Plant Physiol. (1998) 117: 809-820
Induction of Defense-Related Responses in Cf9 Tomato Cells by the
AVR9 Elicitor Peptide of Cladosporium fulvum Is
Developmentally Regulated1
Guy Honée,
Julia Buitink2,
Thorsten Jabs3,
José De Kloe,
Fred Sijbolts,
Marion Apotheker,
Rob Weide,
Titia Sijen4,
Maarten Stuiver, and
Pierre
J.G.M. De Wit*
Department of Phytopathology, Wageningen Agricultural University,
Binnenhaven 9, 6709 PD Wageningen, The Netherlands (G.H., J.B.,
T.J., R.W., T.S., P.J.G.M.D.W.); and Mogen International N.V.,
Einsteinweg 71, 2333 CB Leiden, The Netherlands (J.D.K., F.S.,
M.A., M.S.)
 |
ABSTRACT |
The
AVR9 elicitor from the fungal pathogen Cladosporium
fulvum induces defense-related responses, including cell death,
specifically in tomato (Lycopersicon esculentum Mill.)
plants that carry the Cf-9 resistance gene. To study
biochemical mechanisms of resistance in detail, suspension cultures of
tomato cells that carry the Cf-9 resistance gene were
initiated. Treatment of cells with various elicitors, except AVR9,
induced an oxidative burst, ion fluxes, and expression of
defense-related genes. Agrobacterium
tumefaciens-mediated transformation of Cf9 tomato leaf discs
with Avr9-containing constructs resulted efficiently in
transgenic callus formation. Although transgenic callus tissue showed
normal regeneration capacity, transgenic plants expressing both the
Cf-9 and the Avr9 genes were never
obtained. Transgenic F1 seedlings that were generated from
crosses between tomato plants expressing the Avr9 gene
and wild-type Cf9 plants died within a few weeks. However, callus cultures that were initiated on cotyledons from these seedlings could
be maintained for at least 3 months and developed similarly to callus
cultures that contained only the Cf-9 or the
Avr9 gene. It is concluded, therefore, that induction of
defense responses in Cf9 tomato cells by the AVR9 elicitor is
developmentally regulated and is absent in callus tissue and
cell-suspension cultures, which consists of undifferentiated cells.
These results are significant for the use of suspension-cultured cells
to investigate signal transduction cascades.
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INTRODUCTION |
Plants that defend themselves against fungal pathogen attack
activate several defense responses, including production of
phytoalexins, accumulation of antimicrobial proteins, and reinforcement
of cell walls (Dixon et al., 1994
). Although these responses are
frequently associated with localized collapse of infected tissue,
termed the HR, evidence for a causal link between the HR and resistance is presently lacking (Dangl et al., 1996). Activation of these defense
responses follows fungal pathogen recognition, which involves elicitor
perception and subsequent signal transduction. Most elicitor molecules
originate from the invading pathogens, but chemical plant stimuli
released during infection can also elicit defense responses (Boller,
1995; De Wit, 1995). Pathogen-derived elicitor molecules can be race
specific and produced by the pathogen in planta, or the molecules can
be nonspecific and enzymatically released from the surface of the
pathogen during the infection process (De Wit, 1995). These various
types of elicitor molecules induce biochemical changes as part of the
resistance response. Electrolyte leakage, oxidative burst, production
of phytoalexins and PR proteins, and increased biosynthesis of ethylene
have been described in leaf tissue treated with nonspecific elicitors
(Hahlbrock et al., 1986; Peever and Higgins, 1989) and with specific
elicitors (Yu et al., 1995; Hammond-Kosack et al., 1996; May et al.,
1996; Rustérucci et al., 1996; Wubben et al., 1996).
Many biochemical and physiological aspects of the
defense response can be studied in suspension-cultured plant cells.
Treatment of parsley cell suspensions with an oligopeptide elicitor
from Phytophthora sojae leads to changes in membrane ion
permeability, generation of active oxygen species, production of
phytoalexins, and activation of defense-related genes (Nürnberger
et al., 1994). Glucan elicitors from the same pathogen induce ion
fluxes and phytoalexin production in suspension-cultured soybean cells
(Ebel et al., 1994). Treatment of suspension-cultured tobacco cells with elicitins from P. sojae leads to ion fluxes and
oxidative burst, together with changes in protein phosphorylation and
production of ethylene and phytoalexins (Yu, 1995; Rustérucci et
al., 1996). These responses have also been described for
suspension-cultured tomato (Lycopersicon esculentum Mill.)
cells treated with elicitor preparations derived from different
microorganisms such as yeast, Pseudomonas syringae, and
Cladosporium fulvum (Felix et al., 1991, 1993; Vera-Estrella
et al., 1992, 1994; Chandra et al., 1996).
Tomato plants that are challenged with the fungal pathogen C. fulvum are resistant to infection when carrying a resistance gene
that matches an avirulence gene of the intruding pathogen (De Wit,
1995). Two avirulence genes, Avr4 and Avr9, have
been cloned and the encoded elicitor peptides have been isolated and characterized (Van Kan et al., 1991; Joosten et al., 1994). As soon as
the fungus penetrates the leaf, both avirulence genes are expressed to
high levels and the encoded elicitor molecules, which are secreted as
pre-proteins, are proteolytically processed in the apoplast into
Cys-rich, mature elicitor peptides (Van den Ackerveken et al., 1993;
Joosten et al., 1997). Recently, the three-dimensional structure of the
AVR9 elicitor peptide was elucidated by 1H-NMR
(Vervoort et al., 1997). The AVR9 peptide consists of three antiparallel 
-strands interconnected by three disulfide bonds. The
AVR9 peptide, which is folded as a cystine knot protein, shows structural homology to carboxy peptidase inhibitor (Vervoort et al.,
1997).
