Full text of DOI:10.1093/emboj/19.10.2304

The EMBOJournal Vol. 19 No. 10 pp. 2304±2314, 2000
A bacterial sensor of plant cell contact controls the
transcriptional induction of Ralstonia solanacearum
pathogenicity genes
Didier Aldon, Belen Brito, Christian Boucher
and St e phane Genin
Despite these well-documented examples, little is known
1
about how the process of bacterium±plant cell recognition
occurs in many other interactions, especially for the other
main groups of Gram-negative phytopathogenic bacteria.
With the exception of Agrobacterium, Gram-negative
plant pathogenic bacteria possess key pathogenicity
determinants called hrp genes. These hrp genes are
required both for causing disease symptoms on host plants
and for eliciting a defence reaction, called the hypersen-
sitive response, on resistant or non-host plants. The
molecular characterization of hrp gene clusters has
revealed that several hrp genes are homologous to
pathogenicity determinants of animal pathogens, which
encode a type III secretion machinery (reviewed in Van
Gijsegem et al., 1993; Hueck, 1998; Galan and Collmer,
Laboratoire de Biologie Mol e culaire des Relations Plantes±
Microorganismes, INRA-CNRS, BP27, 31326 Castanet-Tolosan
Cedex, France
1
Corresponding author
e-mail: sgenin@toulouse.inra.fr
The hrp genes of the plant pathogen Ralstonia solana-
cearum are key pathogenicity determinants; they
encode a type III protein secretion machinery involved
in the secretion of mediators of the bacterium±plant
interaction. These hrp genes are under the genetic
control of the hrpB regulatory gene, expression of
which is induced when bacteria are co-cultivated with
plant cell suspensions. In this study, we used hrp±gfp
transcriptional fusions to demonstrate that the expres-
sion of the hrpB and type III secretion genes is specif-
ically induced in response to the bacterium±plant cell
contact. This contact-dependent induction of hrpB
gene expression requires the outer membrane protein
PrhA, but not a functional type III secretion appar-
atus. Genetic evidence indicates that PrhA constitutes
the ®rst example of a bacterial receptor for a non-dif-
fusible signal present in the plant cell wall and which
triggers the transcriptional activation of bacterial
virulence genes.
1999). In animal pathogens, type III secretion pathways
enable the translocation of several effector proteins
directly into eukaryotic cells, where they exert their anti-
host functions (see Cornelis, 1998; Galan and Collmer,
1999). In plant pathogens, multiple reports suggest that the
products of avr genes encode effector proteins, which are
injected into host plant cells by the Hrp type III secretion
machinery. This hypothesis is supported by the fact that
avr gene products are active when they are expressed,
either stably or transiently, within plant cells (reviewed in
Galan and Collmer, 1999). Bacterial type III secretion is
thought to proceed through the formation of a pilus-like
structure connecting the pathogen and host cell. The
structural components of this pilus are encoded by hrp
genes, hrpA for P.syringae (Roine et al., 1997) and hrpY
for Ralstonia solanacearum (Van Gijsegem et al., 2000).
Several other proteins have been shown to be secreted in
the extracellular medium in an Hrp-dependent manner:
harpins from Erwinia amylovora, P.syringae and
R.solanacearum (Wei et al., 1992; He et al., 1993; Arlat
et al., 1994; Charkowski et al., 1998; Kim and Beer, 1998)
and a set of Avr/Dsp proteins from different plant
pathogenic bacteria (Gaudriault et al., 1997; Bogdanove
et al., 1998; Mudgett and Staskawicz, 1999; Rossier et al.,
1999; van Dijk et al., 1999; Gu e neron et al., 2000).
However, the function of these diverse proteins and their
contributions to pathogenicity are still poorly understood.
The hrp genes are induced in plants, but no plant
inducers of hrp gene expression have been characterized
so far. hrp genes are not expressed when bacteria are
grown in rich media; rather, they are most strongly
expressed in various minimal media that mimic plant
apoplastic ¯uids (for references, see Van Gijsegem, 1997),
and in several cases the level of induction found in plants
is comparable to that observed in the synthetic medium.
However, recent results in our laboratory provide evidence
that speci®c plant factor(s) induce(s) R.solanacearum hrp
gene expression. Upon co-culture with Arabidopsis
thaliana or tomato cell suspensions, the expression of
Keywords: Arabidopsis thaliana/green ¯uorescent
protein/hrp genes/signal transduction/type III secretion
pathway
Introduction
Many species of soil bacteria form specialized symbiotic
and parasitic interactions with plant cells. To initiate an
interaction, a bacterium must ®rst recognize its appropriate
host plant cell. This recognition can be used by the
bacterium to activate bacterial genes whose products
mediate the development of the interaction. For >10 years,
it has been known that several small diffusible plant
molecules play a major role in these recognition processes
by inducing a variety of genes in plant-associated bacteria.
This was ®rst observed for Agrobacterium tumefaciens,
which, in wounded tissues, senses a structurally diverse
group of plant phenolics, including acetosyringone
(
Stachel et al., 1985). This perception of phenolic signal
molecules leads to transcriptional activation of virulence
vir) genes necessary for the transfer of T-DNA to host
(
cells. Certain phenolic b-glycosides also serve as signals
that induce phytotoxin production by Pseudomonas
syringae pv. syringae (Mo et al., 1995), while in rhizobia,
speci®c plant ¯avonoids activate nodulation genes essen-
tial for the establishment of symbiosis (Peters et al., 1986).
2
304
ã European Molecular Biology Organization

Host contact-dependent induction of virulence genes
the regulatory hrpB gene is induced up to 20-fold more
than in minimal medium (Marenda et al., 1998). This
speci®c plant cell induction of hrp genes is controlled by
PrhA, a protein that shows homology to outer membrane
siderophore receptors (Marenda et al., 1998). PrhA was
proposed to act as a receptor for a plant-derived signal and
appears to activate a speci®c plant-dependent pathway
controlling the induction of R.solanacearum hrp genes
(Figure 1). Two additional components of this regulatory
cascade, HrpG and PrhJ, have recently been characterized
(
Brito et al., 1999).
