UMR 5546 UPS-CNRS, Pôle de Biotechnologie Végétale, 24 Chemin de Borde-Rouge, BP17, Auzeville, F-31326 Castanet-Tolosan, France
* Author for correspondence (e-mail: bottin{at}smcv.ups-tlse.fr)
Accepted 3 September 2002
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Summary |
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Key words: Oomycete, Transformation, Silencing, Branching, Lobed structures, Cellulose, CBD
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Introduction |
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Owing to the potential involvement of the cell wall in many aspects of
Phytophthora biology, a better knowledge of this compartment is
needed. In particular it is a reservoir of elicitors, a class of molecules
whose identification is based on their ability to induce defense and/or
hypersensitive response (HR) necrosis when externally supplied to plant
tissues (Keen et al., 1972).
This definition does not imply an intrinsic function of elicitors for the
microorganism itself. Whether elicitor molecules do fulfil basic functions in
the biology of the microorganisms which produce them is not known yet. Various
molecules endowed with elicitor activity have been characterized in
Phytophthora, notably glucan fragments
(Keen and Yoshikawa, 1983
;
Sharp et al., 1984
;
Waldmüller et al., 1992
),
small polypeptides from the elicitin family
(Ricci et al., 1989
;
Ponchet et al., 1999
),
glycoproteins (Farmer and Helgeson,
1987
; Parker et al.,
1991
; Séjalon-Delmas et
al., 1997
) and fatty acids
(Bostock et al., 1981
). Among
possible roles, one might conceive their involvement in cell wall structure,
adhesion, signal perception, development or nutrition.
Working on Phytophthora parasitica var. nicotianae
(P.p.n.), a root pathogen which is the causal agent of the black
shank disease of tobacco, led us to the purification of a glycoprotein which
elicits HR-like necrosis and defense gene expression in tobacco
(Séjalon-Delmas et al.,
1997). This glycoprotein was localized by immunogold-labelling to
the external and internal layers of the hyphal cell wall. Early deposition
from secretory vesicles was recorded at the onset of cell wall synthesis
during encystement of zoospores. Labelling was associated with the living
microorganisms in vitro and in planta, and declined with mycelium cell death
(Séjalon-Delmas et al.,
1997
). Cloning of the corresponding gene indicated that the
protein portion is composed of two cysteine-rich domains separated by a
threonine/proline-rich linker region
(Villalba Mateos et al.,
1997
). Interestingly, each repeated domain contains one sub-domain
homologous to cellulose binding domains (CBDs) of glucan hydrolases
(Tormo et al., 1996
). CBDs are
well characterized in microbial cellulolytic enzymes, and are supposed to
enhance the efficiency of hydrolysis notably by docking the enzymes to their
substrate (Gilkes et al.,
1991
). Further studies demonstrated that CBEL i) binds crystalline
cellulose without showing enzyme activity on cellulose and various glycans,
and ii) has lectin activity. Accordingly, the elicitor was named CBEL for
Cellulose-Binding Elicitor Lectin
(Villalba Mateos et al.,
1997
). With respect to its cell wall localization, expression
pattern and multiple activities, it was proposed that CBEL is involved in cell
surface properties during the life cycle of P.p.n. In order to check
this hypothesis, it is necessary to generate P.p.n. strains with
altered levels of CBEL production. So far, only a few Phytophthora
(P.) species, comprising P. infestans, P. sojae, P. capsici
and P. palmivora, have been transformed
(Judelson et al., 1991
;
Judelson et al., 1993a
;
Ersek et al., 1994
;
van West et al., 1999b
). We
formerly established that P.p.n. is amenable to transformation with a
reporter gene (Bottin et al.,
1999
). In the present study, this method was used to generate
transgenic strains containing antisense and sense copies of CBEL
cDNA. The obtained transformants were characterized with respect to
CBEL gene expression and to their phenotype during saprophytic growth
and upon inoculation of the tobacco host plant.
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Materials and Methods |
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Phytophthora parasitica var. nicotianae (P.p.n.)
transformation
DNA vectors used for P.p.n. transformation were pTH210
(Judelson et al., 1991), and
plasmid constructs derived from it. In pTH210, an oomycete hygromycin B
resistance cassette has been inserted into the pUC19 vector. This cassette is
composed of the coding sequences from the hygromycin phosphotransferase gene
HPH from Escherichia coli under the control of a
HSP70 gene promoter and a HAM34 gene terminator from the
oomycete Bremia lactucae (Fig.
