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: [email protected]; 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).
hrpG-regulon 1277
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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.
hrpG-regulon 1279
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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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