ARTICLE IN PRESS

Journal of Plant Physiology 163 (2006) 256—272

0176-1617/$ - sdoi:10.1016/j.

AbbreviationAD, activationtion factor; HRrepeat; NB, nusignal; PIC, preR, resistance;peptide repeatXoo, Xanthomo�Correspond

fax: +49 345 55E-mail addr

(T. Lahaye).

www.elsevier.de/jplph

Gene-for-gene-mediated recognition ofnuclear-targeted AvrBs3-like bacterial effectorproteins

Sebastian Schornack, Annett Meyer, Patrick Romer, Tina Jordan,Thomas Lahaye�

Institut fur Genetik, Martin-Luther-Universitat Halle-Wittenberg, 06099 Halle (Saale), Germany

Received 9 November 2005; received in revised form 2 December 2005; accepted 2 December 2005

KEYWORDSAvrBs4;Guard model;Receptor–ligandmodel;Tetratricopeptiderepeats;Transcriptionalactivation

ee front matter & 2005jplph.2005.12.001

s: 35S, Cauliflower modomain; Avr, avirulence, hypersensitive responcleotide binding site; Ninitiation complex; PP5TFIIA, transcription facts; Xcv, Xanthomonas canas oryzae pv. oryzaeing author. Tel.: +49 34527151.ess: thomas.lahaye@ge

SummaryPlant disease resistance (R) genes mediate specific recognition of pathogens viaperception of cognate avirulence (avr) gene products. The numerous highly similarAvrBs3-like proteins from the bacterial genus Xanthomonas provide together withtheir corresponding R proteins a unique biological resource to dissect the molecularbasis of recognition specificity. A central question in this context is if R proteins thatmediate recognition of structurally similar Avr proteins are themselves functionallysimilar or rather dissimilar. The recent isolation of rice xa5, rice Xa27 and tomatoBs4, R genes that collectively mediate recognition of avrBs3-like genes, provides afirst clue to the molecular mechanisms that plants employ to detect AvrBs3-likeproteins. Their initial characterization suggests that these R proteins are structurallyand functionally surprisingly diverge. This review summarizes the current knowledgeon R-protein-mediated recognition of AvrBs3-like proteins and provides workingmodels on how recognition is achieved at the molecular level.& 2005 Published by Elsevier GmbH.

Published by Elsevier GmbH.

saic virus 35S promoter;; GTF, general transcrip-se; LRR, leucine-richLS, nuclear localization, protein phosphatase 5;or IIA; TPR, tetratrico-mpestris pv. vesicatoria;

55 26345;

netik.uni-halle.de

Introduction

Plants have evolved efficient defense systems toward off a phylogenetically broad diversity ofpathogens including bacteria, fungi, oomycetes,nematodes, viruses and even insects (Dangland Jones, 2001). Conceptually, we distinguishat present two major layers of resistance,that mediate protection against non-host and

ARTICLE IN PRESS

Gene-for-gene-mediated recognition of nuclear-targeted AvrBs3-like bacterial effector proteins 257

host-adapted pathogens, respectively. The basal(non-host) defense system is triggered by function-ally indispensable and structurally conserved mi-crobial structures. Recognition of these generalelicitors (also defined as ‘‘pathogen-associatedmolecular patterns’’) is probably brought about bya limited number of receptors residing in theplasma membrane (Boller, 2005; Nurnberger andLipka, 2005; Zipfel and Felix, 2005). Despite thepresence of the basal defense system, microbialphytopathogens are capable of multiplying incertain host plants to which they are adapted.Analysis of bacterial pathogens suggests thatsuppression of the basal defense system ismediated by the synergistic action of numerousso-called effector proteins that are injected intothe plant by the type III secretion system (Abramo-vitch and Martin, 2004; Espinosa and Alfano, 2004).In some cases, the effectors that suppress the basaldefense trigger a second, superimposed defenselayer. The genes that encode these effectors havebeen functionally defined as avirulence (avr) genesbecause their presence renders strains expressingthem avirulent (Staskawicz et al., 1984). Recogni-tion of microbial avr gene products is dependent onthe simultaneous expression of corresponding Rgene products in the plant host (Flor, 1971) andoften associated with a hypersensitive response(HR; Greenberg and Yao, 2004). Since this defenselayer is genetically defined by correspondingpathogen avr and plant R genes, it has been coinedgene-for-gene resistance. The simplest biochemicalmodel that explains this genetic interaction is thatavr-gene encoded ligands bind to and activate amatching R gene-encoded receptor (Gabriel andRolfe, 1990). Isolation of numerous R genesrevealed that most encode proteins with a pre-dicted nucleotide binding site (NB) and leucine-richrepeats (LRRs) (Belkhadir et al., 2004; Gomez-Gomez, 2004; Jones and Takemoto, 2004; Meyerset al., 2005). The fact that (i) LRR domains areknown to participate in protein–protein interac-tions and (ii) the structural uniformity of R proteinssupport the assumption that the receptor–ligandmodel best represents the unifying biochemicalbasis of genetically defined gene-for-gene interac-tions. However, experimental data that support thereceptor–ligand model are rare (Deslandes et al.,2003; Jia et al., 2000; Scofield et al., 1996; Tanget al., 1996) and have inspired formulation of theguard model. This model predicts that Avr proteinsbind to and interfere with components of the basaldefense system which themselves are monitored(guarded) by complementary R proteins (Van derBiezen and Jones, 1998). Recently, many variationsof the guard model have been proposed, all

collectively stressing that R proteins seem todetect (virulence) activity rather than the struc-ture of a respective Avr protein (Bonas and Lahaye,2002; Dangl and Jones, 2001; Martin et al., 2003).Consequently, detailed knowledge of the virulencefunction of a given Avr protein is a crucialprerequisite to study its perception by cognate Rproteins.

In the past, we have studied recognition ofAvrBs3 and AvrBs4, two nearly identical bacterialeffector proteins from Xanthomonas campestris pv.vesicatoria (Xcv) that are specifically detected bythe cognate pepper Bs3 and tomato Bs4 resistanceproteins, respectively. AvrBs3 and AvrBs4 belong toa large family of highly similar, nuclear-targetedAvr proteins. Availability of numerous highly simi-lar, but structurally and functionally distinct,avrBs3 homologs and complementary plant R genesprovides an exceptional biological resource tostudy the molecular basis of recognition specificity.Molecular analysis of these gene-for-gene interac-tions should provide valuable information on howplants manage to distinguish between nearlyidentical microbial Avr proteins. Another intriguingquestion in this context is if the molecularmechanisms of R proteins that mediate recognitionof AvrBs3-like proteins are similar or dissimilar.