We developed a green ¯uorescent protein (GFP)
reporter gene system to monitor the in situ expression of
bacterial genes upon co-cultivation of R.solanacearum
with plant cells. In this paper, we show that the strong
induction of hrp genes detected in the presence of plant
cells speci®cally requires a physical contact between the
bacterium and its target cell. We demonstrate that this
plant cell contact-dependent induction of hrp genes is
abolished in a prhA mutant strain, indicating that PrhA is
the ®rst example of a bacterial receptor for a non-diffusible
plant cell wall molecule that induces the expression of
pathogenicity genes.
Fig. 1. Model of regulation of the R.solanacearum Hrp type III
secretion system in response to plant signal(s). Above, genetic
organization of the hrp gene cluster, with grey arrows showing the
orientation and length of the hrp transcriptional units. A new
nomenclature has distinguished hrp and hrc (for hrp-conserved)
genes, the latter are homologues of type III secretion genes of animal
pathogens and are predicted to encode components of the type III
secretion apparatus. Four proteins, the harpin PopA, PopB, PopC and
HrpY have been described to be secreted by the Hrp pathway into the
extracellular medium. HrpY is the structural component of the Hrp
pilus, an essential extracellular structure involved in PopA secretion.
The outer membrane protein PrhA controls the plant-responsive
regulatory cascade composed of PrhJ, HrpG and HrpB, the ®nal
activator of hrp transcription units 1±4 and 7. Two additional
regulatory components (dotted lines) have recently been identi®ed
Results
Construction of hrp±gfp transcriptional fusions
We assayed the functional expression of the gfp gene in
R.solanacearum using a mini-transposon located on the
suicide delivery plasmid pAG408 (Suarez et al., 1997), in
order to generate several chromosomal fusions. One of
them, GMI1600, appeared to be highly and constitutively
expressed; the corresponding strain was subsequently used
as a positive GFP control strain for studying bacterium±
plant cell interactions. We used polymerase chain reaction
(
B.Brito and S.Genin, unpublished). The HrpG protein integrates
another hrp-inducing signal pathway, which is dependent on the
nutrient/metabolic status of the bacterium. This model is based on data
compiled from Arlat et al. (1994), Marenda et al. (1998), Brito et al.
(
PCR) cloning techniques to construct a transcriptional
fusion between a 250 bp DNA fragment containing the
functional promoter of the hrpB regulatory gene (Genin
et al., 1992) and the gfp gene from pAG408 (Suarez et al.,
(
1999), Gu e neron et al. (2000) and Van Gijsegem et al. (2000).
conditioned medium (i.e. medium in which plant cells
have been grown and then removed by ®ltration) is not
able to induce the transcription of hrpB (Marenda et al.,
1
997) (see Materials and methods). This hybrid hrpB±gfp
gene fusion was expressed in R.solanacearum on the low
copy number plasmid pLAFR6 (Huynh et al., 1989)
between two synthetic trp terminator sequences to ensure
that the expression of the cloned fragment was driven from
its own promoter region. The resulting plasmid, pSG261,
was transferred by conjugation into the wild-type strain
GMI1000 and various hrp/prh mutants (Table I). The
recombinant GMI1000/pSG261 strain gave slightly ¯uor-
escent colonies on minimal medium plates after incubation
at 28°C, but only background activity was detected after
growth on rich medium; this is the expression pattern
expected for the hrpB gene (Genin et al., 1992). Similarly,
another GFP reporter fusion was generated by PCR
ampli®cation with the hrpY gene promoter and introduced
into R.solanacearum on a pLAFR6 plasmid.
1998) indicates that this induction either speci®cally
requires plant cells or involves a very labile diffusible
plant signal.
By cytological GFP ¯uorescence detection, we used a
GMI1000/pSG261 hrpB±gfp transcriptional fusion to
monitor the expression of the hrpB gene when bacteria
were co-cultivated with Arabidopsis cells. Plasmid reten-
tion studies indicated that the population of bacteria
present in the co-culture showed no net loss of plasmid-
borne tetracycline resistance during the experiments (data
not shown). Because the auto¯uorescence of plant cell
wall material at 395 nm (the wavelength at which GFP
absorbs) interferes with the observation of bacterium±
plant cell interaction we used the dye Evans blue, which
binds to plant cells and causes a shift in the ¯uorescence
emission of plant cell material, which thus appears red.
This method allowed easy detection of gene activation in
individual bacteria co-cultivated with host plant cells.
Strong ¯uorescence was detected when bacteria were
physically associated with Arabidopsis cells (Figure 2A),
whereas only a weak signal (comparable to that detected
Expression of the regulatory hrpB gene and type
III secretion genes is speci®cally induced in
response to bacterium±plant cell contact
Expression of the regulatory hrpB gene is strongly induced
(
15- to 20-fold) when R.solanacearum are co-cultured
with A.thaliana or tomato plant cell suspensions (Marenda
et al., 1998; Brito et al., 1999). The observation that
2305

D.Aldon et al.
