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Plant Physiol. (1999) 120: 1137-1146
Early Events in the Signal Pathway for the Oxidative Burst in
Soybean Cells Exposed to Avirulent Pseudomonas
syringae
pv glycinea1
Vinagolu K. Rajasekhar2, *,
Chris Lamb3, and
Richard A. Dixon
Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam
Noble Parkway, Ardmore, Oklahoma 73401 (V.K.R., R.A.D.); and Plant
Biology Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037 (C.L.)
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ABSTRACT |
Soybean
(Glycine max) cv Williams 82 suspension cultures exhibit
an oxidative burst approximately 3 h after challenge with Pseudomonas syringae pv glycinea
(Psg) harboring the avrA (avirulence) gene. Pretreatment with the tyrosine (Tyr) kinase inhibitor herbimycin A or the serine/threonine kinase inhibitor K252a abolished the burst and subsequent induction of glutathione
S-transferase. However, imposition of a 45-min rest
period between pathogen challenge and subsequent addition of the kinase
inhibitors resulted in escape from inhibition by herbimycin A, whereas
inhibition by K252a persisted. Suramin, a G-protein inhibitor,
inhibited the burst if added up to 90 min after pathogen challenge. The
burst was also induced by the ion channel generator amphotericin B, and
this induction was sensitive to suramin and K252a. Conversely, the ion
channel blocker anthracene-9-carboxylate inhibited the
Psg:avrA-induced burst.
Psg:avrA rapidly induced Tyr
phosphorylation of several proteins, and this was inhibited by
herbimycin A or anthracene 9-carboxylic acid. These data suggest that
the activation of ion channels is followed by an upstream Tyr kinase
before the serine/threonine kinase-dependent steps in the signal
pathway leading to the oxidative burst.
Psg:avrA-dependent induction of phenylalanine
ammonia-lyase was not inhibited by herbimycin or suramin, suggesting
the operation of different signal pathways for the oxidative burst and
phenylpropanoid-derived defense responses.
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INTRODUCTION |
Plant cells respond to challenge with avirulent pathogens by
mounting a multi-component defense response called the HR.
Characteristic features of the HR include an oxidative burst leading to
generation of H2O2 (Lamb
and Dixon, 1997 ), localized cell death and cell wall cross-linking
(Bradley et al., 1992; Levine et al., 1994), and synthesis of
antimicrobial phytoalexins (Dixon and Harrison, 1990). The HR results
from direct or indirect recognition of a microbial avirulence gene
product by the product of a host resistance gene (de Wit, 1997; Parker
and Coleman, 1997). Many avirulence and resistance genes have now been
identified at the molecular level (Staskawicz et al., 1995; de Wit,
1997). In addition, biochemical studies have defined a number of
microbial elicitor molecules that induce some or all of the responses
induced by avirulent microbes (Ebel and Mithöfer, 1998). Such
elicitors may or may not be direct products of microbial avirulence
genes.
The oxidative burst in challenged plant cells resembles that exhibited
by human neutrophils (Baggiolini and Wymann, 1990), producing
H2O2 that originates from
superoxide generated by a plasma membrane-associated NADPH oxidase
(Lamb and Dixon, 1997). The major component (a gp91 homolog) of the
plant oxidase complex has been cloned (Groom et al., 1996; Keller et
al., 1997; Torres et al., 1998), and pharmacological experiments have
shown that activation of the oxidase involves a protein kinase cascade
that can be blocked by the Ser/Thr kinase inhibitor K252a (Levine et al., 1994; Yang et al., 1997).
Many studies over the past several years have reported the effects of
pharmacological agents known to affect signaling in mammalian cells on
the defense responses of cultured plant cells (for review, see Dixon et
al., 1994; Yang et al., 1997; Ebel and Mithöfer, 1998). On the
basis of such studies, ion fluxes (Jabs et al., 1997), calcium uptake
(Stäb and Ebel, 1987), G-proteins (Legendre et al., 1992, 1993a),
kinase cascades (Schwacke and Hager, 1992; Levine et al., 1994; Suzuko
and Shinshi, 1996), and polyphosphoinositides (Legendre et al., 1993b)
have been implicated as signal transduction components in the induction
of phytoalexins or the oxidative burst. More recently, studies have
reported the involvement of plant functional homologs of mammalian MAP
kinases in plant defense signal transduction (Suzuki and Shinshi, 1995; Adam et al., 1997; Ligterink et al., 1997; Stratmann and Ryan, 1997;
Zhang and Klessig, 1998). A feature of these kinases is their
phosphorylation on both Ser/Thr and Tyr residues. However, little is
known concerning Tyr phosphorylation in plants.