Due to the concerted efforts of several researchgroups, we have detailed knowledge on thevirulence function of AvrBs3-like proteins and mostrecently three R genes corresponding to AvrBs3-likeproteins have been cloned. This review summarizesour current knowledge on R-gene-mediated recog-nition of AvrBs3-like proteins. We also discuss thecurrently cloned R genes corresponding to AvrBs3-like proteins and postulate working models of howthese R gene products mediate recognition at themolecular level. Readers that are primarily inter-ested in the virulence functions of AvrBs3 andAvrBs3-like proteins are referred to the review byGurlebeck et al. (this issue), which provides a morecomprehensive review of the subject.

AvrBs3-like proteins differ primarily intheir repeat domain

AvrBs3 was the first-described member of theAvrBs3-family, identified more than 17 years ago(Bonas et al., 1989). Since then, more than 40AvrBs3-like proteins from different Xanthomonasspecies and pathovars and Ralstonia solanacearumhave been deposited in GenBank (Table 1). Themost striking structural feature of AvrBs3 andhomologous proteins is the so-called repeat domain

ARTICLE IN PRESS

Table

1.

Mem

bersof

theAv

rBs3

family

Designa

tion

Gen

Ban

kac

cession

Repea

tstructure

Referenc

eaRge

neSp

ecies

Referenc

eb

Xantho

mon

ascampe

strispv.

vesicatoria

AvrBs3

X16

130,

CAA34

257

17.5�34

Bon

aset

al.(198

9)Bs3

Pepper

Pierre

etal.(200

0)Av

rBs4

X68

781,

CAA48

680

17.5�34

Bon

aset

al.(199

3)Bs4

Tomato

Scho

rnac

ket

al.(200

4)*

X.oryzae

pv.

oryzae

AvrXa3

AY12

9298

,NC_0

0683

4,AAN01

357,

YP_1

9987

8.1

8.5�34

Leeet

al.(200

5);Li

etal.

(200

4)Xa

3Rice

Ezuk

aet

al.(197

5);Oga

wa

etal.(198

6);Yoshim

uraet

al.

(199

2)Av

rxa5

AY37

7126

,AAQ79

773

5.5�34

Bai

etal.(200

0);Hop

kins

etal.(199

2)xa

5Rice

Blair

etal.(200

3);Iyer

and

McC

ouch

(200

4)*;

Zhon

get

al.(200

3)Av

rXa7

AF2

6293

3,AAF9

8332

,AAF9

8343

25.5�34

Hop

kins

etal.(199

2);Ve

raCruzet

al.(200

0)Xa

7Rice

Porter

etal.(200

3);Sidhu

etal.(197

8)Av

rXa1

0U50

552,

AAA92

974

15.5�34

Hop

kins

etal.(199

2);Zh

uet

al.(199

8)Xa

10Rice

Yoshim

uraet

al.(198

3);

Xingh

uaet

al.(199

6);

Yoshim

uraet

al.(199

5)Av

rXa2

7AY

9864

94,AAY

5416

816

.5�34

Guet

al.(200

5)Xa

27Rice

Guet

al.(200

5)*

X.oryzae

pv.

oryzicola

Avr/pth3

AY87

5712

,AAW

5949

215

.5�34

Avr/pth13

AY87

5711

,AAW

5949

15.5�34

Avr/pth14

AY87

5713

,AAW

5949

319

.5�34

X.campe

strispv.

armoraciae

Hax

2AY

9939

3721

.5�35

Kay

etal.(200

5)Hax

3AY

9939

3811

.5�34

Kay

etal.(200

5)Bs4

Tomato

Kay

etal.(200

5)Hax

4AY

9939

3914

.5�34

Kay

etal.(200

5)Bs4

Tomato

Kay

etal.(200

5)

X.campe

strispv.

malva

cearum

Avrb6

L066

34,AAB00

675

13.5�34

DeFe

yter

andGab

riel

(199

1);

DeFe

yter

etal.(199

3)B1

Cotton

DeFe

yter

etal.(199

3);

Gab

riel

etal.(198

6)PthN

AF0

1622

1,AAB69

865

13.5�34

Cha

krab

arty

etal.(199

7)

X.ax

onop

odis

pv.

citri

Apl1

AB02

1363

,BAA37

119

17.5�34

Apl2

AB02

1364

,BAA37

120

15.5�34

Apl3

AB02

1365

,BAA37

121

23.5�34

PthA

U28

802,

AAC43

587

17.5�34

Gab

riel

etal.(198

6);Sw

arup

etal.(199

1)

S. Schornack et al.258

ARTICLE IN PRESS

PthA1

XACa0

022,

AAM39

226

16.5�34

Bruning

san

dGab

riel

(200

3);

DaSilvaet

al.(200

2)PthA2

XACa0

039,

AAM39

243

15.5�34

Bruning

san

dGab

riel

(200

3);

DaSilvaet

al.(200

2)PthA3

XACb00

15,AAM39

261

15.5�34

Bruning

san

dGab

riel

(200

3);

DaSilvaet

al.(200

2)PthA4

XACb00

65,AAM39

311

17.5�34

Bruning

san

dGab

riel

(200

3);

DaSilvaet

al.(200

2)PthB

AY22

8335

,AAO72

098

17.5�34

X.campe

strispv.

man

ihotis

pTHB

AF0

1232

5,AAD01

494

12.5�34

Restrepoan

dVe

rdier(199

7)

Ralstoniasolana

cearum

Brg11

NC_0

0329

5,NP_5

1993

616

.5�35

Salano

ubat

etal.(200

2);

Cun

nacet

al.(200

4)

aRe

ferenc

esdescribeisolationan

dch

arac

terization

ofav

irulen

cege

nesan

dproteins,

respec

tive

ly.

bRe

ferenc

esdescribege

neticmap

ping

,isolation(ind

icated

byasterisk)of

plant

resistan

ce(R)ge

nes,

respec

tive

ly.Pleaseno

tethat

xa5isform

ally

notprove

nto

med

iate

reco

gnitionof

anAv

rBs3-likeprotein.