The mechanisms of AVR9 perception and subsequent intracellular signal
transduction events are not well understood. The Cf-9 resistance gene encodes a putative membrane-anchored glycoprotein with
a large extracellular loop consisting of 28 imperfect Leu-rich repeats
(Jones et al., 1994). Based on the structural features of the CF9
protein, it has been speculated that it functions as a receptor for the
AVR9 elicitor peptide. However, a high-affinity binding site for AVR9
was found to be present on the plasma membranes of tomato and other
solanaceous plants, irrespective of the presence of the Cf-9
gene (Kooman-Gersmann et al., 1996). Injection of the AVR9 elicitor
peptide into the leaves of Cf9 tomato plants induces an oxidative
burst; electrolyte leakage; production of ethylene, salicylic acid, and
PR proteins; and a hypersensitive cell death at the injected area
(Scholtens-Toma and De Wit, 1988; Hammond-Kosack et al., 1996; May et
al., 1996; Wubben et al., 1996). Leaves of tomato plants that do not
carry the Cf-9 gene do not respond upon injection of AVR9
elicitor. Seedlings obtained from crosses between wild-type Cf9 tomato
plants and transgenic Cf0 tomato plants that express the
Avr9 gene showed delayed growth, necrosis, and eventually
complete plant death (Hammond-Kosack et al., 1994; Honée et al.,
1995).
To study the biochemical mechanism of AVR9-induced plant defense in
detail we initiated suspension cultures of tomato cells carrying the
Cf-9 gene. These suspension-cultured cells responded upon
addition of various nonspecific elicitors. However, treatment of
cell-suspension cultures with AVR9 elicitor did not induce ion fluxes,
oxidative burst, or activation of PR-protein genes. Using transgenic
Cf9 tomato cells expressing the Avr9 gene, we show that
undifferentiated Cf9 tomato cells, like callus tissue and
cell-suspension cultures, are insensitive to AVR9 elicitor protein.
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MATERIALS AND METHODS |
Elicitor Preparations
The mature AVR9 elicitor peptide of 28 amino acids was isolated
from apoplastic fluid, which was obtained from leaves of the tomato
(Lycopersicon esculentum Mill.) genotype MM-Cf5 infected by race 5 of Cladosporium fulvum, according to the
protocol described by Van den Ackerveken et al. (1993). The
concentration of the AVR9 peptide was determined by low-pH PAGE
(Reisfeld et al., 1962) using a well-defined AVR9 batch as a reference.
The Pmg elicitor was kindly provided by Dr. D. Scheel (Institute of
Plant Biochemistry, Halle, Germany). Xylanase from Trichoderma
viride, chitosan, and chitopentaose were kindly provided by Dr.
T. Boller (Friedrich Miescher Institute, Basel, Switzerland).
Fusicoccin from Fusicoccum amygdali was purchased from
Sigma.
Avr9-Containing Constructs for Plant Transformation
A 440-bp CaMV 35S promoter fragment was amplified by PCR using
construct pMOG410 (Hood et al., 1993) as the template and the two
oligonucleotides 5
-CTTGGATCCCTGCAGGTCAAC-3 and
5-CTGGAATTCACGTGTCCTCTCCAAATG-3 as forward and reverse primers,
respectively. The reverse primer in combination with the
oligonucleotide 5-TTGACTGGATCCAAAATCTAAC-3 as the forward primer and
construct pETVgus27-1 as the template (Martini et al., 1993) were used
for amplification by PCR of a gst1 promoter fragment.
This gst1 promoter fragment consists of 340-bp
gst1 promoter sequences from 
402 to 130 fused to CaMV 35S promoter sequences from 46 to +8 encompassing the TATA box. The
oligonucleotide primers were constructed in such a way that the
amplified CaMV 35S and gst1 promoter fragments contained a BamHI site at the 5 terminus and an EcoRI site
at the 3 terminus to enable cloning in pBluescript and to generate
pFM1 and pFM2, respectively. Except for the EcoRI sites, the
3 end of the cloned promoter fragments also contained a
Pml1 site. After restriction of pFM1 and pFM2 with
Pml1 and EcoRI, a
SnaB1-EcoRI fragment containing the synthetic
tobacco mosaic virus-untranslated omega-leader sequence (Gallie et al.,
1987)
5-TACGTATTTTTACAACAATTACCAACAACAACAAACAACAAACAACATTACAATTACTATTTACAATTACCATGGTGAATTC-3 was inserted downstream of the CaMV 35S and gst1 promoter
fragments, generating constructs pFM4 and pFM5, respectively. In
constructs pFM4 and pFM5, a NcoI site is present immediately
upstream of the EcoRI site.
Two oligonucleotide primers, 5-CCAGGTACCATCCATGGGATTTGTTC-3 and
5-CGATAAAAGAGCTCAATGTACACATTGG-3, and construct potato virus
X:Avr9 (Hammond-Kosack et al., 1995) as the template, were used to
amplify by PCR a fragment encoding the PR1a signal sequence fused to
the mature AVR9 elicitor peptide of 28 amino acid residues. These
primers also generated a PR1a-Avr9(R8K) fragment by PCR amplification
using potato virus X:Avr9(R8K) (Kooman-Gersmann et al., 1997) as the
template. Both amplified fragments contain KpnI and
NcoI sites at the 5 terminus and a SacI site at
the 3 terminus. Using the KpnI and SacI sites,
the fragments were cloned in pBluescript-derived vectors, which placed
the PR1a-Avr9 and PR1a-Avr9(R8K) fragments upstream of the TPI-II
terminator sequences (An et al., 1989), thereby generating constructs
pFM8 and pFM9, respectively. In pFM8 and pFM9 EcoRI sites
are present downstream of the TPI-II fragment. The
NcoI-EcoRI fragment of pFM8 containing the
PR1a-Avr9 coding sequences fused to the TPI-II terminator sequences was
cloned in NcoI and EcoRI linearized pFM4 and pFM5
generating constructs pFM10 and pFM12, respectively. The
NcoI-EcoRI fragment of pFM9 containing the
PR1a-Avr9(R8K) coding sequences fused to the TPI-II terminator
sequences was also cloned in NcoI- and
EcoRI-linearized pFM4, generating construct pFM11.