1A). Plasmids derived from pTH210 were constructed according to
standard procedures (Sambrook et al.,
1989
) using Life Technologies (France) restriction and
modification enzymes. The CBEL cDNA was isolated as a 918 bp
EcoRI-XhoI fragment
(Villalba Mateos et al.,
1997
), and DNA ends were filled in by the Klenow fragment enzyme.
The 1.2 kb HPH gene insert in pTH210 was released by SmaI
digestion, and the remaining vector fragment was ligated with the
EcoRI-XhoI CBEL cDNA blunt fragment. This resulted
in either plasmid pTHEX3 or plasmid pTHEX11, both of 4.7 kb size, where the
CBEL cDNA is inserted in place of the HPH gene in sense or
antisense orientation respectively (Fig.
1A).
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Genetic transformation was carried out as described
(Bottin et al., 1999). Briefly,
P.p.n. protoplasts were prepared by enzymatic digestion of mycelium
and incubated with a mixture of BamHI-linearized pTH210 (15 µg)
and either pTHEX3 or pTHEX11 (15 µg) in the presence of CaCl2
and polyethylene glycol. Regeneration was carried out on V-8 agar medium
containing 70 µg ml-1 hygromycine B.
Molecular characterization of P.p.n. transformants
Genomic DNA and total RNA were extracted from the mycelium according to
Dellaporta et al. and Villalba Mateos et al., respectively
(Dellaporta et al., 1983;
Villalba Mateos et al., 1997
).
Nucleic acid concentrations were measured spectrophotometrically at 260 nm.
For Southern blot analysis, 10 µg of EcoRI-digested genomic DNA
were electrophoresed on a 1% (w/v) agarose gel. After alkaline denaturation,
DNA fragments were transferred onto a positive nylon membrane (Hybond N+,
Amersham, France) and subjected to Southern hybridization
(Sambrook et al., 1989
).
Northern blots were performed with 10 µg of total RNA as described
previously (Rickauer et al.,
1997
). The 918 bp EcoRI-XhoI CBEL cDNA
fragment (Villalba Mateos et al.,
1997
) was labelled with
-[32P]dCTP by random
priming using the RadPrime DNA Labelling System (Life Technologies, France)
and used as the probe in Southern and northern blot analyses. A radiolabelled
PCR fragment corresponding to 18S ribosomal DNA was used as a control of RNA
amount in northern blot analysis. After hybridization and washing, the
membranes were exposed to Hyperfilm MP films (Amersham, France) at
-80°C.
For western blot analysis, protein extracts were prepared following a
procedure adapted from Séjalon-Delmas et al.
(Séjalon-Delmas et al.,
1997). Briefly, 1 g of ground frozen mycelium was suspended in 1.5
ml of acidic ethanol, the extract was clarified by centrifugation and proteins
were precipitated by cold acetone and air-dried. Protein samples (100 µg
dry weight) were analyzed by sodium-dodecylsulfate polyacrylamide gel
electrophoresis (Laemmli,
1970
) on a 12% (w/v) resolving gel. After transfer onto a
nitrocellulose membrane (Protran, Schleicher & Schuell, Germany), proteins
were probed with a polyclonal antiserum directed against CBEL as described
(Séjalon-Delmas et al.,
1997
).
Characterization of P.p.n. transformant phenotypes
For adhesion assay and microscopy analysis of mycelium, a washed,
autoclaved cellophane (Cellophane octaframe, MERCK-Eurolab, France) or
polycarbonate (Polycarbonate cyclopore, MERCK-Eurolab, France) membrane was
put down onto V-8 agar medium and subsequently inoculated on its center with a
mycelium plug. In some experiments, a bed of depectinised flax cellulose
fibres (a gift from C. Morvan, Rouen, France), boiled for 2 hours in 0.1 M
NaOH to remove hemicellulose contaminants, was spread onto the cellophane
sheet prior to inoculation. After incubation for 7 days at 25°C in the
dark, the membrane together with the mycelium was gently removed from the
underneath medium and cut into 8 sectors of similar sizes. These samples were
either directly processed for microscopy observation, or incubated under
gentle horizontal shaking at room temperature in a 50 ml polypropylene tube
containing 30 ml of distilled water in the case of adhesion assay. After 2
hours, the presence of remaining adherent mycelium was checked with bright
field microscopy.