Gene-for-gene-mediated recognition of nuclear-targeted AvrBs3-like bacterial effector proteins 259

ARTICLE IN PRESS

S. Schornack et al.260

made up of 5.5–25.5 nearly identical, tandemlyarranged copies of a 34 amino-acid (aa) repeat unit(Lahaye and Bonas, 2001). Two members of theAvrBs3 family, Brg11 from R. solanacearum (Cunnacet al., 2004) and Hax2 from Xanthomonas campes-tris pv. armoraciae (Kay et al., 2005), are structu-rally exceptional and contain repeat units that are35 aa in length due to insertion of a prolinebetween repeat residues 32 and 33. Variability

Figure 1. Comparison of AvrBs3 and the AvrBs3-likeAvrBs4 protein. The central parts of the AvrBs3 andAvrBs4 polypeptide chains are composed of 17.5 tan-demly arranged, nearly identical 34-aa repeat units. TheC-terminus contains functional NLS and a transcriptionalactivation domain (AD). Differences between AvrBs3 andAvrBs4 are confined to the repeat region (residues289–882) with exception of a 4 aa deletion in the AvrBs4C-terminus (D4 aa). The AvrBs3 and AvrBs4 repeat unitsare numbered and depicted in single-letter code belowthe structural representation in the upper and lowerlines. The last two lines represent the AvrBs3 and AvrBs4consensi, respectively. Residues identical to, or differingfrom the repeat unit consensus are depicted by dashes orsingle-letter code, respectively.

among the imperfect repeat units of a given AvrBs3-like protein is not randomly distributed but foundprimarily in repeat unit positions 4, 12, 13 and 24(Fig. 1). At the majority of polymorphic residuesonly two alternatives occur. For example, in AvrBs3the repeat unit residue 12 is either histidine orasparagine (Fig. 1). In contrast, repeat unit residue13 generally displays a much higher variability. Forexample, four different aa’s can be found in repeatunit residue 13 of AvrBs3 (Fig. 1). Some AvrBs3-likeproteins even have a deletion of this residue insome of their repeat units (e.g. AvrXa7; (Yanget al., 2000) and AvrXa27 (Gu et al., 2005)).Comparison of different AvrBs3-like proteins fromXanthomonas reveals 80–97% overall sequenceidentity (Table 2) with most differences confinedto the repeat domain. For example, AvrBs3 and theAvrBs3-like AvrBs4 protein differ exclusively in theirrepeat regions, with the exception of a four aadeletion in the C-terminus of AvrBs4 with respect toAvrBs3 (Fig. 1).

In silico analysis predicts atetratricopeptide repeat-like structurefor AvrBs3-homologous proteins

Although more than 40 genes encoding AvrBs3-like proteins have been isolated, as yet there is nocrystal structure available for any member of thisprotein class. In the absence of a crystal structure,we employed 3D-Jury metapredictor (Ginalskiet al., 2003) to identify potential templates formodeling a three-dimensional structure of AvrBs3and to generate a 3D model (Fig. 2). In silicoanalysis revealed that the AvrBs3 repeat domainstructurally resembles tetratricopeptide repeat(TPR) containing proteins (D’Andrea and Regan,2003). The TPR is a structural motif that consists of3–16 tandem repeats of 34-aa residues and whichmediates protein–protein interactions and theassembly of multiprotein complexes (Blatch andLassle, 1999). Based on its homology to the TPRdomain, each repeat unit of AvrBs3 is predicted toform two a-helices. The sum of the repeat units issupposed to generate a right-handed a–a helicalsuperstructure (Fig. 2). In this helical superstruc-ture, the polymorphic repeat unit residues arearranged in a string that winds itself along the axisof the helix.

In agreement with the known functions of TPRdomains, the TPR-like repeat domain of AvrBs3 hasbeen shown to be crucial to dimerization of AvrBs3(Gurlebeck et al., 2005). Although the structuralbasis of this dimerization remains to be clarified,

ARTICLE IN PRESS

Table

2.