Subsequently, the Avr9 expression cassettes of constructs pFM10, pFM11, and pFM12 were as BamHI-EcoRI
fragments cloned in the binary vector pMOG800, which differs from
pMOG402 (Jongedijk et al., 1995) by an extra KpnI site in
the multiple cloning site, revealing pMOG978, pMOG979, and pMOG980.
The full-length Cf-9 coding sequence was amplified by PCR
using Cf-9 cDNA as the template (kindly provided by
J. Jones, Norwich, UK; Jones et al., 1994) and the forward
oligonucleotide primer 5 TGCTCTAGAGCATGCCATGGATTGTGTAAAACTTG-3
and the reverse oligonucleotide primer
5-AGACTGCAGCTAATATCTTTTCTTGTG-3. These oligonucleotide primers
were constructed in such a way that the amplified fragment at the
5 terminus contained the XbaI and NcoI sites and
at the 3 terminus a PstI site. The XbaI and
PstI sites were used for cloning of the Cf-9
fragment in a pBluescript-derived vector, which placed the
Cf-9 coding sequence upstream of TPI-II terminator sequences, resulting in construct pCf9.4. Construct pCf9.4 contained a
BamHI site downstream of the TPI-II sequences. Subsequently, the NcoI-XbaI fragment of construct pFM4
encompassing gst1:
sequences was placed in front of the
Cf-9 coding sequence in construct pCf9.4, which revealed
construct pCf9.5. The Cf-9 expression cassette of construct
pCf9.5 was cloned as a BamHI fragment in
BamHI-linearized pMOG979, which resulted in construct
pMOG1043.
Tissue Culture
Tomato seeds of lines Sonato and Sonatine, harboring the
C. fulvum resistance genes Cf-2 and
Cf-4, and Cf-2, C-f4, and
Cf-9, respectively, were surface sterilized for 20 min in 2% (w/v) NaOCl and subsequently were allowed to germinate on synthetic Murashige-Skoog medium (Murashige and Skoog, 1962) supplemented with
1% (w/v) agar and 2% (w/v) Suc at 25°C. After 10 d, explants of cotyledons were transferred to synthetic medium containing Murashige-Skoog salts and B5 vitamins (Gamborg et
al., 1968) supplemented with 1 mg/L 2,4-D, 0.1 mg/L
6-furfurylaminopurine (kinetin), 3% (w/v) Suc, and 1% (w/v) agar,
pH 5.7, and incubated in the dark at 25°C to induce callus formation.
Callus tissue was transferred onto fresh medium every 4 weeks. After 4 to 6 months, friable calli were transferred into liquid Murashige-Skoog
medium (as above) and incubated under continuous shaking (120 rpm, at
25°C in the dark). Every 7 d, cells growing in the log phase
were transferred into fresh medium. Suspension-cultured cells used for
all experiments were 5 to 6 d old. Cell viability was determined
by the use of fluorescein diacetate according to the method of Widholm
(1972).
For transformation of MM genotypes Cf0 and Cf9 constructs pMOG978,
pMOG979, and pMOG980 were transferred to Agrobacterium tumefaciens strain EHA105 (Hood et al., 1993). Plant
transformation was performed as described by Van Roekel et al. (1993).
Primary transformant MM-Cf0 978-16 was selfed and crossed with
wild-type MM-Cf9. Seedlings were grown on synthetic Murashige-Skoog
medium containing 20% (w/v) Suc and 1% (w/v) agar, at 25°C and with
16 h of light. One cotyledon from each seedling was cut off and
used to induce callus (at 25°C in the dark) on modified R3B medium (Meredith, 1979; Koornneef et al., 1987), which is a Murashige-Skoog medium containing 2 mg/L 1-naphthaleneacetic acid, 1 mg/L
6-benzylaminopurine, and 3% (w/v) Suc.
GUS Assay
Histochemical localization of GUS activity in transgenic callus
was performed with
5-bromo-4-chloro-3-indolyl--D-glucuronide as described
by Jefferson (1987). Upon vacuum infiltration of explants with a
solution consisting of 100 mM sodium phosphate and 0.5 mg/mL 5-bromo-4-chloro-3-indolyl--D-glucuronide, the enzymatic reaction was allowed to proceed for 16 h at 37°C.
Finally, leaf explants were cleared of pigment by incubation in ethanol to make blue color more apparent.
Binding Assays
Microsomal membrane fractions were prepared from 6-d-old Sonato
and Sonatine cell-suspension cultures following the protocol for the
isolation of microsomal membranes from leaf tissue described by
Kooman-Gersmann et al. (1996). Protein concentration was determined by
the method of Bradford (1976) with BSA as a standard. Binding assays
with 125I-AVR9 were performed according to the protocol
described by Kooman-Gersmann et al. (1996).