A large proportion of the pharmacological studies on plant defense
signal transduction have utilized non-race-specific elicitor molecules
as primary inducing agents. With the exception of the tomato:Pseudomonas syringae pv tomato system
(Lamb, 1994; Zhou et al., 1995, 1997), little biochemical information
is available on signal transduction leading to the HR or defense gene
induction in response to resistance-gene-mediated recognition of
bacterial avirulence genes. We have developed a soybean cell culture
system that responds, in a race-specific manner, to Pseudomonas
syringae pv glycinea (Psg) carrying the
avrA avirulence gene that is recognized by the corresponding
Rpg2 resistance gene in the soybean (Glycine max) cv Williams 82. Treatment of the cells with
Psg:avrA results in a strong oxidative burst and
isoflavonoid phytoalexin accumulation (Levine et al., 1994; Shirasu et
al., 1997; Guo et al., 1998). The burst can be potentiated by
physiological concentrations of the endogenous signal molecule
salicylic acid (Shirasu et al., 1997), and also by chemical inhibitors
of Ser proteases (Guo et al., 1998). Thus, this system is an excellent
one for pharmacological dissection of avrA-mediated signal
transduction.
In the present study, we investigate the potential involvement of Tyr
phosphorylation in signal transduction utilizing the soybean:
Psg cell culture system. We also examine the relative position in the signal transduction pathway of Tyr phosphorylation in
relation to ion channel activity, G-protein activation, and Ser/Thr
kinase(s). Our results indicate that Tyr phosphorylation is an early
event in the induction of the oxidative burst by avirulent bacteria in
soybean cells, and that it precedes the events requiring Ser/Thr
phosphorylation.
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MATERIALS AND METHODS |
Chemicals
All protein kinase inhibitors were obtained from Calbiochem. GTP
analogs, amphotericin B, and anthracene-9-carboxylate were purchased
from Sigma. Protein kinase inhibitors, GTP analogs, and
anthracene-9-carboxylic acid were dissolved in DMSO and maintained in
100× concentrated stocks. Control cultures were always treated with an
equivalent amount of DMSO.
Maintenance and Inoculation of Soybean Cell Suspension
Cultures
Soybean (Glycine max cv Williams 82) cell suspensions
were subcultured every 7 d by 1:5 dilution in fresh Murashige and
Skoog medium (Murashige and Skoog, 1962) containing 3% (w/v)
Suc, 0.5 mg/L 2,4-D, and 0.5 mg/L 6-benzylaminopurine, pH 5.7. All
experiments were performed starting 3.5 d post subculture.
One-milliliter aliquots of suspension culture were transferred to
12-well tissue culture plates (1 mL/well) and maintained with circular
rotation at 80 to 90 rpm.
Pseudomonas syringae pv glycinea (Psg)
race 4 harboring the plasmid pLAFR1 carrying the avrA or
avrC avirulence genes (Keen and Buzzell, 1991) was grown
overnight in King's B medium (20 g of protease peptone, 10 mL of
glycerol, 2.25 g of
K2HPO4, and 1.5 g of
MgSO4·7H2O
L 1, pH 7.2) supplemented with streptomycin (30 µg/mL) or kanamycin (50 µg/mL), respectively. The bacteria were
then centrifuged, re-suspended in sterile water, and added to soybean
suspension cultures at a final inoculum of 108
cfu/mL.
For conditioning experiments, the bacteria grown overnight were
centrifuged gently, washed once with sterile distilled water, and
re-suspended in filter-sterilized conditioned medium from 3.5-d-old
soybean suspension cultures at 108 cfu/mL. The
bacteria were grown in this medium in the presence or absence of
various inhibitors for 90 min. They were then collected by
centrifugation, washed twice with sterile distilled water, re-suspended
in the sterile distilled water, and used as conditioned bacteria.
Measurement of H2O2 Accumulation and Cell
Death
H2O2 was measured by
monitoring the destruction of scopoletin fluorescence, and cell death
was measured by Evan's blue staining, as described previously (Shirasu
et al., 1997). All data represent the mean and SE from
three or four independent experiments.
Protein Extraction, Immunoprecipitation, and Western Analysis
Total proteins were extracted from 1 mL of suspension cultures in
ice-cold cell lysis buffer containing 50 mM Tris, pH 7.5, 2 mM EDTA, 2 mM EGTA, 150 mM NaCl,
1% NP40, 0.25% sodium deoxycholate, 1 mM DTT, 1 mM PMSF, 10 µg/mL each of leupeptin, pepstatin,
aprotenin, 4 mM each of
Na3VO4 and NaF, and one
protease inhibitor cocktail tablet per 25 mL (Boehringer Mannheim). The
protein extracts were clarified by centrifugation at 1,000g
and were then either used for immunoprecipitations and/or directly for
western analysis. In the immunoprecipitation reactions, 4 µg of mouse
anti-(phospho-Tyr) monoclonal IgG2b 4G10 (Upstate Biotechnology,
Lake Placid, NY) were employed in 1-mL reactions with gentle rocking
overnight in the cold room. Immuno complexes were captured using
protein A-agarose (Santa Cruz Biotech, Santa Cruz, CA), washed once
with lysis buffer followed by three times with PBS, and re-suspended in
2× Laemmli sample buffer (Laemmli, 1970). Equal amounts of proteins were processed for SDS-PAGE analysis using Tris-Gly gels (Novex, San Diego).