Seque

nceco

mparison

ofAv

rBs3-likeproteins

Apl1

Apl2

Apl3

Avrb6

AvrBs3

AvrBs4

Avr-Pth3

Avr-Pth13

Avr-Pth14

AvrXa3

Avrxa5

AvrXa7-1M

AvrXa7-2M

AvrXa7-3M

AvrXa7-4M

AvrXa7

AvrXa10

AvrXa27

Hax2

Hax3

Hax4

PthA

PthA1

PthA2

PthA3

PthA4

PthB*

pTHB*

PthN

Brg11

Apl

1**

*99

,498

,597

,996

,696

,390

,190

,590

,488

,588

,791

,591

,291

,991

,791

,690

,790

,692

,494

,094

,399

,798

,098

,498

,099

,886

,8

81,8

93

,1

41,5

Apl

20,

6 **

* 98

,997

,197

,496

,089

,790

,790

,488

,688

,891

,491

,592

,191

,791

,591

,291

,492

,694

,194

,699

,498

,399

,298

,399

,486

,8

81,4

93

,2

43,5

Apl

31,

4 0,

9 **

* 98

,294

,594

,690

,390

,190

,688

,588

,891

,190

,792

,091

,491

,291

,190

,690

,494

,295

,098

,698

,398

,798

,398

,687

,0

81,3

93

,7

40,0

Avr

b62,

9 2,

6 2,

5 **

* 97

,796

,389

,590

,990

,188

,988

,991

,791

,292

,192

,091

,891

,291

,293

,393

,996

,897

,797

,497

,497

,197

,987

,2

81,2

92

,5

43,2

Avr

Bs3

3,4

3,0

3,7

2,5

***

96,6

89,6

90,5

88,0

90,8

88,7

88,8

89,0

89,5

89,1

88,9

90,6

90,3

90,2

97,1

97,1

96,6

97,2

97,2

96,9

96,6

86,6

81

,3

92,6

39

,9

Avr

Bs4

3,4

3,4

3,4

3,0

3,4

***

89,8

89,8

88,4

90,2

88,5

89,6

89,8

90,0

89,9

89,7

90,7

90,7

90,0

96,2

96,4

96,6

96,6

96,4

96,5

96,4

86,0

81

,0

92,0

39

,7

Avr

-Pth

3

10,7

10

,8

10,6

11

,2

11,2

11

,0

***

95,2

99,9

90,6

90,6

93,6

92,6

93,2

93,5

93,6

92,6

92,4

87,8

88,0

87,4

90,3

90,3

90,2

90,3

90,3

86,5

81

,3

90,5

42

,7

Avr

-Pth

13

10,4

10

,4

10,4

9,

6 10

,5

10,3

4,

7 **

* 94

,094

,790

,994

,093

,594

,494

,394

,293

,593

,988

,790

,189

,790

,590

,691

,390

,390

,686

,2

79,7

91

,0

38,4

Avr

-Pth

14

10,7

10

,7

10,6

11

,1

11,1

10

,7

0,1

4,5

***

90,2

90,5

93,5

91,9

93,2

93,5

93,6

93,0

92,3

87,3

88,0

87,6

90,5

90,9

90,6

90,5

90,5

86,3

81

,1

91,3

39

,4

Avr

Xa3

10

,1

10,0

9,

9 8,

5 9,

8 9,

7 6,

9 6,

3 6,

7 **

* 93

,393

,893

,794

,094

,593

,993

,193

,386

,390

,890

,688

,588

,588

,888

,088

,6

84,1

77

,8

89,0

39

,3

Avr

xa5

11,8

11

,8

11,8

10

,8

11,8

11

,2

10,0

8,

2 9,

8 6,

7 **

* 93

,694

,094

,394

,293

,893

,593

,386

,887

,988

,188

,788

,488

,988

,788

,7

84,4

78

,4

89,5

37

,2

Avr

Xa7

-1M

9,

2 8,

7 9,

3 9,

6 10

,3

9,3

7,4

7,0

7,4

4,4

6,7

***

96,6

97,0

99,5

99,9

95,8

95,5

86,2

88,9

89,0

91,5

91,7

92,0

91,0

91,7

86,4

81

,7

91,5

39

,3

Avr

Xa7

-2M

9,

0 9,

0 9,

1 8,

7 9,

1 8,

5 8,

5 7,

1 8,

3 4,

2 6,

4 3,

2 **

* 96

,296

,796

,795

,894

,285

,988

,688

,691

,491

,891

,591

,891

,486

,1

81,5

91

,5

39,3

Avr

Xa7

-3M

9,

6 9,

2 9,

3 9,

3 9,

4 9,

3 7,

2 6,

7 7,

3 4,

0 7,

0 3,

4 4,

3 **

* 97

,597

,097

,095

,086

,989

,589

,391

,892

,191

,992

,092

,087

,2

82,1

92

,2

39,4

Avr

Xa7

-4M

8,

9 8,

4 9,

0 9,

2 10

,0

9,0

7,5

6,7

7,4

3,7

6,1

0,5

3,1

2,9

***

99,6

96,4

95,5

86,4

89,1

89,2

91,7

92,0

92,2

91,2

92,0

86,6

82

,0

92,0

39

,3

Avr

Xa7

9,

1 8,

6 9,

2 9,

5 10

,2

9,2

7,4

6,8

7,3

4,3

6,5

0,1

3,1

3,3

0,4

***

95,9

95,6

86,2

88,9

89,1

91,6

91,8

92,1

91,1

91,8

86,5

81

,8

91,6

39

,3

Avr

Xa1

0 9,

3 9,

0 9,

0 9,

3 9,

3 9,

4 7,

2 6,

4 7,

1 4,

2 7,

0 5,

0 4,

6 3,

2 4,

4 4,

9 **

* 94

,688

,188

,788

,690

,890

,891

,391

,690

,885

,4

81,6

91

,6

42,7

Avr

Xa2

7 9,

5 9,

6 9,

4 9,

0 9,

3 9,

0 7,

9 6,

2 7,

8 5,

4 7,

0 5,

1 4,

9 4,

9 5,

0 5,

0 5,

4 **

* 87

,588

,488

,890

,891

,291

,491

,190

,885

,7

81,8

91

,4

42,2

Hax

27,

0 6,

7 13

,6

6,7

7,3

7,2

13,9

12

,9

13,9

13

,0

14,2

13

,0

11,8

12

,6

12,8

13

,0

12,8

12

,3

***

92,0

90,5

92,6

92,6

92,8

92,2

92,4

84

,8

80,7

89

,8

42,8

Hax

34,

2 4,

1 3,

7 4,

2 2,

9 3,

4 11

,3

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Gene-for-gene-mediated recognition of nuclear-targeted AvrBs3-like bacterial effector proteins 261

ARTICLE IN PRESS

Figure 2. AvrBs3-like proteins are predicted to form a helical superstructure that resembles a tetratricopeptide repeat(TPR) fold. Variable repeat unit residues 4 (yellow), 12+13 (red) and 24 (blue) are depicted. (A) 3D Jury/MODELLERsurface model was predicted based on the crystal structure of the TPR domain of the O-linked GLCNAC transferase(Protein Database (PDB) entry: 1w3b_A; Jinek et al., 2004) and is displayed as lateral and top view. (B) Schematicillustration of the predicted right-handed a–a helical superstructure of AvrBs4 and its structural hierarchy. A secondAvrBs3-like protein (blue) illustrates the postulated dimerization of this protein type.

S. Schornack et al.262

it is tempting to speculate that two AvrBs3 proteinshelically intertwine and form a right-handed doublehelix (Fig. 2). Apart from its contribution todimerization, surprisingly little is known on themolecular function of the repeat domain.

AvrBs3-like proteins and their virulenceaction

More than a decade ago in planta expression ofthe AvrBs3-like PthA and AvrB6 proteins revealedthat these prokaryotic proteins contain nucleartargeting signals that are functional in theireukaryotic hosts (Yang and Gabriel, 1995b). Thisfinding was quite remarkable since at that time itwas not even known that bacteria, which colonizethe spaces between plant cells (apoplast), employa molecular needle (type III secretion system) todeliver so-called effector proteins into the plant

cell. In silico analysis of PthA, AvrB6 and otherAvrBs3-like proteins predicted C-terminally locatedmonopartite nuclear localization signals (NLSs; Vanden Ackerveken et al., 1996; Yang and Gabriel,1995b) that are known to mediate nucleo-cytoplas-mic trafficking via interaction with importin a(Goldfarb et al., 2004). Successive studies onAvrBs3 showed indeed that the NLSs are crucialfor its interaction with importin a (Szurek et al.,2001) and thus confirmed the suspicion that AvrBs3-like proteins recruit the host’s nuclear importmachinery to reach the nucleus.

Yeast one-hybrid assays revealed an acidic AD atthe far C-terminus of AvrBs3 (Szurek et al., 2001)and the AvrBs3-like AvrXa10 protein (Zhu et al.,1998, 1999). Since AvrBs3-like proteins containfunctional NLS and AD domains, it was postulatedthat members of this protein family act astranscription factors (Lahaye and Bonas, 2001).Indeed, transcriptome analysis revealed thatAvrBs3 induces host transcripts and that this gene

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Gene-for-gene-mediated recognition of nuclear-targeted AvrBs3-like bacterial effector proteins 263

activation is strictly dependent on the presence ofthe AD domain (Marois et al., 2002). However,AvrBs3-like proteins have no recognizable DNAbinding motif and thus it was unclear if theseproteins bind DNA directly or if they form acomplex with DNA-binding proteins. Gel mobilityshift assays with the AvrBs3-like AvrXa7 proteindemonstrated that this protein binds to randomdouble-stranded oligonucleotides (Yang et al.,2000). This dsDNA binding was efficiently competedby poly dA/dT oligonucleotides, but only poorlycompeted by poly dC/dG oligonucleotides. These invitro assays might suggest that AvrXa7 binds inplanta DNA in a sequence-specific manner. How-ever, the identification of respective promoterconsensus sequences that bind specifically toAvrXa7 or another AvrBs3-like protein has not beenreported until now.