Ion Flux Measurements
Changes in the concentration of extracellular
H+ were determined using a micro-pH electrode
(Radiometer, Copenhagen, Denmark). Changes of the extracellular pH
induced by chitopentaose, xylanase, AVR9, or fusicoccin were registered
for 4 h in 5-mL suspension aliquots, which were incubated on a
rotary shaker at 120 rpm (Felix et al., 1993). Alternatively, changes
of the extracellular pH induced by Pmg elicitor, AVR9, or fusicoccin
were followed according to the method of Conrath et al. (1991). An
assay suspension was prepared that contained 5- to 6-d-grown
cells washed with fresh medium and subsequently resuspended to a cell
density of 2 to 6 × 105 cells/mL in 1 mM Mes buffer containing 4% of the liquid growth medium
and 3% (w/v) Suc, pH 5.7 adjusted with Bis-Tris. The assay suspension
was preincubated for 2 h and subsequently elicitor treated.
Changes of the extracellular pH AVR9 or fussicoccin were also measured
according to the protocol of Vera-Estrella et al. (1994). Cells were
resuspended in 0.3 µM Tris/Mes buffer with 3% (w/v) Suc,
pH 6.5, to a cell density of 2 to 6 × 105
cells/mL and preincubated on a rotary shaker at 120 rpm for 2 h.
Changes of the extracellular H+ concentration
were recorded for 4 h after addition of AVR9 or fusicoccin.
Uptake of [45Ca]2+ into
tomato cells was monitored according to the method of Conrath et al.
(1991). Tomato cells were preincubated for 2 h in an assay
suspension as described above. Subsequently, elicitor-induced
[45Ca]2+ uptake was
measured for 2 h.
Determination of Active Oxygen Species
Generation of H2O2 was
monitored in a fluorescence transition assay (Apostol et al., 1989). In
an assay suspension, suspension-cultured tomato cells were preincubated
for 3 to 4 h under continuous shaking at 120 rpm. Subsequently, a
2-mL aliquot of the assay suspension was transferred to a 3-mL quartz
cuvette and 4 µL of 1 mg/mL pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt; Molecular Probes, Leiden, The Netherlands) was added. To prevent sedimentation of
cells, the cells were continuously stirred at a slow speed without
mechanically disrupting or eliciting them. Subsequently, elicitor was
added and the production of
H2O2 was followed up to
6 h after elicitation by monitoring the quenching of fluorescence of pyranine (emission 512 nm and excitation 405 nm).
For measurement of O2
generation Cyt c reduction was followed according to the
method described by Doke (1985), using 50-µL suspension-cultured
cells in a final volume of 1 mL.
Oxygen consumption was monitored on 2.5-mL aliquots of
suspension-cultured cells using a Clark O2
electrode (Rank Bros., Bottisham, Cambridge, UK) and recorded
continuously for 30 min.
RNA Isolation and RNA Gel-Blot Analysis
Suspension-cultured tomato cells were collected by filtration and
subsequently frozen in liquid N2. Total RNA was
isolated according to the protocol described by Verwoerd et al. (1989). Thirty micrograms of total RNA was separated on a 1.5% agarose gel
containing formaldehyde and subsequently transferred to a Hybond
N+ membrane (Amersham) as described by Maniatis
et al. (1982). The blot was hybridized with cDNA probes labeled with
-32P using the Ready.To.Go labeling kit from
Pharmacia, according to the manufacturer's instructions. The blot was
washed at 65°C in 2× SSC.
| |
RESULTS |
Injection of the AVR9 elicitor peptide in leaves of the tomato
line Sonatine, which carries the resistance genes Cf-2,
Cf-4, and Cf-9, results in a typical HR at the
injected area. Leaves of line Sonato, which carries the resistance
genes Cf-2 and Cf-4, do not respond upon AVR9
injection, indicating that this HR is highly specific. In leaves of
Sonatine plants, necrosis can be induced by AVR9 at a concentration of
1 µg/µL. To study defense-related responses that were specifically
induced by the AVR9 elicitor, Sonato and Sonatine cell-suspension
cultures were established.
Elicitation of Suspension-Cultured Tomato Cells
Addition of AVR9 elicitor as high as 10 µg/µL to
suspension-cultured Sonatine cells that contain the Cf-9
gene and to Sonato cells without the Cf-9 gene did not
induce increase or decrease of the extracellular pH (Fig.
1, A and B). Treatment of
suspension-cultured Sonato and Sonatine cells with the nonspecific elicitor preparation Pmg (Parker et al., 1991) at a final concentration of 50 µg/mL raised the extracellular pH (Fig. 1, A and B).
Suspension-cultured Sonato and Sonatine cells responded identically to
Pmg-elicitor treatment. The time course of Pmg-elicitor-induced
extracellular alkalization was similar, as reported for
suspension-cultured parsley cells (Nurnberger et al., 1994).
Alkalization of the extracellular medium of Sonato and Sonatine cell
cultures was also observed after addition of the nonspecific elicitors
chitopentaose, at a final concentration of 10 nM, and
xylanase, at a concentration of 10 µg/mL (results not shown). Both
elicitors have been reported to induce an increase of the pH in tomato
MsK8 cell-suspension medium (Felix et al., 1993). Suspension-cultured
Sonato and Sonatine cells responded also upon the addition of the
phytotoxin fusicoccin. Fusicoccin stimulates
H+-ATPase activity, thereby inducing
extracellular acidification (Marrè et al., 1993). At a final
concentration of 2 µM, fusicoccin induced extracellular
acidification in both Sonato and Sonatine cell cultures (results not
shown). In addition to H+ fluxes,
elicitor-induced changes in the Ca2+ permeability
of plasma membranes of the suspension-cultured cells were analyzed.
Treatment of suspension-cultured Sonato and Sonatine cells with Pmg
elicitor resulted in a Ca2+ influx (Fig. 1, C and
D), whereas addition of AVR9 elicitor up to 10 µg/mL did not
stimulate Ca2+ uptake (Fig. 1, C and D).