Table I. Bacterial strains and plasmids
Designation
Relevant characteristics
Reference/source
R.solanacearum strains
GMI1000
GMI1410
GMI1462
GMI1475
GMI1525
GMI1575
GMI1578
GMI1579
GMI1600
GMI1601
Plasmids
wild-type strain
hrpY mutant
hrcC mutant
hrpB-Tn5B20 mutant
hrpB mutant
prhA mutant
hrpG mutant
prhJ mutant
GMI1000 with constitutive GFP expression
GMI1600 carrying a prhA mutation
Boucher et al. (1985)
Van Gijsegem et al. (1995)
Gough et al. (1992)
Genin et al. (1992)
Genin et al. (1992)
Marenda et al. (1998)
Brito et al. (1999)
Brito et al. (1999)
this study
this study
pAG408
pLAFR6
pSG261
pSG282
suicide plasmid containing a promotorless gfp reporter gene on a mini-transposon
pLAFR1 with trp terminators, carries tetracycline resistance gene
pLAFR6 carrying hrpB±gfp transcriptional fusion
Suarez et al. (1997)
Huynh et al. (1989)
this study
pLAFR6 carrying hrpY±gfp transcriptional fusion
this study
after growth in conditioned medium, Figure 2D) was
observed from bacteria present in the medium, but not in
close contact with plant cells. Confocal laser scanning
microscopy was used to con®rm that the strongly ¯uor-
escent bacteria were attached to the plant cells, often in a
polar fashion (Figure 2F). Under the experimental condi-
tions used, distinctly ¯uorescent bacteria in contact with
plant cells were ®rst detected after 90 min of co-
cultivation. Measurements of the b-galactosidase activity
of strain 1475 (carrying a chromosomal hrpB±lacZ fusion)
con®rmed that after 4 h the activity found in bacteria
attached to plant cells was 6-fold higher than that observed
in bacteria recovered from the culture medium (data not
shown).
et al., 1999). In order to determine whether the contact-
dependent induction of hrpB expression was also con-
trolled by PrhA, the hrpB±gfp fusion carried by the
plasmid pSG261 was introduced into strain GMI1575
(prhA mutant). After growth of the resulting strain in the
presence of Arabidopsis cells, only a weak level of
¯uorescence, corresponding to the basal level of hrpB
expression, could be detected after 16 h, even when
bacteria were found in the vicinity of plant cells (Figure 3A
and B). These observations demonstrate that PrhA is
speci®cally involved in the recognition of the bacterium±
plant cell contact, con®rming that the plant signal that
induces hrpB expression is a non-diffusible ligand mol-
ecule.
To determine whether the expression of type III
secretion genes followed the same expression pattern, we
monitored expression of an hrpY±gfp transcriptional
fusion in a wild-type strain. The hrpY gene was recently
shown to encode a component of an essential extracellular
pilus structure of the secretion apparatus, required for the
secretion of PopA (Van Gijsegem et al., 2000). We
observed that this hrpY±gfp fusion was also strongly
induced upon contact of bacteria with plant cells
Plasmid pSG261 was also introduced into strains
carrying disruptions of regulatory genes encoding other
components of the plant signal transduction pathway:
GMI1578 (hrpG::W) and GMI1579 (prhJ::W), and into
strain GMI1525 (hrpB::W). In a prhJ mutant, a basal level
of ¯uorescence was observed, but no transcriptional
induction could be detected upon bacterium±plant cell
contact, as described above in a prhA mutant background
(Figure 3C and D). Expression of the hrpB±gfp fusion was
undetectable in an hrpG mutant co-cultivated with plant
cells, whether bacteria were in contact with plant cells or
not (Figure 3E and F). As expected, plant cell contact-
dependent expression of the hrpB±gfp fusion was observed
in the hrpB mutant strain (data not shown), con®rming the
speci®city of the upstream components of the PrhA-
dependent regulatory cascade in transducing the signal
triggered by the bacterium±plant cell contact.
(
Figure 2G), but was not expressed in an hrpB mutant
strain (data not shown), con®rming the previous report that
hrpY is HrpB regulated. From these observations, we
conclude that transcription of the hrpB gene and of type III
secretion genes is strongly induced in R.solanacearum
only after contact with plant cells has been established,
indicating that the pathogen is able to recognize a cell
surface ligand and responds accordingly.
Contact-dependent induction of hrpB gene
expression requires the outer membrane protein
PrhA
PrhA is required for recognition,but not for
attachment to plant cells
The above data indicate that the putative PrhA receptor
recognizes a ligand upon contact with the plant cell
surface, which leads to a speci®c up-regulation of the hrpB
gene promoter. Because cytological studies revealed that
R.solanacearum are tightly attached to plant cells, we
wanted to ®nd out whether PrhA is required for bacteria to
become attached to plant cells. Strain GMI1575 (prhA
mutant) bearing the pSG261 plasmid was only weakly
¯uorescent and therefore not suitable for accurate study of
The high level of induction of hrpB gene transcription
observed when bacteria are co-cultivated with Arabidopsis
cells depends on a functional prhA gene, the product of
which has been proposed to act as a receptor for plant-
speci®c signal(s) (Marenda et al., 1998). Genetic evidence
indicates that the PrhA protein is at the top of a regulatory
cascade involved in the transduction of plant-dependent
signal(s) to the hrpB gene (Marenda et al., 1998; Brito
2
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Host contact-dependent induction of virulence genes
Fig. 2. Induction of hrp gene expression in response to bacterium±plant cell contact. Interaction of R.solanacearum and A.thaliana cells observed by
epi¯uorescence microscopy (A±E and G±I) or by confocal microscopy (F) after 16 h of co-cultivation. The ¯uorescence and phase contrast images
have been overlaid in (B) and (H) [(A) + (C) and (G) + (I), respectively] in order to co-localize induced bacteria and plant cell surfaces. Images were
acquired with the same parameters to allow comparisons between the emitted ¯uorescence level in the different frames. The bar represents 20 mm,
except in F (10 mm). (A±C) Co-cultivation of GMI1000/pSG261 with Arabidopsis cells. Strongly induced bacteria expressing the hrpB±gfp fusion are
found in contact with plant cells; some uninduced bacteria present in the medium are indicated by arrowheads (B). The few strongly induced bacteria
observed in the medium are assumed to have previously been in close association with plant cells. (D and E) Basal level of hrpB±gfp expression
obtained when strain GMI100/pSG261 is grown in Arabidopsis-conditioned medium. (F) Detail of the interaction between strain GMI1000/pSG261
and Arabidopsis cell surface observed by confocal microscopy. The bacteria in contact with the plant cell surface exhibit a high level of GFP
¯uorescence (red colour); non-induced bacteria are visible at the upper right. (G±I) Co-cultivation of strain GMI1000/pSG282 (hrpY±gfp) with
Arabidopsis cells, showing that the hrpY gene follows the same expression pattern as hrpB.