In summary, the presence of functional NLS andAD domains and their DNA binding activity stronglysuggests that AvrBs3-like proteins act as transcrip-tion factors that modulate the host’s transcriptometo the benefit of the bacterial intruder.

The Avr activity of AvrBs3-like proteinsrelies in most cases on functional NLSand AD domains

The guard model postulates that Avr-inducedmodifications of a host target protein triggeractivation of matching plant R proteins (Dangl andJones, 2001; Van der Biezen and Jones, 1998). Thisimplies that the (virulence) activity rather than thestructure of a respective Avr protein is beingdetected by a corresponding R protein. If thismodel holds true for a given gene-for-gene inter-action, avirulence and virulence activities of arespective Avr protein should be inseparable.Mutational analysis of many avrBs3-like genesprovided initially compelling evidence that thevirulence and Avr function are interconnected inthis avr family (Lahaye and Bonas, 2001). Forexample, mutations in the AvrBs3 NLS or AD domainabolish nuclear targeting or host gene induction,respectively (Marois et al., 2002; Szurek et al.,2001; Van den Ackerveken et al., 1996). The verysame mutations also abrogate recognition of AvrBs3by the corresponding pepper Bs3 resistance gene(Marois et al., 2002; Szurek et al., 2001; Van denAckerveken et al., 1996). Mutations in the NLSs andADs of the AvrBs3-like AvrXa7 or AvrXa10 proteinsalso abolish recognition by the corresponding riceXa7 or Xa10 R genes, respectively (Yang et al.,2000; Zhu et al., 1998, 1999). Notably, the

endogenous NLS and AD motifs in AvrBs3, AvrXa7and AvrXa10 can be functionally replaced by theheterologous NLS from the large T-antigen of simianvirus SV40 and the heterologous AD from the Herpessimplex protein VP16, respectively (Van den Ack-erveken et al., 1996; Yang et al., 2000; Zhu et al.,1999). Hence, the loss of avirulence activity in NLSand AD mutants is due to a loss of function ratherthan due to structural changes in the respectiveAvrBs3-like proteins. In summary, the functionalityof Bs3, Xa7 and Xa10 encoded R proteins reliescollectively on the presence of functional NLSs andADs in their cognate Avr proteins, suggesting thatthey employ similar perception mechanisms.

In some gene-for-gene interactions thatinvolve avrBs3-like genes the virulenceand Avr activities were shown to beseparable

Even though mutational studies revealed thatvirulence and avirulence activity are mostly inter-connected in AvrBs3-like proteins, the separation ofthese functions has been recently demonstrated forthe Xanthomonas oryzae pv. oryzae (Xoo) avrXa7gene (Yang et al., 2005). The avrBs3-like avrXa7gene triggers a defense response in rice linescontaining the matching Xa7 disease resistancegene (Porter et al., 2003). In contrast, avrXa7contributes to aggressiveness of Xoo in rice linesthat lack the Xa7 resistance gene (Bai et al., 2000).The virulence contribution of AvrXa7 can be easilymonitored by measuring the lesion length that isobserved upon Xoo infection. Mutational analysis ofthe avrXa7 repeat domain uncovered derivativesthat lost their contribution to aggressiveness butthat retained their avirulence activity (Yang et al.,2005). Given that mutations in the above-describedavrXa7 derivatives were restricted to the repeatdomain, it is conceivable that these derivativeshave still functional NLSs and ADs and that each ofthese avrXa7 derivatives directs induction orrepression of a defined set of host genes. Thus itis tempting to speculate that the repertoire ofgenes that needs to be induced or repressed inorder to either trigger Xa7-mediated resistance oralternatively to support aggressiveness of Xoo arenot identical. Transcriptome studies will possiblyshed light on this interesting question.

The Xcv AvrBs4 protein, which triggers a defenseresponse in tomato (Lycopersicon esculentum)genotypes that express the matching Bs4 resistancegene (Ballvora et al., 2001a), represents anotherexample of an AvrBs3-like protein in which

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S. Schornack et al.264

virulence and avirulence seem to be separable.Analysis of Xcv mutant strains revealed that AvrBs4is a virulence factor that promotes bacterial growthin planta (Wichmann and Bergelson, 2004). Nota-bly, the virulence contribution of AvrBs4 was onlydetectable in Xcv strains that contain no functionalcopy of the widespread avrBs2 gene. The growth-promoting activity of avrBs4 mutant derivatives hasnot been studied yet. However, mutational analysisof other AvrBs3-like proteins infers that theC-terminal NLS and AD domains are crucial to thevirulence activity of AvrBs4. In contrast, the Bs4-dependent avirulence activity of AvrBs4 is notcompromised by deletion of its NLS nor AD domains.Remarkably, a C-terminal truncation of AvrBs4 thatlacks the C-terminal AD and NLSs and whichcontains only 3.5 of 17.5 repeats (AvrBs4D230) isnot only capable of triggering the Bs4-mediated HR(Ballvora et al., 2001a; Bonas et al., 1993), but haseven stronger elicitor activity than the full-lengthAvrBs4 protein. Many reports have pointed out thatAvrBs4 is seemingly exceptional since it is the onlyknown member of the AvrBs3 family that showselicitor activity even when the NLSs or the AD aredeleted. However, further studies revealed thatAvrBs4 triggers not only HR in tomato but also inC. pubescens (Minsavage et al., 1999) and potato(Schornack et al., 2004). Notably, AvrBs4 NLS- orAD-mutant derivatives, which elicit the tomato Bs4response, have no Avr activity in C. pubescens(Gurlebeck, 2001) and potato (Schornack et al.,2004). These data suggest tomato Bs4 protein isfunctionally exceptional, rather than the Xantho-monas AvrBs4 protein.

In summary the AvrBs4–Bs4 and the AvrXa7–Xa7interactions represent two cases in which thevirulence and avirulence of an AvrBs3-like proteinwere shown to be separable. Even though virulenceand avirulence are not strictly coupled in theAvrXa7–Xa7 interaction, it seems likely that AvrXa7derivatives that trigger Xa7 resistance are stillcapable to modulate the hosts transcriptome. Incontrast, Bs4 also mediates recognition of severelytruncated AvrBs4 derivatives lacking the functionalNLS and AD domains (Bonas et al., 1993; Schornacket al., 2004) and which are most likely not capableof modulating the host’s transcriptome.

Recognition of AvrBs3-like proteins –how is specificity defined?