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| Figure 1.
Time courses of elicitor-stimulated
H+ and Ca2+ fluxes across the plasma membrane
and H2O2 production in suspension-cultured tomato cells. Extracellular alkalization of Sonato (A) and Sonatine (B)
cells, Ca2+ influx of Sonato (C) and Sonatine cells (D),
and H2O2 production of Sonatine cells (E) are
shown.  , untreated;  , AVR9 elicitor (10 µg/mL); and  , Pmg
elicitor (50 µg/mL). FW, Fresh weight.
|
|
The oxidative burst, measured by the release of
H2O2, was investigated by
monitoring decreasing fluorescence of pyranin due to oxidation after
challenging suspension-cultured Sonatine and Sonato cells with
different elicitor preparations. Addition of the Pmg elicitor to
Sonatine cells induced the formation of
H2O2 (Fig. 1E). The same
response was recorded for Sonato cells treated with Pmg elicitor
(results not shown). Treatment of suspension-cultured Sonatine cells
with AVR9 elicitor up to 10 µg/mL did not induce H2O2 production (Fig. 1E).
Release of H2O2 by Sonato
cells was also not induced by addition of AVR9 elicitor (results not
shown). Treatment of Sonato and Sonatine cell suspensions with AVR9 did not induce O2 uptake or the generation of the
oxygen radicals O2, which was
measured by Cyt c reduction (results not shown). In conclusion, suspension-cultured Sonatine cells did not respond by an
oxidative burst upon AVR9 elicitor treatment.
To investigate elicitor-induced transcriptional activation of
defense-related genes, total RNA isolated from elicitor-treated suspension cells was hybridized with cDNA clones of tomato PR-protein genes that encode basic and acidic isoforms of chitinase and
-1,3-glucanase. The gene encoding the basic isoform of chitinase was
found to be constitutively expressed in suspension-cultured Sonato and Sonatine cells (Fig. 2). Expression of
genes that encoded the acidic and basic isoforms of -1,3-glucanase
and the acidic isoform of chitinase was increased in Pmg
elicitor-treated suspension-cultured Sonato and Sonatine cells (Fig.
2). Addition of AVR9 elicitor to suspension-cultured Sonato and
Sonatine cells did not increase expression of any of these PR-protein
genes.
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| Figure 2.
Elicitor-induced PR-protein gene expression.
Northern-blot analysis of total RNA (20 µg) isolated from
suspension-cultured Sonato cells (left panel) and Sonatine cells (right
panel) at different time points (0, 8, 12, and 24 h) after
treatment with AVR9 elicitor (10 µg/mL) and Pmg elicitor (50 µg/mL)
or from untreated cells. Hybridization was performed with
32P-labeled cDNA probes from acidic 1,3 -glucanase (glu
A), basic 1,3 -glucanase (glu B), acidic chitinase (chi A), and
basic chitinase (chi B).
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Cell Death Induced in Transgenic Cf9 Tomato Cells Expressing the
Avr9 Gene
Sonatine cell-suspension cultures, which were established from
independently initiated calli, responded upon treatment with various
elicitors except AVR9. The inability of suspension-cultured Sonatine
cells to respond to AVR9 treatment is therefore not due to a lack of a
general biochemical mechanism involved in the induction of
defense-related responses. Elicitor binding to a cellular receptor is
required for elicitor perception. Binding studies using microsomal membranes isolated from suspension-cultured Sonato and Sonatine cells
showed Kd and receptor concentration values
of 0.08 pM and 0.5 pmol/mg microsomal protein,
respectively, which are similar to those reported for microsomal
membranes obtained from tomato leaves (Kooman-Gersmann et al., 1996).
Conditions in the extracellular medium of suspension-cultured Sonato
and Sonatine cells, such as pH, ionic strength, and temperature, allow
optimal AVR9 binding (Kooman-Gersmann et al., 1996). Thus, the fact
that suspension-cultured Sonatine cells do not respond to AVR9
challenge is not due to a defect in the AVR9 binding of these cells.
In contrast to Sonatine cell suspensions, leaves of Sonatine plants
respond to AVR9 injection, which suggests that induction of
defense-related responses by the AVR9 elicitor is developmentally regulated. Three different Avr9-containing constructs, pMOG978, pMOG980, and pMOG1043, were designed for A. tumefaciens-mediated transformation of tomato plants (Fig.
3). A synthetic sequence encoding the
mature AVR9 peptide of 28 amino acid residues, fused to the PR1a signal
sequence to target the AVR9 peptide to the apoplast, was placed behind
the tobacco mosaic virus omega-leader. In construct pMOG978, the
-PR1a-Avr9 cassette was put under the transcriptional
control of the constitutive CaMV 35S promoter and terminator sequences
of the proteinase inhibitor II gene (PI-II) from potato (An et al.,
1989). In construct pMOG980, the -PR1a-Avr9 cassette of
pMOG978 is put under the transcriptional control of the
pathogen-inducible gst1 promoter sequences from potato
(Martini et al., 1993) and the PI-II terminator sequences. Construct
pMOG1043 harbors a 35S:-PR1a-Avr9:TPI-II cassette
together with the Cf-9 coding sequence under the
transcriptional control of gst1 promoter and PI-II
terminator sequences. The Avr9 coding sequence of pMOG1043 contains one nucleotide substitution, which results in Arg (R) changed
into a Lys (K) at position 8 of the 28-amino acid AVR9 peptide. The
AVR9(R8K) mutant elicitor peptide is more active on Cf9 tomato leaves
than the wild-type AVR9 elicitor (Kooman-Gersmann et al., 1997).
