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Evidence that the caterpillar salivary enzyme glucose oxidase provides herbivore offense in solanaceous plants.

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128
Musser et al.
Archives of Insect Biochemistry and Physiology 58:128–137(2005)
Evidence That the Caterpillar Salivary Enzyme
Glucose Oxidase Provides Herbivore Offense in
Solanaceous Plants
Richard O. Musser,1 Don F. Cipollini,2 Sue M. Hum-Musser,1 Spencer A. Williams,1
Judith K. Brown,3 and Gary W. Felton4*
The insect salivary enzyme glucose oxidase (GOX) can inhibit wound-inducible nicotine production in tobacco, Nicotiana tabacum.
We examined whether salivary gland extracts of Helicoverpa zea lacking active GOX could still suppress nicotine in tobacco,
Nicotiana tabacum, and whether GOX could suppress wound-inducible defenses of another Solanaceous plant, tomato
Lycopersicon esculentum. Tobacco leaves were wounded with a cork borer and treated with water, salivary gland extracts with
active GOX (SxG), or salivary gland extracts with inactive GOX (SxI). After three days, leaves treated with SxG had significantly
less nicotine than all other wounded treatments. Neonates that fed on the terminal leaves of tobacco plants treated with SxG
had significantly higher survival than neonates that fed on leaves treated with either SxI or water. This evidence supports the
assertion that GOX is the salivary factor responsible for the suppression of tobacco plant nicotine production by H. zea saliva.
Results for the NahG tobacco plants, which lack salicylic acid (SA) due to a transgene for bacterial SA hydroxylase, indicate
that suppression of nicotine by GOX does not require SA. However, tobacco leaves that were wounded and treated with SxG
had significantly higher levels of the SA-mediated PR-1a protein than leaves treated with SxI or water. Leaves of tomato
plants wounded with scissors and then treated with SxG had trypsin inhibitor levels that were moderately lower than plants
wounded and treated with purified GOX, water, or SxI. However, all the wounded tomato leaves irrespective of treatment
resulted in lower caterpillar growth rates than the non-wounded tomato leaves. Glucose oxidase is the first insect salivary
enzyme shown to suppress wound-inducible herbivore defenses of plants. Arch. Insect Biochem. Physiol. 58:128–137,
2005. © 2005 Wiley-Liss, Inc.
KEYWORDS: Helicoverpa zea; reactive oxygen species; induced resistance; hydrogen peroxide; oxidative burst;
elicitor; systemic acquired resistance; glucose oxidase; jasmonate; salicylate
INTRODUCTION
Emerging evidence indicates that herbivore oral
secretions are important mediators of inducible
defenses of plants (Musser et al., 2002a,b, 2004;
Alborn et al., 1997, 2000; Spiteller et al., 2000;
McCloud and Baldwin, 1997; Mattiacci et al., 1995;
1
Department of Biological Sciences, Western Illinois University, Macomb
2
Department of Biological Sciences, Wright State University, Dayton, Ohio
3
Department of Plant Science, Center of Insect Science, University of Arizona, Tucson
4
Department of Entomology, Pennsylvania State University, University Park
Na and Chenzhu, 2004; Voelckel and Baldwin,
2004). Most studies on this subject have reported
the identity and effects of oral secretions that act
as elicitors of plant anti-herbivore defenses. These
include β-glucosidase from the saliva of Pieris
brassicae (Linnaeus), and the fatty acid-amino acid
conjugates identified in various lepidopteran lar-
Paper presented at the 51st Annual Meeting of the Entomological Society of America, October 2003. Symposium entitled Insect Saliva: An Integrative Approach.
Contract grant sponsor: Illinois Department of Agriculture; Contract grant sponsor: University Research Council, Western Illinois University; Contract grant sponsor:
Center for Insect Science, University of Arizona.
*Correspondence to: Gary W. Felton, Department of Entomology, Pennsylvania State University, University Park, PA 16802. E-mail: gwf10@psu.edu
© 2005 Wiley-Liss, Inc.