Most of our current knowledge on gene-for-geneinteractions involving AvrBs3-like proteins was ob-served by the analysis of modified Avr derivatives,

since the corresponding plant R genes have beenisolated only very recently. The first insights of howrecognition specificity is determined in AvrBs3-likeproteins came from in vitro generated randomdeletions of a variable amount repeat units of theAvrBs3 protein and demonstrated that recognitionspecificity is defined by the number and the order ofAvrBs3 repeat units (Herbers et al., 1992). Manysuccessive studies on different AvrBs3-like proteinsconsistently showed that exchanging the repeatdomain generally alters the Avr specificity of theprotein to the specificity of the gene from which thedomain was derived (Yang et al., 1994; Zhu et al.,1998). In contrast, the non-repeat regions are forthe most part functionally interchangeable betweendifferent AvrBs3-like proteins. However, an AvrBs3-derivative that contains an AvrXa7-derived fragmentin its C-terminal non-repeat region was recentlyshown to trigger Xa7 resistance thus indicating thatAvr specificity is not exclusively defined by therepeat domain (Yang and White, 2004).

Recombinational shuffling generates newavrBs3-like genes

The analysis of in vitro generated avrBs3 deriva-tives with a modified repeat domain uncoveredderivatives that no longer triggered HR in the Bs3-resistant pepper cultivar ECW-30R but insteadshowed Avr activity in the isogenic bs3 genotypeECW (Herbers et al., 1992). These data suggestedthat the bs3 genotype ECW contains R genes thatmediate recognition of avrBs3 derivatives. From anevolutionary point of view, the presence of an Rgene that mediates recognition of an in vitrogenerated AvrBs3 repeat derivative (bs3 in ECW)suggests that such rearrangements may also occurin nature. Indeed, analysis of the avrBs3-like pthAand avrXa7 genes revealed that repeat domainderivatives occur in vivo most likely due to intra-and intergenic recombination (Yang and Gabriel,1995a; Yang et al., 2005). Not unexpectedly, theserepeat derivatives showed novel virulence andavirulence activities.

Consequently, a strain possessing multiple AvrBs3homologs may benefit since recombinational shuf-fling of avrBs3-like genes could allow the genera-tion of avrBs3-like genes with modified avirulenceand virulence functions. This might explain whymany xanthomonads contain multiple avrBs3 homo-logs, although these have no obvious contributionto pathogenicity. However, it remains to beclarified why other xanthomonads have only oneor even no avrBs3-like gene (Da Silva et al., 2002).

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Gene-for-gene-mediated recognition of nuclear-targeted AvrBs3-like bacterial effector proteins 265

The relationship of recognitionspecificity and expression levels ofAvrBs3-like proteins

It is a well-known fact that an antiserum raisedagainst a given antigen tends to cross-react withstructurally related antigens especially if these arepresent at high levels. In this context it seemsremarkable that some plant R proteins are capableof discriminating between almost identical AvrBs3-like proteins. For example, the pepper Bs3 and thetomato Bs4 proteins mediate specific recognition ofavrBs3- and avrBs4-expressing xanthomonads, re-spectively, although AvrBs3 and AvrBs4 are 96.6%identical (Ballvora et al., 2001a). One may wonderif plant R proteins retain their recognition specifi-city if confronted with high levels of a structurallyrelated Avr protein instead of the matching Avrprotein. Indeed, constitutive in planta expressionof avrBs3 via the strong constitutive cauliflowermosaic virus 35S (35S) promoter triggers a Bs4-dependent HR while delivery of AvrBs3 into theplant by Xanthomonas does not (Schornack et al.,2004). However, 35S-driven expression of hax2,another avrBs3-like gene that is 92% identical withavrBs4 at the aa level, does not trigger a Bs4-dependent HR (Kay et al., 2005), indicating that35S expression leads to only a partial, but notcomplete loss of recognition specificity. Notably,the relaxation of recognition specificity upon 35S-driven expression of an avrBs3-like gene has beenobserved only with tomato Bs4, but not withother R genes that mediate recognition of AvrBs3-like proteins. For example, the pepper Bs3gene retains its recognition specificity even ifavrBs4 was expressed in planta from the strong35S promoter (Schornack et al., 2005). Similarly,the cotton R genes B4, b7, and BIn retain theirrecognition specificity even if other highly relatedavrBs3-like genes were expressed in planta undercontrol of the strong 35S promoter (De Feyteret al., 1998).

The finding that the tomato Bs4 HR is triggeredonly upon delivery of a 35S-driven avrBs3 transgenebut not by AvrBs3-delivering xanthomonads sug-gests that quantitative differences between inplanta-produced and type III-delivered AvrBs3protein account for the observed differences.However, it is also possible that qualitative ratherthan quantitative alterations in AvrBs3 accountedfor the observed differences in Bs4-dependentrecognition of AvrBs3. To clarify this issue, binaryvectors have been constructed in which the T-DNAinsert is not driven by the strong 35S promoter, butby the Bs4 promoter. The Bs4 promoter leads to

substantially lower transcript and protein levels inplanta than the 35S promoter (Schornack et al.,2004, 2005). Bs4 promoter driven avrBs4, but notavrBs3 constructs, triggered a Bs4-dependent HR.Thus, the recognition specificity of Bs4 is retained ifthe in planta expression of the avrBs3-like gene isdriven by the weak Bs4 promoter instead of thestrong 35S promoter. Notably, the recognitionspecificity of Bs4 seems to be insensitive to itsown expression levels since Bs4 under its nativepromoter or the 35S promoter show identicalreaction patterns (Schornack et al., 2004, 2005).One might speculate that internal control mechan-isms do not permit the accumulation of high,potentially toxic Bs4 levels in the plant cell evenif the strong 35S promoter drives Bs4. However,immunoblot analysis confirmed that the Bs4 pro-moter directs the generation of substantially lowerBs4 levels than the 35S promoter (Schornack andLahaye, unpublished) and supports our postulatethat only high levels of AvrBs3-like proteins but notof Bs4 interfere with recognition specificity.

Recent studies revealed that tomato Bs4 may bepromiscuous as it mediates not only recognition ofavrBs4-containing Xanthomonas strains but also ofxanthomonads expressing hax3 or hax4. hax3 andhax4 are recently isolated avrBs3-homolog encod-ing proteins with 96.1% and 96.2% identity toAvrBs4, respectively (Kay et al., 2005). Hax3 andHax4 triggered also a Bs4-dependent HR when thecorresponding genes were expressed under controlof the weak Bs4 promoter (Schornack et al., 2005).Hax3 and Hax4 are less similar to AvrBs4 than toAvrBs3, which does not trigger a Bs4-dependent HRwhen expressed under control of the weak Bs4promoter. Given that overall sequence homology toAvrBs4 does not define elicitor activity of a givenAvrBs3-like protein, we assume that short peptidemotifs are crucial in the determination of Bs4-dependent Avr specificity.