For microscopy analysis of germlings, P.p.n. zoospores were
obtained by starvation of mycelial cultures followed by cold shock
(Gooding and Lucas, 1959),
adjusted to 5x104 cell µl-1 in water, and
vortexed at maximum speed for 1 minute in order to provoke encystment. Fifteen
microliters aliquots were then dropped either directly onto the surface of a
polystyrene Petri dish or onto cellophane sheets placed on top of a 4% (w/v)
water agar. The samples were incubated at 24°C for 16 to 48 hours in the
dark and observed under differential interference contrast microscopy.
Pathogenicity was assessed on tobacco 46-8 and 49-10 isolines, susceptible
and resistant to P.p.n. race 0 respectively. Inoculation assays were
performed on stem sections of 10 week-old 46-8 plants
(Rancé et al., 1998),
and on the root system of 3 week-old 49-10 seedlings. For this assay, 12
surface-sterilized seeds were sowed on water agar (1.2% w/v, pH 6.5) in square
12 cm-Petri dishes set up vertically in order for the roots to grow on the
surface of the medium. After 3 weeks of culture at 25°C with 16 hours
daily illumination at 30 µE/m2s, mycelium plugs taken up from
the periphery of P.p.n. colonies grown on V-8 agar medium were placed
in contact with the roots beneath the collar. The dishes were further
incubated in the same conditions and symptoms were scored during 12 days,
according to the following notation: 0, no symptom; 1, leaf discoloration and
black shank; 2, brown dead seedling with visible mycelium. For microscopy
studies, infected seedlings were observed with an inverted light microscope
either directly in the assay Petri dish, or after removal of the seedling and
staining.
Microscopy techniques
Inoculated seedlings, as well as mycelium grown on cellophane or
polycarbonate membranes, were observed without prior fixation with an inverted
light microscope (Leitz DMIRBE, Leica, Germany). Microscopy views were
obtained with a Color Cool View CCD camera (Photonic Science, UK) linked to an
Image Acquisition Photolite software (Photonic Science, UK). In some cases,
inoculated seedlings were bleached for 24 hours at 30°C in 10% KOH (w/v),
washed in water, stained in rosazurin (3% w/v in water) and mounted in water
for microscopy observation.
For transmission electron microscopy, the samples were fixed for 2 hours at
room temperature in 0.1 M sodium cacodylate buffer (pH 7.2) containing 1.5%
(w/v) glutaraldehyde (Oxford Agar, UK), then washed in the same buffer without
glutaraldehyde. They were dehydrated in a series of aqueous solutions of
increasing ethanol concentration (10, 20, 40, 50, 60, 70, 80, 90, 100% v/v, 20
minutes each). Progressive infiltration with LR White resin (Oxford, UK) was
carried out by serial incubation in ethanolic solutions of increasing LR White
resin concentration (10, 30, 50, 70, 90% v/v, at least 1 hour each), followed
by several incubations in undiluted LR White resin. The infiltrated samples
were then embedded in gelatine capsules and allowed to polymerize for 24 hours
at 60°C. Ultrathin sections (90 nm thickness) were prepared using an
UltraCut E ultramicrotome (Reichert-Leica, Germany) and collected on gold
grids. They were submitted to the periodic acid, thiocarbohydrazide
silver-proteinate reaction (PATAg) according to Thiéry
(Thiéry, 1967).
Sections were floated on a 1% (w/v) periodic acid aqueous solution for 30
minutes and rinsed twice in distilled water for 15 minutes. They were then
maintained overnight at 4°C on a 20% (v/v) acetic acid aqueous solution
containing 2 g l-1 thiocarbohydrazide, washed in solutions of
acetic acid of decreasing concentrations, and finally in pure water. Sections
were then treated with 1% (w/v) silver proteinate in water for 30 minutes in
the dark. Grids were rinsed and air dried before examination with a
transmission electron microscope operating at 75 kV (Hitachi, H-600, Japan)
and photographs were taken on Kodak-Electron films (Kodak, France).
For scanning electron microscopy, the samples were fixed and dehydrated as described above and critical-point dried with CO2 as transitional fluid. The dried samples were sputter-coated with gold-palladium using a Jeol JFC1100 apparatus (JEOL, Japan). Observations were made with a Hitachi C-450 (Japan) scanning electron microscope operating at 15 kV, and photographs were taken on Illford 125 ISO film.