DOI: 10.1002/arch.20039
Published online in Wiley InterScience (www.interscience.wiley.com)
Archives of Insect Biochemistry and Physiology
Caterpillar Saliva Provides Herbivore Offense
vae (Mattiacci et al., 1995; Alborn et al., 1997;
McCloud and Baldwin, 1997; Alborn et al., 2000;
Spiteller et al., 2000). Only recently has it been
demonstrated that herbivore saliva can suppress
wound-induced anti-herbivore defenses of their
host plants in a manner analogous to that of saliva from blood-feeding arthropods suppressing the
defenses of their animal host (Musser et al., 2002a,
2004; Kahl et al., 2000; Na and Chenzhu, 2004).
Glucose oxidase (GOX), a salivary enzyme from
the caterpillar Helicoverpa zea (Boddie), was the first
characterized salivary suppressor of nicotine, an
inducible anti-herbivore defense of tobacco (Musser et al., 2002a; Musser et al., 2004; Na and
Chenzhu, 2004). Before this role of GOX was identified, most researchers studying inducible plant
defenses had viewed herbivores as passive victims
of plant responses.
Ribonuclease found in the regurgitant of the
Mexican bean beetle, Epilachna varivestis (Mulsant),
was recently shown to stimulate plant defenses
against viruses that are vectored by this beetle
(Musser et al., 2002b). Glucose oxidase, while inhibiting some plant defense responses, has also
been suggested to be an elicitor of plant pathogen
defenses. Soybean plants that were mechanically
wounded and treated with GOX had a reduction
in the severity of disease caused by the bacterium,
Pseudomonas syringae pv. glycinea, and had higher
levels of the phytoalexin daidzein (a marker of salicylic acid-mediated systemic acquired resistance)
than plants that were mechanically wounded and
treated with a buffer (Felton and Eichenseer, 1999).
Inhibitory signal cross talk can exist between insect- and pathogen-inducible defenses in plants
(Felton and Korth, 2001). Indeed, the induction
of salicylic acid-mediated pathogen defenses by
GOX may explain why it can suppress some
wound- and herbivore-inducible defense responses.
Regurgitant from M. sexta was shown to inhibit
wound-induced increases in nicotine in wild tobacco plants, Nicotiana sylvestris, despite increasing
levels of the wound signal, jasmonic acid (McCloud and Baldwin, 1997; Kahl et al., 2000). This
effect was apparently caused by increased ethylene
levels induced by oral secretions of M. sexta that
February 2005
129
attenuated wound-inducible nicotine production
(Kahl et al., 2000; Voelckel et al., 2001; Winz and
Baldwin, 2001; Baldwin, 2001). Specific components of herbivore saliva that mediate such effects
have not been well characterized (Felton and
Eichenseer, 1999; Ribeiro, 1995; Mattiacci et al.,
1995; Alborn et al., 1997, 2000; Voelckel et al.
2001; Baldwin, 2001; Winz and Baldwin, 2001).
Here we provide additional evidence that GOX
is a primary salivary protein from the caterpillar,
H. zea, that suppresses the induction of nicotine
in wounded tobacco leaves and we demonstrate
that this effect does not require salicylic acid. In
addition, we demonstrate that labial saliva may alter trypsin inhibitor levels, an inducible plant defense in tomato and tobacco plants. Glucose
oxidase converts D-glucose and oxygen to D-gluconic acid and hydrogen peroxide (H2O2). Because
GOX is commonly occurring among caterpillar species (Felton and Eichenseer, 1999), we suggest that
our findings reveal a new facet in the co-evolutionary struggle between plants and their insect herbivores.
MATERIALS AND METHODS
Plant Maintenance
Tobacco (N. tabacum) seeds were sown in 4-L
plastic pots filled with a Redi Earth Peat-Lite soil
mixture (Scott-Sierra Horticulture Products Company, Marysville, OH) in a greenhouse at the University of Arkansas, Fayetteville, Arkansas. The
greenhouse was maintained on a 14-h photoperiod, with high-pressure sodium lights (1,000 W)
and day and night temperatures between 33° and
25°C. The plants were watered at least every two
days (or as required) and fertilized (N:P:K =
15:30:15, American Plant Food Co., Creve Coeur,
MO) weekly. Tobacco plants were used in bioassays when they were approximately 8 weeks old
and 0.33 meters tall.