Altogether the observations that Bs4 (i) mediatesrecognition of at least three distinct AvrBs3-likeproteins and (ii) that its recognition specificity isAvr-level dependent but (iii) does not requirefunctional NLS or AD motifs in the cognate Avrcomponent marks Bs4 as functionally peculiarwithin the R proteins that mediate recognition ofAvrBs3-like proteins.

xa5 – a pathogenicity target rather thana resistance gene

Considerable effort has been devoted to theidentification, genetic mapping and positional

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S. Schornack et al.266

cloning of R genes that mediate recognition ofAvrBs3-like proteins (Ballvora et al., 2001a, b; Blairet al., 2003; Gu et al., 2004, 2005; Iyer andMcCouch, 2004; Pierre et al., 2000; Porter et al.,2003; Schornack et al., 2004; Yang et al., 1998;Zhong et al., 2003). Since these R genes have been

Figure 3. R gene-mediated recognition of AvrBs3-like proteinas described in Fig. 1. All AvrBs3-like proteins are injected bcytoplasm and translocated into the nucleus. (A) xa5-mediatmediates activation of disease-promoting genes via interactiopreinitiation complex (PIC). PIC interaction of AvrXa5 is aboTFIIA. (B) Xa27-mediated recognition of AvrXa27. AvrXa27 intdisease promoting genes. Xa27 resistant plants possess a proactivated by AvrXa27. (C) Bs4-mediated recognition of AvrBs4AvrBs3-like Hax3 and Hax4 proteins. Bs4 associates with cognuclear import, which leads to activation of the host’s defen

cloned only recently, little is known about themolecular mechanisms that they employ to med-iate recognition. Yet, their structural featuresallow some speculations on how these R geneproducts mediate recognition at the molecularlevel.

s. Structural features of AvrBs3-like proteins are displayedy Xanthomonas type III secretion system into the plant’sed recognition of the AvrBs3-like Avrxa5 protein. Avrxa5n with the TFIIA which itself is part of the transcriptionallished in xa5 plants due to an amino-acid difference ineracts directly or indirectly with promoter sequences ofmoter upstream of the Xa27 R gene, which is specifically, its C-terminal deletion derivative AvrBs4D230, and thenate AvrBs3-like proteins in the cytoplasm prior to theirse system.

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Gene-for-gene-mediated recognition of nuclear-targeted AvrBs3-like bacterial effector proteins 267

xa5 is a recessive, race-specific rice R gene thatprovides resistance to races of Xoo that express thecognate avrxa5 gene. Avrxa5 is likely to be amember of the AvrBs3 family (Bai et al., 2000;Hopkins et al., 1992; Iyer and McCouch, 2004)although this has not been formally proven, so far.xa5 encodes the g subunit of transcription factor IIA(TFIIAg) and belongs to the so-called generaltranscription factors (GTFs; Iyer and McCouch,2004). RNA polymerase II and its associated GTFsform a preinitiation complex (PIC) at the TATA boxof cognate promoters and thereby initiate tran-scription (Asturias, 2004). PIC assembly and thustranscription can be stimulated by transcriptionalactivators like the VP16 Herpes simplex virusprotein. Notably, the VP16 AD has been shown tobind to TFIIAg and to a lesser extent to TFIIAab(Kobayashi and Hohn, 2004). Analysis of VP16 ADdeletion derivatives indicated that their ability tobind to TFIIA correlates with their ability tostimulate PIC assembly. Thus, VP16-mediated tran-scriptional activation is most likely brought aboutvia interaction with TFIIA.

Comparative sequencing of TFIIAg in multiplerice cultivars uncovered an aa polymorphism atresidue 39 that correlated perfectly with resistanceand susceptibility, respectively. Given that position39 resides in a predicted solvent-exposed surface,it is tempting to speculate that xa5 and Xa5encoded TFIIAg isoforms have different affinitiesto the transcriptional AD of the Xoo AvrXa5 protein.Given that Xa5 genotypes are susceptible to Xoo,we propose a model in which AvrXa5 binds with itsAD to TFIIAg and thereby modulates the host’stranscriptome to the benefit of the bacterialinvader. Furthermore, our model predicts thatAvrXa5 is incapable to bind to and interfere withthe xa5-encoded TFIIAg isoforms in Xoo resistantcultivars (Fig. 3A).

Xa27 – not an R gene but an‘‘R-promoter’’!

The rice R gene Xa27 mediates recognition ofxanthomonads that express avrXa27, a new mem-ber of the avrBs3 family (Gu et al., 2005).Mutational analysis of avrXa27 uncovered that theNLSs and the ADs are crucial for Xa27-mediatedrecognition. Thus, Xa27 functionally resemblespepper Bs3 and many other R genes that mediaterecognition of AvrBs3-like proteins in an NLS- andAD-dependent fashion.

Xa27 has no apparent sequence homology toproteins from organisms other than rice and its

structural analysis provided no hints about possiblefunctions. Unexpectedly, the resistant and suscep-tible parental lines of the mapping populationencode identical Xa27 proteins. Yet, nucleotidedifferences between the presumed Xa27/xa27promoters raised the possibility that the two allelesdiffer in their expression. Indeed, only the Xa27 butnot the xa27 allele was detectable by northern blotanalysis. Further studies uncovered that expressionof the Xa27 allele occurs only when a rice plant ischallenged by bacteria harboring avrXa27, but notby isogenic strains lacking avrXa27. These datasuggest that Xa27-resistant plants evolved a pro-moter sequence that is ‘‘inadvertently’’ switchedon by the bacterial AvrXa27 protein (Fig. 3B). Thusthe Xa27 promoter is conceptually a molecularhyperparasite as it exploits AvrXa27, the transcrip-tional activator of a bacterial parasite, to activatethe plants defense system. It remains to be seenwhether other AvrBs3-like proteins might also‘‘inadvertently’’ target the promoters of thecognate R proteins.