bacterial population were bound to plant cell surfaces. In
the nature of the bacterium±plant cell interaction, so a
prhA mutation was introduced into strain GMI1600, which
constitutively expresses GFP activity. We observed by
confocal laser microscopy (Figure 4A and B) that mutant
strain GMI1601 interacts qualitatively with plant cells,
like the wild-type strain GMI1000 (Van Gijsegem et al.,
conclusion, PrhA does not appear to be involved in the
attachment of bacteria to plant cells, supporting the view
that it acts instead as a receptor for a non-diffusible plant
signal.
The up-regulation of hrpB in response to
pathogen±plant cell contact does not require a
functional type III secretion system
The hrp regulon controlled by HrpB comprises at least 20
genes encoding components of the type III secretion
machinery, along with a secreted protein (PopA) and other
candidate proteins exported by the Hrp apparatus. In
Yersinia, the expression of virulence genes induced by the
sensing of target cell contact depends on the secretion of
2
of attachment of P-radiolabelled bacterial populations to
000). To con®rm this ®nding, we determined the kinetics
3
2
Arabidopsis cells. Figure 4C shows that strain GMI1575
(
prhA mutant) was still able to bind to plant cells as
ef®ciently as the parental strain. In both cases, only 5% of
the bacteria present in the co-culture were attached to plant
cells after 1 h (when cultures were shaken), but this
proportion increased gradually so that after 7 h, 40% of the
2307

D.Aldon et al.
Fig. 3. Contact-dependent induction of hrpB gene expression requires
components of the plant-responsive regulatory cascade. Epi¯uorescence
microscopy study after 16 h of co-cultivation of R.solanacearum and
Arabidopsis cells. The ¯uorescence and phase contrast images have
been overlaid in (B), (D) and (F). The bar represents 20 mm. (A and
B) Strain GMI1575/pSG261 carrying a prhA mutation. (C and D) Strain
GMI1579/pSG261 carrying a prhJ mutation. (E and F) Strain
GMI1425/pSG261 carrying an hrpG mutation.
Fig. 4. PrhA is not involved in the attachment of bacteria to plant cell
surfaces. (A and B) Confocal analysis of the interaction between
Arabidopsis cells and (A) strain GMI1600 carrying a constitutive gfp
reporter fusion or (B) strain GMI1601, which carries, in addition, a
prhA mutation. In both cases, adherence and polar attachment of the
bacteria (in red) to the plant cell surface can be observed, as has been
described for the wild-type strain GMI1000 (Van Gijsegem et al.,
2
000). The bar represents 15 mm. (C) Time course of attachment of
P-radiolabelled bacteria (wild-type strain GMI1000 and prhA mutant
strain GMI1575) to Arabidopsis cells grown under continuous
3
2
agitation.
the negative regulator LcrQ, through the type III secretion
pathway (Pettersson et al., 1996). This strategy, based on
the sensing of secretion itself, allows the coupling of the
transcription and secretion processes. We investigated
whether the Hrp pilus of R.solanacearum could in¯uence
the transcription of hrpB, either by being directly involved
in the sensing of the host plant cell or by promoting the
translocation of proteins into plant cells. The hrpB±gfp
fusion carried on pSG261 was introduced into strain
GMI1410, which carries a Tn5 insertion within the hrpY
gene coding sequence. Microscopic detection of ¯uores-
cent bacteria grown in co-culture with Arabidopsis cell
suspensions clearly revealed that the absence of the Hrp
pilus in this strain does not affect the contact-dependent
up-regulation of the hrpB gene (Figure 5A). This also
con®rmed the observation that bacterial attachment is not
impaired in this hrpY mutant strain (Van Gijsegem et al.,
encodes the R.solanacearum secretin (Figure 5B). An
hrpY mutant strain carrying an hrpY±gfp transcriptional
fusion was also up-regulated upon contact of the bacteria
with plant cells; up-regulation thus still occurred in a strain
unable to synthesize the Hrp pilus (Figure 5C). From these
data, we conclude that the Hrp pilus is not involved in the
sensing of host plant cells and that the inability to secrete
virulence factors does not modulate the transcriptional
activation of the hrpB and hrpY genes in response to
contact with the target cell.
The signal that induces hrp gene expression
during pathogen±plant cell contact is a ubiquitous
and non-diffusible molecule present in the
Arabidopsis cell wall
In order to de®ne the nature of the hrp-inducing plant
signal, we investigated whether it could be found in plant
2
000). A contact-dependent induction of the hrpB gene
was also observed when the pSG261 plasmid was
introduced into another hrp secretion mutant strain,
GMI1462, carrying a disruption of the hrcC gene, which
2
308

Host contact-dependent induction of virulence genes
Fig. 5. Transcriptional induction of hrpB gene expression in response to plant cell contact is not type III secretion dependent. Observations by
epi¯uorescence microscopy after 16 h of co-cultivation of Arabidopsis cells with the R.solanacearum hrpY mutant strain GMI1410/pSG261 (A) and
the hrcC mutant strain GMI1462/pSG261 (B), both carrying an hrpB±gfp reporter fusion. The same experimental conditions were used for strain
GMI1410/pSG282, carrying an hrpY±gfp reporter fusion (C). The bar represents 20 mm.