Tomato (Lycopersicon esculentum L.) seeds were
sown in 4-L plastic pots containing Redi Earth PeatLite Mix soil mixture. The plants were grown in a
greenhouse for 4 weeks at the University of Ari-
130
Musser et al.
zona, Tucson, AZ, with a 14-h photoperiod, using
natural light conditions, and day and night temperatures between 33° and 25°C. Plants were watered daily and fertilized weekly as above.
under 33 µM/mg/min for the autoclaved extracts.
These preparations were immediately used in experiments. When necessary, commercial GOX
(Sigma, St. Louis, MO) was used for follow-up experiments.
Insect Rearing and Labial Gland Extraction
Neonates of H. zea caterpillars were reared on
a wheat germ and soy protein-based artificial diet
at 28°C on a 15-h photoperiod until the 6th instar (Chippendale, 1970; Broadway and Duffey,
1986). Intact labial salivary glands were removed
from actively feeding 2-day-old 6th instar H. zea
and stored in chilled distilled water in 1.5 mL micro-centrifuge tubes as described by Eichenseer et
al. (1999). After collection, the salivary glands were
frozen at –80°C until use.
Purification of Labial Salivary Gland
Glucose Oxidase
Glucose oxidase was purified from the labial
salivary glands according to Eichenseer et al.
(1999). Approximately 500 pairs of labial glands
were used for GOX purification. Following enzyme
purification with a Bio-Rad Rotofor system (BioRad Laboratories, Inc., Richmond, CA), ampholytes
were removed from the sample with a 10,000 Molecular Weight Cut-off concentrator. Half of the collected purified GOX was autoclaved to inactivate
the enzyme and the other half was kept active. In
the following experiment, the inactive GOX was
added back to half of the salivary gland extract
from which the GOX had been extracted. Active
GOX was added back to the remaining half of the
salivary gland extract from which GOX had been
extracted. This provided salivary gland extract with
active GOX and salivary gland extract with inactive GOX. Glucose oxidase activity was compared
between these two preparations to ensure activity
was present only in the sample with active GOX.
Glucose oxidase activity was measured by the
change of absorbance of the reaction mixture at
460 nm as described by Eichenseer et al. (1999).
The activity was found to be over 1,684 µM/mg/
min for the preparation containing active GOX and
Role of Active Salivary GOX in Wound-Induced
Responses of Tobacco
To determine the potential role of salivary GOX
in induced herbivore defenses, one leaf per plant
was damaged with a 1.5-cm-diameter cork borer
to simulate insect damage. Six holes were uniformly distributed on the second uppermost fully
expanded leaf with the exception of the nonwounded control. Wounds on individual leaves
were treated with one of three treatments: (1) salivary gland extract with active GOX, (2) salivary
gland extract with inactive GOX, or (3) water.
Wounds that were treated with salivary gland extract received approximately 30 µg of protein, as
determined with the Bradford (1976) method, in
10 µl of their respective solution. After three days,
the wounded leaf was harvested and flash-frozen
in liquid nitrogen, the leaves were homogenized
and extracted in a methanol-water buffer for 24 h,
then analyzed for nicotine with HPLC following
the method described in Saunders and Blume
(1983). There were 8 replicate plants per treatment,
and the mean nicotine concentration was compared among treatments using a one-way ANOVA
with JMP (Sall and Lehman, 1996), followed by a
Tukey-Kramer Honestly Significant Difference
(HSD) test for separation of the means.
