorious pathog

of plants. Oomycetes accomplish parasitic colonization of plantsmodulating host cell defenses through an array of disease effec

proteins. The biology of effectors is poorly understood but tremdous progress has been made in recent years. This review classifi

and catalogues the effector secretome of oomycetes. Two classeseffectors target distinct sites in the host plant: Apoplastic effect

are secreted into the plant extracellular space, and cytoplasmic eff

tors are translocated inside the plant cell, where they target differsubcellular compartments. Considering that five species are und

going genome sequencing and annotation, we are rapidly movtoward genome-wide catalogues of oomycete effectors. Already

is evident that the effector secretome of pathogenic oomycetemore complex than expected, with perhaps several hundred prote

dedicated to manipulating host cell structure and function.

41

8/13/2019 ARP_06

2/20

Effectors: pathogenmolecules thatmanipulate host cellstructure andfunction therebyfacilitating infection

and/or triggeringdefense responses.Effectors can beelicitors and/ortoxins. Unlike these,the term effector isneutral and does notimply a negative orpositive impact onthe outcome of thedisease interaction

Haustoria:

specialized infectionstructures ofbiotrophic oomyceteand fungal pathogensthat invaginate intothe plant cells butremain enveloped bya modified host cellmembrane.

AVR: avirulence

Toxins: pathogenmolecules that causeplant cell deaththereby facilitatingcolonization bynecrotrophicpathogens

Elicitors: pathogenmolecules thattrigger defenseresponses resultingin enhancedresistance to theinvading pathogen

INTRODUCTION

The oomycetes form a phylogenetically dis-

tinct group of eukaryotic microorganismsthat includes some of the most notorious

pathogens of plants (41). Among these, mem-bers of the genus Phytophthora cause enor-

mous economic losses on crop species as well

as environmental damage in natural ecosys-tems (25). SomePhytophthoraspecies, such as

the potato and tomato late blight agentPhy-

tophthora infestans (11, 45, 90), and the soy-

bean root and stem rot agent Phytophthora

sojae (85), have caused longstanding prob-

lems for agriculture, whereas others, such as

the sudden oak death pathogen Phytophthoraramorum, have surfaced in recent epidemics

(80). Other significant oomycetes include thedowny mildews, a heterogeneous and di-

verse group of obligate parasites (3). Somedowny mildews infect economically impor-

tant hosts such as grapevines (35). Hyaloper-

onospora parasitica is a natural pathogen ofArabidopsis thaliana and figures prominently

in research on disease resistance in this modelplant (89).

Phytophthoraand downy mildews establishintimate associations with plants and typi-

cally require livinghost cells to complete their

infection cycle, a process known as biotro-phy (31, 68). An emerging view on oomycete

pathogenesis is that the oomycetes accom-plish parasitic colonization of plants by repro-

gramming the defense circuitry of host cellsthrough an array of disease effector proteins

(38). Two classes of effectors target distinct

sites in the host plant: apoplastic effectorsare secreted into the plant extracellular space,

where they interact with extracellular targetsand surface receptors; and cytoplasmic effec-

tors are translocated inside the plant cell pre-sumably through specialized structures like

infection vesicles and haustoria that invagi-

nate inside living host cells (Figure 1).This review attempts to catalogue the ef-

fector secretome of oomycetes. Because ofour fragmentary knowledge of the mecha-

nisms of pathogenicity of oomycetes, we re-

tain a flexible definition of the term effec-tors that is consistent with previous pub-

lications (38, 41, 99). Effectors are defined

as molecules that manipulate host cell struc-ture and function, thereby facilitating infec-

tion (virulence factors or toxins) and/or trig-gering defense responses (avirulence factors

or elicitors). This dual (and conflicting) activ-ity of effectors has been broadly reported in

plant-microbial pathosystems, such as bacte-

rial and fungal diseases (4, 49, 51, 103). Themajority of the oomycete effectors described

here, including the avirulence (AVR) proteinsare so far known only by their ability to acti-

vate defense responses and innate immunityAn underlying assumption of this review is

that these AVR effectorsand some of theother

defense elicitors have virulence functions of

unknown nature. Indeed, one crucial ques-tion in the study of oomycete pathogenic-ity is how effectors disrupt the activation

and execution of plant defenses. Future func-tional studies will undoubtedly reveal a com-

plex array of virulence functions for oomycete

effectors.

IDENTIFICATION OFOOMYCETE EFFECTORS

Biochemical, genetic, and bioinformaticstrategies, often in combinations, have been

applied to the identification of effector genesfrom oomycetes. Traditionally, effectors have

been identified by biochemical purificationand genetic analyses. With the advent o

genomics, novel strategies have emerged

The implementation of functional genomicspipelines to identify effectors from sequence

data sets has proved particularly successful. Atypical pipeline consists of two major steps

use of data mining tools to identify candi-date genes that fulfill a list of specific cri-

teria, followed by analysis and validation of

these candidate genes by functional assayssuch as expression in planta and evaluation

for effector-like activities. InFigure 2, we il-lustrate two examples of functional genomics

42 Kamoun

8/13/2019 ARP_06

3/20

Germinated cyst

Appressorium

Adhesive material

Infection vesicle Plant cell

Intercellular hypha

Intercellular hypha

Apoplastic space

Apoplastic effector

Apoplasticeffector

Apoplastic effector target

Apoplasticeffector target

Haustorium

Haustorium

Extrahaustorialmatrix

Extrahaustorialmatrix

Plant-derivedhaustorial

membrane

Plant-derivedhaustorialmembrane

Plant cell

Plant cell

Cytoplasmiceffector

Cytoplasmiceffector

Cytoplasmic effector target

Cytoplasmiceffector target

Plantnucleus

b

a

Figure 1

Plant pathogenic oomycetes secrete apoplastic effectors into the plant extracellular space and cytoplasmiceffectors inside the plant cell. (a) Schematic view of the early stages of infection byPhytophthora infestansof a host plant illustrating the sites of action of the apoplastic and cytoplasmic effectors. ( b) Cross-sectionof the view in panel A illustrating the delivery of apoplastic and cytoplasmic effectors to their cellulartarget. One class of cytoplasmic effectors is known to target the plant nucleus (T. D. Kanneganti, J. Win& S. Kamoun, unpublished). Apoplastic effectors interact with extracellular targets or surface receptors.Pathogen structures are shown in purpleand plant structures ingreen. Apoplastic effectors and effectortargets are inblue, whereas cytoplasmic effectors and their targets are in red. Note that for clarity thefigure components were not drawn to scale relative to each other.

www.annualreviews.org Effector Secretome in Oomycetes 43

8/13/2019 ARP_06

4/20

488 ESTs out of 2808 ESTs from infected tomato tissue predicted to originate from

P. infestans using GC content analysis

42 out of 488 ESTs predicted to encode secreted proteins using PexFinder

Domain annotation revealed one EST, PC064G6, with similarity to Kazal-like protease inhibitor proteins

142 putative secreted proteins predicted from 2147 ESTs of P. infestans using PexFinder

BLASTN searches against EST database revealed several polymorphic genes,

including PEX147

Sequence analyses of amplicons from different isolates confirmed polymorphisms and

identified SNPs between alleles and paralogs

Full-length cDNA corresponding to PC064G6 sequenced and corresponding protein, EPI1, expressed inE. coli

Serine protease inhibition assaysrevealed that EPI1 inhibits subtilisin and a

pathogenesis-related apoplastic proteaseof tomato

Co-immunoprecipitations revealed that EPI1interacts with the tomato subtilisin-like serineprotease P69B

100% association in 55 P. infestansisolatesbetween SNPs in PEX147 gene andavirulence on R3a potato

Expression in R3a potato of two PEX147

alleles using biolistic bombardment assay

resulted in cell death with only one of thealleles (Avr3a)

In planta co-expression of PEX147 and R3a inNicotiana benthamianaresulted in hypersensi-tive cell death specifically with the Avr3aallele

Apoplastic effector EPI1 Cytoplasmic effector AVR3a

Candid

ateidentification

Candidat

evalidation

Figure 2

Examples of high-throughput functional genomics pipelines for the identification of effector genes. Theleft panels illustrate the strategy of Tian et al. (96) for the discovery of the apoplastic effector EPI1. Theright panels depict the strategy of Bos et al. (16) as implemented by Armstrong et al. (6) for the cloning ofAvr3aofPhytophthora infestans.