Fig. 6. Different plant cell species induce hrpB gene expression in a PrhA-dependent manner. Illustrations are an overlay of the ¯uorescence and
visible images. The bar represents 20 mm. (A and D) Co-cultivation of tobacco cells with R.solanacearum strain GMI1000/pSG261 (A) or the prhA
mutant strain GMI1575/pSG261 (D). (B and E) Co-cultivation of M.truncatula cells with GMI1000/pSG261 (B) or GMI1575/pSG261 (E). (C and
F) Co-cultivation of tomato cells with GMI1000/pSG261 (C) or GMI1575/pSG261 (F).
species other than A.thaliana. We monitored the expres-
sion level of an hrpB±gfp gene fusion in individual
bacteria co-cultivated with plant cell suspensions from
tomato, which is a host plant for R.solanacearum, and
from tobacco and Medicago truncatula, two non-host
plants in which the bacterium is unable to cause disease. In
order to standardize the culture conditions, these different
plant cell suspensions were maintained and grown for
experiments in the same de®ned culture medium
(Gamborg medium). Results shown in Figure 6 indicate
that contact-dependent induction of hrpB expression was
observed in the presence of all the plant cell types tested
and that this induction was, in each case, dependent on a
functional prhA gene. This latter observation differs from
our previous results in which hrpB gene expression was
only reduced in a prhA mutant strain co-cultivated with
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D.Aldon et al.
Fig. 7. The plant signal recognized by PrhA that induces hrpB gene expression is present in the Arabidopsis cell wall. Epi¯uorescence microscopy
observations of R.solanacearum strains grown in the presence of Arabidopsis cell wall material. Illustrations (C±F) are overlays of the ¯uorescence
and visible light microscopy images. The bar represents 20 mm. (A) Strain GMI1000/pSG261 co-cultivated for 4 h with cell wall material.
(
B±D) Strain GMI1000/pSG261 (B and C) and the prhA mutant strain GMI1575/pSG261 (D) co-cultivated for 16 h with cell wall material. (E) Strain
GMI1000/pSG261 with heat-treated cell wall material (15 min, 100°C). (F) Strain GMI1000/pSG261 with proteinase K-treated cell wall material.
tomato cells (Marenda et al., 1998). This discrepancy is
probably due to the difference between the plant cell
culture media used in the two studies (T-MSMO was used
in the earlier study and Gamborg medium in this one). In
the presence of tomato, tobacco or Medicago cells, the
induction kinetics of hrpB expression were comparable to
those observed in the presence of Arabidopsis cells and
reached a maximum after 4 h of co-cultivation. None of
the conditioned media prepared from these different plant
cell cultures induced hrpB gene expression (data not
shown).
To localize the active plant signal in plant cells, cell
wall material was prepared from Arabidopsis cell suspen-
sions and incubated at 28°C with R.solanacearum. Under
these conditions, bacteria carrying the hrpB±gfp fusion
bound to this plant cell wall material started to ¯uoresce
after 4 h (Figure 7A) and were intensely ¯uorescent after
grown in the absence of bacteria. Furthermore, this
Arabidopsis cell wall material still activated hrpB gene
expression after heating (10 min at 100°C) and proteinase
K treatment before incubation with bacteria (Figure 7E
and F).
Discussion
Bacterial virulence genes are integrated into sophisticated
regulatory networks that allow their control at the
transcriptional level in response to environmental changes
encountered by the pathogen in its various habitats.
Expression of the hrp genes of phytopathogenic bacteria
can be induced in vitro by a variety of environmental
signals, such as pH, osmolarity, and the carbon and
nitrogen sources used in the medium. In this study, we
demonstrate for the ®rst time that contact between
R.solanacearum and host plant cells is a signal triggering
maximal hrp gene expression. It has already been shown
that contact of bacteria with mammalian cells is an
important signal, stimulating the production of several
effector proteins translocated by type III secretion path-
ways (M e nard et al., 1994; Rosqvist et al., 1994; Watarai
et al., 1995; Zierler and Galan, 1995) and also results in
increased transcription of bacterial virulence genes
(Pettersson et al., 1996; Zhang and Normark, 1996;
Jacobi et al., 1998; Taha et al., 1998). Here, we present
1
6 h (Figure 7B and C). The low ¯uorescence detected
with a prhA mutant strain con®rmed that the transcrip-
tional induction of the hrpB gene observed in the presence
of cell wall material follows the same expression pattern as
in the presence of plant cells (Figure 7D), and indicated
that wild-type bacteria were able to recognize a non-
diffusible ligand molecule concentrated in the cell wall
fraction. This signal appears to be a constitutive compon-
ent of the plant cell wall since it was present in the
Arabidopsis cell wall material prepared from plant cells
2
310

Host contact-dependent induction of virulence genes
evidence that this is also the case for plant-pathogenic
bacteria. In R.solanacearum, the transcription of two
essential pathogenicity determinants, namely the hrpB
regulatory gene (controlling both Hrp type III secretion
genes and genes encoding type III-secreted proteins such
as PopA) and hrpY (encoding the structural component of
the Hrp pilus), is strongly induced when the bacterium
comes into contact with plant cells. We show that the plant
signal that triggers hrp gene expression upon bacterium±
plant cell contact is a component of the Arabidopsis plant
cell wall. To our knowledge, this is the ®rst description of
a non-diffusible plant signal inducing bacterial gene
expression. The induction of R.solanacearum hrp genes
detected in response to contact with diverse plant cell
species from three different dicotyledonous plant families
siderophore receptor structures is not necessarily correl-
ated with substrate speci®city (Koebnik et al., 1993).
