cDNA-AFLP analysis unravels a genome-widehrpG-regulon in the plant pathogen Xanthomonascampestris pv. vesicatoria

Laurent Noel, Frank Thieme, Dirk Nennstiel† and

Ulla Bonas*

Institut fur Genetik, Martin-Luther-Universitat

Halle-Wittenberg, D-06099 Halle (Saale), Germany.

Summary

The Hrp type III protein secretion system is essential

for pathogenicity of the Gram-negative plant patho-

gen Xanthomonas campestris pv. vesicatoria.

Expression of the hrp gene cluster is controlled by

HrpG, a two-component response regulator, and

HrpX, an AraC-type transcriptional activator. Using

the cDNA-AFLP technique, 30 hrpG-induced (hgi )

and five hrpG-repressed (hgr ) cDNA fragments

were identified, defining a large hrpG-regulon in

X. campestris pv. vesicatoria. Expression of most

genes in the hrpG-regulon was dependent on hrpX.

Seven cDNA fragments map to the known hrp gene

cluster and flanking regions. All other genes appear to

be scattered over the chromosome and endogenous

plasmids. Sequence analysis identified genes encod-

ing putative extracellular proteases, a putative

transcriptional regulator and XopJ and XopB (Xantho-

monas outer proteins), homologues of YopJ from

Yersinia spp. and the avirulence protein AvrPphD of

Pseudomonas syringae respectively. XopB is

secreted by the Hrp type III secretion system. Analysis

of deletion mutants in several hgi genes revealed a

new virulence locus. This study demonstrates that

cDNA-AFLP is a powerful tool to study prokaryotic

transcriptomes and to identify genes contributing

to Xanthomonas virulence and putative effector

proteins.

Introduction

Pathogens have developed sophisticated strategies to

suppress host defence responses and to successfully

colonize their hosts. Basic pathogenicity of Gram-negative

bacterial plant pathogens belonging to spp. of Erwinia,

Pseudomonas syringae, Ralstonia solanacearum and

Xanthomonas depends on a type III protein secretion

system (TTSS) encoded by hrp genes (hypersensitive

reaction and pathogenicity). hrp genes are essential for

bacteria to grow in planta, cause disease in susceptible

plants and trigger the hypersensitive reaction (HR) in

resistant plants. The HR is a rapid, localized cell death that

is part of the plant’s innate disease resistance response

and which halts pathogen ingress (Klement, 1982). Among

the more than 20 proteins encoded by a given hrp gene

cluster, nine Hrc proteins (hrp-conserved) were found to

be highly conserved not only in the plant pathogens

mentioned above but also in many mammalian pathogens

such as Yersinia spp., enteropathogenic Escherichia coli,

Shigella flexneri and Salmonella spp. (Bogdanove et al.,

1996).

A characteristic feature of the TTSS is that protein

substrates are secreted across both bacterial membranes

in a sec-independent manner, without processing. Some

of the proteins, so-called effectors, appear to be ‘injected’

into the eukaryotic host cell (Galan and Collmer, 1999).

Studies of type III secretion in plant pathogens revealed

secretion of only few proteins in vitro, such as subunits of

the Hrp pili, harpins or avirulence (Avr) proteins (see

Cornelis and Van Gijsegem, 2000 for a recent review).

Avirulence genes determine specific recognition of spp. of

Xanthomonas and P. syringae by a plant that expresses

the corresponding disease resistance (R ) gene (White

et al., 2000).

Xanthomonas campestris pathovar vesicatoria

(X. campestris pv. vesicatoria ) is the causal agent of

bacterial spot disease in pepper and tomato. The

chromosomal 23 kb hrp gene cluster contains six operons,

hrpA to hrpF (Bonas et al., 1991; Fenselau et al., 1992;

Fenselau and Bonas, 1995; Huguet et al., 1998; U. Bonas,

unpublished observations). Expression of hrp genes is

induced in plant leaves and in minimal medium XVM2

(Schulte and Bonas, 1992; Wengelnik and Bonas, 1996)

and is controlled by the regulatory genes hrpG and hrpX,

located outside the hrp gene cluster. The HrpG protein

belongs to the OmpR family of two-component regulatory

systems and activates the expression of hrcC and hrpX

(Wengelnik et al., 1996). HrpX, an AraC-type transcrip-

tional activator, controls expression of the operons hrpB to

hrpF (Wengelnik and Bonas, 1996) and the avirulence

Accepted 4 June, 2001. *For correspondence. E-mail: bonas@genetik.uni-halle.de; Tel. (149) 345 552 6290; Fax: (149) 345 5527277. †Present address: Bayer-AG Zentrale Forschung, D-51368Leverkusen, Germany.

Molecular Microbiology (2001) 41(6), 1271–1281

Q 2001 Blackwell Science Ltd

gene avrXv3, which is located outside the large hrp region

(Astua-Monge et al., 2000).

Recently, we demonstrated Hrp-dependent secretion of

the avirulence proteins AvrBs3 and AvrRxv and of proteins

from other plant and animal pathogenic bacteria (Rossier

et al., 1999). In addition, HrpB2 and HrpF, which are

encoded within the hrp gene cluster, are secreted by the

X. campestris pv. vesicatoria TTSS (Rossier et al., 2000).

Owing to low in vitro secretion efficiency (Rossier et al.,

1999; 2000), the number of proteins that use the TTSS is

not known. Knowledge of the panoply of secreted proteins,

however, is a prerequisite to unravel bacterial plant

pathogenicity. To identify new genes encoding TTSS

effectors, we compared the expression profile of two

isogenic X. campestris pv. vesicatoria strains, 85-10 and

its derivative 85*, which differ in their hrp gene expression

status. Strain 85* expresses a mutated form of the key

regulatory gene hrpG (hrpG*) leading to constitutive

expression of the hrp genes in normally non-inducing

medium (Rossier et al., 1999; Wengelnik et al., 1999).