In competition experiments, the complete immunoprecipitation reactions
were incubated with 1 to 2 mM phospho-DL-Tyr, 1 mM phospho-DL-Thr, or 1 mM
phospho-DL-Ser (Sigma). Western analysis of the proteins
resolved by SDS-PAGE was carried out following transfer to a PVDF
membrane (Immobilon-P, Millipore) and incubating with
phospho-Tyr-specific monoclonal antibody or rabbit antiserum against
maize GST (kindly supplied by Dr. Klaus Kreuz, Ciba Geigy, Basel). The
primary antibody cross-reactions were detected by peroxidase-conjugated
secondary antibodies against rabbit IgG from goat (1:10,000 dilution)
(Bio-Rad) or against mouse IgG from sheep (1:2,000 dilution) (Amersham)
and visualized by chemiluminescence (ECL detection system, Amersham).
The blots were then exposed to x-ray film (X-OMAT, Kodak).
Extraction and Analysis of RNA
Total RNA was isolated from 200 mg fresh weight of tissue using
the TRI-reagent method according to the manufacturer's instructions (Molecular Research Center, Cincinnati). For northern analysis, 20 µg
of total RNA from each sample was run on a 1% agarose-formaldehyde gel
and transferred to a Hybond-N membrane. The membranes were UV
cross-linked in a Stratalinker (Stratagene). Hybridizations were
carried out overnight at 65°C using a
32P-labeled soybean PAL1 probe or a Glomus
versiforme 18S rRNA probe using standard protocols (Sambrook et
al., 1989). The 929-bp PAL1 fragment (Frank and Vodkin, 1991) was
amplified (forward primer 5 CCAAGGAACCCCTATTGG3; reverse primer
5CCATTCCACTCCCCAAGG3) from cv Williams 82 genomic DNA using standard
PCR conditions in a Robocycler 96 (Stratagene). A 590-bp G. versiforme 18S rRNA RT-PCR fragment (Simon et al., 1992) was
provided by Dr. Ignacio E. Maldonado-Mendoza (Noble Foundation).
The membranes were washed twice at 65°C in 0.5× SSC and 0.2%
(w/v) SDS, and exposed to x-ray film with intensifying screens
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RESULTS |
Effects of Inhibitors of Tyr and Ser/Thr Kinases on the Oxidative
Burst Induced by Psg:avrA
The soybean cv Williams 82 contains the Rpg2 resistance
gene that recognizes the avrA avirulence gene of
Psg, but not the Rpg3 resistance gene that
recognizes the avrC avirulence gene (Keen and Buzzell,
1991). Thus, challenging Williams 82 suspension cultures with
Psg:avrA results in a sustained oxidative burst and subsequent cell death, whereas Psg:avrC
induces only a very weak, early, nonspecific oxidative burst and no
resultant cell death (Levine et al., 1994). Although somewhat variable
between cell culture batches, the oxidative burst in response to
Psg:avrA usually occurs around 180 min after contact with
the bacteria, and reaches maximum levels of
H2O2 release after 240 to
350 min (Shirasu et al., 1997).
It has previously been shown that the oxidative burst induced in
soybean cv Williams 82 cells by Psg:avrA is
strongly reduced by co-treatment with the protein Ser/Thr kinase
inhibitor K252a (Levine et al., 1994; Guo et al., 1998), a compound
that is highly effective in inhibiting protein Ser/Thr kinases in plant
cell cultures, with an IC50 of 100 nM for in vivo inhibition of tomato cell culture
defense responses, and a Ki of 15 nM for in vitro inhibition of tomato protein kinase (Grosskopf et al., 1990). Herbimycin and the isoflavone genistein have been widely used as
pharmacological agents for inhibition of protein Tyr kinases in
mammalian cells (Akiyama et al., 1987; Uckun et al., 1991; Riordan et
al., 1998). Administration of herbimycin A (1.0-1.5 µM, IC50 0.84 µM) to soybean cells 15 min prior to challenge
with Psg:avrA considerably inhibited the extent
of the subsequent oxidative burst (Fig.
1A). Under the same conditions,
herbimycin A alone had no effect.
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| Figure 1.
A herbimycin-inhibited step precedes a protein
Ser/Thr kinase step in the oxidative burst signal pathway. A, Dose
response for inhibition of the
Psg:avrA-induced oxidative burst by
herbimycin. Soybean suspension cells were treated for 4.5 h with
H2O ( , control), Psg:avrA
( , 108 cfu/mL), or
Psg:avrA and various concentrations of
herbimycin added 15 min prior to exposure to the bacteria. B, Escape
from inhibition by herbimycin but not K252a.
H2O2 accumulation was measured 4.5 h after
treatment of cells with Psg:avrA or
inhibitors (1 µM) alone,
Psg:avrA plus inhibitors added 15 min
prior to bacteria, or Psg:avrA added at
zero time with inhibitors added 45 min later.
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Preliminary experiments with genistein (50-100 µM) gave
a less reproducible inhibition of the oxidative burst, and variable inhibition was also seen with daidzein, the 5-deoxy-derivative of
genistein routinely used as a negative control in mammalian Tyr kinase
studies. Because genistein and daidzein are major isoflavonoid natural
products in soybean, occurring as intermediates in an infection-induced
phytoalexin defense pathway in this species (Graham et al., 1990), we
decided not to pursue genistein further as a pharmacological agent.