For confocal laser scanning microscopy, samples were stained with Congo Red (1% w/v in water) for 15 minutes, then briefly washed in distilled water. Preparations were viewed with a Leica SP-2 (Germany) microscope equipped with a 40x (1.25 NA) oil immersion objective. Excitation was brought about by the 543 nm emission line of the He-Ne laser, and light emitted between 560 and 630 nm was collected. Pictures were computed by projection of 15 plan-confocal images acquired in z dimension with 0.5 µm increment between two focal planes.
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Results |
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CBEL gene expression in the transformants was assessed by northern blot analyses (Fig. 1Bb). The 1.3 kb hybridising band corresponding to the CBEL transcript was observed with RNA from the untransformed strain and some antisense transformants, but it was undetectable in the EX11-4 antisense transformant. Interestingly, the level of CBEL mRNA was not enhanced in any of the sense transformants, but was reduced in most of them, being undetectable in the EX3-3 transformant.
To determine the effect of CBEL mRNA suppression on the level of the CBEL protein, we conducted western blot analyses of protein extracts prepared from the untransformed and from four transformant strains (Fig. 1Bc). A major band of Mr of 34x103, corresponding to the CBEL glycoprotein, was detected in the untransformed, EX11-1 and EX3-5 strains, whereas the protein was not detected in EX11-4 and EX3-3 strains.
Altogether, northern and western blot analyses indicate that expression of
the CBEL gene, and synthesis of the CBEL protein, were suppressed in
transformants EX11-4 and EX3-3, whereas transformants EX11-1 and EX3-5
exhibited similar levels of CBEL expression as the untransformed
control strain. These two expressing (CBEL+) and the two
non-expressing (CBEL-) transformants were selected for further
analyses. By analogy to suppression of gene expression in sense and antisense
transformants of P. infestans
(van West et al., 1999a), it
can be supposed that the CBEL- strains resulted from
homology-dependent gene silencing. It is worth noting that the transformants
were highly impaired in zoospore production independently of CBEL
gene expression. For this reason, all experiments aimed at comparing
transgenic CBEL+ and CBEL- strains were performed with
mycelium.
CBEL silencing has no effect on mycelium growth
Cultures of the EX11-1 CBEL+ and EX11-4 CBEL- strains
were grown on liquid medium and the mycelium dry weight was recorded as a
function of time. Fig. 2A shows
that the two strains grew equally well under these conditions. As it has been
demonstrated that CBEL has affinity for cellulose
(Villalba Mateos et al.,
1997), the growth of CBEL+ and CBEL- strains
was studied on a cellophane membrane placed on top of an agar medium. Mycelium
growth, assessed by measuring the diameter of the colonies, was not affected
under these conditions (Fig.
2B). The untransformed strain and all transformants colonized the
whole surface of the cellophane sheet after 7 days of culture. Thus,
CBEL silencing did not affect mycelium growth in vitro.
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CBEL silencing reduces attachment to cellulosic
surfaces
It has been shown previously that CBEL is localized at the surface of cysts
and hyphae both in vitro and in planta
(Séjalon-Delmas et al.,
1997), exhibits lectin activity and binds to cellulose and tobacco
root cell walls in vitro (Villaba Mateos
et al., 1997
). To gain insight into the intrinsic role for
P.p.n. of the carbohydrate-binding capacity of CBEL, we investigated
attachment of CBEL+ and CBEL- strains to cellulosic or
polycarbonate (PC) membranes. These strains were grown on membrane disks
placed on top of V-8 agar medium for 7 days; the disks were then removed from
the medium, cut in sectors of similar sizes
(Fig. 3A-E), and the sectors
were incubated with moderate shaking for 2 hours in water
(Fig. 3F-J). Microscopic
observation showed that the mycelium of the untransformed and CBEL+
EX11-1 and EX3-5 strains remained attached to the cellophane membrane after
this treatment (Fig. 3F,G,I).
In contrast, most hyphae of the EX11-4 and EX3-3 CBEL- strains were
detached from the membrane (Fig.
3H,J). None of the five strains remained attached to a PC membrane
under the same conditions (data not shown). These results demonstrate that the
ability of P.p.n. to stick to a cellulosic substrate is highly
dependent on the presence of CBEL.
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CBEL silencing hampers differentiation in contact with
cellulosic surfaces
In addition to a difference in adhesion, we observed that the untransformed
strain and transgenic CBEL-expressing strains EX11-1 and EX3-5
differentiated two types of structures when grown on a cellophane membrane.
One type appeared as small regions of densely aggregated hyphae
(Fig. 3A,B,D,F,G,I, arrows).
The thickness of these mycelial aggregates prevented clear visualisation with
the light transmission microscope (Fig.