Using the cDNA-AFLP technique, we identified a large

hrpG-regulon containing at least 33 hrpX-dependent and

two hrpX-independent genes. One of the identified genes

encodes a novel secreted protein.

Results

cDNA-AFLP analysis of X. campestris pv. vesicatoria

identifies a large hrpG-regulon

To identify new X. campestris pv. vesicatoria genes

expressed under the control of hrpG, a cDNA-AFLP

screen (Bachem et al., 1996) was carried out (Fig. 1).

cDNA was synthesized using total RNA from the isogenic

strains 85-10 and 85* (85-10 carrying the hrpG* mutation)

grown in complex medium NYG. The hrpG* mutation leads

to a single amino acid exchange in the regulatory protein

HrpG, rendering hrp gene expression constitutive (Wen-

gelnik et al., 1999). The bacterial cDNA was digested with

Pst I and Taq I to generate cDNA-AFLP templates. All 256

possible Pst I 1 NN/Taq I 1 NN primer combinations were

used to screen for differences in gene expression between

85-10 and 85* (Fig. 1). In total, 52 differentially expressed

cDNA-AFLP fragments were found. Seven fragments

were more abundant in cDNAs from strain 85-10

compared with 85* and were designated hgr (hrpG-re-

pressed). cDNA-AFLP fragments that were present in 85*

but absent or under-represented in 85-10 templates were

designated hgi (hrpG-induced). Thirty-five fragments (five

hgr and 30 hgi ) could be sequenced and were analysed

further. The other 17 fragments caused problems in direct

sequencing and were not studied. As the cDNA fragments

were only 40–386 bp long, they were extended to 0.3–

1.0 kb using ‘Genome Walker’. Sequence analysis

Fig. 1. cDNA-AFLP strategy for the analysis of the hrpG-regulon.cDNA from Xanthomonas campestris pv. vesicatoria (Xcv ) 85-10(1; no hrp gene expression) and strain 85* (2; constitutive hrp geneexpression) was analysed by cDNA-AFLP.A. cDNA digestion by Pst I and Taq I.B. Ligation of adaptors.C. Preamplification of the total cDNA population using non-selectiveprimers.D. Selective amplification of a subset of cDNA fragments usingprimers with two selective nucleotides (NN).E. The Pst I primer is 33P-labelled and allows visualization of theamplicons by autoradiography after separation on a denaturingpolyacrylamide gel. The autoradiogram shows a typical result for sixselective Pst I/Taq I primer combinations. Filled arrows indicatehrpG-induced (hgi ) cDNA fragments and open arrows indicatehrpG-repressed (hgr ) cDNA fragments.

1272 L. Noel et al.

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 41, 1271–1281

revealed that several hgi and hgr fragments correspond to

the same gene. For example, hgi 25, hgi 66 and hgi 80 are

derived from the same cDNA. In such cases, fragments

were renamed accordingly, e.g. hgi 25/66/80. The

expression patterns of all genes were confirmed by

reverse transcription-polymerase chain reaction (RT-

PCR) using the same cDNA as for the AFLP analysis

(Fig. 2). Table 1 summarizes important features of the hgi

and hgr fragments, including regulation of the expression,

average G 1 C content and sequence homologies.

HrpX is a key regulator of the hrpG-regulon

As described earlier, HrpG activates transcription of the

hrp operons hrpB to hrpF through HrpX (Wengelnik et al.,

1996). To test for hrpX-dependent expression of hgi and

hgr genes, we generated a non-polar hrpX mutant in the

85* background, giving 85*DhrpX. cDNA-AFLP patterns of

the 35 differentially expressed genes were compared

between 85* and 85*DhrpX (Fig. 2; Table 1). Expression of

all genes, except hgi 58 and hgi 224, was found to be

hrpX-dependent, indicating that hrpX is essential for

expression of most genes belonging to the hrpG-regulon.

As hrp gene expression is induced in minimal medium

XVM2 (Wengelnik and Bonas, 1996), we also tested all hgi

and hgr genes for their expression in XVM2. Most hgi

fragments were significantly induced in XVM2 although

sometimes expression was weaker than in strain 85*

(Fig. 2; Table 1). hgr genes were poorly repressed in

XVM2 and hgr 10 expression appeared unaffected by

XVM2 in the different experiments performed (Fig. 2;

Table 1).

hgi and hgr fragments are scattered throughout the

X. campestris pv. vesicatoria genome

For further characterization, the hgi and hgr fragments

were hybridized to a genomic cosmid library of

X. campestris pv. vesicatoria. As no genomic library of

strain 85-10 was available and all isolated cDNAs are

highly conserved among different X. campestris pv.

vesicatoria strains (data not shown), a library from strain

75–3 (Minsavage et al., 1990) was used. At least one

cosmid clone per fragment was identified, except for hgi 9

and hgi 11, and confirmed by PCR using gene-specific

primers. Few cosmids were found to contain more than

one hgi or hgr fragment, indicating that most members of

the hrpG-regulon are scattered throughout the genome.