Herbimycin A and K252a are similarly effective in blocking the
oxidative burst if added 15 min prior to exposure to
Psg:avrA (Fig. 1B). However, if the inhibitors
are added 45 min after exposure to Psg:avrA, the cells
completely escape from inhibition by herbimycin A, but the level of
inhibition by K252a remains unaltered. We show later that inhibition by
K252a is retained at least up to the period of
H2O2 release. These data
suggest that the event(s) inhibited by herbimycin A occur earlier in
the signal pathway than the Ser/Thr kinase(s) inhibited by K252a, and
that Ser/Thr phosphorylation is continuously required for the oxidative
burst, whereas the requirement for putative Tyr phosphorylation is more transient.
Involvement of G-proteins in the Oxidative Burst Induced by
Psg:avrA
It has been suggested that heterotrimeric G-proteins function
downstream of elicitor reception in the induction of the oxidative burst (Legendre et al., 1992, 1993a). To assess the involvement of
G-proteins in our system, we first tested the effects of GTP homologs.
GTP- -S, a nonhydrolyzable GTP analog that locks G-proteins in the
active state, reproducibly doubled the amount of
H2O2 released when added at
50 to 100 µM (EC50 37.5 µM) 15 min prior to challenge with
Psg:avrA. This effect of GTP--S was
avr gene dependent, because the compound did not induce or
potentiate H2O2 production when added in conjunction with Psg:avrC. However,
GDP- -S, which locks G-proteins in the inactive state, was totally
inactive in these assays, suggesting that there may be uptake problems
for GTP analogs in DMSO-treated soybean cells, and that the extent of
the response to GTP--S may be an underestimate.
Suramin interferes with the GTP binding site of the alpha subunit of
G-proteins (Chahdi et al., 1998) and also inhibits binding of growth
factors to their receptors in mammalian systems (Mills et al., 1990).
Between 30 and 120 µM (I50 26 µM), suramin caused an approximately 60% decrease in
H2O2 production when added
15 min prior to challenge with Psg:avrA.
Increasing the concentration of suramin did not significantly reduce
H2O2 production further, presumably because suramin itself can induce a detectable oxidative burst at concentrations above 30 µM (Fig.
2).
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| Figure 2.
Dose response for the effects of the G-protein
inhibitor suramin on the oxidative burst. Suramin was added alone or 15 min prior to exposure of soybean cells to
Psg:avrA, and
H2O2 accumulation was determined after 4.5 h. , psg:avrA; , control.
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We next used pharmacological inhibition studies to help delineate the
relationships between potential Tyr phosphorylation, G-protein
activation, and Ser/Thr kinase involvement in the oxidative burst. The
oxidative burst potentiated by GTP--S was completely blocked by 1 µM K252a or 5 µM DPI, the latter being a
suicide inhibitor of the NADPH oxidase (O'Donnell et al., 1993) with
an I50 value of 2 µM for inhibition
of the oxidative burst in soybean cells (Levine et al., 1994). This
suggests that G-protein mediated events are upstream of the Ser/Thr
kinase-mediated steps in the signal transduction pathway to activation
of the NADPH oxidase (Fig. 3).
Potentiation of the Psg:avrA induced oxidative
burst by GTP--S was completely blocked by suramin (Fig. 3), which is consistent with the effect of GTP--S being mediated via a
receptor-coupled G-protein. However, in the presence of GTP--S,
suramin did not reduce the burst below the level observed in the
presence of Psg:avrA alone. Herbimycin A further reduced the
burst potentiated by GTP--S to a level below that obtained with
Psg:avrA alone, suggesting that Tyr
phosphorylation is somehow linked to G-protein action in this system.
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| Figure 3.
Effects of protein kinase inhibitors and suramin
on the oxidative burst induced by
Psg:avrA in the presence of GTP--S.
GTP--S, herbimycin (1 µM), suramin (90 µM), K252a (1 µM), or DPI (5 µM) were added 15 min prior to treatment of cells with
Psg:avrA, and
H2O2 accumulation was determined 4.5 h
after challenge.
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Although the inhibitory effect of herbimycin A was lost when the
compound was added 45 min after challenge with
Psg:avrA (Fig. 1B), suramin inhibited the
inducible oxidative burst when added at least 90 min after pathogen
challenge (Fig. 4). Under the same experimental conditions, K252a and DPI completely inhibited the Psg:avrA-inducible oxidative burst when added at
least 180 min after pathogen challenge. These results suggest that
G-protein-mediated events occur between the Tyr and the Ser/Thr
phosphorylation events in the signal pathway.
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| Figure 4.
Escape of the
Psg:avrA induced oxidative burst from
inhibition by suramin. Suramin (90 µM), K252a (1 µM), or DPI (5 µM) was administered at the
times shown to soybean suspension cultures treated with
Psg:avrA, and accumulation of
H2O2 was determined 4.5 h after exposure
to the pathogen. White bars, Psg:avrA; black bars,
Psg:avrA plus suramin; striped bars,
Psg:avrA plus K252a; stippled bars,
Psg:avrA plus DPI.
