Evidence that the caterpillar salivary enzyme glucose oxidase provides herbivore offense in solanaceous plants.код для вставкиСкачать
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: email@example.com © 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 February 2005 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 132 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. LITERATURE CITED Alborn HT, Turlings TCJ, Jones TH, Stenhagen G, Loughrin JH, Tumlinson JH. 1997. An elicitor of plant volatiles from beet armyworm oral secretion. Science 276:945–949. 136 Musser et al. Alborn HT, Jones TH, Stenhagen GS, Tumlinson JH. 2000. Identification and synthesis of volicitin and related components from beet armyworm oral secretions. J Chem Ecol 26:203–220. 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