Finally, it should be noted that no bacterial sensor of target
cell contact has yet been identi®ed in mammalian patho-
gens. It will therefore be of prime interest to determine
whether pathogenic bacteria have evolved different classes
of external receptors able to recognize contact with their
host cells and to generate a speci®c signal triggering
virulence functions.
Our observations indicate the existence of two distinct
steps in the interaction between bacteria and plant cells.
First, bacteria attach to the plant cell surface (often in a
polar manner); this ®rst step does not require a functional
PrhA protein and is not dependent on hrp-encoded
functions (since this attachment process is still observed
with an hrpB mutant strain). Once this attachment is
established, the PrhA receptor is able to detect an
accessible ligand; this event leads to increased transcrip-
tion of the hrpB regulatory gene. This two-step interaction
is reminiscent of the situation described during the
interaction of enteropathogenic E.coli with its target cell,
which requires initial adhesion, through a bundle-forming
pilus, followed by intimate adhesion mediated by intimin
(Rosenshine et al., 1996). Since the R.solanacearum Hrp
pilus is not involved in the sensing of plant cells (this
study) or in the attachment process, but rather in the
translocation of Hrp-dependent secreted products (Van
Gijsegem et al., 2000), this suggests that speci®c deter-
minants involved in bacterial attachment to the host cell
surface remain to be identi®ed.
This study shows that the contact-dependent induction
of R.solanacearum hrp genes can be observed even in the
absence of a functional type III secretion apparatus. We
have also shown that transcription of the type III-secreted
protein HrpY is not regulated by the secretion status, as is
also probably the case for PopA, expression of which is
hrpB dependent (Arlat et al., 1994). This situation is
clearly different from that observed in Yersinia, where
export of the negative regulator LcrQ via the type III
secretion pathway is a prerequisite for achieving full
induction of yop gene expression (Pettersson et al., 1996).
In Shigella ¯exneri, transcription of the secreted protein
VirA also requires the integrity of the type III secretion
machinery (Demers et al., 1998). In R.solanacearum,
control of the type III secretion process appears to be
regulated differently, suggesting that this regulation oper-
ates mainly at a post-transcriptional level, probably by
ensuring the coupling of translation and secretion at the
mRNA level (Anderson and Schneewind, 1997). This
observation, along with the fact that contact-dependent
induction of hrpB expression occurs with a wide range of
host and non-host plant cell cultures, favours the view that
the speci®city of the plant±bacterium interaction does not
reside in the transcriptional activation of type III secretion
genes, but rather at the level of the Hrp-dependent secreted
products.
(
Solanaceae, crucifers and legumes) and including both
host and non-host plants strongly suggests that this
inducing signal involves a ubiquitous component of the
plant cell wall. Two additional arguments support this
hypothesis: (i) the elicitation of the hypersensitive
response on non-host plants by phytopathogenic bacteria
is speci®cally dependent upon hrp genes, which argues
against host-speci®c hrp gene induction; and (ii) the
biochemical composition of the plant cell wall is widely
conserved among dicotyledonous plants (Varner and Lin,
1
989). This hrp-inducing signal appears to be heat stable
and resistant to proteinase K treatment. This suggests the
involvement of a complex carbohydrate macromolecule
present in the hemicellulosic/pectic fraction, although the
involvement of a proteinaceous component protected in
the pectic gel matrix cannot be ruled out completely.
This work provides evidence that the recently char-
acterized plant-responsive regulatory cascade that induces
hrp gene expression in R.solanacearum in the presence of
plant cells (Marenda et al., 1998; Brito et al., 1999) is
activated in response to pathogen±plant cell contact.
Accordingly, contact-dependent induction of hrpB gene
expression is strongly reduced or abolished, respectively,
in mutant strains disrupted in prhA or hrpG, which both
encode components of this regulatory cascade. Genetic
evidence and structural features of PrhA indicate that PrhA
de®nes a novel class of receptor proteins involved in the
sensing of plant cell contact by the recognition of a non-
diffusible ligand molecule present in the plant cell wall.
PrhA is homologous to siderophore receptors belonging to
the FecA/PupB subclass (Marenda et al., 1998). FecA and
PupB are the receptor components of the so-called `three-
compartment signal transduction systems', which are
involved in transcriptional regulation of, respectively,
the Escherichia coli and Pseudomonas putida iron trans-
port genes (Koster et al., 1994; H aÈ rle et al., 1995). This
newly discovered signal transduction system allows Gram-
negative bacteria to rapidly modulate transcription in
response to a stimulus from the cell surface. Preliminary
data suggest that in R.solanacearum PrhA might, along
with two other components, form an analogous three-
compartment signal transduction system, which speci®c-
ally induces the expression of downstream genes in the
presence of plant cells (our unpublished data). The
phylogenetic relationship between PrhA and hydroxamate
siderophore receptors suggests that PrhA can recognize a
ligand molecule related to ferrichrome derivatives.
Nethertheless, it has been shown that co-evolution of
The plant cell contact-dependent induction of hrp gene
expression is probably very rapid: hrpB gene induction is
clearly detected within 90 min, which is shorter than the
generation time of R.solanacearum grown under optimal
conditions and also represents the time required for
attachment of bacteria to plant cells (Figure 4C) and the
synthesis of a detectable amount of GFP protein under our
2311

D.Aldon et al.