4A). The second type appeared in contact with cellophane under the
form of lobed granular structures (Fig.
4A,B). These structures were not in the same focus plane as most
hyphae (Fig. 4B) but seemed to
develop within the cellophane sheet. They were clearly distinguishable from
protoplasm leaking out of damaged hyphae
(Fig. 4C). These
differentiations were substrate-specific since they did not occur when the
strains were grown on a PC membrane (data not shown). The
CBEL-silenced strains EX11-4 and EX3-3 never presented either
mycelial aggregates or lobed structures
(Fig. 4D).
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In order to get insight into the biological significance of the lobed structures, we looked for their occurrence during P.p.n. germling development on various substrates. Since the transgenic strains were impaired in zoospore production, experiments were conducted with the untransformed strain. Encysted zoospores germinated and produced globular appressoria within a few hours on a solid surface (Fig. 5). When the substrate was a cellophane membrane, lobed structures emerged from some appressoria (Fig. 5A). In contrast, only repeated appressorium formation was observed along the germ tube growing on a polystyrene surface (Fig. 5B) or on a glass slide (data not shown). The data indicate that lobed structures develop subsequently to appressorium formation and confirm that their formation is substrate-specific.
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Additional experiments using scanning electron microscopy were undertaken in order to better characterize the interaction between P.p.n. and cellulosic substrates. In addition to cellophane, flax cellulose fibres were used as a more natural cellulose substrate. When grown on either substrate, the EX11-1 CBEL+ strain formed highly intricate and branched hyphae that likely corresponded to the above-mentioned mycelial aggregates (Fig. 6A,C). These aggregates were localised in close vicinity of the cellulose fibres (Fig. 6C). No lobed structures could be detected in these conditions, a result which is in agreement with the hypothesis that they do not develop at the surface of a substrate, but within it. In accordance with light microscopy observations, the EX11-4 CBEL- transformant showed only loosely branched hyphae and no mycelial aggregates on either substrate (Fig. 6B,D).
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Taken together, the data show that CBEL is required for differentiation of lobed structures and hyphal aggregates in contact with cellulosic surfaces.
CBEL silencing results in cell wall thickenings
Because CBEL is localised in the cell wall of P.p.n., we
hypothesized that it might also be involved in the cell wall architecture.
Hyphae from the various strains were stained with Congo Red, a dye with high
affinity for ß-1,4 polysaccharides
(Wood, 1980), and observed
with a confocal laser scanning electron microscope. Strongly stained cell wall
patches were detected along the hyphae of the CBEL- strains grown
either on a cellophane or a polycarbonate membrane, as illustrated with the
EX11-4 strain in Fig. 7B,D. In
contrast, the hyphal walls of CBEL+ strains did not exhibit any
thickening and were labelled homogeneously regardless of the chemical nature
of the substrate (Fig.
7A,C).
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Further investigations were performed on ultra-thin sections of hyphae
which were submitted to the PATAg reaction. This staining reveals the
polysaccharides of the cell wall which contain vicinal hydroxyl groups
(Roland and Vian, 1991). In
transformants expressing CBEL, the hyphae were surrounded by a
PATAg-stained cell wall of regular thickness, as illustrated with the EX11-1
CBEL+ strain (Fig.
7E). However, in the two silenced strains, numerous hyphae
exhibited paramural deposits (Fig.
7F,G), a pattern which was never observed with the untransformed
and CBEL+ transgenic strains. These appositions were stained with
PATAg and likely corresponded to the above-described Congo Red-stained
material. In conclusion, the data indicate that the absence of CBEL results in
abnormal cell wall deposition.
CBEL silencing has no major effect on the
tobacco-P.p.n. race-cultivar specific interactions
The effect of CBEL silencing on pathogenicity was assessed on
susceptible and resistant plants from two near-isogenic tobacco lines using
mycelial explants as inoculum. When CBEL+ or CBEL-
transgenic strains were inoculated onto the stems of plants from the resistant
line, a dark-brown HR-like necrosis was observed and disease symptoms did not
develop further (data not shown), indicating that avirulence on the resistant
tobacco line was retained in the strains silenced for CBEL
expression. When the susceptible line was inoculated with the same strains,
the CBEL-silenced strains were still pathogenic. A root inoculation
assay with the susceptible line confirmed these data; the
CBEL-silenced strains EX11-4 and EX3-3 were either more or less
aggressive than the EX11-1 and EX3-5 CBEL+ control strains in two
independent experiments (Table
1). Light microscopy observation of inoculated roots did not allow
detection of the presence of lobed structures. However, it showed that the
untransformed and EX11-4 CBEL- strains differentiate appressoria
and hyphal aggregates in contact with the root surface
(Fig. 8). Taken together, the
data show that CBEL silencing does not greatly alter the outcome of
the race-cultivar specific interaction between tobacco and P.p.n.
when the source of inoculum is a mycelial explant.