One exception was the large hrp gene cluster to which

seven hgi fragments hybridized. In summary, the 35

fragments identified map to 20 distinct loci. Southern

hybridization performed for 17 loci revealed that 4 (hgr

10, hgi 1, hgi 127 and hgi 128/130 ) map to the largest

(,200–250 kb) of the two endogenous plasmids present

in strain 85-10 (Canteros, 1990; Table 1). Here, we

describe the analysis of several of these hgi and hgr

genes.

hgi 11 encodes a new member of the AvrRxv/YopJ family

The open reading frame (ORF) encompassing the hgi 11

fragment is 1119 bp long and contains an imperfect PIP-

box (plant inducible promoter; TTCGT-N15-TTCGG;

consensus TTCGC-N15-TTCGC; Wengelnik and Bonas,

1996) 65 bp upstream of the putative translational start

codon. The predicted protein contains 373 amino acids

(40 kDa) and shares 45% identity/64% similarity with Y4l0

from Rhizobium sp. NGR234 and 33% identity/48%

similarity with AvrRxv from X. campestris pv. vesicatoria

which belongs to the AvrRxv/YopJ family of type III-

effector proteins. Owing to the sequence similarity, the

gene corresponding to hgi 11 was renamed xopJ (encodes

a putative Xanthomonas outer protein).

Expression of extracellular protease homologues is

suppressed in 85*

DNA sequence analysis of three cDNAs, hgr 10, hgr 70

and hgr 71, revealed that the deduced amino acid

sequences share similarity with bacterial extracellular

proteases (Table 1). The genes were expressed in the

hrpX deletion mutant, 85*DhrpX, grown in NYG. However,

expression was undetectable in 85* (hgr 71: see Fig. 2),

indicating that hrpX is required for hrpG-mediated

repression. To investigate whether the repression of the

expression correlates with a reduced extracellular pro-

tease activity of X. campestris pv. vesicatoria, a protease

assay was performed on agar plates containing 1%

skimmed milk (Tang et al., 1987). As shown in Fig. 3,

degradation of milk proteins by extracellular proteases

resulted in haloes surrounding bacterial colonies of strains

85-10, 85-10DhrpG and 85*DhrpX, whereas only a small

halo was observed for strain 85*. This finding is consistent

with the cDNA-AFLP results.

Mutational analysis of hgi genes located in the regions

flanking the hrp gene cluster

As mentioned above, seven hgi fragments hybridized to

the large hrp region. Four cDNAs correspond to genes

within the known hrp gene cluster: hgi 58 (hrcC ), hgi 3 and

hgi 206 (both in hrcU ), and hgi 34 (hrpF ) (Table 1).

Interestingly, three hgi fragments are located in regions

flanking the hrp gene cluster: hgi 27 and hgi 81 map to the

region left of hrpA, and hgi 203 to the right of hrpF.

Previous analyses of transposon insertions in these

regions did not reveal the presence of any hrp genes

(Bonas et al., 1991). For further studies of these regions,

hrpG-regulon 1273

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 41, 1271–1281

Tab

le1.

Featu

res

of

the

hprG

-repre

ssed

(hgr)

and

hrp

G-induced

(hgi)

cD

NA

fragm

ents

dete

rmin

ed

by

cD

NA

-AF

LP

and

RT

-PC

Ranaly

ses.

cD

NA

fragm

ents

hrp

X-d

ependency

XV

M2

aG

1C

conte

nt

Pla

sm

idlo

cation

bC

luste

ring

with

Hom

olo

gy

[Org

anis

m]

accessio

nnum

berc

Bla

stX

E-v

alu

e

hg

rhgr

10

1–

64%

1E

ndopro

tein

ase

Arg

-Cpre

curs

or

[L.

enzym

ogenes]

(AA

D11571)

10

246

hgr

70

11

/–68%

–hgr

90/9

5P

uta

tive

serine

pro

tease

[X.

c.

pv.

pela

rgonii]

(AA

F63395)

10

273

hgr

71

11

/–65%

–hgi224

Extr

acellu

lar

pro

tease

[A.

hydro

phila

](O

05485)

10

264

hgr

90/9

51

1/–

68%

–hgr

70

Puta

tive

oute

rm

em

bra

ne

pro

tein

XadA

[X.

ory

zae

pv.

ory

zae]

(AA

G01335)

YadA

adhesin

fam

ily10

268

hg

iin

the

larg

eh

rpg

en

eclu

ste

r64%

hgi27

11

50%

–H

pa1

[X.

o.

pv.

ory

zae]

(AA

C95121)

10

213

hgi81

11

59%

–H

pa2

[X.

o.

pv.

ory

zae]

(AA

F61278)

10

266

hgi58

–1

64%

–H

rcC

[X.

c.

pv.

vesic

ato

ria]

(P80151)

0.0

hgi3/2

06

11

60%

–H

rcU

[X.

c.

pv.

vesic

ato

ria]

(U.

B.,

unpublis

hed

observ

ations)

0.0

hgi34

11

60%

–H

rpF

[X.

c.

pv.

vesic

ato

ria]

(AA

B86527)

0.0

hgi203

11

64%

––

–O

ther

hg

i–

–hgi1

11

57%

1–

–hgi9

11

70%

nd

d–

–hgi11

11

54%

–Y

4lO

[Rhiz

obiu

msp.

NG

R234]

(P55555)

YopJ

fam

ily10

249

hgi16/9

8/2

27

11

49%

––

–hgi25/6

6/8

01

145%

nd

AvrP

phD

[P.

s.

pv.

phaseolic

ola

](C

AC

16699)

0.0

hgi37/4

11

147%

––

–hgi40

11

62%

––

–hgi63/2

19

11

66%

––

–hgi69

11

57%

––

–hgi94

11

62%

––

–hgi100

11

65%

––

–hgi127

11

/–67%

1P

CZ

A361.1

3hypoth

eticalpro

tein

[A.

orienta

lis]

(T17479)

10

29

hgi128/1

30

11

/–67%

1P

seudom

onapepsin

epre

curs

or

(pepsta

tin-insensitiv

ecarb

oxyl

pro

tein

ase)

[Pseudom

onas

sp.