In order to assay for the presence of pathogenesis related protein-1a (PR-1a), leaf samples from
each treatment were pooled together into four replications and analyzed by Western blot for the
pathogenesis-related PR-1a. Total soluble protein
was extracted from tobacco leaves following the
protocol of Conrad and Fiedler (1998). Protein was
quantified according to the protocol of Bradford
(1976) using a microplate reader (Cambridge
7520, Cambridge, MA). Protein was mobilized with
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a 4% stacking gel and
Archives of Insect Biochemistry and Physiology
Caterpillar Saliva Provides Herbivore Offense
12% resolving gel in Tris-Glycine-SDS buffer (BioRad, Hercules, CA) and blotted onto polyvinylidene fluoride membrane (BioTrace™ PVDF, PALL
Gelman Laboratory, Ann Arbor, MI). Western blotting was performed according Sheng and Schuster
(1992). The blot was probed with anti-PR-1a diluted 1:1,000 obtained from Dr. Yinong Yang, University of Arkansas. The blot was also probed with
antibody and horseradish peroxidase conjugates
and Supersignal West Pico solutions (Pierce, Rockford, IL). Bands were digitally visualized and quantified with a Fujifilm LAS-1000 Intelligent Dark Box
II Image Reader (Fujifilm, Edison, NJ).
In addition, leaf samples from each treatment
were pooled together into four replications and
analyzed by Western blot for the pathogenesis related protein-1a (PR-1a). Band intensity was analyzed with a scanner, and mean band intensities
were compared among treatments with a one-way
ANOVA with JMP (Sall and Lehman, 1996), followed by a Tukey-Kramer Honestly Significant Difference (HSD) test for means separation.
In addition, the terminal top of each treated
tobacco plant was removed and the stem was
placed into a 25-mL medicine cup filled with distilled water. Twenty neonate H. zea were placed
onto each plant terminal and allowed to feed for
one week, and survival was recorded. Survival of
neonates was compared among treatments using
logistic regression.
Requirement for Salicylate in the Suppression of
Nicotine by GOX
A transgenic line of tobacco plants expressing
bacterial SA hydroxylase (NahG) that suppresses SA
levels in planta was used in this experiment (Gaffney
et al., 1993; Felton et al., 1999). We have shown
that surgery can be performed on caterpillars to
block labial salivary secretions and specifically GOX
secretion (Musser et al., 2002a, 2004, 2005), which
were still capable of feeding. To examine the role
of salicylate in the suppression of wound-induced
increases in nicotine levels by GOX, caterpillars
with intact or ablated salivary glands were caged
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131
onto the NahG tobacco leaves in a manner described by Musser et al. (2002a, 2004). Three days
later, the wounded tobacco leaves were collected
and analyzed for nicotine as described above. There
were four replicates of each treatment. Means were
compared among treatments, as described above.
Effect of GOX on Wound-Inducible Defenses
in Tomato
In this experiment, we examined the growth of
H. zea caterpillars on excised leaves from nonwounded tomato plants or excised leaves from
wounded plants treated with: water, labial salivary
extract (with active GOX), purified GOX, or autoclaved salivary extract (with inactive GOX). Each
tomato plant was mechanically wounded by clipping the distal half of three leaflets on three of the
upper most fully expanded compound leaves. The
wounded leaves were then dipped into either a 1
mg protein per mL solution (protein concentration was analyzed with Bradford assay) of their respective treatment, water, or were untreated. We
have determined that H. zea has proteases in the
saliva. To prevent addition of salivary proteases in
the trypsin inhibitor (TI) assay, immediately before harvesting tomato leaves for the chemical assay the wounded edge was clipped a second time
to remove the treated edge. The first set of leaves
were harvested 4 days post treatment and analyzed
for TI and fed to neonate caterpillars. After 6 days,
the caterpillars were weighed. The caterpillars were
then offered a "fresh" set of leaves from plants
treated in the same manner, but the leaves were
excised from plants that were wounded with scissors and treated 10 days prior to excision. After 4
more days of caterpillar feeding, for a total of 10
days, the caterpillars were weighed a final time. The
mean weight of 20 caterpillars was determined for
each treatment and analyzed with a Tukey-Kramer
Honestly Significant Difference (HSD) test in a oneway ANOVA with JMP (Sall and Lehman, 1996).
Trypsin Inhibitor Assay
Trypsin inhibitors are anti-nutritive proteins that
competitively inhibit digestive serine proteases of
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Musser et al.