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Effect of Ion Channel Generators and Blockers on the Oxidative
Burst Induced by Psg:avrA
Defense responses can be induced in parsley suspension cultures by
treatment with amphotericin B, a compound that forms artificial ion
channels. Furthermore, a MAP kinase is phosphorylated in this system in
a manner reversible by an ion channel blocker (Jabs et al., 1997;
Ligterink et al., 1997). To test the involvement of ion channels in
signaling in the soybean cells, we first studied the effect of
amphotericin B as an inducer of the oxidative burst in the absence of
bacteria. Preliminary experiments indicated that 50 to 100 µM amphotericin B was optimal for inducing the oxidative
burst, with an EC50 of approximately 25 µM. Herbimycin A, suramin, K252a, and DPI each inhibited
the induction of the oxidative burst by 50 µM
amphotericin B in a manner comparable to their inhibition of the
Psg:avrA induced burst (Fig.
5). Anthracene 9-carboxylate, an ion
channel blocker, inhibited the Psg:avrA inducible oxidative
burst at concentrations between 300 and 400 µM
(I50 340 µM). These
results are consistent with changes in ion channel activity preceding
the early Tyr phosphorylation-dependent step(s) in the signal pathway
to the oxidative burst.
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| Figure 5.
Inhibition of the oxidative burst induced by
amphotericin B. Soybean suspension cells were treated with
Psg:avrA alone, amphotericin B alone
(Ampho-B, 50 µM), or amphotericin B preceded 15 min
earlier by herbimycin (1 µM), suramin (90 µM), K252a (1 µM), or DPI (5 µM). The accumulation of H2O2 was
determined 4.5 h after inducer treatment.
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To ensure that the effects of the above inhibitors were not simply the
results of cellular toxicity, we measured the level of cell death in
the soybean suspensions by Evan's blue staining 5 h post
application for each inhibitor (herbimycin, suramin, K252a, DPI, or
anthracene 9-carboxylate) at the concentrations used in the above
studies (1.2-10 times the I50 value depending on
the inhibitor) in the presence of Psg:avrA
relative to control levels. At this time, the level of cell death in
bacterially induced cells was approximately 20% above control values,
and the level of cell death in all bacteria plus inhibitor treatments
was within this value and the control value (data not shown). However,
higher concentrations of the inhibitors did exhibit cellular toxicity within the time periods of the experiments, and were not used.
Pre-Conditioning of Bacteria and Effects of Pharmacological Agents
on Psg:avrA
Psg:avrA pre-incubated in conditioned
soybean cell culture medium for 90 min induced an earlier onset of the
oxidative burst (by approximately 90 min) than bacteria pre-incubated
in water (Fig. 6). This effect was not
observed if Psg:avrA were incubated in fresh Murashige and
Skoog medium (data not shown). To verify that the inhibitors employed
in the present investigation do not exert their effects on
Psg:avrA itself, we grew bacteria in conditioned medium from soybean suspension cultures for 90 min in the presence of
the same concentrations of herbimycin, suramin, K252a, and anthracene
9-carboxylate shown to inhibit the oxidative burst in the cell
cultures, and then washed the bacteria prior to addition to soybean
cells. Addition of inhibitors during the incubation did not affect the
ability of the bacteria to induce an early oxidative burst (all values
for H2O2 production were
75%-100% of the value obtained with non-pretreated bacteria).
However, treatment with the bacterial protein synthesis inhibitor
lincomycin strongly inhibited the ability of the bacteria to induce the
burst (H2O2 production was
reduced to 15% of the value obtained with non-pretreated bacteria).
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| Figure 6.
Effect of preconditioning
Psg:avrA with culture medium from soybean
suspension cultures. Bacteria were re-suspended in culture medium
filtrate from 3.5-d-old soybean cells, and incubated for 90 min (,
conditioned). Unconditioned bacteria () were re-suspended in water
and incubated for 90 min. Bacteria were then added to soybean
suspensions, and H2O2 production was measured
at the times shown.
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Effects of Inhibitors on Downstream Defense Response Gene Induction
GST is an anti-oxidant-response enzyme that is induced directly in
soybean cells by the H2O2
produced in the oxidative burst (Levine et al., 1994). We utilized an
antiserum against maize GST (Flury et al., 1995) to determine the
effects of the various pharmacological reagents on a downstream
response enzyme as further confirmation of their effects on signal
transduction in soybean. Figure 7 shows a
western blot of total proteins extracted from soybean cells 4.5 h
after exposure to Psg:avrA in the presence of the
different inhibitors. Control cells and cells exposed to Psg:avrC showed no signal at 39 kD following
development of blots with anti-(GST) serum and secondary antibody,
whereas a strong signal was seen in cells treated with
Psg:avrA. This response was inhibited by
herbimycin A, suramin, and K252a. Perhaps surprisingly, amphotericin B
alone did not induce GST, whereas anthracene 9-carboxylate increased
the level of GST induction in response to avirulent bacteria.