101]

(P42790)

10

218

hgi208

11

68%

––

–hgi213

11

64%

nd

Tra

nscriptionalre

gula

tor

XF

0216

[X.

fastidio

sa]

(D82833)

MarR

fam

ily10

219

hgi224

–1

62%

–hgr

71

Meta

llopeptidase

XF

0576

[X.

fastidio

sa]

(B82788)

10

263

a.

(1)

indic

ate

sin

duction

of

expre

ssio

nin

XV

M2.

(–)

indic

ate

sno

XV

M2

eff

ect

on

gene

expre

ssio

n.

(1/–

)re

fers

tonon-r

epro

ducib

leX

VM

2eff

ects

.b

.T

he

pla

sm

idlo

cation

was

dete

rmin

ed

by

South

ern

hybridiz

ation.

c.

Clo

sest

hom

olo

gues

were

identified

by

Bla

stX

(htt

p:/

/ww

w.n

cbi.nlm

.nih

.gov/b

last/

).d

.N

ot

dete

rmin

ed.

1274 L. Noel et al.

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 41, 1271–1281

the clone pXV331 corresponding to hgi 27 and hgi 81 was

isolated from the genomic library of X. campestris pv.

vesicatoria 75-3 (Minsavage et al., 1990). pXV4 containing

hgi 203 was isolated earlier (Wengelnik and Bonas, 1996).

In order to analyse the contribution of the hgi 27, hgi 81

and hgi 203 genes to virulence, the following deletions

were generated and introduced into strain 85-10: in DL, a

3.8 kb sequence containing hgi 27 and hgi 81 was deleted,

and in DR, a 2.5 kb sequence encompassing hgi 203 was

deleted. The mutant strains were tested for symptom

formation and in planta growth in susceptible pepper

plants ECW.

In ECW pepper plants, the appearance of water-soaking

symptoms was delayed by one day for strain 85-10DL

compared with 85-10 and 85-10DR (data not shown). In

addition, we determined bacterial growth in the plant

(Fig. 4). The wild-type strain 85-10 multiplied to approxi-

mately 3� 107 colony-forming units (cfu)/cm2, whereas 85-

10DhrpG, which was included as a typical hrp mutant, only

reached 3� 104 cfu/cm2. Growth of the strain 85-10DL in

ECW plants was reduced by a factor of 10 compared with

85-10, whereas growth of strain 85-10DR was indistinguish-

able from 85-10. These results indicate that the region

containing hgi 27 and hgi 81 contributes to virulence but is

not essential for the interaction with the plant.

XopB is similar to the Pseudomonas syringae AvrPphD

family

Three hgi fragments (hgi 25, hgi 66 and hgi 80) mapped

to cosmid pXV229, which was isolated for further

study. The DNA sequence of pXV229 was determined

using a shotgun approach. Most predicted ORFs have no

clear homologues or show homologies to putative house-

keeping genes, e.g. of Xylella fastidiosa. The hgi 25/66/80

sequences correspond to a single ORF of 1839 bp (Fig. 5).

In contrast to all known hrpX-regulated genes (Wengelnik

and Bonas, 1996; Astua-Monge et al., 2000), there is no

PIP-box present upstream of this ORF. Southern analysis

showed that the gene is a conserved single copy gene in

different X. campestris pv. vesicatoria strains (data not

shown). The predicted protein contains 613 amino acids

(65 kDa). Its last 535 residues show 77% identity/82%

similarity to the C-terminus of the avirulence protein

AvrPphD of the bean pathogen Pseudomonas syringae

pv. phaseolicola (Arnold et al., 2001), suggesting that

Fig. 2. Role of hrpX and hrp gene-inducing medium XVM2 in theexpression of members of the hrpG-regulon. Expression profiles ofselected cDNA fragments analysed by AFLP and RT-PCR are shown:hgi 11, hgi 25, hgi 40, hgi 58 (hrcC ), hgi 94 and hgr 71. Expression wasanalysed in different genetic backgrounds. The bacteria were grown inNYG or XVM2 medium. cDNA-AFLP products were visualised byautoradiography; reverse-transcribed-PCR amplicons (RT) weredetected by ethidium bromide staining. Polymorphic bands areindicated by an arrow. hgi, hrpG-induced gene; hgr, hrpG-repressedgene. The 16S rRNA was used as a constitutive control for the RT-PCR. For further information on all the hgi and hgr fragments, seeTable 1.

Fig. 3. X. campestris pv. vesicatoria strain 85* has a reduced proteaseproduction. Strains 85–10, 85*, 85–10DhrpG and 85*DhrpX werespotted on NYG-agar plates containing 1% skimmed milk andincubated at 308C for 4 d. Clear haloes around bacterial coloniesindicate the presence of extracellular proteases.

hrpG-regulon 1275

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 41, 1271–1281

the protein might be a TTSS effector. The protein was

therefore renamed XopB.