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Discussion |
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Reverse genetics to address CBEL function in Phytophthora
parasitica var. nicotianae
Attempts to inactivate or overexpress genes in Phytophthora have
been limited by the difficulty to transform most species. In addition, unlike
the situation in fungi, gene inactivation in this organism cannot be performed
by a single round of gene disruption because of the diploid nature of
oomycetes and the low frequency of homologous DNA recombination
(Judelson, 1997). The
feasibility to suppress gene expression was first illustrated in P.
infestans where a transgenic strain expressing the ß-glucuronidase
(gus) reporter gene was further transformed with gus
antisense constructs resulting in suppression of gus expression
(Judelson et al., 1993b
). So
far, this technology has been applied only once to inactivate an endogenous
gene: the inf1 gene of P. infestans, which encodes a peptide
elicitor from the elicitin protein family
(Kamoun et al., 1998
;
van West et al., 1999a
).
However, the presence of multiple inf genes in the pathogen did not
allow complete suppression of elicitin production. Since CBEL is encoded by a
single gene (Villalba Mateos et al.,
1997
), it should be easier to significantly suppress its
production. We therefore transformed P.p.n. with an antisense
construct of CBEL. In parallel, we attempted to obtain overexpressing
strains by introducing a CBEL sense construct. Both strategies only
allowed recovery of strains in which CBEL expression was suppressed.
This is in accordance with data obtained in P. infestans, where
silencing resulted from both sense and antisense constructs
(van West et al., 1999a
).
At first sight, the phenotype of the transformants was normal when they
were grown on standard media. Further analysis showed however that the
transformants, expressing CBEL or not, were impaired in zoospore
production. A similar sporulation defect was also reported by Ersek et al. in
P. capsici transformants containing the pTH210 plasmid used in this
study (Ersek et al., 1994).
Our unpublished data, obtained with P.p.n. strains resistant to
either hygromycin or G418 antibiotic, indicate that suppression of sporulation
is due to conjugated effects of the presence of the antibiotic in culture
medium and of the antibiotic resistance cassette in the genome of the
transformants (E.G. and A.B., unpublished).
CBEL is necessary for adhesion and differentiation in contact with
cellulosic surfaces
The presence of two cellulose binding domains in CBEL and the ability of
the protein to bind to cellulose in vitro suggested that CBEL participates in
adhesion of P.p.n. to cellulosic substrates. Adhesion to solid
surfaces is a common feature of both saprophytic and parasitic microorganisms.
In fungi and oomycetes, it is mediated by secreted adhesins that are part of
the cell wall or physically associated with it
(Nicholson, 1996;
Carzaniga et al., 2001
;
Tucker and Talbot, 2001
).
Since the first report over 20 years ago
(Sing and Bartnicki-Garcia,
1975a
; Sing and
Bartnicki-Garcia, 1975b
), various proteins and glycoproteins have
been putatively associated with adhesion in Phytophthora, notably
during zoospore encystment and cyst germination. Among them, the Vsv1 protein
of P. cinnamomi (Hardham and
Mitchell, 1998
), and mucin-like proteins from cyst germination
fluids of P. infestans (Gornhardt
et al., 2000
) have been best characterized. The availability of
P.p.n. strains suppressed in CBEL expression provides here
the first genetic evidence that a cell wall glycoprotein of
Phytophthora is required for adhesion to a cellulosic substrate in
vitro.