Construction of hrp±gfp transcriptional fusions
experimental conditions. In Yersinia, the up-regulation of
yopE gene expression is also very rapid, being detected
within 30 min after infection of eukaryotic cells (Jacobi
et al., 1998). It is noteworthy that a basal level of GFP
activity can be detected in bacteria before the contact with
plant cells. This basal level is comparable to the level of
hrpB expression observed after growth of the bacteria in
minimal medium in vitro and it is known that protein
secretion via the Hrp type III secretion pathway can be
observed in these conditions (Arlat et al., 1994). This
observation indicates that contact of the bacteria with plant
cells does not trigger de novo expression of hrp genes, but
rather triggers a strong (full?) induction of hrp gene
expression. Several recent reports also support the view
that, even for mammalian pathogens, type III secretion can
occur in the absence of contact with eukaryotic cells
Escherichia coli DH5a (Bethesda Research Laboratory) was used as a
host for plasmid constructs. Transcriptional fusions between the
R.solanacearum hrpB and hrpY genes and gfp were constructed by a
PCR cloning procedure using the Expand Long Template PCR kit
(Boehringer). A 727 bp fragment of the gfp gene was ampli®ed from the
plasmid pAG408 (Suarez et al., 1997) using the forward primer
5¢-CATATGAGTAAAGGAGAAG-3¢ and the reverse primer 5¢-GAA-
TTCATTATTTGTAGAGC-3¢. The hrpB gene promoter was ampli®ed
with the forward primer 5¢-CAGGTCAAGGGTACGCTC-3¢ and the
reverse primer 5¢-GGAATTCCATATGAATGCTCCTGAAGCGTCA-
3
¢. The resulting 236 bp ampli®ed fragment was cloned upstream of the
gfp gene, resulting in plasmid pSG257. This hrpB±gfp transcriptional
fusion was then cloned in the low copy number plasmid pLAFR6 (Huynh
et al., 1989), generating plasmid pSG261.
The hrpY promoter was ampli®ed with the following pair of primers:
5¢-GAATTCGAGCAGCGCGCC-3¢ and 5¢-GCTAGCCATGATAGT-
TTCCTTTGATGG-3¢. The resulting 249 bp ampli®ed fragment was
cloned upstream of the gfp gene, creating plasmid pSG278. This hrpY±gfp
transcriptional fusion was cloned in the pLAFR6 vector to generate
plasmid pSG282.
(
1
Blocker et al., 1999; Dae¯er, 1999; Lee and Schneewind,
999) and that some environmental factors, such as pH of
Detailed descriptions of plasmid constructs can be obtained on request
from S.G.
the growth medium, strongly in¯uence the secretion
process. A contact-dependent signal leading to full and
rapid activation of hrp genes could serve to produce more
Hrp proteins and Hrp-dependent effectors in the vicinity of
plant cells, ensuring the release of these pathogenicity
factors at the appropriate time and place. Another attract-
ive possibility is that this contact-dependent signal could
result in a differential control of virulence gene expres-
sion, suggesting the existence of a hierarchy in the type III
secretion process (Collazo and Galan, 1996; Demers et al.,
Construction of an R.solanacearum strain with constitutive
GFP expression
We used the promoter±probe gfp-based mini-transposon pAG408 located
on a suicide delivery plasmid (Suarez et al., 1997) to generate
chromosomal fusions. Escherichia coli S17-1 (lpir) cells (Suarez et al.,
1
997) transformed with pAG408 were used as donor in mating
experiments to transfer the suicide plasmid into the R.solanacearum
wild-type strain GMI1000. Transconjugants were recovered on minimal
medium plates supplemented with gentamycin. Several colonies that
¯uoresced bright green were examined after growth on both complete and
minimal media, and strain GMI1600 was selected on the basis of its
constitutive high GFP expression. The pathogenicity of strain GMI1600
was investigated by its ability to induce the hypersensitive response on
tobacco leaves and to cause wilting symptoms in tomato plants as
described by Brito et al. (1999). Plant tests showed that, under our
experimental conditions, strain GMI1600 behaved in the same way as the
wild-type strain GMI1000, indicating that the random chromosomal
insertion of the pAG408 mini-transposon did not alter the pathogenicity
of the wild-type strain.
1
998). Our observations are consistent with the hypothesis
that this contact-dependent signal could contribute to
triggering the polarized translocation of some speci®c
type III effector proteins into the eukaryotic cell, even
though this translocation of bacterial effectors has not yet
been formally demonstrated in the case of plant pathogens
(
Galan and Collmer, 1999). As mentioned above, how-
ever, this translocation step is certainly a ®nely tuned
process that also involves additional controls at the post-
transcriptional level.
Strain GMI1601 was generated after transformation of strain GMI1600
by total genomic DNA of strain GMI1575, which carries a prhA::W
mutation (Marenda et al., 1998). The occurrence of a double
recombination event in strain GMI1601 leading to disruption of the
prhA gene was checked by Southern analysis.
In summary, we have identi®ed a new type of non-
diffusible plant signal inducing bacterial virulence gene
expression and we have presented genetic evidence that
prhA encodes the candidate receptor protein of this
contact-dependent signal. Our next goal is to characterize
the nature of the PrhA ligand, which was shown to be a
component of the plant cell wall, in order to demonstrate
that PrhA is the speci®c receptor of this ligand. Since this
hrp-inducing signal was found to be present in a wide
range of plant species, it is plausible that it may induce the
expression of genes encoding type III secretion systems in
the main groups of non-tumorigenic Gram-negative bac-
teria.
Plant cell cultures and co-cultivation procedure
Cell suspensions of A.thaliana At-202 (accession Col 0), tomato
(Lycopersicon esculentum) MSK8, Nicotiana tabacum L. var.
Wisconsin no. 38 and M.truncatula were grown in Gamborg B5 medium
(
1
ICN Biomedicals, Aurora, OH) supplemented with 20 g/l sucrose and
mM a-naphthalene acetic acid at 23°C under continuous illumination
and shaking. Samples of 0.5 g of plant cell suspensions in the exponential
phase were inoculated with R.solanacearum as described previously
(Marenda et al., 1998; Brito et al., 1999) and grown at 28°C on a rotary
shaker.
The term `conditioned medium' refers to culture medium from a
1
-week-old plant cell suspension from which plant cells have been
removed by ®ltration; the medium is then sterilized by ®ltration through
.22 mm Millipore ®lters.