A deletion of xopB does not affect pathogenicity or

bacterial growth in planta

In order to test the contribution of xopB to virulence of

X. campestris pv. vesicatoria strain 85-10, we generated

the 85-10DxopB mutant strain in which a 1.9 kb deletion

encompassing the hgi 25/66/80 fragments was deleted

(Fig. 5). Inoculation of 85-10DxopB revealed no difference

in timing or appearance of water-soaking symptoms in

susceptible pepper ECW. Growth of 85-10DxopB

in susceptible pepper plants was similar to that of the

wild type (Fig. 4). Therefore, xopB does not appear to

contribute significantly, under the conditions tested, to

X. campestris pv. vesicatoria virulence.

XopB is secreted by the Hrp TTSS

To study XopB expression, a new integrative vector was

constructed (pIC1). The C-terminal sequence of xopB

(367 bp) was translationally fused with a triple c-myc

epitope, followed by a transcriptional fusion to the

promoterless uidA gene to generate pIC25 (Fig. 5B).

pIC25 was conjugated into X. campestris pv. vesicatoria

strains 85* and 85*DhrcV (Rossier et al., 2000) giving

85*::pIC25 and 85*DhrcV::pIC25 respectively. To com-

pare expression of xopB in both 85* and the wild-type

strain 85-10, the wild-type hrpG gene was cloned into the

suicide vector pOK1 (Huguet et al., 1998) and conjugated

into 85*::pIC25, resulting in 85-10::pIC25. b-glucuronidase

(GUS) activity of strain 85*::pIC25 was approximately 100

times higher than of strain 85-10::pIC25, confirming the

regulation of the xopB gene by hrpG at the transcriptional

level (Fig. 6A).

To detect the XopB protein, total protein extracts and

culture supernatants of different X. campestris pv.

vesicatoria strains were separated using SDS–PAGE

and analysed by Western blot analysis using a specific

c-myc antibody. As shown in Fig. 6B, a 65 kDa protein was

detected in total protein extract of 85*::pIC25. Expression

of the protein was hrpG-dependent. As the last 52 residues

of XopB (5 kDa) were replaced by the triple c-myc epitope

(5 kDa), the c-myc-tagged protein has a predicted size of

65 kDa, which is consistent with our observation (Fig. 6B).

The c-myc-tagged protein was also detected in culture

supernatants of strain 85*::pIC25 but not in supernatants

of 85*DhrcV::pIC25 (Fig. 6C), demonstrating that XopB is

secreted by the Hrp TTSS.

Fig. 5. xopB maps to an atoll of low G 1 Ccontent.A. The G 1 C content of pXV229 was calculatedfor 100 bp windows and displayed usingGENEQUEST (DNASTAR). Only the central 12 kbsegment of the insert of pXV229 is shown. Thelocation of the three hgi fragments is indicatedby grey circles.B. Restriction map of the 6.1 kb BamHI–EcoRVfragment encompassing xopB. The large arrowrepresents xopB. DxopB indicates the extent ofthe xopB deletion generated in 85-10. Greycircles indicate the location of the hgi fragments.The black arrow indicates the fusion point to thec-myc epitope in construct pIC25. Therestriction sites indicated are A (Apa I), B(Bam HI), V (Eco RV), S (Sal I) and X (Xho I).

Fig. 4. Deletion of the region containing hgi 27 and hgi 81 affectsbacterial growth in planta. X. campestris pv. vesicatoria strains wereinoculated at 104 cfu ml21 in 1 mM MgCl2 into the intracellular space offully expanded leaves of susceptible pepper ECW plants. Growth of85-10, 85-10DhrpG, 85-10DR, 85-10DL and 85–10DxopB wasmonitored over 8 d. Values represent the mean of three samples fromthree different plants. Error bars represent the standard deviations.For the sake of clarity, the 85-10DR and 85-10DxopB error bars wereomitted. Results are from one representative experiment.

1276 L. Noel et al.

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Discussion

To date, the cDNA-AFLP technique has been mainly

applied to eukaryotes except for a recent report on the

Erwinia transcriptome (Dellagi et al., 2000). Here, we

demonstrate the power of this technique for the

identification of pathogenicity-related genes. cDNA-AFLP

has several advantages: (i) it allows the simultaneous

identification of up- and downregulated genes in contrast

to protocols based on subtractive hybridization, promoter

trap or in vivo expression technology; (ii) cDNA-AFLP

analysis is highly reproducible and can differentiate

between expression patterns of gene family members

that would not be detected by hybridization-based

approaches; (iii) cDNA-AFLP is based on the presence

of restriction sites within the cDNA population and can

therefore be applied to non-sequenced organisms, for

which, for instance, the microarray technology is not

available; and (iv) cDNA-AFLP can be performed with high

throughput in sequenced organisms as a partial cDNA

sequence unambiguously identifies the corresponding

gene.

Although our initial cDNA-AFLP screen was not

saturated, more than 50 differentially expressed cDNA

fragments belonging to at least 20 loci were identified in

X. campestris pv. vesicatoria. HrpG is a member of the

OmpR family of transcriptional activators and has been

suggested to be at the top of the hrp regulatory cascade

(Wengelnik et al., 1996; 1999). As shown in this study,

transcriptional activation of most new members of the

hrpG-regulon also depends on HrpX, an AraC-type

transcriptional regulator. The putative sensor of a plant

signal has not been identified in X. campestris pv.