Signal peptides:amino acidsequences that direct

the posttranslationaltargeting of proteinsto the generalsecretory pathway

pipelines that led to the discovery in P. infes-

tansof the protease inhibitor EPI1 (96) and

the avirulence protein AVR3a (6).

A critical milestone in the identification ofoomycete effector genes was the validation of

the concept that effector proteins must be se-creted in order to reach their cellular targets

at theintercellular interfacebetween theplantandpathogenor inside thehost cell (99). Iden-

tification of candidate secreted proteins was

facilitated by the fact that in oomycetes asin other eukaryotes, most secreted proteins

are exported through the general secretory

pathway via short, N-terminal, amino-acid se-quences known as signal peptides (99). Sig-

nal peptides are highly degenerate and can-

not be identified using DNA hybridization orPCR-based techniques. Nonetheless, compu-

tational tools, particularly the SignalP pro-gram that wasdevelopedusing machine learn

ing methods (62), can assign signal peptideprediction scores and cleavage sites to un-

known amino acid sequences with a high de-

gree of accuracy (57, 86). Therefore, withthe accumulation of oomycete cDNA and

genome sequences, lists of candidate secreted

44 Kamoun

8/13/2019 ARP_06

5/20

proteins could be readily generated using

bioinformatics tools. For instance, Torto et al.(99) developed PexFinder (with Pex stand-

ing forPhytophthoraextracellular protein), analgorithm based on SignalP v2.0 (62) to iden-

tify proteins containing putative signal pep-tides from expressed sequence tags (ESTs).

PexFinder was then applied to ESTs inP. in-festans. Proteomic identification of secretedproteins collected from culture filtrates of

P. infestans matched PexFinder predictions,convincingly validating the algorithm perfor-

mance (99). Similar high degrees of accuracyof SignalP were also reported (57, 86).

Computational signal peptide predictions

help to identify secreted proteins with no sig-nificant matches to known sequences. Anno-

tation of cDNA and gene sequences is rou-

tinelydone by similarity searches to sequencesin public databases. However, this approachis limited in that it identifies only proteins

with known functions and thus a large number

of proteins with unknown functions are ig-nored. Computational predictions single out

unknown secreted proteins that might oth-erwise be ignored. This results in a manage-

able list of candidates that can be further pro-cessed by sequence annotation and functional

analyses.

Building up an annotated collection ofcomputationally predicted candidate proteins

that feed into a functional analysis pipelineis by no means a one-directional process.

Rather, criteria for annotating and prioritiz-ing candidate genes are continuously refined

as new findings emerge and novel sequence-

to-function linksare established. One illustra-tive example is the discovery that all known

avirulence proteins of oomycetes carry a con-served motif (RXLR) closely following a sig-

nal peptide (see below for more details) (78).Straightforward sequence pattern search tools

can thus be used to discover an additional

number of candidate effectors of the RXLRfamily from severalPhytophthoraspecies.

Identification of genes under diversifyingselection (especially when coupled with se-

lection criteria such as presence of secre-

Pex: Phytophthoraextracellular prote

Diversifyingselection: (a.k.a.positive selection)

natural selection tfavors the fixationadvantageousmutations resultinin adaptivemolecular evolutio

PR:pathogenesis-rela

GIP: glucanaseinhibitor protein

tory signals and in planta expression) can also

help to select candidate effector genes (53).Based on the arms race model, adaptation

and counteradaptation between pathogen andhost is likely to drive their antagonistic coevo-

lution and generate the evolutionary forcesthat shape effectors and their host targets (24,

92). For instance, diversifying selection (alsoknown as positive selection) can be an indi-cator of functionally important loci that con-

tribute to the fitness of the organism. Indeed,some oomycete effector genes, e.g., ATR13

andscr74, exhibit extreme levels of amino acidpolymorphism with evidence of diversifying

selection (5, 53). In the future, evolutionary

analyses will have an even larger role in theidentification of effector genes as genome se-

quences from additional oomycete species be-

come available.Table 1lists major effectors of oomycetes

and the corresponding strategies that led to

their identification. These effectors are de-

scribed in more detail in the remainder of thisreview.

APOPLASTIC EFFECTORS

Enzyme Inhibitors

Several plant pathogenesis-related (PR) pro-teins, such as glucanases, chitinases, and pro-teases, are hydrolytic enzymes. Fungal and

bacterialplant pathogens haveevolved diversemechanisms for protection against the activ-

ities of these PR proteins (2, 75). Similar to

these pathogens, plant pathogenic oomycetes,such asPhytophthora, have also evolved mech-

anisms to escape the enzymatic activity of PRproteins. Oomycetes contain little chitin in

their cell wall and are therefore resistant to

plant chitinases (41).Phytophthora alsoevolvedactive counterdefense mechanisms by secret-

ing inhibitory proteins that target host glu-canases and proteases.

Glucanase inhibitors GIP1 and GIP2.

The glucanase inhibitors GIP1 and GIP2

are secreted proteins ofP. sojae that inhibit


8/13/2019 ARP_06

6/20

Table 1 Disease effectors of plant pathogenic oomycetes

Effector Species Identification strategy Reference

Apoplastic effectors

Enzyme inhibitors

Glucanase inhibitors

GIP1 and GIP2

P. sojae Biochemical purification (81)

Serine protease

inhibitors EPI1 and EPI10

P. infestans Bioinformatic prediction of secreted and

in planta induced proteins coupled with

protein domain annotation and followed

by biochemical analyses

(65, 66)

Cysteine protease inhibitors

EPIC1 and EPIC2

P. infestans Bioinformatic prediction of secreted and

in planta induced proteins coupled with

protein domain annotation

(M. Tian &

S. Kamoun,

unpublished)

Small cysteine-rich proteins

INF1 elicitin P. infestans Biochemical purification (79, 46)

INF2A and INF2B elicitins P. infestans Sequence similarity to INF1 (44)

PcF P. cactorum Biochemical purification (65)

PcF-like SCR74 and SCR91 P. infestans Bioinformatic prediction of secreted andin planta induced proteins coupled with

analyses of polymorphic diversity

(16, 53)

Ppat12, 14, 23, and 24 H. parasitica Suppression subtractive hybridization (13)

Nep1-like (NLP) family

PaNie Pythium aphanidermatum Biochemical purification (105)

NPP1 P. parasitica Biochemical purification (27)

PsojNIP P. sojae Bioinformatic prediction of secreted

proteins coupled with in planta

expression

(76)

PiNPP1 P. infestans Bioinformatic prediction of secreted


expression

(T. D.

Kannegenati

& S. Kamoun,unpublished)

GP42 (PEP13)

Transglutaminase

P. sojae Biochemical purification (63, 83)

CBEL P. parasitica Biochemical purification (87, 106)

Cytoplasmic effectors

RXLR protein family

ATR1NdWsB H. parasitica Positional cloning (78)

ATR13 H. parasitica Suppression subtractive hybridization

combined with positional cloning

(5)

Avr1b-1 P. sojae Positional cloning and candidate gene

approach

(88)

AVR3a P. infestans Bioinformatic prediction of secreted and

polymorphic proteins coupled with

association genetics

(6, 16)

CRN protein family (CRN1 and

CRN2)

P. infestans Bioinformatic prediction of secreted


expression

(99)

46 Kamoun

8/13/2019 ARP_06

7/20

the soybean endo--1,3 glucanase EGaseA

(81). These inhibitor proteins share signif-icant structural similarity with the trypsin

class of serine proteases, but bear mutatedcatalytic residues and are proteolytically non-

functional. GIPs are thought to functionas counterdefensive molecules that inhibit

the degradation of -1,3/1,6 glucans inthe pathogen cell wall and/or the releaseof defense-eliciting oligosaccharides by host-1,3 endoglucanases. There is some degreeof specificity in inhibition because GIP1 does

not inhibit another soybean endoglucanase,EGaseB. Positive selection has acted on -

1,3 endoglucanases in the plant legume genusGlycineand may have been driven by coevolu-tion with glucanase inhibitors inP. sojae(12).

Four genes with similarity to GIPs have been

identified inP. infestans, and their ability to in-hibit tomato endoglucanases is under investi-gation (23).