TABLE 1. Suppression of Wound-Induced Nicotine Production and Induced Resistance by Salivary Gland
Extract With Active GOX*
Tobacco leaf treatment
Salivary gland extract
Plant response
Nicotine (mg/g leaf)
% Neonate survival
Water
0.79 ± 0.06*a
29 ± 5b
Inactive GOX
Active GOX
N.W.
0.80 ± 0.05a
29 ± 3b
0.56 ± 0.04b
42 ± 4a
0.48 ± 0.04b
48 ± 4a
*Values represent standard effor. Different letters represent statistically significant differences between treatments at P
< 0.05. N.W. = non-wounded.
RESULTS
active GOX had significantly lower levels of nicotine than wounded leaves treated with either salivary gland extract with inactive GOX or water
(Table 1). Neonates that fed on tops of either nonwounded plants or plants treated with salivary
gland extract with active GOX had significantly
higher survival than those that fed on wounded
plants treated with either salivary gland extract with
inactive GOX or water (Table 1). In addition,
leaves treated with salivary gland extract with active GOX had PR-1a levels that were at least 2.5
times higher than wounded leaves in any other
treatment (Fig. 1).
The Role of Active GOX in the Suppression of WoundInduced Defense Responses in Tobacco
The Requirement of Salicylate for the Suppression of
Wound-Induced Defense Responses by GOX
Non-wounded leaves and mechanically wounded
leaves treated with labial salivary gland extract with
Leaves of transgenic NahG tobacco plants damaged by caterpillars with intact labial salivary glands
Fig. 1. Salivary gland extracts with active GOX increase
PR-1a levels in wounded tobacco leaves. Tobacco leaves
were mechanically damaged and treated with water, salivary gland extracts with inactive GOX, or salivary gland
extracts with active GOX. A Western blot for PR-1a shows
that leaves treated with salivary gland extracts with active
GOX had at least a 2.5 times higher level of PR-1a protein than non-wounded leaves or leaves treated with water or salivary gland extracts with inactive GOX.
animals and other organisms. Trypsin inhibitors
have been shown to be inducible by wounding,
insect feeding, and jasmonic acid in several plant
species, including tomato (Thaler et al. 1999;
Cipollini and Bergelson, 2000, 2001). The TI content of treated tomato leaves was determined by examining the radial diffusion of soluble protein
extracts through a trypsin-containing agar as in
Cipollini and Bergelson (2000). The mean TI content per mg extract protein was expressed as ng treatments as in the first experiment.
Archives of Insect Biochemistry and Physiology
Caterpillar Saliva Provides Herbivore Offense
133
Fig. 2. Average fresh weight of Helicoverpa zea caterpillars after feeding on excised leaves from non-wounded tomato plants or excised leaves from wounded tomato plants
treated with water, labial salivary extract with GOX, purified GOX, or autoclaved salivary extract. Different letters
represent statistically significant differences (TukeyKramer) between treatments at P < 0.05. The mean weight
of 20 caterpillars was determined for each treatment. Bars
indicate mean ± s.e.
had significantly lower levels of nicotine than
leaves damaged by caterpillars with ablated salivary glands. Mean nicotine concentration for leaves
fed on by caterpillars with ablated salivary glands
and mock treated caterpillars was 0.25 ± 0.01 mg
nicotine/g of leaf and 0.15 ± 0.02 mg nicotine/g
of leaf, respectively. The difference in nicotine levels in leaves with and without the application of
GOX was similar to results observed in nontransgenic tobacco with normal levels of salicylic
acid (Musser et al., 2002a, 2004).
treated with purified GOX, labial salivary extract
with GOX, water, or autoclaved salivary extract were
significantly higher than the non-treated plants.
Trypsin inhibitor levels in plants treated with salivary extract with active GOX were moderately lower
than levels in wounded plants treated with purified
GOX water or autoclaved salivary extract (Fig. 3).