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| Figure 7.
Effects of inhibitors on induction of GST by
Psg:avrA. Cells were treated with
Psg:avrA alone or
Psg:avrA following pretreatment 15 min
earlier with herbimycin (1 µM), suramin (90 µM), K252a (1 µM), amphotericin B (Ampho-B,
50 µM), or anthracene 9-carboxylic acid (A9C, 400 µM). Cells were untreated (control), or were treated with
amphotericin B alone or with Psg:avrC.
Cells were harvested 4.5 h after challenge, and proteins were
extracted and resolved by SDS-PAGE, followed by western blotting and
development of the blot with anti-(maize GST) serum.
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Northern analysis revealed that, of all the inhibitors used in the
present work, only K252a significantly inhibited induction of PAL
transcripts in response to Psg:avrA (Fig.
8). This indicates that the herbimycin-
and suramin-sensitive events are in a separate signal pathway from that
which activates expression of phenylpropanoid-based defenses. However,
unlike GST, PAL induction was stimulated by the addition of
amphotericin B alone.
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| Figure 8.
Effects of inhibitors on induction of PAL
transcripts by Psg:avrA. Cells were
treated with Psg:avrA alone or
Psg:avrA following pretreatment 15 min
earlier with anthracene 9-carboxylic acid (A9C, 400 µM),
herbimycin (1 µM), suramin (90 µM), K252a
(1 µM), or amphotericin B (50 µM). Cells
were untreated (control), or were treated with amphotericin B (Ampho-B)
alone. Cells were harvested 4.5 h after challenge, and total RNA
was extracted and subjected to northern analysis using soybean PAL as a
probe. Blots were then stripped and re-hybridized with G. versiforme rRNA (loading and transfer control).
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Effects of Inhibitors on Protein Tyr Phosphorylation
To provide direct evidence that
Psg:avrA-mediated signal transduction involves
Tyr phosphorylation, we used anti-(phospho-Tyr) monoclonal antibodies
to immunoprecipitate and western blot proteins from soybean cells
treated with Psg in the presence or absence of the various
inhibitors. Four Tyr-phosphorylated proteins were revealed by this
analysis. One approximately 19-kD protein was present at low levels in
extracts from untreated cells and was strongly induced by
Psg:avrA (Fig. 9).
A slightly larger protein of approximately 20 kD was not present in
uninduced cells, and was induced to relatively low levels in response
to Psg:avrA. The two other proteins,
approximately 28 and 55 kD, were present in control cells at low and
high levels, respectively, and were induced to approximately twice the
control levels by bacteria. To test specificity, the
immunoprecipitations were also carried out in the presence of
phospho-Thr, phospho-Ser, or phospho-Tyr at 1 and 2 mM. Phospho-Tyr competed out the
immunoprecipitation of the three lower
Mr proteins and strongly reduced the
amount of precipitated 55-kD protein, whereas phospho-Ser and
phospho-Thr did not. This confirms the specificity of the antibodies
for proteins with Tyr phosphorylation (Suzuki and Shinshi, 1995;
Stratmann and Ryan, 1997).
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| Figure 9.
Immunoprecipitation/western blot analysis of
phospho-Tyr-containing proteins. Cells were treated for 30 min with
Psg:avrA alone or
Psg:avrA following pretreatment 15 min
earlier with anthracene 9-carboxylic acid (400 µM),
herbimycin (1 µM), or suramin (90 µM).
Extracts were prepared and immunoprecipitated with anti-(phospho-Tyr)
monoclonal antibodies. Immunoprecipitations were also carried out on
extracts from cells treated with Psg:avrA
alone, in the presence of the competitor amino acids phospho-Ser (1 mM), phospho-Thr (1 mM), or phospho-Tyr (1 mM or 2 mM). Immunoprecipitates were
resolved by SDS-PAGE and the gels were subjected to western analysis
using anti-(phospho-Tyr) monoclonal antibodies.
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Pretreatment of Psg:avrA treated cells with
herbimycin A or anthracene 9-carboxylic acid completely prevented the
appearance of the two lower Mr
phosphoprotein bands and inhibited the increase in the two higher
Mr proteins (Fig. 9). This confirms
that these inhibitors do indeed block protein Tyr phosphorylation in
the soybean cells, and also suggests that ion channel activity may be
upstream of the Tyr kinase inhibitor-sensitive step(s). Suramin also
reduced the levels of these proteins, but did not completely block the
increases.
| |
DISCUSSION |
Most pharmacological studies on defense response signal
transduction in plant cell cultures have used microbial or synthetic elicitors as the inducer (Dixon et al., 1994; Ebel and Mithöfer, 1998). In these cases, the oxidative burst is almost immediate (Legendre et al., 1993a; Jabs et al., 1997), indicating rapid elicitor-receptor recognition and signaling of downstream events. In
contrast, if the inducing agent is a bacterial strain harboring a
recognized avirulence gene, the oxidative burst does not start until
after a lag period of around 3 to 4 h post infection (Levine et
al., 1994; Shirasu et al., 1997; Guo et al., 1998). This lag period is
presumably necessary for the bacteria to synthesize the
avirulence gene product and to deliver this to the plant cells via the
hrp (hypersensitive response and
pathogenicity)-gene-mediated type III secretory system (Van den
Ackerveken and Bonas, 1997). Specific nutritional conditions that
satisfy requirements for induction and overcoming catabolite
repression, rather than host-specific factors, are required for
expression of the avrB gene (Huynh et al., 1989). This is
presumably also the case for the avrA gene; thus, in the
present system, a 90-min pre-incubation of bacteria in conditioned
plant culture medium reduced the lag period for induction of the
oxidative burst by a corresponding 90 min, whereas growth in fresh
Murashige and Skoog medium did not.