Serine protease inhibitors EPI1 and

EPI10. EPI1 and EPI10 are multidomain se-

creted serine protease inhibitors of the Kazalfamily (MEROPS family I1) (77) that are

thought to function in counterdefense (9597) (Figure 3a). They inhibit and interact

with the PR protein P69B, a subtilisin-like

serine protease of tomato that is thought tofunction in defense (95, 96). The epi1,epi10,

and P69B genes are concurrently expressedand up-regulated during infection of tomato

byP. infestans. The mechanism by which inhi-bition of P69B by EPI1 and EPI10 may affect

the late blight disease is not yet elucidated.

Recent findings indicate that EPI1 protectsseveral secreted proteins in P. infestans from

degradation by P69B thereby directly con-tributing to virulence (M. Tian & S. Kamoun,

manuscript submitted).Inhibition of host proteases by Kazal-like

proteins could be a common virulence strat-

egy between plant and mammalian parasites(96). Kazal-like proteins have also been impli-

cated in virulence of the apicomplexans Tox-

oplasma gondiiand Neospora caninum, a group

of mammalian parasites that transit through

the digestive tract (22, 52, 60, 74). Four pu-tative proteins with Kazal-like domains were

also identified in the genome sequence of

another apicomplexan animal parasite, Cryp-tosporidium parvum (1).In oomycetes, secreted

Kazal-like proteins form a diverse family of atleast 35 members from five plant pathogenic

oomycetes including the downy mildewPlas-mopara halstedii(96). Future biochemical stud-

ies of these proteins will address the extent

to which inhibition of host proteases is awidespread virulence function in oomycetes.

Cysteine protease inhibitors EPIC1 and

EPIC2. The cystatin-like cysteine proteaseinhibitors EPIC1 and EPIC2 (InterPro

IPR000010, MEROPS family I25) (77) form

another class of secreted protease inhibitors

ofP. infestans (M. Tian & S. Kamoun, sub-mitted). Recent findings suggest that thesetwo inhibitors target an apoplastic papain-

like cysteine protease of tomato (M. Tian &S. Kamoun, submitted). This suggests mul-

tifaceted inhibitions of tomato proteases by

secreted effectors ofP. infestans.

Small Cysteine-Rich Proteins

Many eukaryoticAvrgenes, such asCladospo-

riumfulvumAvr2,Avr4,andAvr9,Rhynchospo-rium secalis nip1, and Phytophthora elicitins,

encode small (

8/13/2019 ARP_06

8/20

1 21 26 D 70 96 D 140 162 D 208 224

3516 D 79 94 D 139 1491

EPI1

EPI10

ATR1NdWsB

b

a

AVR3a

Avr1b-1

ATR13

Targeting Function

Targeting Function

CC C C CC C C C C

1

1

1 21 41 65 138

19 38 41 187

1 21 44 59 147

15 48 62 311

CCC C C C CC C C CC C C C C

Apoplastic effectors

Cytoplasmic effectors

Signal peptide

Kazal protease inhibitor domain

RXLR motif

Effector domain

Figure 3

Domain organization of apoplastic and cytoplasmic effectors. (a) Schematic drawings of the apoplasticeffectors EPI1 and EPI10 ofPhytophthora infestans(9596). The two-disulfide bridge Kazal domains ofEPI1 and EPI10 that target the tomato protease P69B (95, 97) are shown in dark blue. P1 residues thatare critical for the inhibitor specificity are marked by an arrow. (b) Schematic drawings of the cytoplasmicRXLR effectors ATR1NdWsB and ATR13 ofHyaloperonsopora parasitica(5, 78), Avr1b-1 ofPhytophthorasojae(88), and AVR3a ofP. infestans(6). In both panels, the numbers under the sequences indicate

amino-acid positions. Thegray arrows distinguish the regions of the effector proteins that are involvedin secretion and targeting from those involved in effector activity.

Elicitins: INF1 and others. Elicitins are a

family of structurally related extracellularpro-teins that induce hypersensitive cell death and

otherbiochemical changes associated withde-

fense responses inNicotianaspp. (46, 48, 73,

79, 84). Strains ofP. infestansdeficient in the

elicitin INF1 cause disease lesions in Nico-

tiana benthamiana, suggesting that INF1 con-

ditions avirulence in this species (47). InP. in-

festans, a complex set of elicitin-like genes was

48 Kamoun

8/13/2019 ARP_06

9/20

identified using PCR amplification with de-

generate primers, low-stringency hybridiza-tions, and sequencing of cDNAs and BAC

clones (26, 43, 44, 46). All elicitin genes en-code putative extracellular proteins that share

a 98-amino-acid elicitin domain with a core of6 conserved cysteines (InterPro IPR002200).

Some infgenes (inf2A,inf2B,inf5,inf6,inf7,and M-25) encode predicted proteins witha C-terminal domain in addition to the N-

terminal elicitin domain. Sequence analysisof these C-terminal domains revealed a high

frequency of serine, threonine, alanine, andproline. The amino-acid composition and the

distribution of these four residues suggest the

presence of clusters ofO-linked glycosylationsites (44). These proteins may form a lol-

lipop on a stick structure in which the O-

glycosylated domain forms an extended rodthatanchorstheproteintothecellwallleavingthe extracellular N-terminal domain exposed

on the cell surface (39). Therefore, these atyp-

ical INF proteins may be surface- or cell wallassociated glycoproteins that could interact

with plant cells during infection.The intrinsic biological function of elic-

itins in Phytophthora has long been subjectto investigation. Conclusive evidence finally

emerged when it was demonstrated that class

I elicitins can bind sterols, such as ergos-terol, and function as sterol-carrier proteins

(15, 58, 59, 104). Consequently, elicitins werehypothesized as having a biological function

of essential importance to Phytophthora spp.because they cannot synthesize sterols and

must assimilate them from external sources

(34). In addition, phospholipase activity wasassigned to elicitin-like proteins from Phy-tophthora capsiciwith significant similarity toINF5 and INF6 (61), suggesting a general

lipid binding/processing role for the variousmembers of the elicitin family (66). Other

work by Osman et al. (67) using elicitin mu-

tants altered in sterol binding suggests thatsterol loading is important for specific bind-

ing to a plasma membrane receptor and in-duction of the hypersensitive response (HR)

in tobacco. However, the exact relationship

between sterol binding and cell death induc-tion remains to be clarified. More recently,

another gene with similarity to elicitins, M-

25, has been reported to be induced duringmating inP. infestans(26).

PcF. PcF is a secreted 52-amino-acid pep-

tide of Phytophthora cactorum that contains 6cysteines and a 4-hydroxyproline at residue

49 (65). PcF triggers necrosis in tomato and

strawberry, an economically important hostfor P. cactorum. Recombinant expression of

PcF inPichia pastorisresulted in a functionalprotein that induces necrosis and elicits ac-

tivity of the defense enzyme phenylalanineammonia lyase (64). PcF was proposed to

function as a toxin considering that it trig-

gers responses on host plants that are similar

to disease symptoms (65). However, its exactmode of action is unclear.

PcF-like SCR74 and SCR91. Two classesof PcF-like genes, scr74 (53) and scr91 (16),

are known in P. infestans. The expression ofscr74 is up-regulated approximately 60-foldtwo to four days after inoculation of tomato

and is also significantly induced during earlystages of colonization of potato (53).scr74be-

longs to a highly polymorphic gene family

withinP. infestanswith 21 different sequencesidentified. Using the approximate and max-

imum likelihood (ML) methods, Liu et al.(53) showed that diversifying selection likely

caused the extensive polymorphism observedwithin thescr74 gene family. These results led

to an evolutionary model that involves gene

duplication and recombination, followed byfunctional divergence ofscr74 genesinP. infes-

tans. Based on the expression pattern ofscr74

and its similarity to a necrosis inducing pro-

tein, the selective forces that shaped the evo-lution of this gene could well be related to

host-pathogen coevolution (53).

PPAT12, 14, 23, and 24. Bittner-Eddy et al.

(13) used suppression subtractive hybridiza-tion to identify genes from the obligate

pathogen Hyaloperonospora parasitica that are


8/13/2019 ARP_06

10/20

highly expressed during infection ofArabidop-

sis thaliana. Of the 25 genes identified, four,Ppat12,14,23, and 24, encode cysteine-rich

peptides ranging in size from 70 to 127 aminoacids. All four peptides carry predicted signal

peptides which suggests that they are likely tobe secreted. They share no sequence similar-

ity to other known cysteine-rich proteins andtheir role in virulence remains unknown.