In addition to their ability to attach to a cellophane membrane, the
untransformed and transgenic CBEL+ strains differentiated mycelial
aggregates and flattened lobed structures when grown on this substrate. The
aggregates were composed of intricate and branched hyphae best visualised by
scanning electron microscopy. When flax cellulose fibres were used as a
substrate, aggregates developed in close vicinity of the fibres, suggesting
that they are produced in response to the contact of exogenous cellulose. The
formation of lobed structures was clearly distinguishable from the formation
of appressoria, from which they emerged in the untransformed P.p.n.
strain. We assume that the lobed structures grew within the cellophane
membrane because light microscopy observations indicated that they were
localized beneath the focus plane of appressoria and of most hyphae; in
addition, they were not detected by scanning electron microscopy, which
visualises only aerial or surface structures. Interestingly, similar lobed
structures, referred to as `fronds', were described for dermatophytic fungi
growing within keratin or cellophane sheets
(English, 1965). It was
proposed that they represent an adaptation to the layered structure and
physical resistance of these substrates. The formation of polymorphic
multilobed hyphae was also observed in P.p.n.-infected tobacco roots
when the parasite was growing intramurally
(Benhamou and Côté,
1992
). Since the plant cell wall represents a multilayered and
physically resistant environment, it is hypothesized that the lobed structures
that we observed in vitro are related to the morphological changes that occur
in planta when the parasite colonizes the host cell wall. Lobed structures
were not convincingly identified when infected tobacco rootlets were observed
with a light microscope, possibly as a result of difficulties in vizualising
internal polymorphic flattened structures whose shape might depend on root
tissue topography which is much more complex than a cellophane sheet.
Neither lobed structures nor mycelial aggregates could be observed in CBEL-silenced strains grown in vitro on a cellophane membrane or on flax cellulose fibres, indicating that CBEL is necessary for the morphological differentiation that occurs in response to these substrates. As lobed structures and mycelial aggregates were shown to be physically associated with cellophane or flax cellulose fibres respectively, they might be directly involved in attachment of P.p.n. to cellulosic substrates.
Adhesion and differentiation are important processes for fungal and
oomycete pathogenicity. In a number of systems, it has been reported that
hyphal branching, aggregate formation, and appressorium differentiation are
induced in response to the presence of host plants (Garrett, 1970;
Kolattukudy et al., 1995;
Nicholson, 1996
;
Morris et al., 1998
;
Hardham and Mitchell, 1998
;
Buée et al., 2000
).
Although CBEL-silenced strains are impaired in their ability to
respond to cellulose in vitro, the EX11-4 CBEL- strain was still
able to form hyphal aggregates in vivo, in contact with the host plant. This
indicates that, besides cellulose, other plant components are able to induce
mycelium differentiation, and that CBEL is not directly involved in the hyphal
aggregate morphogenesis per se. It is worth mentioning that cellulose is
embedded in the cell wall of the root epidermis, and not exposed to the
rhizosphere. Interestingly, a similar difference between in vitro and in vivo
behaviour has been recently reported in Magnaporthe grisea, where
null mutants of a putative chitin-binding protein failed to differentiate
appressoria normally on an artificial surface, but succeeded in
differentiating them on the plant leaf surface
(Kamakura et al., 2002
).
CBEL is required for proper deposition of cell wall polymers
Knowledge about identity and role of cell wall proteins in oomycetes is
still very limited, and no structural protein has been identified to date.
CBEL-defective strains, though growing normally on standard media, formed
frequent thickenings on the inner side of the cell wall. This suggests that
CBEL is involved in the proper deposition of cell wall polymers, a property
that might be related to the presence of cellulose-binding domains in the
protein. In contrast to the cell wall of fungi, the oomycete cell wall is
non-chitinous in nature but contains important amounts of cellulose that may
participate in its scaffolding
(Bartnicki-Garcia and Wang,
1983). CBEL, which has no enzyme activity but binds cellulose
probably via its two CBDs, might be able to crosslink two cellulose chains and
thus be involved in the organisation of the cell wall network. Such a role has
also been suggested for a gametophytic cell wall protein of the red alga
Porphyrea purpurea that contains four CBDs of the fungal type
(Liu et al., 1996
).
Alternatively, CBEL might serve as a shuttle protein as has been suggested for
AGPs (Arabino-Galactan-Protein) in plants
(Gibeaut and Carpita, 1991
).
It is interesting to note that AGPs and CBEL share common features, notably
their cell wall localization, the presence of hydroxyproline in their protein
moiety, and the fact that their suppression (in the case of CBEL;
this study) or inhibition [in the case of AGPs
(Lord et al., 2000
)] result in
abnormal wall appositions. The reactivity to Congo Red and PATAg stainings of
the cell wall thickenings in CBEL- strains suggests the presence of
amorphous ß-1,4-glucans such as non-crystalline cellulose. These
thickenings are reminiscent of cellulosic cell wall appositions formed by
P. parasitica in response to parasitism by Pythium
oligandrum (Picard et al.,
2000
), and of papillae formed in plant cells in response to
pathogen attack (Benhamou,
1995
). Thus, absence of CBEL could either lead to deregulation of
cellulose synthesis or packaging in the cell wall, or it might locally alter
the cell wall in a way that mimicks pathogen attack and induces
P.p.n. to respond by the production of papillae. In the oomycete
Saprolegnia ferax, it has been reported that drug-induced
disorganization of the cytoskeleton results in patterns of abnormal cell wall
deposition (Bachewich and Heath,
1998
) which resemble the cell wall thickenings of CBEL-
strains. With regard to these observations, it will be of interest to compare
the organization of the cytoskeleton in the P.p.n. CBEL+
and CBEL- transgenic strains.