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| Figure 3.
Physical map of the Avr9 and the
Cf-9 gene expression cassettes of the transformation
vectors pMOG978, pMOG980, and pMOG1043. CaMV 35S, CaMV 35S promoter
fused to tobacco mosaic virus-untranslated omega-leader sequences;
gst1, pathogen-inducible gst1 promoter fragment fused to
the TATA box of the 35S CaMV promoter and tobacco mosaic
virus-untranslated omega-leader sequences; 3PI-II, proteinase inhibitor terminator sequences; Avr9, coding sequences of the PR1a
signal peptide fused to the mature AVR9 elicitor peptide of 28-amino
acid residues; Avr9(R8K), coding sequences of the PR1a signal peptide
fused to the mutant, mature AVR9 elicitor peptide of 28-amino acid
residues containing an Arg-8-Lys substitution; and Cf-9, sequence
encoding the CF9 protein.
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In potato transcriptional activity of the gst1 promoter was
shown to be silent in noninfected plant tissue, except for root apices
and senescing leaves (Strittmatter et al., 1996). These expression
patterns were also found in transgenic tomato plants transformed with a
pMOG980-derived construct in which the Avr9 gene was
replaced by the uidA locus of Escherichia coli
encoding GUS (results not shown). In addition, transgenic callus tissue showed GUS activity, indicating that the gst1 promoter, like
the CaMV 35S promoter, is highly active in callus (Fig.
4).
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| Figure 4.
Histochemical detection of GUS activity in
transgenic MM-CF0 callus that contains the uida locus
under the transcriptional control of the CaMV 35S promoter (A) or the
gst1 promoter (B).
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Co-cultivation of leaf discs of tomato MM-Cf0 with A. tumefaciens strains that carry either pMOG978, pMOG980, or
pMOG1043 resulted in an average of one callus per leaf disc on
selective medium (Table I).
Co-cultivation of MM-Cf9 leaf discs with these A. tumefaciens strains resulted in similar callus-formation
efficiencies: 1 to 1.4 calli per leaf disc (Table I). Growth of
transgenic MM-Cf9 callus tissue that express the Avr9 gene
or mutant Avr9(R8K) gene was similar to growth of transgenic
MM-Cf0 callus tissue that express Avr9 (Fig.
5). Transgenic MM-Cf0 callus, obtained after transformation with pMOG1043 expressing both the Avr9
and the Cf-9 gene, also showed normal growth (Fig. 5). From
MM-Cf0 calli obtained after transformation with constructs pMOG978,
pMOG980, and pMOG1043, transgenic plants were obtained after shoot
regeneration (Table I). Transgenic plants were also obtained from
pMOG980-transformed MM-Cf9 calli after shoot regeneration (Table I). On
transgenic MM-Cf9 calli transformed with pMOG978 containing the
35S:Avr9 construct, only a few shoots regenerated but these shoots
appeared unable to root on media containing kanamycin, indicating that they had escaped kanamycin selection and thus were not true
transformants (Table I). Thus, constitutive expression of the
Avr9 gene by the CaMV 35S promoter inhibited regeneration of
shoots on transgenic MM-Cf9 callus. When in transgenic MM-Cf9 callus,
expression of the Avr9 gene was controlled by the
gst1 promoter, which is inactive in shoots, transgenic
shoots regenerated and transgenic plants were obtained. These
transgenic plants showed a necrotic response upon induction of the
gst1 promoter by external stimuli (results not shown). These
results suggest that Avr9 gene expression has no effect on
growth of undifferentiated Cf9 tissue, whereas upon shoot formation
Avr9 gene expression has deleterious effects.
View this table:
[in this window]
[in a new window]
|
Table I.
Transformation of tomato genotypes MM-Cf0 and MM-Cf9
with Avr9- and Cf-9-containing constructs
Data obtained from transformation experiments of MM-Cf0 and MM-Cf-9
leaf discs with the constructs pMOG978 (35S:Avr9), pMOG980 (gst1:Avr9),
and pMOG1043 (gst1:Cf-9 35S:Avr9[R8K]), respectively. All
transformations were performed in parallel except for MM-Cf0 transformed with pMOG1043.
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View larger version (98K):
[in this window]
[in a new window]
| Figure 5.
A. tumefaciens-mediated leaf-disc
transformation of cv MM with Avr9- and
Cf-9-expressing constructs. Leaf discs of MM-Cf0 on
kanamycin-containing medium after co-cultivation with A. tumefaciens strains carrying either construct pMOG978, which
contains the 35S:Avr9 expression cassette (top row), or construct
pMOG1043, which contains the gst1:Cf-9 and 35S:Avr9(R8K) expression
cassettes (bottom row). Leaf discs of MM-Cf9 on kanamycin-containing
medium after co-cultivation with an A. tumefaciens
strain carrying construct pMOG978, which contains the 35S:Avr9
expression cassette (middle row). Pictures were taken 5 weeks after
co-cultivation (left panels), showing callus formation at the edges of
the explants, and after 8 weeks, showing shoot regeneration (right
panels).
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The transgenic plant MM-Cf0 978-16, heterozygous for one intact T-DNA
integration of construct pMOG978, was crossed with wild-type MM-Cf9.