NEP1-Like Family (PaNie, NPP1,PsojNIP, PiNPP1)

Nep1-like proteins (NLPs) are circa 25-kDa

proteins that are widely distributed in bac-

teria, fungi, and oomycetes, particularly inplant-associated species (70). The canoni-

cal 24-kDa necrosis- and ethylene-inducing

protein (Nep1) was originally purified fromculture filtrates of the fungus Fusarium oxys-

porumf. sp.erythroxyli(7). NLPs have subse-

quently been described in species as diverse asBacillus(94),Erwinia (9, 71), Verticillium (107),

Pythium(105), andPhytophthora(27, 76). De-

spite their diverse phylogenetic distribution,NLPs share a high degree of sequence simi-

larity and several members of the family havethe remarkable ability to induce cell death in

as many as 20 dicotyledonous plants (70). The

wide phylogenetic conservation and broad-spectrum activity of NLPs distinguish them

from the majority of cell death elicitors andsuggest that the necrosis-inducing activity is

functionally important.

In oomycetes, NLPs are also broadly dis-tributed inPhytophthoraand Pythium. PaNIe

of Pythium aphanidermatum (105), NPP1of Phytophthora parasitica (27), PsojNIP of

P. sojae (76), and PiNPP1 ofP. infestans (T.D. Kanneganti & S. Kamoun, unpublished)

have been studied in most detail. The contri-bution of these NLPs to disease remains un-clear. NLPs induce defense responses in both

susceptible and resistant plants. InP. sojaeandP. infestans, theNLPgenes were expressed late

during host infection and thus may functionin triggering the host tissue necrosis observed

during the necrotrophic phase of infection

thereby facilitating colonization (76; T.DKanneganti & S. Kamoun, unpublished). A

role of NLPs in virulence is supported by the

recent finding that disruptions of the NIPecandNIPecagenes inErwinia carotovorasubspcarotovora and E. carotovora subsp. atrosep-

tica, respectively, resulted in reduced virulence

phenotypes on potato tubers (71).

GP42 (PEP13) Transglutaminase

GP42 is an abundant cell wall glycoprotein

ofP. sojae that triggers defense gene expres-sion and synthesis of antimicrobial phytoalex-

ins in parsley through binding to a plasma

membrane receptor (63, 83).A 13-amino-acidpeptide fragment (Pep-13) is necessary and

sufficient for activation of defenses in pars-

ley and also triggers cell death in potato (32)Biochemical analyses indicated that GP42 isa Ca2+-dependent transglutaminase (TGase)

that is highly conserved inPhytophthora(21)

The Pep-13 motif was invariant in all exam-ined Phytophthora TGases. Mutational anal-

yses indicated that the same residues withinthe Pep-13 motif are important for activa-

tion of plant defenses and TGase activityThis suggests that plants evolved receptors

to recognize an essential epitope within

the TGase proteins and that GP42 func-tions as a pathogen-associated molecular pat-

tern (PAMP) (21). However, it is not knownwhetherPhytophthora GP42 TGases play an

essential role in virulence or fitness of the

pathogen.

CBEL

CBEL (Cellulose Binding, Elicitor, andLectin-like) is a 34-kDa cell wall protein that

was first isolated from Phytophthora parasit-icavar.nicotianae. CBEL has a dual function(a) it elicits necrosis and defense gene ex-

pression in tobacco plants; (b) it is requiredfor attachment to cellulosic substrates such

as plant surfaces (87, 106). Strains ofP. par-

asitica silenced for the CBEL gene were im-

paired in their ability to attach to cellophane

50 Kamoun

8/13/2019 ARP_06

11/20

membranes, although they remained able to

infect tobacco plants(28). CBEL contains tworegions with similarity to the PAN module

(InterPro: IPR000177), a conserved domainthat includes the Apple domain and functions

in protein-protein or protein-carbohydrateinteractions. PAN module proteins have been

implicated in attachment of apicomplexanparasites to host tissues (1820). The CBEL-like PAN module is particularly diverse in

oomycetes, with 52 different sequences iden-tified in 5 species, including the fish pathogenSaprolegnia parasitica (100). The role of thePAN/cellulose binding domain of CBEL in

induction of defense responses is under inves-

tigation (29).

CYTOPLASMIC EFFECTORSRXLR Protein Family

Race-specific resistance to Phytophthora spp.

follows the gene-for-gene model, which im-plies that Avrgenes from the pathogen are

perceived directly or indirectly by matchingresistance (R) genes from the plant (33, 93).

NumerousR genes that function against theoomycetesH. parasitica,P. infestans, andP. so-

jaehave been cloned (8, 10, 14, 17, 37, 56, 69,

91, 98, 101, 102). They belong to three dif-ferent classes of cytoplasmic and extracellular

leucine-rich repeat (LRR) disease-resistanceproteins, although the great majority belong

to the cytoplasmic class of NBS-LRR (nu-

cleotide binding site, and leucine-rich repeatdomain) proteins. Race-specific Avr genes

from oomycetes have been cloned only re-cently (5, 6, 78, 88). All four oomycete Avr

proteins (ATR1, ATR13, AVR3a, and Avr1b)carry a signal peptide followed by a con-

served motif (RXLR) that occurs in a largenumber of secreted oomycete proteins (78)(Figure 3b). The RXLR motif is similar to

a host-targeting signal that is required fortranslocation of proteins from malaria para-

sites (Plasmodiumspecies) into the cytoplasmof host cells (36, 55), leading to the hypothesis

that RXLR functions as a signal that mediates

NBS-LRR:nucleotide bindinsite and leucine-rirepeat

trafficking into host cells (78). This finding

raises the possibility that plant and animal eu-karyotic pathogens share similar mechanisms

for effector delivery into host cells.

ATR1NdWsB. ATR1NdWsB was identified bymap-based cloning as a single dominant gene

that segregates in a cross between the iso-late Emoy2 (avirulent onArabidopsisecotypeNiederzenz carrying theRPP1-Ndresistance

gene) and isolate Maks9 (virulent) ofH. para-

sitica (78). ATR1NdWsB encodes a 311-amino-

acid protein that is detected not only byRPP1-Ndbut also by theRPP1-WsBgene in

accession Wassilewskija. A construct derived

fromATR1NdWsB triggered cell death when ex-pressed in the cytoplasm ofArabidopsiscells

following transient expression using particle

bombardment.ATR1NdWsB

is highly polymor-phic among isolates ofH. parasitica. Six allelesfrom eight isolates were polymorphicin about

onethirdofallresidues.Diversifyingselection

and recombination apparently drove the evo-lution of theATR1NdWsB locus. Although the

region encompassing the signal peptide andthe RXLR motif show nonsynonymous se-

quence variation, theremainder of theproteinis under positive selection, suggesting that

this C-terminal region is involved in coevo-

lution with plant factors. Complex patternsof perception by the R proteins were identi-

fied. WhereasRPP1-Ndrecognizes only a sin-glealleleofATR1NdWsB,RPP1-WsB recognizes

four different alleles and confers resistance to

a broader range of isolates.

ATR13. Ppat17, one of the genes identifiedinH. parasitica by Bittner-Eddy et al. (13) as

highly expressed during infection ofArabidop-

sis,wasshowntobetheAvrgeneATR13 based

on cosegregation and in planta expressionanalyses (5). ATR13 encodes a 187-amino-acid protein that triggers RPP13-dependent

cell death following transient biolistic ex-pression or induced expression in transgenic

Arabidopsisplants (5). Besides the signal se-quence and RXLR motif, ATR13 has a heptad

leucine/isoleucine repeat motif followed by


8/13/2019 ARP_06

12/20

an imperfect direct repeat of 4 11 residues.

Similar to its cognate resistance geneRPP13

(82),ATR13 exhibits extreme levels of poly-

morphism with evidence of diversifying se-lection. Six different alleles were identified in

isolates ofH. parasitica. The alleles from iso-lates Aswa1, Emco5, and Goco1 carry a sin-

gle 11-amino-acid repeat unit but still trig-ger RPP13 response. As with ATR1, evidenceof recombination and an uneven distribution

of nucleotide variation throughout the codingsequence suggest that the effector activity is

localized to the C-terminal region of ATR13.