vesicatoria. However, for the R. solanacearum hrp gene

cluster, which has a similar organisation and regulation as

X. campestris pv. vesicatoria, it has been shown that plant

and metabolic signals are transduced by pathways

converging at hrpG (Aldon et al., 2000). Genome-wide

regulons, which extend beyond the genes for components

of the TTSS (i.e. the hrp gene cluster), have been found in

several bacterial plant pathogens, e.g. in R. solanacearum,

the popA, popB and popC genes belong to the hrp regulon

and encode proteins secreted by the TTSS (Arlat et al.,

1994; Gueneron et al., 2000). Similarly, in P. syringae, the

key regulator HrpL controls transcription of genes within

the hrp gene cluster and flanking regions as well as avr

genes that are often located on endogenous plasmids

(Jackson et al., 1999; Alfano et al., 2000). Similarly, in the

animal pathogen Salmonella enterica SsrB regulates

expression of the TTSS encoded in pathogenicity island

2 and genes located outside (Worley et al., 2000). In

X. campestris pv. vesicatoria, all identified hrpX-depen-

dent promoters known to date are characterized by a PIP-

box (Wengelnik and Bonas, 1996; Astua-Monge et al.,

2000). The xopB promoter, which is hrpX-dependent, is

unusual as it does not contain any PIP-box. In addition, we

showed that hrpX mediates not only gene induction but

also repression of five hgr fragments. Taken together,

these results suggest that there could be additional

transcriptional regulators involved in the regulatory

cascade such as the protein encoded by the hgi 213

fragment.

Although the majority of the genes in the hrpG-regulon

are induced by hrpG*, four genes are downregulated

(hgr ). Poor repression of hgr fragments expression in

XVM2 could be explained by the fact that bacteria grown in

XVM2 reach the stationary phase at a lower cell density

than in complex medium NYG. Three hgr fragments

correspond to genes encoding putative extracellular

Fig. 6. Expression analysis of the xopB gene.A. Activity of the X. campestris pv. vesicatoria xopB promoter in thepresence of hrpG and hrpG*. Strains 85-10::pIC25 (1) and 85*::pIC25(2) were grown for 16 h in NYG. Specific GUS activities are theaverage of two cultures with duplicates. Values are displayed using alogarithmic scale; error bars represent the standard deviation. GUSactivities below 0.1 U/1010 CFU are considered as background. Oneunit is defined as 1 nM of 4-methylumbelliferone released min21 perbacterium.B. Protein analysis of the c-myc-tagged XopB protein. Total proteinextracts of 85-10::pIC25 (1) and 85*::pIC25 (2) grown for 16 h in NYGwere separated using SDS–PAGE (8% polyacrylamide) and analysedby immunoblotting using the anti-c-myc antibody.C. XopB is secreted by the Hrp TTSS. Strains 85*::pIC25 (1 and 3) and85*DhrcV::pIC25 (2 and 4) were incubated in secretion medium. Totalprotein extracts (TE) and filtered supernatants (SN) were precipitatedwith TCA and concentrated 20 and 200 times respectively. Proteinswere analysed by immunoblotting as in (B) using anti-c-myc antibody.The membrane was reprobed with a specific antibody against thecytoplasmic protein HrcN to ensure that no bacterial lysis hadoccurred (data not shown).

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proteases, presumably secreted by the type II pathway.

The reduced expression level of these genes in

X. campestris pv. vesicatoria hrpG* strain correlated with

a reduced extracellular protease activity. It remains to be

investigated which protease is responsible for the

extracellular activity as well as their biological relevance

for virulence of X. campestris pv. vesicatoria. hgr 90/95

corresponds to a gene coding for XadA, a homologue of

the adhesin YadA from Yersinia. YadA is required for

bacterial adhesion to macrophages and bacterial virulence

(reviewed in Cornelis et al., 1998). The downregulation of

XadA and protease homologues in hrp gene-inducing

conditions is intriguing and would need mutant analysis

and studies of gene expression during plant infection.

The product of xopJ, identified here as a hrpG-induced

gene, belongs to the AvrRxv/YopJ family which has now

been found in almost all plant bacterial pathogens

containing a TTSS, i.e. R. solanacearum, P. syringae

and Xanthomonas and also the symbiont Rhizobium fredii

(White et al., 2000). Recently, YopJ was proposed to act

as a cystein protease (Orth et al., 2000). It is worth noting

that the putative catalytic triad essential for activity is also

conserved in XopJ. In X. campestris pv. vesicatoria,

AvrRxv, and the related protein AvrBsT, were identified

as avirulence proteins recognized in resistant tomato

and pepper plants respectively (Minsavage et al., 1990;

Whalen et al., 1993). AvrRxv and AvrBsT are secreted by

the TTSS and AvrBsT recognition inside the plant cell

suggests that this protein at least is probably injected

into the plant cell by the TTSS (Rossier et al., 1999;

Escolar et al., 2001). Whether XopJ also is secreted by the

TTSS, translocated inside the plant cell and whether it

functions as an avirulence or virulence protein remains to

be investigated.

XopB is another hrpG-induced protein with sequence

similarity to a known avirulence protein, namely AvrPphD

from P. syringae pv. phaseolicola (Arnold et al., 2001). A

mutation in avrPphD did not affect bacterial growth in the

plant (Arnold et al., 2001). Similarly, the xopB mutant was

indistinguishable from the wild type in plant infection

studies. As demonstrated here, XopB is secreted by

the Hrp TTSS. The sequence similarity of XopB to an

avirulence protein suggests that XopB is a novel type III

effector, i.e. is translocated, as all bacterial avirulence

proteins tested to date exert their activity inside the plant

cell. Proteins similar to XopB and AvrPphD are wide-

spread in plant pathogens containing a TTSS but absent

from mammalian pathogens: XopB is a conserved single

copy gene in the four X. campestris pv. vesicatoria strains

analysed. Homologues were identified in almost all

P. syringae pathovars tested (Arnold et al., 2001), as

well as in the genome sequence of P. syringae pv.

syringae DC3000 and R. solanacearum, in which

three and one homologue of AvrPphD, respectively,

were found (http://www.tigr.org/cgi-bin/BlastSearch/blast.

cgi?organism¼p_syringae and http://sequence.toulouse.

inra.fr/ralsto/public/doc/RalstoForm.html).