Hints to a receptor–ligand model for theBs4–AvrBs4 interaction

The tomato Bs4 gene mediates resistance to-wards strains of the bacterial spot pathogen Xcvthat express the avrBs3-like avrBs4 gene (Ballvoraet al., 2001a, b; Schornack et al., 2004). Bs4 is amember of the largest class of R proteins function-ally characterized to date, the so-called NB-LRRclass. Among the known resistance proteins of theNB-LRR family, the Bs4 protein has highest se-quence similarity to the tobacco N and potato Y-1resistance proteins (Vidal et al., 2002; Whithamet al., 1994). Despite intensive efforts, there is yetno generalizable model that would explain how NB-LRR type R proteins mediate recognition of cognateAvr proteins. Some NB-LRR proteins have beenshown to interact physically with their cognate Avrproteins (Deslandes et al., 2003; Jia et al., 2000).In many other cases, NB-LRR proteins seem todetect the virulence activity rather than thestructure of their cognate Avr proteins (Innes,2004). Apparently, Bs4 does not belong to thelatter type as it mediates not only perception ofAvrBs4, but also of severely truncated AvrBs4derivatives that have most likely lost their virulenceactivity (Schornack et al., 2004). Another informa-tive aspect in the elucidation of the molecular basisof Bs4 resistance is the finding that Bs4 mediates notonly recognition of AvrBs4, but also of the 96.6%identical AvrBs3 protein. However, AvrBs3 needs to

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be expressed to higher levels than AvrBs4 in order totrigger Bs4 resistance (Schornack et al., 2005). Thissituation is reminiscent of an antibody-mediateddetection which generally shows reduced specificityif the antigen is present at high concentrations andsuggests a direct interaction between Bs4 andAvrBs4 (Fig. 3C).

Figure 4. Structure prediction of the Bs4-leucine-richrepeat (LRR) domain and its potential association withthe AvrBs4 repeat domain. (A) 3D Jury/MODELLER ribbonmodel of the Bs4-LRR was predicted based on homologyto the porcine ribonuclease inhibitor (PDB entry: 2bnh_;Kobe and Deisenhofer, 1996). a-helical stretches andb-sheets are depicted as spiral ribbons and flat arrows,respectively. LRR repeats are numbered and theirputatively solvent-exposed hypervariable residues arehighlighted in red. (B) Possible association of the Bs4-LRR(blue) and the AvrBs4 repeat domain. Surface models ofthe Bs4-LRR (blue) and the AvrBs4 repeat domains aredisplayed to scale. The dimensions of the putativelyinteracting Bs4 and AvrBs4 domains are compatible with areceptor–ligand model.

In silico modeling using the 3D-Jury metapredic-tor (Ginalski et al., 2003) predicts that the Bs4 LRRforms a rod-like structure (Fig. 4A) which, based onits dimensions, would be able to enclose thepredicted a–a superhelical structure of the AvrBs4repeat domain (Fig. 4B). The predicted stericrelationships imply that the Bs4 LRR interacts onlywith three repeat units and not with the completerepeat domain of AvrBs4. In agreement with thisfinding, Bs4 mediates recognition of an AvrBs4derivative (AvrBs4D230) that contains 3.5 out of17.5 repeat units (Schornack et al., 2004). Incontrast, an AvrBs4 derivative that contains onlyone complete repeat unit (AvrBs4D233) no longertriggers a Bs4-dependent HR (Schornack et al.,2004).

Another piece of evidence in support of a directBs4–AvrBs4 interaction comes from the finding thatthe protein phosphatase 5 (PP5) TPR domain, whichstructurally resembles the AvrBs4 repeat domain,was shown to interact with the LRR of tomato I-2and several other NB-LRR-type R proteins (de laFuente Bentem et al., 2005). Notably, this interac-tion was only detectable with the I-2 LRR but notthe full length I-2 protein, which might explainwhile previous studies failed to detect a directinteraction between AvrBs4 and the full-length Bs4protein (Schornack et al., 2004; Schornack andLahaye, unpublished).

Consideration of the in planta target compart-ments of Bs4 and AvrBs4 adds yet another level ofcomplexity to the proposed interaction model. Bs4contains no apparent N-terminal signal sequencesand was predicted to be cytoplasmatic (Schornacket al., 2004). In contrast, AvrBs4 contains NLSs and isthus likely to be a nuclear protein. Confocalmicroscopy of GFP-tagged Bs4 and AvrBs4 deriva-tives confirmed these predictions (Schornack andLahaye, unpublished) and raises the question of howBs4 and AvrBs4 can interact if they are not located inthe same cellular compartment. However, AvrBs3-like proteins must interact transiently with cytoplas-matic host shuttle proteins in order to reach theplant’s nucleus (Szurek et al., 2001). Thus it seemspossible that Bs4 interacts with and is activated bycytoplasmatic AvrBs4 (Hax2 or Hax3) that has notreached its destination compartment. Notably, theamount or the speed of the nuclear import of AvrBs4is not significantly affected by coexpression of Bs4(Schornack and Lahaye, unpublished), which mightindicate that the proposed Bs4–AvrBs4 interaction iseither transient or unstable.

In summary, we favor a model in which AvrBs4and Bs4 interact physically, despite the fact that wehave not been able to validate this assumptionexperimentally.

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Conclusions

Although the AvrBs3 family is undoubtedly one ofthe most intensively studied Avr families, somecentral questions still remain unresolved. Onemajor challenge will certainly be the identificationof a promoter to which AvrBs3 binds either directlyor indirectly. The fact that such an interaction hasnot yet been identified might suggest that this is anexperimentally challenging task or alternativelythat AvrBs3-like proteins do not interact with DNA.Thus an indirect strategy which does not rely onDNA-binding activity might be the most promisingapproach to identify DNA elements that potentiallybind to AvrBs3-like proteins. We envisage thatgenes that are induced by a given AvrBs3-likeprotein represent a sought-after experimental hookto fish for promoters that potentially bind toAvrBs3-like proteins.

Given that AvrBs3-like proteins share extensivesequence similarity, one might expect that thecorresponding R genes would encode structurallyand functionally similar proteins. However, theopposite seems to be the case, as the currentlycloned R genes complementary to AvrBs3-likeproteins are extraordinarily dissimilar with respectto their mode of inheritance and structure. We areeagerly awaiting the cloning of additional R genesthat mediate recognition of AvrBs3-like proteins inorder to learn more about the arsenal of molecularstrategies that plants have evolved to detect nearlyidentical microbial proteins.

Acknowledgments

We are deeply indebted to U. Bonas and hercoworkers who started the scientific analysis of thepepper Bs3 and tomato Bs4 disease resistancegenes. We are grateful to L. Rose for helpfulcomments on earlier versions of the manuscript.We would like to acknowledge contributions andtechnical support in the course of the SFB363 byA. Fick, C. Kretschmer, K. Peter, J. Piprek, B.Rosinsky, M. Schulze and R. Szczesny. This work wassupported by grants of the Deutsche Forschungsge-meinschaft (SFB 363/648 and LA 1338/2-2) and bythe Kultusministerium des Landes Sachsen-Anhalt(3470A/902M).

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