Biological significance of CBEL
Although CBEL was initially isolated and characterized in race 0 from
P. parasitica var. nicotianae, Southern and western blot
analyses indicated that CBEL homologues are also present in race 1 of
P.p.n., in a tomato isolate of P. parasitica, in strains of
P. citricola and P. sojae and in Pythium irregulare
(F.V. and M.R., unpublished). In addition to these species, homologues of CBEL
have been detected in Expressed Sequence Tag libraries of P.
infestans and P. sojae (available at
https://xgi.ncgr.org/pgc).
This distribution is consistent with a general role of CBEL in the biology of
Phytophthora and related Pythiaceae.
According to its properties and localization in the innermost and outermost
layers of the cell wall, CBEL might fulfil several functions. One of them
relates to its elicitor activity. A few microbial elicitors have been shown to
participate in virulence on susceptible hosts, or to avirulence on resistant
plants (Rohe et al., 1995;
Kamoun et al., 1998
;
Laugé and De Wit, 1998
;
White et al., 2000
). The data
reported in this work indicate that CBEL is not a primary determinant of the
tobacco-P.p.n. racecultivar specific interactions, since
CBEL- strains remained virulent and avirulent on susceptible and
resistant cultivars respectively. This is in accordance with unpublished
results showing that infiltration of purified CBEL induces defence reactions
in the two tobacco isolines (F.V. and M.R., unpublished). The question as to
whether CBEL acts as an elicitor upon colonization of the host plant cannot be
readily answered, mainly because CBEL suppression may have adverse effects on
pathogenicity. Thus, lowering the elicitor effect might increase
pathogenicity, whereas abnormal cell wall appositions might weaken the
mycelium, hence the agressiveness of CBEL- strains. Opposite
effects on elicitation and agressivity might then account for apparent
unchanged pathogenicity.
Specific assay conditions and plant species or cultivars are often required
in order to detect modifications in plant-parasite interactions, particularly
when multiple virulence or avirulence factors are involved in the interaction
(Kamoun et al., 1998;
Isshiki et al., 2001
). For
example, the Ecp2 gene of Cladosporium fulvum was first
claimed not to be essential for pathogenicity on tomato
(Marmeisse et al., 1994
),
whereas improvement of the infection assay in order to better mimick natural
conditions allowed to demonstrate subsequently that it was a virulence gene
(Laugé et al., 1997
).
Since the P.p.n. transformants used in this study do not release
zoospores, the production of standardized inocula for fine quantitation of
virulence is difficult, and the natural infection mode cannot be mimicked.
Another major characteristic of CBEL relates to adhesion to cellulose and
associated differentiation of CBEL+ strains as compared to
CBEL-suppressed strains. This suggests that CBEL, which contains two
cellulose-binding domains, acts as a sensor of exogenous cellulose either in
the soil during the saprophytic life of P.p.n., and/or in the host
plant after penetration has occurred. Besides the presence of two CBDs, a
striking feature of CBEL is its high cysteine content, a structural property
it shares with proteins of the hydrophobin family identified in filamentous
fungi (Templeton et al.,
1994). The fact that hydrophobins are cell wall proteins involved
in development, attachment, and cell wall structure
(Wösten, 2001
) extends
the parallel between CBEL and hydrophobins to functional properties.
In conclusion, the finding that cell surface properties are altered in
CBEL- strains and that abnormal cell wall appositions occur in
these strains, illustrates the concept that elicitor molecules have other
functions than those related to their effect when externally supplied to host
plants. Microbial cell walls are the subject of increasing interest as
potential targets in the search for new antimicrobial compounds
(Goldman and Branstrom, 1999).
CBEL is the first cell wall protein of an oomycete microorganism which has
been molecularly characterized. The demonstration that it is involved in the
proper deposition of cell wall polymers represents a major step towards the
understanding of cell wall biogenesis in this organism.
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