Sequence analysis did not provide further information

on the putative function of most hrpG-regulated genes

identified in this study. However, it is interesting to note

that the G 1 C content of several genes (hgi 27, xopB,

xopJ and hgi 37/41; Table 1) differs from the average

X. campestris pv. vesicatoria genomic G 1 C content (64%

average over 100 kb, L. Noel and U. Bonas, unpublished

observations), suggesting acquisition of DNA segments by

horizontal gene transfer. Particularly striking is xopB which

has a G 1 C content of 45% and is embedded into a high

G 1 C content region (66%; Fig. 5). Finally, the hgi 27

fragment located in the region flanking the hrp gene cluster

has a G 1 C content of 50%, which is significantly lower

than that of the hrp gene cluster (62%). Similar findings

were reported for the EEL region (for exchangeable

effector locus; Alfano et al., 2000) flanking the hrp gene

cluster of P. syringae, which was proposed to be a

pathogenicity island (Alfano et al., 2000). In different

pathogens, genes encoding Hrp-secreted proteins map to

regions flanking the hrp gene clusters. Although previous

studies did not reveal hrp genes in regions flanking the

X. campestris pv. vesicatoria hrp gene cluster (Bonas

et al., 1991), the identification of hgi genes in this location

suggests the presence of non-essential virulence genes.

We could show that the DL deletion encompasses genes

necessary for full virulence of X. campestris pv.

vesicatoria. This is corroborated by the identification of

the hpa1 gene (homologous to hgi 27 ) which is located in a

region flanking the hrp gene cluster of X. oryzae pv. oryzae

and plays a role in bacterial growth in the host plant (Zhu

et al., 2000). Detailed studies of the genes contained in the

DL deletion will determine exactly which genes contribute

to virulence and whether they encode effector proteins

secreted by the TTSS.

Experimental procedures

Bacterial strains, growth conditions and plasmids

Bacterial strains used in this study were Escherichia colistrains DH5a or DH10B (Life Technologies GmbH) and DH5a

lpir (Menard et al., 1993). Xanthomonas campestris pv.vesicatoria strains 85-10 and 85-10DhrpG, which carries a

deletion of hrpG, 85* (85-10 hrpG*) and 85*DhrcV, whichcarries a deletion of hrcV, were described previously (Rossier

et al., 1999; 2000; Wengelnik et al., 1999). E. coli cells werecultivated at 378C in Luria-Bertani (LB) medium and

X. campestris pv. vesicatoria at 308C in NYG broth or agar(Daniels et al., 1984) or in hrp gene induction medium XVM2

(Wengelnik and Bonas, 1996). Plasmids were introduced intoE. coli by electroporation and into X. campestris pv.

vesicatoria by conjugation, using pRK2013 as a helper

plasmid in triparental matings (Figurski and Helinski, 1979;

1278 L. Noel et al.

Q 2001 Blackwell Science Ltd, Molecular Microbiology, 41, 1271–1281

Ditta et al., 1980). Antibiotics were added to the media inthe following concentrations: ampicillin, 100mg ml21; tetra-

cyclin, 10mg ml21; rifampicin, 100mg ml21; spectinomycin,100mg ml21.

Generation of hrpX deletion strain 85*DhrpX

To create a non-polar deletion of hrpX, the pRX1 plasmid was

constructed as follows. The 7 kb Bam HI fragment containing

hrpX was deleted from pBX1 (Wengelnik and Bonas, 1996)and the remaining insert ligated into the suicide vector pRO1

(Rossier et al., 1999), giving pRX1. Strain 85*DhrpX wasgenerated by introduction of pRX1 into strain 85*. The mutant

has a typical hrp phenotype in tomato and pepper plants andcould be complemented by pSX2 (Wengelnik and Bonas,

1996), which carries the hrpX but not the hrpG gene.Molecular biology experiments were performed according to

standard procedures (Ausubel et al., 1996).

RNA extraction and cDNA synthesis

Bacteria were harvested by centrifugation at an OD600nm¼0.6.RNA was extracted by the hot phenol procedure (Aiba et al.,

1981). RNA (5mg) and 150 ng random hexamers were used togenerate random primed cDNAs with the TimeSaver cDNA

Synthesis kit (Amersham Pharmacia Biotech). At least twoindependent experiments were performed.

cDNA-AFLP analysis

cDNA-AFLP analysis was performed as described (Bachemet al., 1996). cDNA (100–200 ng) was digested with Pst I and

Taq I and ligated to Pst I adaptors (Pierre et al., 2000) andTaq I adaptors (Bachem et al., 1996). First, 19 cycles of non-

radioactive polymerase chain reaction (PCR) preamplificationwith primers Pst I 1 0/Taq I 1 0 without selective nucleotides

were carried out on one third of the ligation mixture. Analysisof the preamplification products revealed major bands ranging

from 0.15 kb to 0.9 kb. For the selective amplification, a33P-labelled Pst I primer and a Taq I primer, both with two

selective nucleotides, were used. The size of the observedamplicons varied from 70 bp to more than 400 bp. Repro-

ducible results were obtained with cDNA-AFLP templatesproduced from independent RNA extractions and cDNA

synthesis reactions. Sequences of the selective primers usedfor the amplification of the different hgi and hgr fragments are

available upon request.