Assuming that the onset of the oxidative burst starts very soon after
perception of the avrA gene product in the plant cells, events occurring in the host cells within the 1st h post infection may
not be dependent on avrA gene product recognition. For
example, an early, small, and transient oxidative burst occurs in
soybean cv Williams 82 cells in response to exposure to Psg
harboring avrC, which is not recognized by this cultivar
(Levine et al., 1994). This early, nonspecific burst is strongly
potentiated by Ser protease inhibitors (Guo et al., 1998). It also
occurs in cells exposed to Psg:avrA, and is
presumably activated via a signaling pathway that operates prior to
avirulence gene product recognition. It is known that plants recognize
and respond to common microbial surface components such as chitin in
fungal cell walls (Baureithel et al., 1994), and such recognition may
lead to early defense responses (Felix et al., 1993). However, it is
not known whether such early responses that do not involve
avr gene product recognition by the host cells are necessary
for subsequent responses that do require avr gene product
recognition.
Pharmacological experiments should always be interpreted with caution.
In particular, nonspecific effects are always possible, particularly if
reagents are used at relatively high concentrations. The
I50 values in the soybean cell system for the
effects of the various inhibitors on the oxidative burst were all
within the concentration ranges previously employed in signal
transduction studies in plant or mammalian cells. Furthermore, we can
in some cases rule out blanket effects on cell metabolism in view of
inhibitor specificity. For example, suramin is an effective inhibitor
of the oxidative burst and resulting GST induction, but does not inhibit PAL expression. DPI is not only an inhibitor of the NADPH oxidase, but also inhibits macrophage nitric oxide synthase at concentrations similar to those reported here (Stuehr et al., 1991). Nitric oxide is now known to function as a signal
molecule in plant defense responses (Delledonne et al., 1998; Durner et al., 1998). We cannot therefore formally rule out the possibility that
DPI affects nitric oxide production in soybean cells. However, nitric
oxide, while potentiating induction of downstream defense genes such as
PAL and chalcone synthase, is not an effective inducer of
GST, a gene that is responsive to
H2O2 (Delledonne et al., 1998). Thus, it is unlikely that the effects of DPI on the oxidative burst are mediated via inhibition of NOS.
The only paradoxical results we obtained from the inhibitor studies
concerned the effects of ion channel reagents on GST induction. Amphotericin B appeared unable to induce GST, although this reagent strongly induced the oxidative burst, and GST induction in soybean cells is known to be linked causally to
H2O2 generation (Levine et
al., 1994). We could not evaluate higher concentrations of amphotericin
B in this experiment because of cellular toxicity. Likewise, anthracene
9-carboxylate appeared to increase the level of GST protein in response
to Psg:avrA. Clearly, GST protein levels cannot be viewed
simply as a reporter for active oxygen generation in soybean cells.
The inhibition escape kinetics for herbimycin A, to which the plant
cells are only sensitive within the first 45 min following exposure to
bacteria, indicate that this compound inhibits a signaling event that
is necessary for the avrA-mediated oxidative burst but which
may be upstream of avrA gene product recognition. Similarly, the effectiveness of suramin as an inhibitor of the oxidative burst
diminishes gradually when the compound is added at different times
during the 3-h lag period prior to the onset of the burst. In contrast,
K252a is an effective inhibitor of the burst for a much longer period,
which is consistent with the conclusion that a Ser/Thr kinase cascade
operates downstream of avirulence gene product recognition (Levine et
al., 1994) and has to remain in the "on position" for downstream
defense responses to be expressed (Felix et al., 1991, 1994).
The observation that induction of GST and PAL is differentially
affected by signal transduction inhibitors confirms previous results
indicating that phytoalexin production, which is associated with
increased PAL activity in soybean (Bhattacharyya and Ward, 1986), and
the oxidative burst are regulated via distinct signal transduction
pathways. Although the oxidative burst is necessary for
avrA-dependent phytoalexin accumulation in soybean cells, treatments that induce a strong oxidative burst do not necessarily induce a phytoalexin response (Guo et al., 1998), which is consistent with the previous genetic demonstration that the overall HR and phytoalexin production are not causally linked (Jakobek and Lindgren, 1993). As mentioned above, the differential effects on gene expression also provide an internal control for blanket effects of herbimycin and
suramin, the activities of which have not been described extensively in
plant systems.