Avr1b-1. Fine structure genetic mapping

of avirulence of P. sojae on soybean plantscarrying the resistance gene Rps1b identified

a complex locus with at least two functional

genes,Avr1b-1 and Avr1b-2(88). The iden-tity ofAvr1b-2is not known, although it wasproposed to be required for the accumulation

ofAvr1b-1 mRNA. Avr1b-1 encodes a 138-

amino-acid protein that belongs to a complexgene family inP. sojae. Polymorphic alleles or

paralogs ofAvr1b-1 occur and exhibit highrates of nonsynonymous substitutions indica-

tive of diversifying selection. Virulent isolatessuch as P6497 and P9073 contain an intact

Avr1b-1gene but fail to accumulate Avr1b-1

mRNA, in contrast to avirulent isolates. In-filitration into soybean leaves of recombinant

Avr1b-1 protein produced in the yeastPichia

pastorisresulted in Rps1b-specific cell death.

Avr1b-1 also induced limited cell deathresponses on Rps1k plants, suggesting weak

recognition by this resistance gene. Because

the Rps1 gene cluster encodes cytoplasmicNBS-LRR proteins (10), Avr1b-1 is thought

to function in host cytoplasm (88). However,still unclear is whether the infiltrated Avr1b-1

protein acts extracellularly or can entersoybean cells in the absence of the pathogen.

AVR3a. Avr3a was identified using associ-ation analyses of polymorphisms in candi-

date effector genes with avirulence on potatoR genes (6, 16). Avr3a encodes at least two

polymorphic secreted proteins of 147 amino

acids that differ in only three residues, two ofwhich are in the mature protein (6). Isolates

ofP. infestansthat are avirulent onR3apotato

carry the avirulence gene Avr3a C19K80I103

whereas virulent isolates carry only the

virulence allele avr3a S19E80M103. Transientbiolistic and agroinfiltration coexpression as-

says showed thatR3a-mediated cell death wasspecifically induced by theAvr3aallele, con-firming interaction between the gene pair

Theavr3aallele is conserved among all iso-lates examined inP. infestans, suggesting that i

plays an important function in the pathogenOn the other hand, the lack of allelic diver-

sity in the Avr3a gene may reflect genetic

bottlenecks that populations ofP. infestansex-perienced as they spread out worldwide (30)

AVR3a does not require a signal peptide se-

quence for triggering R3a-mediated HR intransient assays and is therefore recognizedinside the plant cytoplasm (6). Remarkably

Avr3aofP. infestansandATR1NdWsB ofH. par-

asitica reside in syntenic regions, suggestingan ancestral effector locus in these oomycete

pathogens.

Other RXLR proteins. Bioinformatic anal-

yses indicated that the RXLR motif is fre-quent among secreted proteins ofP. infestans

P. sojae, andP. ramorumwith at least 100 pro-teins identified in each genome ( J. Win, C

Young & S. Kamoun, unpublished). This sug-gests that these pathogensdeliver a large seto

effectors to the host cytoplasm. Some of the

RXLR proteins, such as IPIO (In Planta In-duced) and several Pexs, have been identified

as candidate effectors prior to the discovery othe RXLR motif (72, 76, 99). One RXLR pro-

tein ofP. infestanscarries a functional nuclearlocalization signal and may therefore accumu-

late in host nuclei (T. D. Kanneganti, J. Win& S. Kamoun, unpublished).

CRN Protein Family (CRN1,CRN2. . .)

CRN1 and CRN2 were identified following

an in planta functional expression screen of

52 Kamoun

8/13/2019 ARP_06

13/20

candidate secreted proteinsofP. infestans(Pex)

based on a vector derived from Potato virusX(99). Expression of both genes inNicotiana

spp. and in the host plant tomato results ina leaf-crinkling and cell-death phenotype ac-

companied by an induction of defense-relatedgenes. Torto et al. (99) proposed that CRN1

and CRN2 function as effectors that per-turb host cellular processes based on anal-ogy to bacterial effectors, which typically

cause macroscopic phenotypes such as celldeath, chlorosis, and tissue browning when

expressed in host cells (49). The two crn genesare expressed inP. infestansduring coloniza-

tion of the host plant tomato.In planta expres-

sion of a collection of deletion mutants ofcrn2

indicate that this protein activates defense

responses in the plant cytoplasm (T. Torto-

Alalibo & S. Kamoun, unpublished).Database searches revealed that the CRNs

form a complex family of relatively largeproteins (about 400850 amino acids) inPhy-

tophthora(99). Sequence analyses revealed ev-idence of gene conversion and/or recombina-

tioninthe crn genefamilyofP. infestans(Z.Liu& S. Kamoun, unpublished). One of the crn

genes,crn8, encodes a secreted protein with

a predicted RD kinase domain that may tar-get host factors (C. Cakir & S. Kamoun, un-

published), whereas most crn genes have no

similarity to known sequences.

CONCLUSION

Our understanding of the biology of effec-tors is incomplete despite the tremendous

progress made in recent years. The discov-ery of diverse apoplastic and cytoplasmic ef-fectors facilitated an initial attempt at clas-

sifying and cataloguing these molecules. Asfive species, H. parasitica, P. capsici, P. infes-tans,P. ramorum,andP. sojae, undergo genomesequencing and annotation, we are moving

rapidly toward genome-wide catalogues of

oomycete effectors. Already it is evident thatthe effector secretome of plant pathogenic

oomycetes is much more complex than ex-

pected, with perhaps several hundred proteinsdedicated to reprogramming host cells. Com-

prehensive knowledge of the structure andfunction of pathogen effectors and the per-

turbations they cause in plants is a precon-dition for understanding the molecular ba-

sis of pathogenesis and disease. As molecularanalyses become more detailed, novel strate-

gies for manipulating plants toward resistance

to oomycete pathogens will undoubtedly be-come apparent.

SUMMARY POINTS

1. Plant pathogenic oomycetes modulate host cell defenses through a complex array of

effector proteins.

2. Functional genomics pipelines have greatly facilitated the identification of effectorsfrom cDNA and genomic sequences.

3. Apoplastic effectors are secreted into the plant extracellular space where they interact

with their host targets.

4. Several apoplastic effectors function as inhibitors of the enzymatic activities of plantPR proteins, such as glucanases and proteases.

5. Cytoplasmic effectors are translocated inside the plant cell where they target differentsubcellular compartments.

6. Several cytoplasmic effectors carry the RXLR motif, which is similar to a signal re-quired for translocation of proteins from malaria parasites to host cells.


8/13/2019 ARP_06

14/20

FUTURE ISSUES TO BE RESOLVED

1. What are the biochemical activities of oomycete effectors? How do they operate tomodulate host defenses? What are their host substrates?

2. How does the RXLR motif function in translocation of effectors inside host cells?

What is the translocation machinery? Is it derived from the pathogen or the host?

3. To what extent are effector secretion systems conserved in pathogenic eukaryotes?

ACKNOWLEDGMENTS

I thank Jorunn Bos, Catherine Bruce, Bill Morgan, Jing Song, Joe Win, and Carolyn Young

for critical reviews of the manuscript, Jonathan Walton for a thorough review of the textas well as many members of my laboratory and other colleagues for insightful discussions

Research in my laboratory is supported by NSF Plant Genome grant DBI-02 11659, USDA-

NRI project OHO00963-SS, and State and Federal Funds appropriated to OARDC, the OhioState University.

LITERATURE CITED

1. Abrahamsen MS, Templeton TJ, Enomoto S, Abrahante JE,ZhuG, et al. 2004. Complete

genome sequence of the apicomplexan,Cryptosporidium parvum.Science304:441452. Abramovitch RB, Martin GB. 2004. Strategies used by bacterial pathogens to suppress

plant defenses.Curr. Opin. Plant Biol.7:35664

3. Agrios GN. 2004.Plant Pathology. San Diego, CA: Academic. 952 pp. 5th ed.

4. Alfano JR, Collmer A. 2004. Type III secretion system effector proteins: double agents

in bacterial disease and plant defense. Annu. Rev. Phytopathol.42:385414

5. Similar to itscognate resistancegeneRPP13,ATR13exhibitsextreme levels ofpolymorphismswith evidence ofdiversifyingselectionsuggesting a

coevolutionaryarms race betweenH. parasitica andArabidopsis.