The Effect of GOX on Wound-Induced Defense
Responses of Tomato
Caterpillar weights were not drastically different when weighed on day 6, but caterpillars that
fed on non-wounded plants had the highest body
weights. However, after 10 days of feeding, caterpillars that fed on non-wounded tomato leaves
were double the weight of caterpillars that fed on
leaves from wounded plants treated with water,
purified GOX, labial salivary extract with GOX, or
autoclaved salivary extract (Fig. 2). In addition, effects of treatments on larval growth appear associated with trypsin inhibitor levels in treated tomato
plants. Trypsin inhibitor levels in wounded plants
February 2005
DISCUSSION
It is well documented that salivary proteins of
blood-feeding arthropods circumvent the defenses
of their vertebrate hosts (Ribeiro, 1995). In stark
contrast, the saliva of phytophagous, chewing insects has been virtually ignored as an element involved in overcoming their host plant defenses
(Felton and Eichenseer, 1999; Ribeiro, 1995).
Musser et al. (2002a, 2004) characterized glucose
oxidase (GOX) as the first insect-derived suppressor
of host plant defenses. The salivary enzyme GOX
can suppress nicotine, an inducible anti-herbivore
defense of the host tobacco plant (Musser et al.,
2002a, 2004; Na and Chenzhu, 2004). In our previous studies, we demonstrated that mechanically
wounded tobacco leaves treated with purified active GOX or leaves fed on by caterpillars with normal salivary secretion had lower levels of nicotine
134
Musser et al.
Fig. 3. Measurement of trypsin inhibitors, an inducible
plant defense. Trypsin inhibition was measured in excised
tomato leaves from previously mechanically wounded tomato plants treated with: labial salivary gland extract with
glucose oxidase, purified glucose oxidase, autoclaved sali-
vary extract, or water. Trypsin inhibitor (TI) content of
extracts was expressed as µg TI per mg leaf protein extract.
Different letters represent statistically significant differences
(Tukey-Kramer) between treatments at P < 0.05. Bars indicate mean ± s.e.
than tobacco plants treated with inactive GOX or
fed on by caterpillars with blocked salivary secretions. Furthermore, Na and Chenzhu (2004)
showed that the noctuid caterpillars H. armigera
(Hübner) and H. assulta (Guenee) with GOX
present in their salivary glands dramatically suppressed the induction of the nicotine in tobacco
plants compared to noctuid caterpillar Spodoptera
litura that had no detectable levels of GOX in their
salivary glands.
In this current study, we confirm that GOX appears to be the primary labial salivary enzyme responsible for this suppression of nicotine, and that
active GOX is necessary for this response. Tobacco
plants that were mechanically wounded and treated
with salivary glands extracts with active GOX had
significantly less nicotine than tobacco leaves that
were wounded and treated with salivary gland extracts containing inactive GOX (autoclaved) or water. No significant difference was observed in
nicotine levels between mechanically wounded tobacco treated with salivary gland extract with inactive GOX or water, indicating that the other active
components of the labial salivary extract have little
effect on levels of nicotine. Furthermore, neonates
of H. zea that subsequently consumed leaves from
plants treated with salivary gland extracts having
active GOX exhibited higher rates of survival than
neonates that consumed leaves treated with either
water or salivary gland extracts with inactive GOX.
Glucose oxidase may inhibit the induction of
nicotine via several possible mechanisms including direct inhibition of the wound signal jasmonic
acid and/or through antagonistic cross talk from
other signal pathways. Musser et al. (2002a) demonstrated that the by-products of GOX, hydrogen
peroxide (H2O2), and gluconic acid appeared to be
responsible for the suppression of the inducible
nicotine levels in wounded tobacco. Hydrogen peroxide is known to activate WIPK, a MAP-kinase involved in the regulation of systemic acquired
resistance (SAR) to pathogens in tobacco (Romeis
et al., 1999; Seo et al., 1995). Expression of a fungal GOX in potato plants triggers systemic resistance to bacterial and fungal pathogens (Wu et al.,
1995). In some cases, SAR inhibits the expression
of jasmonate-dependent induced resistance to insect herbivores (Felton et al., 1999; Preston et al.,
1999; Felton and Korth, 2000; Cipollini et al.,
2004). Salicylic acid (SA) is an important component of SAR (Gaffney et al., 1993). A potential
mechanism for the suppression of nicotine by GOX
is through the induction of SA by H2O2 , a by-product of GOX activity (Bi et al., 1995; Wu et al., 1995;
Alvarez et al., 1998; Chamnongpol et al., 1998).