AFLP fragment sequencing, extension and RT-PCR

analysis

AFLP fragments of interest were recovered, reamplified asdescribed previously (Pierre et al., 2000) and sequenced

using the non-selective Pst I primer. Specific primers were

designed and used to extend the DNA sequence withthe Genome Walker kit (Clontech Laboratories). Sequence

analysis was performed using the DNASTAR package(DNASTAR) and the BlastX algorithm (http://www.ncbi.nlm.

nih.gov/blast/).For reverse transcription (RT)-PCR analysis, the cDNA

prepared for cDNA-AFLP was used as template in a 1:2000dilution. Twenty (16S rDNA) to 44 amplification cycles were

used. 16S rDNA was used as a constitutive control.

Mapping of the hgi and hgr fragments to a X. campestris

pv. vesicatoria cosmid library

hgi- and hgr-fragment probes were generated using the

PCR digoxygenin (DIG) Probe Synthesis kit (Roche). AX. campestris pv. vesicatoria genomic library from strain 75-3

(Minsavage et al., 1990) containing 1000 clones in thepLAFR3 vector was replicated on nylon filters, hybridized and

washed at high stringency according to the manufacturer(Roche).

Southern hybridization

For Southern hybridization, Bam HI-digested genomic DNA

and non-digested plasmid DNA of X. campestris pv.vesicatoria were separated on a 0.7% agarose gel,

transferred to a nylon membrane and hybridized using hgi-

and hgr fragment-specific DIG probes (see above).

Deletions of regions containing hgi fragments in strain

85-10

To create a deletion of the hgi 27 and hgi 81 genes, the 7.5 kbBam HI fragment of pXV331 was cloned into pUC118 and

pBluescript II SK1 (pB-SK), giving pULB1 and pBLB1respectively. pXV331 was identified in the X. campestris pv.

vesicatoria genomic library from strain 75–3 (Minsavage et al.,1990) probed with the large hrp gene cluster. pULB1 was

digested with Bgl II and religated, generating a 3.8 kb deletiongiving pULB2. The Bam HI insert of pULB2 was then cloned

into pOK1 (Huguet et al., 1998), giving pOLB, and introducedinto strain 85-10, creating 85-10DL. The deletion of the

chromosomal region was verified by Southern analysis usingpULB1 as a probe.

To achieve deletion of hgi 203, a 3 kb Cla I fragment

containing hrpF and a 3.4 kb Xho I fragment containing theregion downstream of hgi 203 fragment were cloned from

cosmid clone pXV4 into pB-SK, giving pB3Cla and pB3.4Xhorespectively. pXV4 was identified previously from the

X. campestris pv. vesicatoria genomic library from strain75–3 of X. campestris pv. vesicatoria probed with hrp genes

(Wengelnik and Bonas, 1996). The insert of pB3.4Xho wasreleased by Xho I digestion and cloned into Eco RV-digested

pB3Cla, giving pBDR. The 5.7 kb Xba I/Xho I insert from pBDRwas cloned into Xba I/SalI-digested pOK, giving pOR. pOR

was introduced into X. campestris pv. vesicatoria 85-10,

giving 85-10DR.To delete xopB, the 6.1 kb Bam HI/Eco RV fragment

containing xopB was subcloned into pB-SK (Fig. 5). A 1.9 kbdeletion of xopB was created by partial Xho I/Sal I digestion,

giving pBDxopB. The 2.2 kb Apa I/Bam HI fragment ofpBDxopB was subcloned into pOK1 (Huguet et al., 1998),

giving pOXB, and conjugated into 85-10 to generate 85-

10DxopB.

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Q 2001 Blackwell Science Ltd, Molecular Microbiology, 41, 1271–1281

Plant material and plant inoculations

Inoculations of the pepper cultivar Early Cal Wonder (ECW)were performed as described (Bonas et al., 1991). For

macroscopic tests, bacteria were infiltrated into the leaf at 108

cfu ml21. In planta growth curves were performed in ECW

plants as described (Bonas et al., 1991).

b-glucuronidase assays

b-glucuronidase (GUS) assays were performed with expo-nentially growing X. campestris pv. vesicatoria as described

(Rossier et al., 1999). One unit is defined as 1 nM of4-methylumbelliferone released min21 and per bacterium.

Epitope tagging of XopB

To generate the integrative vector pIC1, the genes panB andlacZY were deleted from pIVETP (Rainey, 1999) and replaced

by the triple c-myc tag followed by a promoterless uidA gene(encodes b-glucuronidase). Further details are available upon

request. A 367 bp PCR product containing the 30 part of xopB,

amplified from 85-10, was cloned into pIC1 in frame withc-myc, giving pIC25. The gene is therefore fused in frame with

three c-myc tags followed by a transcriptional fusion with theuidA gene. For technical reasons, the last 156 bp of the xopB

coding sequence are missing in pIC25 (Fig. 5).

Protein analysis and secretion experiments

Protein separation, Western blot analysis and secretion

experiments were performed as described (Rossier et al.,1999). The following antibodies were used: monoclonal anti-c-

myc antibody (Roche; 1:10000 dilution), polyclonal anti-HrcNantiserum (Rossier et al., 2000).

Accession numbers

The sequences of xopJ and xopB have been submitted

to the DDBJ/EMBL/GenBank under accession numbersAY036108 and AY036109 respectively.

Acknowledgements

We thank Kai Wengelnik for providing pRX1 and Carola

Kretschmer for technical assistance. We are grateful to RalfKoebnik and Daniela Buttner for critical reading of the

manuscript. L. Noel was supported by the Ecole NormaleSuperieure de Lyon (France) and the Deutsche Forschungs-

gemeinschaft (SFB 363). This work was funded by a grantfrom the SFB 363 to U. Bonas.

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