5. Allen RL, Bittner-Eddy PD, Grenville-Briggs LJ, Meitz JC, Rehmany AP, et al

2004. Host-parasite coevolutionary conflict between Arabidopsis and downy

mildew.Science306:195760

6.P. infestans Avr3aandH. parasiticaATR1NdWs B areRXLR effectorsthat reside insyntenic regionssuggesting anancestral effectorlocus in theseoomycetepathogens.

6. ArmstrongMR, Whisson SC, Pritchard L, Bos JI, Venter E, et al. 2005. An ancestra

oomycete locus contains late blight avirulence geneAvr3a, encoding a protein tha

is recognized in the host cytoplasm.Proc. Natl. Acad. Sci. USA 102:776671

7. Bailey BA, Jennings JC, Anderson JD. 1997. The 24-kDa protein fromFusarium oxys-porum f.sp.erythroxyli: occurrence in related fungi and the effect of growth medium onits production.Can. J. Microbiol.43:4555

8. Ballvora A, Ercolano MR, Weiss J, Meksem K, Bormann CA, et al. 2002. The R1 genefor potato resistance to late blight (Phytophthora infestans) belongs to the leucine zip-

per/NBS/LRR class of plant resistance genes.Plant J.30:36171

9. Bell KS, Sebaihia M, Pritchard L, Holden MT, Hyman LJ, et al. 2004. Genome sequence

of the enterobacterial phytopathogenErwinia carotovorasubsp.atrosepticaand character-ization of virulence factors.Proc. Natl. Acad. Sci. USA101:1110510

10. Bhattacharyya MK, Narayanan NN, Gao H, Santra DK, Salimath SS, et al. 2005. Iden

tification of a large cluster of coiled coil-nucleotide binding siteleucine rich repeat-type

genes from theRps1region containingPhytophthoraresistance genes in soybean.Theor

Appl. Genet.111:7586

11. Birch PRJ, Whisson S. 2001.Phytophthora infestansenters the genomics era. Mol. PlanPathol.2:25763

54 Kamoun

8/13/2019 ARP_06

15/20

12. Bishop JG, Ripoll DR, Bashir S, Damasceno CM, Seeds JD, Rose JK. 2004. Selection onGlycinebeta-1,3-endoglucanase genes differentially inhibited by aPhytophthoraglucanaseinhibitor protein.Genetics169:100919

13. Bittner-Eddy PD, Allen RL, Rehmany AP, Birch P, Beynon JL. 2003. Use of suppressionsubtractive hybridization to identify downy mildew genes expressed during infection of

Arabidopsis thaliana.Mol. Plant Pathol.4:501714. Bittner-Eddy PD, Crute IR, Holub EB, Beynon JL. 2000. RPP13 is a simple locus in

Arabidopsis thaliana for alleles that specify downy mildew resistance to different avirulencedeterminants inPeronospora parasitica.Plant J.21:1778815. Boissy G, ODonohue M, Gaudemer O, Perez V, Pernollet JC, Brunie S. 1999. The

2.1 A structure of an elicitin-ergosterol complex: a recent addition to the Sterol Carrier

Protein family.Protein Sci8:11919916. Bos JIB, Armstrong M, Whisson SC, Torto T, Ochwo M, et al. 2003. Intraspecific com-

parative genomics to identify avirulence genes fromPhytophthora.New Phytol.159:637217. Botella MA, Parker JE, Frost LN, Bittner-Eddy PD, Beynon JL, et al. 1998. Three genes

of theArabidopsis RPP1complex resistance locus recognize distinctPeronospora parasitica

avirulence determinants.Plant Cell10:18476018. Brecht S, Carruthers VB, Ferguson DJ, Giddings OK, Wang G, et al. 2001. The toxo-

plasmamicronemal protein MIC4 is an adhesin composed of sixconserved apple domains.J. Biol. Chem.276:411927

19. Brown PJ, Billington KJ, Bumstead JM, Clark JD, Tomley FM. 2000. A micronemeprotein fromEimeria tenellawith homology to the Apple domains of coagulation factor

XI and plasma pre-kallikrein.Mol. Biochem. Parasitol.107:9110220. Brown PJ, Gill AC, Nugent PG, McVey JH, Tomley FM. 2001. Domains of invasion

organelle proteins from apicomplexan parasites are homologous with the Apple domains

of blood coagulation factor XI and plasma pre-kallikrein and are members of the PANmodule superfamily.FEBS Lett.497:3138

21. Brunner F, Rosahl S, Lee J, Rudd JJ, Geiler C, et al. 2002. Pep-13, a plant defense-inducing pathogen-associated pattern from Phytophthora transglutaminases. EMBO J.

21:66818822. Bruno S, Duschak VG, Ledesma B, Ferella M, Andersson B, et al. 2004. Identification

and characterization of serine proteinase inhibitors fromNeospora caninum.Mol. Biochem.

Parasitol.136:101723. Damasceno CM. 2005. Cynthia Damasceno. http://labs.plantbio.cornell.edu/rose/

people/cynthia.htm24. de Meaux J, Mitchell-Olds T. 2003. Evolution of plant resistance at the molecular level:

ecological context of species interactions.Heredity91:3455225. Erwin DC, Ribeiro OK. 1996.Phytophthora Diseases Worldwide. St. Paul, MN: APS Press.

562 pp.26. Fabritius AL, Cvitanich C, Judelson HS. 2002. Stage-specific gene expression during

sexual development inPhytophthora infestans.Mol. Microbiol.45:105766

27. Fellbrich G, Romanski A, Varet A, Blume B, Brunner F, et al. 2002. NPP1, aPhytophthora-associated trigger of plant defense in parsley and Arabidopsis.Plant J.32:37590

28. Gaulin E, Jauneau A, Villalba F, Rickauer M, Esquerr e-Tugay e M-T, Bottin A. 2002.

The CBEL glycoprotein ofPhytophthora parasiticavar.nicotianaeis involved in cell walldeposition and adhesion to cellulosic substrates.J. Cell Sci.115:456575

29. Gaulin E, Torto T, Martinez Y, Khatib M, Bottin A, et al. 2003. Structure-functionrelationship studies on the CBEL glycoprotein ofPhytophthora parasiticavar.nicotianae,

by PVX expression inN. benthamiana.Fungal Genet. Newsl.50(Suppl.):119


8/13/2019 ARP_06

16/20

30. Goodwin SB, Cohen BA, Fry WE. 1994. Panglobal distribution of a single clonal lineage

of the Irish potato famine fungus. Proc. Natl. Acad. Sci. USA91:1159195

31. Grenville-Briggs LJ, Van West P. 2005. The biotrophic stages of oomycete-plant inter-actions.Adv. Appl. Microbiol.57:21743

32. HalimVA,HungerA,MacioszekV,LandgrafP,NurnbergerT,etal.2004.Theoligopep

tide elicitor Pep-13 induces salicylic acid-dependent and -independent defense reactionsin potato.Physiol. Mol. Plant Pathol.64:31118

33. Hammond-Kosack KE, Jones JDG. 1997. Plant disease resistance genes.Annu. Rev. PlanPhysiol. Plant Mol. Biol. 48:575607

34. Hendrix JW. 1970. Sterols in growth and reproduction of fungi.Annu. Rev. Phytopathol

8:11130

35. Hewitt WB, Pearson RC. 1988. Downy mildew. InCompendium of Grape Diseases, ed. RCPearson, AC Goheen, pp. 1113. St. Paul, MN: APS Press

36. Hiller NL, Bhattacharjee S, van Ooij C, Liolios K, Harrison T, et al. 2004. A host-

targeting signal in virulence proteins reveals a secretome in malarial infection. Science

306:193437

37. Huang S, van der Vossen EHK, Vleeshouwers V, Zhang N, et al. 2005. Comparative

genomics enabled the isolation of the R3a late blight resistance gene in potato. Plant J

42:2516138. Huitema E, Bos JIB, Tian M, Win J, Waugh ME, Kamoun S. 2004. Linking sequence to

phenotype inPhytophthora-plant interactions.Trends Microbiol.12:193200

39. Jentoft N. 1990. Why are proteinsO-glycosylated?Trends Biochem.Sci.15:29194

40. Joosten MHAJ, Vogelsang R, Cozijnsen TJ, Verberne MC, de Wit PJGM. 1997. The

biotrophic fungusCladosporium fulvumcircumvents Cf-4 mediated resistance by produc-ing unstable AVR4 elicitors.Plant Cell9:36779