The appearance of necrosis in F1 seedlings
segregated with the presence of the Avr9 transgene and
developed as described previously (Honée et al., 1995). In the
greenhouse seedlings containing both the Cf-9 and
Avr9 genes responded quickly and died within 18 d after
sowing (data not shown). Necrosis development was also followed in
vitro. On artificial selective medium that contained kanamycin, 29 F1 and 20 MM-Cf0 978-16 seedlings were germinated. On artificial medium without kanamycin, 20 MM-Cf9 seedlings
were germinated. After 10 d one cotyledon was cut off from each
seedling and transferred to artificial medium containing 2 mg/L
1-naphthaleneacetic acid and 2 mg/L 6-benzylaminopurine to induce
callus formation. Within the following 19 d all 29 F1 seedlings, which contain both the
Cf-9 and the Avr9 genes, developed a strong
necrotic response, resulting in growth inhibition and complete plant
death, whereas the MM-Cf9 and MM-Cf0 line 978-16 seedlings developed
normally (Fig. 6A). However, callus
formation on the explants was similar for all three genotypes. Explants derived from plantlets that expressed both the Avr9 and the
Cf-9 genes initiated callus with the same efficiency as
explants originating from plantlets that expressed only the
Cf-9 or the Avr9 gene. Initially, growth of
callus tissue on explants that contained both the Cf-9 and
the Avr9 genes appeared retarded compared with callus growth
on explants that contained only one of the two genes (Fig. 6B).
Probably, stress responses induced in cotyledons that contained both
the Avr9 and the Cf-9 genes negatively influenced callus growth. Growth of callus tissue separated from the explants was
identical between all three genotypes. Established callus cultures
containing Avr9, Cf-9, or both genes developed
identically for at least 3 months (Fig. 6C).
View larger version (90K):
[in this window]
[in a new window]
| Figure 6.
Development of necrosis in F1 seedling
from a cross between a transgenic MM-Cf0 978-16 plant expressing
Avr9 and a wild-type MM-Cf9 plant. A, In vitro-grown
plantlets 4 weeks after seed germination of MM-Cf9 (left), MM-Cf0
978-16 (middle), and F1 progenies of MM-Cf9 crossed with
MM-Cf0 line 978-16 (right). B, Callus, 2.5 weeks after initiation on
cotyledons from the plantlets shown in A: left, callus on a cv MM-Cf9
explant; middle, callus on a cv MM-Cf0 978-16 explant; and right,
callus on an explant of a F1 progeny from MM-Cf9 crossed
with MM-Cf0 line 978-16. C, Several independently established callus
cultures 3 months after initiation on MM-Cf9 explants (left), MM-Cf0
explants (middle), and on explants of F1 progenies from MM-Cf9 crossed
with MM-Cf0 978-16 (right).
|
|
| |
DISCUSSION |
Undifferentiated tomato cells, like cell-suspension cultures and
callus that contain the Cf-9 resistance gene, do not respond upon AVR9 challenge. However, mechanisms that are involved in elicitor-induced defense responses are functional in these
undifferentiated tomato cells. An oxidative burst, ion fluxes, and
expression of PR-protein genes were induced in suspension-cultured
Sonato and Sonatine cells upon treatment with various nonspecific
elicitors, as has been reported for other plant cell-suspension
cultures (Felix et al., 1993; Nurnberger et al., 1994). Addition of the race-specific elicitor AVR9 to suspension-cultured Sonatine cells did
not induce oxidative stress, ion fluxes, or gene activation. In
contrast, when Cf9 tomato leaves are injected with AVR9 elicitor preparations, an oxidative burst, expression of PR-protein genes, production of ethylene and salicylic acid, and necrotic cell death are
induced (De Wit and Spikman, 1982; Hammond-Kosack et al., 1996; May et
al., 1996; Wubben et al., 1996). This suggests that induction of
defense responses by AVR9, which is restricted to tomato cells carrying
the Cf-9 gene, depends on the developmental stage of the
cells and is absent in undifferentiated tomato cells.
Regeneration capacity of transgenic Cf9 callus is not negatively
influenced by the expression of the Avr9 transgene. Normal shoot regeneration was observed from transgenic Cf9 callus containing Avr9 under the transcriptional control of the
gst1 promoter, which is active in callus and inactive in
plantlets. In contrast, regeneration was unsuccessful from transgenic
callus, which expressed the cytotoxic compound barnase under control of
the gst1 promoter (Strittmatter et al., 1995). No transgenic
plants were regenerated from transformed Cf9 callus tissue when the
CaMV 35S promoter, which is constitutively active in callus tissue and
in plantlets, was used to drive the expression of the Avr9
gene. Normally developing Cf9 callus tissue that expressed the
Avr9 gene was not only obtained by A. tumefaciens-mediated transformation of Cf9 leaf discs with
Avr9-containing constructs, but could also be generated from
cotyledons from seedlings obtained from crosses between
Cf-9- and Avr9-expressing parent plants. Established callus cultures were, even after 3 months of culture, indistinguishable from those containing only one of the two genes, Avr9 or Cf-9. However, from these established Cf9
callus cultures that express the Avr9 gene, no shoots could
be regenerated. The inability to regenerate shoots was independent of
the presence of the Avr9 and Cf-9 genes and is
more likely to be characteristic for the species tomato. Callus
cultures from tomato genotypes that display a very high regeneration
capacity lose the ability to regenerate shoots when they are cultured
for more than 1 month (Koornneef et al., 1993).
F1 seedlings in which the Cf-9 gene and 35S:Avr9 transgene were combined developed a severe
necrotic response and the plants died within 3 weeks after germination. Progress of the necrotic response in these seedlings was similar to
that described previously (Hammond-Kosack et al., 1994; Honée et
al., 1995). In conclusion, in undifferentiated Cf9 tomato cells, as in
callus tissue, expression of the Avr9 gene has no
deleterious effects on growth and regeneration capacity, whereas in
differentiated Cf9 tissue such as shoots and plantlets, Avr9
gene expression induces cell death. Necrosis was not observed in
AVR9-challenged root tissue of Cf9 plants (G. Honée, unpublished
data), which also points to developmental regulation of AVR9-induced
necrosis in this organ.