Herbimycin A is a potent inhibitor of Tyr phosphorylation and
subsequent downstream events in mammalian cells, and as such has been
widely used as a reagent to demonstrate Tyr kinase involvement in
signaling (Einsphar et al., 1991; Uckun et al., 1991; Asslan et al.,
1998; Riordan et al., 1998). Based on its effects on soybean phosphoproteins, we conclude that herbimycin A also inhibits Tyr kinase
activity in soybean cells. Although the vast majority of studies on
plant protein kinases have dealt with Ser/Thr kinases (Stone and
Walker, 1995), inhibitor studies have suggested a role for Tyr
phosphorylation in auxin transport and ABA-mediated events in plants
(Bernasconi, 1996; Heimovaara-Dijkstra et al., 1996), and several
recent reports have documented the involvement of Tyr-phosphorylated
proteins in plant defense signaling (Suzuki and Shinshi, 1995; Adam et
al., 1997; Ligterink et al., 1997; Stratmann and Ryan, 1997; Zhang and
Klessig, 1998). In each case, the proteins were closely related in
properties to the mammalian MAP kinases, which are phosphorylated on
Tyr and Ser/Thr residues and which can themselves phosphorylate myelin
basic protein as an in vitro substrate. Their natural plant substrates
are not known.
The MAP kinase-related proteins are all 45 to 49 kD, whereas the
Tyr-phosphorylated proteins observed in the present study to be rapidly
induced at least 2.5 h prior to the avrA-mediated oxidative burst were of 19, 20, 28, and 55 kD. These proteins (with the
possible exception of the 20 kD protein, which could be of bacterial
origin) are therefore novel plant phosphoproteins. It must be stressed
that, although our results clearly indicate that herbimycin inhibits
Tyr phosphorylation of proteins in soybean cells, they do not prove
that any of the Tyr-phosphorylated proteins revealed in Figure 9 is
causally involved in regulation of the oxidative burst.
Based on the above observations, we propose the following model for the
early signaling events in the oxidative burst in cultured soybean cells
challenged with avirulent Psg. First, specific nutritional conditions associated with the conditioned plant culture medium that
mimic similar conditions in planta induce expression of the avrA gene. Before or during the transfer of the
avrA gene product to the host cells, recognition of a
bacterial component(s) initiates a signal transduction pathway
involving activation of ion channels and Tyr phosphorylation on one or
more critical proteins. This is followed by coupling of G-proteins to
receptors, which may be directly or indirectly associated with
Tyr-phosphorylated proteins.
These events are essential for the full oxidative burst response
following recognition of the avrA gene product, but not for phenylpropanoid-derived defenses such as the isoflavonoid
phytoalexin response. Thus, Tyr-phosphorylated proteins may function as
upstream components in an avrA gene product reception
process that, assuming avrA product recognition occurs after
45 min post infection in the soybean cell system, is "pre-primed"
by an earlier recognition process. Interestingly, these events find
parallels in mammalian neutrophils, where herbimycin inhibits an early
signaling step for the activation of NADPH oxidase following
stimulation by peptides (Zhang et al., 1998), and Tyr kinase activity
may be necessary for activation of downstream kinases involved in
phosphorylation of the oxidase component p47-phox (Yaname et al.,
1999).
There are two caveats to the above model. First, we cannot exclude the
possibility that the Tyr-phosphorylation events are avrA
gene dependent, even though their timing is suggestive of their
occurring prior to recognition of the avrA gene product in
the host cells. In this regard, we have not been able to demonstrate increased protein Tyr phosphorylation in soybean cv Williams 82 cells
in response to Psg:avrC. Second, we do not know exactly when
the avrA gene product first activates the signal pathway for
the oxidative burst; the above model is based on assumptions supported
by comparisons of the kinetics of the burst in response to bacteria or
isolated elicitors (Guo et al., 1998). The sequence of events proposed
above can only be rigorously tested once reagents become available to
determine directly the exact timing of production of the
avrA gene product and its transfer to or association with the host cells.
| |
FOOTNOTES |
1
This work was supported by The Samuel Roberts
Noble Foundation.
2
Present address: Department of Molecular
Oncology, M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box
317, Houston, TX 77030.
3
Present address: Institute of Cell and Molecular
Biology, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JH,
Scotland, UK.
*
Corresponding author; e-mail rajbvkrj{at}hotmail.com.
Received February 2, 1999;
accepted April 23, 1999.
| |
ABBREVIATIONS |
Abbreviations:
DPI, diphenylene iodonium.
GST, glutathione
S-transferase.
HR, hypersensitive response.
MAP, mitogen-activated protein.
PAL, Phe ammonia-lyase.
Psg, Pseudomonas syringae pv glycinea.
| |
ACKNOWLEDGMENTS |
We thank Dr. K. Kreutz for anti-(GST) serum, Dr. I. Maldonado-Mendoza for the rRNA probe, Cuc Ly for artwork, Drs. Toshiro Shigaki and Ken Shirasu for helpful discussions and sharing unpublished data, and Drs. Madan K. Bhattacharyya and Christopher L. Steele for
critical review of the manuscript.
| |
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Top
Abstract
Introduction