41. Kamoun S. 2003. Molecular genetics of pathogenic oomycetes.Eukaryot. Cell2:19199

42. Kamoun S, Honee G, Weide R, Lauge R, Kooman-Gersmann M, et al. 1999. The funga

geneAvr9and the oomycete gene inf1confer avirulence to potato virus X on tobacco

Mol. Plant Microbe. Interact.12:4596243. Kamoun S, Hraber P, Sobral B, Nuss D, Govers F. 1999. Initial assessement of genediversity for the oomycete pathogenPhytophthora infestansbased on expressed sequences

Fungal Genet. Biol.28:94106

44. Kamoun S, Lindqvist H, Govers F. 1997. A novel class of elicitin-like genes fromPhy-

tophthora infestans.Mol. Plant Microbe. Interact.10:102830

45. Kamoun S, Smart CD. 2005. Late blight of potato and tomato in the genomics era.PlanDis.89:69299

46. Kamoun S, van West P, de Jong AJ, de Groot K, Vleeshouwers V, Govers F. 1997. A geneencoding a protein elicitor ofPhytophthora infestansis down-regulated during infection of

potato.Mol. Plant Microbe. Interact.10:1320

47. Kamoun S, van West P, Vleeshouwers VG, de Groot KE, Govers F. 1998. ResistanceofNicotiana benthamiana to Phytophthora infestansis mediated by the recognition of theelicitor protein INF1.Plant Cell10:141326

48. Kamoun S, Young M, Glascock C, Tyler BM. 1993. Extracellular protein elicitors fromPhytophthora: host-specificity and induction of resistance to fungal and bacterial phy-topathogens.Mol. Plant Microbe Interact.6:1525

49. Kjemtrup S, Nimchuk Z, Dangl JL. 2000. Effector proteins of phytopathogenic bacteria

bifunctional signals in virulence and host recognition.Curr. Opin. Microbiol.3:7378

56 Kamoun

8/13/2019 ARP_06

17/20

50. Kooman-Gersmann M, Vogelsang R, Hoogendijk ECM, de Wit PJGM. 1997. Assign-

ment of amino acid residues of the Avr9 peptide ofCladosporium fulvumthat determineelicitor activity.Mol. Plant Microbe. Interact.10:82129

51. Lauge R, De Wit PJ. 1998. Fungal avirulence genes: structure and possible functions.Fungal Genet. Biol.24:28597

52. Lindh JG, Botero-Kleiven S, Arboleda JI, Wahlgren M. 2001. A protease inhibitor asso-ciated with the surface ofToxoplasma gondii.Mol. Biochem. Parasitol.116:13745

53. Thescr74geencodes a highlypolymorphic smacysteine-richprotein. It isup-regulatedduring hostinfection and isunder diversifyinselection possiblas a consequencea coevolutionaryarms race with h

plants.

53. Liu Z, Bos JIB, Armstrong M, Whisson SC, da Cunha L, et al. 2005. Patternsof diversifying selection in the phytotoxin-likescr74 gene family ofPhytophthora

infestans.Mol. Biol. Evol.22:65972

54. Luderer R, de Kock MJD, Dees RHL, de Wit PJM, Joosten MH. 2002. Functionalanalysis of cysteine residues of ECP elicitor proteins of the fungal tomato pathogenCladosporium fulvum.Mol. Plant Pathol.2:9195

55. Marti M, Good RT, Rug M, Knuepfer E, Cowman AF. 2004. Targeting malaria virulence

and remodeling proteins to the host erythrocyte.Science306:193033

56. McDowell JM, Dhandaydham M, Long TA, Aarts MG, Goff S, et al. 1998. Intragenicrecombination and diversifying selection contribute to the evolution of downy mildew

resistance at theRPP8locus ofArabidopsis.Plant Cell10:186174

57. Menne KM, HermjakobH, Apweiler R. 2000.A comparison of signalsequencepredictionmethods using a test set of signal peptides.Bioinformatics16:74142

58. Mikes V, Milat ML, Ponchet M, Panabieres F, Ricci P, Blein JP. 1998. Elicitins, pro-

teinaceous elicitors of plant defense, are a new class of sterol carrier proteins. Biochem.

Biophys. Res. Commun.245:1333959. Mikes V, Milat ML, Ponchet M, Ricci P, Blein JP. 1997. The fungal elicitor cryptogein

is a sterol carrier protein.FEBS Lett.416:1909260. Morris MT, Cheng WC, Zhou XW, Brydges SD, Carruthers VB. 2004.Neospora caninum

expresses an unusual single-domain Kazal protease inhibitor that is discharged into theparasitophorous vacuole.Int. J. Parasitol.34:693701

61. Nespoulous C, Gaudemer O, Huet JC, Pernollet JC. 1999. Characterization of elicitin-

like phospholipasesisolated fromPhytophthora capsiciculture filtrate.FEBS Lett452:400662. Nielsen H, Brunak S, vonHeijne G. 1999.Machinelearningapproachesfor theprediction

of signal peptides and other protein sorting signals.Protein Eng.12:3963. Nurnberger T, Nennstiel D, Jabs T, Sacks WR, Hahlbrock K, Scheel D. 1994. High

affinity binding of a fungal oligopeptide elicitor to parsley plasma membranes triggersmultiple defense responses.Cell78:44960

64. Orsomando G, Lorenzi M, Ferrari E, de Chiara C, Spisni A, Ruggieri S. 2003. PcF

protein fromPhytophthora cactorumand its recombinant homologue elicit phenylalanineammonia lyase activation in tomato.Cell. Mol. Life Sci.60:147076

65. Orsomando G, Lorenzi M, Raffaelli N, Dalla Rizza M, Mezzetti B, Ruggieri S. 2001.Phytotoxic protein PcF, purification, characterization, and cDNA sequencing of a novel

hydroxyproline-containing factor secreted by the strawberry pathogenPhytophthora cac-torum.J. Biol. Chem.276:2157884

66. Osman H, Mikes V, Milat ML, Ponchet M, Marion D, et al. 2001. Fatty acids bind to

the fungal elicitor cryptogein and compete with sterols.FEBS Lett.489:555867. Osman H, Vauthrin S, Mikes V, Milat ML, Panabieres F, et al. 2001. Mediation of elicitin

activity on tobacco is assumed by elicitin-sterol complexes. Mol. Biol. Cell12:28253468. Panstruga R. 2003. Establishing compatibility between plants and obligate biotrophic

pathogens.Curr. Opin. Plant Biol.6:32026


8/13/2019 ARP_06

18/20

69. Parker JE, Coleman MJ, Szabo V, Frost LN, Schmidt R, et al. 1997. The Arabidop-

sis downy mildew resistance gene RPP5shares similarity to the toll and interleukin-1receptors withNandL6.Plant Cell9:87994

70. Pemberton CL, Salmond GPC. 2004. The Nep1-like proteins a growing family of mi-

crobial elicitors of plant necrosis.Mol. Plant Pathol.5:35359

71. Pemberton CL, Whitehead NA, Sebaihia M, Bell KS, Hyman LJ, et al. 2005. Novelquorum-sensing-controlled genes in Erwinia carotovora subsp. carotovora: identification

of a fungal elicitor homologue in a soft-rotting bacterium. Mol. Plant Microbe. Interact18:34353

72. Pieterse CMJ, Derksen AMCE, Folders J, Govers F. 1994. Expression of thePhytophthorainfestans ipiBandipi0genesin plantaandin vitro.Mol. Gen. Genet.244:26977

73. Ponchet M, Panabieres F, Milat M-L, Mikes V, Montillet J-L, et al. 1999. Are elicitins

cryptograms in plant-Oomycete communications?Cell.Mol. Life Sci.56:102047

74. Pszenny V, Angel SO, Duschak VG, Paulino M, Ledesma B, et al. 2000. Molecularcloning, sequencing and expression of a serine proteinase inhibitor gene fromToxoplasma

gondii.Mol. Biochem. Parasitol.107:24149

75. Punja ZK. 2004. Fungal Disease Resistance in Plants. Binghamton, NY: Food ProductsPress. 266 pp.

76. QutobD,KamounS,GijzenM.2002.ExpressionofaPhytophthora sojae necrosis-inducingprotein occurs during transition from biotrophy to necrotrophy.Plant J.32:36173

77. Rawlings ND, Tolle DP, Barrett AJ. 2004. MEROPS: the peptidase database. Nucleic

Acids Res. 32:D16044

78. ATR1NdWs B is ahighly polymorphicRXLR effectorgene that exhibitscomplex patterns ofrecognition onArabidopsisplants

carrying theRPP1gene.