The fact that undifferentiated Cf9 tomato cells are specifically
nonresponsive to the AVR9 elicitor peptide suggests that a cellular
component at the beginning of the AVR9 signaling cascade is absent or
inhibited. Two components of the AVR9 signaling cascade are known and
at least partially characterized: the AVR9-binding site and the
Cf-9 resistance gene (Jones et al., 1994; Kooman-Gersmann et
al., 1996). Normal AVR9 binding has been observed with microsomal membranes isolated from suspension-cultured tomato cells, which suggests that AVR9 perception is not due to the absence of the AVR9-binding site. Recently, it has been shown that the Cf-9
resistance gene does not encode the high-affinity binding site for AVR9
present in plasma membranes of tomato and other plant species
(Kooman-Gersmann et al., 1996; M. Kooman-Gersmann, unpublished data).
A. tumefaciens-mediated transformation of line MM-Cf0 leaf
discs with construct pMOG1043 containing both the Cf-9 and
Avr9 genes resulted in normal development of transgenic
callus (Table I). The two promoters, CaMV 35S and gst1, that
drive Avr9 and Cf-9 expression, respectively, are
highly active in callus. However, growth and regeneration capacity of this callus were not inhibited, indicating that expression of the
Avr9 and Cf-9 genes had no deleterious cellular
effects on these cell types. These observations make it unlikely that
absence of functional CF9 protein explains the absence of AVR9-induced defense responses in undifferentiated tomato cells.
Cell-suspension cultures are handled relatively easily, which make them
valuable and attractive for standardized experiments to study
elicitor-induced defense responses. For different cell-suspension cultures, a variety of elicitor molecules have been shown to induce defense-related responses that are similar to the responses in elicited
plants. However, the physiological condition and developmental stage
between suspension-cultured cells and cells of intact plants differ.
Therefore, conclusions drawn from studies on suspension-cultured cells
have to be taken with some caution to explain mechanisms involved in
the defense of intact plants. For instance, in suspension-cultured Sonato and Sonatine cells the gene encoding the basic isoform of
chitinase is constitutively expressed (Fig. 2). For several defense-related genes differential expression patterns have been reported to be developmentally regulated (Cordero et al., 1994; Logemann et al., 1995). Elicitation of Cf9 tomato cells by AVR9 is also
under developmental regulation. In contrast to tomato, suspension-cultured transgenic tobacco cells expressing the
Cf-9 gene have been reported to respond specifically upon
AVR9 treatment (Jones et al., 1996). Apparently, in undifferentiated
transgenic tobacco cells, components involved in the AVR9 signaling
cascade are actively present. Studying AVR9-induced defense responses in tomato Cf9 cells at different developmental stages will reveal insight in developmental regulation of these defense responses, which
is possibly specific for AVR9-Cf9-mediated resistance in tomato.
| |
FOOTNOTES |
1
This work was supported by grants to J.B. from
the Life Science Foundation, which is subsidized by the Netherlands
Organization for Scientific Research; and from the Ministry of Economic
Affairs; the Ministry of Education, Culture, and Science; and the
Ministry of Agriculture, Nature Management, and Fishery in the
framework of the Industrial Relevant Research Program of The
Netherlands Association of Biotechnology Centers in The Netherlands to
G.H.
2
Present address: Department of Plant Physiology,
Wageningen Agricultural University, Arboretumlaan 4, 6703 BD
Wageningen, The Netherlands.
3
Present address: Institut für Biologie
III, RWTH Aachen, Worringer Weg, 52074 Aachen, Germany.
4
Present address: Department of Genetics, Free
University, de Boelelaan 1087, 1081 HV Amsterdam, The Netherlands.
*
Corresponding author; e-mail pierre.dewit{at}medew.fyto.wau.nl; fax
31-317-483412.
Received October 23, 1997;
accepted March 19, 1998.
| |
ABBREVIATIONS |
Abbreviations:
CaMV, cauliflower mosaic virus.
HR, hypersensitive response.
MM, MoneyMaker.
Pmg, partial acid hydrolysate
from the cell walls of Phytophthora megasperma f. sp.
glycinea (Phytophthora sojae) .
PR, pathogenesis-related.
| |
ACKNOWLEDGMENTS |
The authors acknowledge Prof. Dr. D. Scheel for the kind gift of
partial acid hydrolysate of cell walls of P. sojae; Prof. Dr. T. Boller for his kind gift of xylanase, chitosan, and
chitopentaose; and Dr. M. Kooman-Gersmann for technical assistance on
AVR9-binding assays. Bert Essenstam and Rick Lubbers are acknowledged
for excellent horticulture assistance. Drs. Matthieu Joosten, Sietske
Hoekstra, and Ronelle Roth are acknowledged for helpful discussions on
the manuscript. Financial support by the Landbouw Export Bureau
fund is greatly appreciated.
| |
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[Abstract]
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Plant Physiol
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[Abstract/Free Full Text]
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The race-specific elicitor AVR9 of the tomato pathogen Cladosporium fulvum: a cystine knot protein. Sequence-specific 1H-NMR assignments, secondary structure and global fold of the protein.
FEBS Lett
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[CrossRef][ISI][Medline]
Verwoerd TC,
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Nucleic Acids Res
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2362
[Free Full Text]
Widholm JM
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The use of fluorescein diacetate and phosafranine for determining viability of cultured plant cells.
Stain Technol
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[ISI][Medline]
Wubben JP,
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Differential induction of chitinase and 1,3-beta-glucanase gene expression in tomato by Cladosporium fulvum
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Abstract
Introduction