0
Materials and methods
Time course of attachment of bacteria to plant cells
Bacteria were labelled by growth overnight in a phosphate-depleted
3
2
Bacterial strains and conjugation procedure
Bacterial strains used in this study are listed in Table I. Ralstonia
solanacearum strains were grown in B medium or minimal medium
liquid medium supplemented with 100 mCi of P-radiolabelled NaPO
4
(NEN Life Science Products, Boston, MA). Bacteria were washed four or
®ve times to eliminate unincorporated radioactivity before co-culture
with an Arabidopsis cell suspension. Independent 200 ml aliquots taken
from co-cultures were ®ltered through 20 mm pore size nylon membranes
and washed thoroughly three times with sterile culture medium to
eliminate bacteria that were not speci®cally bound to plant cells. The
radioactivity of this washed plant cell fraction was measured by Cerenkov
determination (1 min) on a liquid scintillation analyser (Tri-Carb
2100TR; Packard Instrument Co., Meriden, USA).
(Boucher et al., 1985) supplemented with glutamate (20 mM ®nal
concentration). When needed, antibiotics were added to the media at the
following concentrations (mg/l): kanamycin, 50; spectinomycin, 40;
gentamycin, 25; tetracycline, 10. Plasmids were introduced in
R.solanacearum by triparental mating using the helper strain E.coli
K12 carrying plasmids pRK2013or pRK2073as conjugative helper
plasmids (references and method in Brito et al., 1999).
2
312

Host contact-dependent induction of virulence genes
Cytological analysis of the plant cell±bacterium interactions
Microscopic examination of the plant cell±bacteria co-culture was
performed after the addition of the dye Evans blue at a ®nal concentration
of 0.001% (w/v). Plant cells treated with Evans blue emit a red
regulatory cascade controlled by PrhA in Ralstonia solanacearum.
Mol. Microbiol., 31, 237±251.
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protein has domains similar to harpins and pectate lyases and can elicit
the plant hypersensitive response and bind to pectate. J. Bacteriol.,
180, 5211±5217.
Collazo,C.M. and Galan,J.E. (1996) Requirement for exported proteins
in secretion through the invasion-associated type III system of
Salmonella typhimurium. Infect. Immun., 64, 3524±3531.
Cornelis,G.R. (1998) The Yersinia deadly kiss. J. Bacteriol., 180, 5495±
5504.
Dae¯er,S. (1999) Type III secretion by Salmonella typhimurium does not
require contact with a eukaryotic host. Mol. Microbiol., 31, 45±51.
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¯
(
uorescence when observed in the GFP excitation wavelength range
480 6 30 nm) with a 520 nm long-pass glass emission ®lter. This
procedure decreases the plant cell wall auto¯uorescence and facilitates
the discrimination between GFP-tagged bacteria and plant cells.
Observations were performed using 10 ml of the co-culture mounted on
a glass slide previously coated with poly-L-lysine (Sigma, St Louis, MO).
Observations were made using an epi¯uorescence microscope (Leitz
DMIRBE; Leica, Wetzlar, Germany; Fluotar objective 633, n.a. 0.7) or a
confocal laser scanning microscope (Axiovert; Carl Zeiss, Oberkochen,
Germany) with a Neo¯uar objective (633, n.a. 1.3) equipped with dual
detectors and an argon±helium laser for simultaneous scanning of
¯
uorescence and DIC-like images. Fluorescence images were acquired
using a CCD camera (Colour Coolview; Photonic Science, Robertsbridge,
UK) connected to the Leitz microscope. Images in the same ®gure were
acquired with the same parameters to allow comparisons between the
emitted ¯uorescence level in the different samples. Figures were edited
using Adobe Photoshop 5.0 software.
Preparation of cell wall material from Arabidopsis cell
suspensions
A 1-week-old Arabidopsis cell culture removed from the medium by
®
ltration was placed in 90% cold ethanol to inactivate degradative
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that the hrpB gene encodes a positive regulator of pathogenicity genes
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Gough,C.L., Genin,S., Zischek,C. and Boucher,C.A. (1992) hrp genes of
Pseudomonas solanacearum are homologous to pathogenicity
determinants of animal pathogenic bacteria and are conserved
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novel proteins, PopB, which has functional nuclear localisation
signals, and PopC, which has a large leucine-rich repeat domain, are
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enzymes. Cell walls were isolated according to the methods described by
Selvendran and O'Neill (1987) and Morvan et al. (1991). Brie¯y, cells
were homogenized with an Ultraturrax for 10 min. The resulting
suspension was washed twice with distilled water and insoluble material
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resuspended in 5 vols of chloroform±methanol 1:1 (v/v), washed three
times on the sinter with this mixture and then twice more with acetone.
The resulting material was called `cell wall material'. The ability of this
cell wall material to trigger hrpB±gfp gene expression was tested after
growth of the bacteria for 16 h in minimal medium containing cell wall
material at a ®nal concentration of 1% (w/v). Similar conditions were
used with cell wall material that had been heat treated (15 min at 100°C)
or treated with proteinase K (50 mg/ml for 90 min at 40°C).
H aÈ rle,C., Kim,I., Angerer,A. and Braun,V. (1995) Signal transfer
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We thank A.Jauneau and P.Cochard for expert technical assistance with
microscopy, C.Guzman for providing the plasmid pAG408, S.Charlier
and J.Vasse for their contribution in developing GFP in R.solanacearum,
P.Barberis for technical assistance, and M.Arlat. We are indebted to
M.-C.Roland and C.Gough for critical reading of the manuscript. This
work was funded by projects BIO4-CT-97-2244 from the European
Commission and AIP-188 Microbiologie from the Institut National de la
Recherche Agronomique. B.B. was the recipient of a Marie Curie TMR
postdoctoral grant from the European Commission.
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