78. Rehmany AP, Gordon A, Rose LE, Allen RL, Armstrong MR, et al. 2005. Differen-

tial recognition of highly divergent downy mildew avirulence gene alleles byRPP1

resistance genes from two Arabidopsis lines.Plant Cell17:183950

79. Ricci P, Bonnet P, Huet J-C, Sallantin M, Beauvais-Cante F, et al. 1989. Structure and

activity of proteins from pathogenic fungi Phytophthora eliciting necrosis and acquiredresistance in tobacco.Eur. J. Biochem.183:55563

80. Rizzo DM, Garbelotto M, Hansen EM. 2005.Phytophthora ramorum: integrative researchand management of an emerging pathogen in California and Oregon forests.Annu. Rev

Phytopathol.43:30935

81. Glucanaseinhibitor proteinsGIP1 and GIP2 ofP. sojae inhibit thesoybeanendo--1,3glucanase EgaseAsuggesting acounterdefense

mechanism.

81. Rose JK, Ham KS, Darvill AG, Albersheim P. 2002. Molecular cloning and charac-

terization of glucanase inhibitor proteins: coevolution of a counterdefense mech-

anism by plant pathogens.Plant Cell14:132945

82. Rose LE, Bittner-Eddy PD, Langley CH, Holub EB, Michelmore RW, Beynon JL. 2004

The maintenance of extreme amino acid diversity at the disease resistance gene,RPP13

inArabidopsis thaliana.Genetics166:151727

83. Sacks W, Nurnberger T, Hahlbrock K, Scheel D. 1995. Molecular characterization o

nucleotide sequences encoding the extracellular glycoprotein elicitor from Phytophthora

megasperma.Mol. Gen. Genet.246:455584. Sasabe M, Takeuchi K, Kamoun S, Ichinose Y, Govers F, et al. 2000. Independent path-

ways leading to apoptotic cell death, oxidative burst and defense gene expression in re-

sponse to elicitin in tobacco cell suspension culture. Eur. J. Biochem.267:500513

85. Schmitthenner AF. 1985. Problems and progress toward control of Phytophthora roorot of soybean.Plant Dis.69:36268

86. Schneider G, Fechner U. 2004. Advances in the prediction of protein targeting signalsProteomics4:157180

58 Kamoun

8/13/2019 ARP_06

19/20

87. Sejalon-Delmas N, Villalba Mateos F, Bottin A, Rickauer M, Dargent R, Esquerr e-

Tugay e MT.1997. Purification, elicitor activity, and cellwall localization of a glycoproteinfromPhytophthora parasiticavar.nicotianae, a fungal pathogen of tobacco. Phytopathology

87:899909

88.Avr1b-1, aRXLR effector,occurs in a comp

locus with at leastwo functionalgenes that confeavirulence onsoybean plantscarrying theresistance geneRps1.

88. Shan W, Cao M, Leung D, Tyler BM. 2004. The Avr1b locus of Phytophthora

sojaeencodes an elicitor and a regulator required for avirulence on soybean plants

carrying resistance geneRps1b.Mol. Plant Microbe. Interact.17:394403

89. Slusarenko AJ, Schlaich NL. 2003. Downy mildew ofArabidopsis thaliana caused byHyaloperonospora parasitica (formerlyPeronospora parasitica).Mol. Plant Pathol.4:1597090. Smart CD, Fry WE. 2001. Invasions by the late blight pathogen: renewed sex and en-

hanced fitness.Biol. Invas.3:2354391. Song J, Bradeen JM, Naess SK, Raasch JA, Wielgus SM, et al. 2003. Gene RB cloned

fromSolanum bulbocastanumconfers broad spectrum resistance to potato late blight.Proc.

Natl. Acad. Sci. USA100:912823392. Stahl EA, Bishop JG. 2000. Plant-pathogen arms races at the molecular level.Curr. Opin.

Plant Biol. 3:29930493. Staskawicz BJ, Ausubel FM, Baker BJ, Ellis JG, Jones JDG. 1995. Molecular genetics of

plant disease resistance.Science268:6616794. Takami H, Horikoshi K. 2000. Analysis of the genome of an alkaliphilic Bacillusstrain

from an industrial point of view. Extremophiles4:9910895. Tian M, Benedetti B, Kamoun S. 2005. A second Kazal-like protease inhibitor fromPhy-

tophthora infestansinhibits and interacts with the apoplastic pathogenesis-related protease

P69B of tomato.Plant Physiol.138:178593

96. EPI1 interacwith and inhibitsthe PR proteinP69B, asubtilisin-likeserine protease otomato, suggesti

a counterdefensemechanism.

96. Tian M, Huitema E, da Cunha L, Torto-Alalibo T, Kamoun S. 2004. A Kazal-

like extracellular serine protease inhibitor fromPhytophthora infestanstargets the

tomato pathogenesis-related protease P69B.J. Biol. Chem.279:263707797. Tian M, Kamoun S. 2005. A two disulfide bridge Kazal domain fromPhytophthora exhibits

stable inhibitory activity against serine proteases of the subtilisin family. BMC Biochem.

6:1598. Tor M, Brown D, Cooper A, Woods-Tor A, Sjolander K, et al. 2004. Arabidopsis downy

mildew resistance gene RPP27 encodes a receptor-like protein similar to CLAVATA2and tomato Cf-9.Plant Physiol.135:110012

99. Computationpredictions ofsecreted proteinwere combinedwithin plantaexpression assayresulting in theidentification of crnfamily of

Phytophthoraeffectors.

99. Torto T, Li S, Styer A, Huitema E, Testa A, et al. 2003. EST mining and func-

tional expression assays identify extracellular effector proteins fromPhytophthora.

Genome Res.13:167585100. Torto-Alalibo T, Tian M, Gajendran K, Waugh ME, van West P, Kamoun S. 2005.

Expressed sequence tags from the oomycete fish pathogen Saprolegnia parasitica revealputative virulence factors.BMC Microbiol.5:46

101. vanderVossenE,SikkemaA,HekkertBL,GrosJ,StevensP,etal.2003.AnancientR genefrom the wild potato speciesSolanum bulbocastanumconfers broad-spectrum resistance to

Phytophthora infestansin cultivated potato and tomato.Plant J.36:86782

102. van der Vossen EA, Gros J, Sikkema A, Muskens M, Wouters D, et al. 2005. TheRpi-blb2gene fromSolanum bulbocastanumis anMi-1gene homolog conferring broad-spectrum

late blight resistance in potato.Plant J.44:20822103. vant Slot KAE, Knogge W. 2002. A dual role for microbial pathogen-derived effector

proteins in plant disease and resistance.Crit. Rev. Plant Sci.21:22971104. Vauthrin S, Mikes V, Milat ML, Ponchet M, Maume B, et al. 1999. Elicitins trap and

transfer sterols from micelles, liposomes and plant plasma membranes. Biochim. Biophys.Acta1419:33542


8/13/2019 ARP_06

20/20

105. Veit S, Worle JM, Nurnberger T, Koch W, Seitz HU. 2001. A novel protein elici-

tor (PaNie) fromPythium aphanidermatuminduces multiple defense responses in carrotArabidopsis, and tobacco.Plant Physiol.127:83241

106. Villalba Mateos F, Rickauer M, Esquerre-Tugaye MT. 1997. Cloning and characteriza-tion of a cDNA encoding an elicitor ofPhytophthora parasiticavar.nicotianaethat shows

cellulose-binding and lectin-like activities.Mol. Plant Microbe Interact.10:104553107. Wang JY, Cai Y, Gou JY, Mao YB, XuYH, etal. 2004. VdNEP, anelicitorfrom Verticillium

dahliae, induces cotton plant wilting.Appl. Environ. Microbiol.70:498995

Documents

ARP_06