Salicylic acid has been shown to suppress increases
Archives of Insect Biochemistry and Physiology
Caterpillar Saliva Provides Herbivore Offense
in either jasmonic acid levels or jasmonate-mediated defenses in several plant species, including tobacco and tomato (Felton and Korth, 2000; Preston
et al., 1999; Felton et al., 1999; Thaler et al., 1999;
Cipollini et al. 2004). Induction of nicotine by
wounding in tobacco is mediated by jasmonate
(Kahl et al., 2000; Baldwin et al. 1994; Baldwin et
al., 1997). However, our findings show that caterpillars with intact salivary glands, with normal levels of salivary GOX, suppressed the wound-induced
production of nicotine in salicylate-deficient NahG
tobacco plants just as well as on non-transgenic
tobacco plants (Musser et al., 2002a, 2004). Thus,
GOX suppression of nicotine does not appear to
be integrally mediated by SA.
However, it appears that SA levels or responses
are stimulated by GOX. For example, salicylic acid
is known to stimulate the synthesis of pathogenesis-related proteins in tobacco (Bi et al., 1995).
Wounded leaves treated with salivary gland extracts
with GOX had significantly higher levels of the salicylate-mediated PR-1a than non-wounded leaves
or wounded leaves treated with water or salivary
gland extract with autoclaved GOX.
Alternatively, the suppression of nicotine by
GOX could be mediated by ethylene biosynthesis.
Hydrogen peroxide can stimulate ethylene (Chamnongpol et al., 1998) and ethylene was recently
found to suppress nicotine induction in N. sylvestris
(Kahl et al., 2000; Voelckel et al., 2001a; Baldwin,
2001; Winz and Baldwin, 2001). Additional studies will be required to determine if GOX alters the
levels of plant stress-related compounds or plant
hormones, including ethylene.
Tomato plants wounded and treated with salivary gland extract (with active GOX) had moderately lower levels of trypsin inhibitor compared to
plants wounded and treated with purified GOX,
water, or autoclaved salivary gland extract (with
inactive GOX). However, irrespective of treatment
wounded tomato plants had significantly higher levels of trypsin inhibitor and lower caterpillar growth
rates than non-wounded plants. Our findings suggest that GOX does not appear to markedly alter
trypsin inhibitor levels in our experimental system.
February 2005
135
Orozco-Cárdenas et al. (2001) showed that GOX
in the presence of glucose increased protease inhibitors due to hydrogen peroxide production,
while GOX in the absence of glucose increased protease inhibitor levels over non-wounded control
plants. Their experiments were conducted quite differently from those in our experiments. For instance, we did not treat the entire plant with
peroxide by cutting the main stem and placing the
plant in a solution of GOX. Also, we did not add
additional glucose to stimulate further peroxide
formation. It is quite possible that there may be
dose-dependent effects of hydrogen peroxide. Further studies are warranted to determine the role of
saliva, GOX, and hydrogen peroxide as signaling
components in tomato.
In addition to the results reported here, we have
obtained evidence that caterpillars’ labial salivary
gland extracts and GOX may be involved in the
suppression of the pathogenocity of bacterial
pathogens (Musser et al., 2005). Because GOX occurs ubiquitously in Lepidopteran insects (Felton
and Eichenseer, 1999), these findings may have
broader significance towards understanding the coevolutionary struggle between insect herbivores and
their host plants.
ACKNOWLEDGMENTS
We thank the Illinois Department of Agriculture and Mike Rahe for funding through a Sustainable Agriculture Grant. We acknowledge Dr. Vicki
L. Chandler at the University of Arizona for providing space in her lab for some of the experiments
performed, and we thank the students, faculty, and
staff at Western Illinois University for their support. We are grateful to Dr. Yinong Yang at the University of Arkansas for providing PR-1a anti-body
used in these experiments and the faculty and staff
at the University of Arkansas and Wright State University for their support on this project.
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